Updated on 2024/12/12

写真a

 
ITOH Kimiko
 
Organization
Academic Assembly Institute of Science and Technology NOUGAKU KEIRETSU Professor
Faculty of Agriculture Department of Agriculture Professor
Title
Professor
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Degree

  • Ph.D (Science) ( 1994.8   Toho University )

Research Areas

  • Life Science / Applied biochemistry  / Applied GlycoScience

  • Life Science / Food sciences  / Starch Science

  • Life Science / Applied molecular and cellular biology

  • Life Science / Plant molecular biology and physiology

Research History (researchmap)

  • Niigata University   Faculty of Agriculture Department of Agriculture   Professor

    2017.4

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  • Niigata University   Faculty of Agriculture Department of Applied Biological Chemistry Applied Biological Chemistry   Professor

    2016.4 - 2017.3

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  • Niigata University   Graduate School of Science and Technology Life and Food Sciences   Associate Professor

    2004.4 - 2016.3

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  • Niigata University   Faculty of Agriculture Department of Applied Biological Chemistry   Associate Professor

    2004.4 - 2016.3

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  • Niigata University   Faculty of Agriculture   Associate Professor (as old post name)

    1999.7 - 2000.3

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  • オーストラリア科学産業機構(CSIRO)   文部科学省在外研究員

    1998.11 - 1998.12

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    Country:Australia

    Notes:高等植物における遺伝子不活性化のメカニズムとその発生における役割の研究

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  • Niigata University   Faculty of Agriculture   Assistant

    1995.10 - 1999.6

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  • Mitsubishi Chemical Cooperation Plantech Research Institute

    1986.10 - 1995.9

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Research History

  • Niigata University   Faculty of Agriculture Department of Agriculture   Professor

    2017.4

  • Niigata University   Abolition organization Applied Biological Chemistry   Professor

    2016.4 - 2017.3

  • Niigata University   Graduate School of Science and Technology Life and Food Sciences   Associate Professor

    2004.4 - 2016.3

  • Niigata University   Faculty of Agriculture Department of Applied Biological Chemistry   Associate Professor

    2004.4 - 2016.3

  • Niigata University   Faculty of Agriculture   Associate Professor (as old post name)

    1999.7 - 2000.3

  • Niigata University   Faculty of Agriculture   Research Assistant

    1995.1 - 1999.6

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Professional Memberships

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Committee Memberships

  • 一般社団法人 日本応用糖質科学会   評議員  

    2021.8 - 2023.9   

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    Committee type:Academic society

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  • 日本応用糖質科学会   編集委員  

    2010.7   

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    Committee type:Academic society

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  • 日本植物生理学会   評議委員  

    2006.1 - 2009.12   

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    Committee type:Academic society

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  • 日本植物生理学会   2005年度 第46回年会 大会実行委員  

    2004.3 - 2005.7   

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    Committee type:Academic society

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Qualification acquired

  • Work Environment Measurement Person (first and second kind)

 

Papers

  • Genome-wide characterization and expression analysis of cyclic nucleotide-gated ion channels (CNGCs) gene family in Arabidopsis thaliana L. and Helianthus annuus L. for drought stress

    Sadaf Oranab, Hafiz Muhammad Ahmad, Sajid Fiaz, Athar Hussain, Muhammad Rizwan, Shazia Arif, Saira Ishaq, Shahnaz Zakia, Asmaa M. Abushady, Itoh Kimiko, Kotb A. Attia

    Brazilian Journal of Botany   47 ( 3 )   885 - 900   2024.9

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    Publishing type:Research paper (scientific journal)   Publisher:Springer Science and Business Media LLC  

    DOI: 10.1007/s40415-023-00957-x

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    Other Link: https://link.springer.com/article/10.1007/s40415-023-00957-x/fulltext.html

  • Identification of abiotic stress responsive genes: a genome wide analysis of the cytokinin response regulator gene family in rice

    Setu Rani Saha, S. M. Shahinul Islam, Kimiko Itoh

    Genes & Genetic Systems   2024.6

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    Authorship:Corresponding author   Publishing type:Research paper (scientific journal)   Publisher:Genetics Society of Japan  

    DOI: 10.1266/ggs.24-00068

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  • Comprehensive insights into the regulatory mechanisms of lncrna in alkaline-salt stress tolerance in rice. International journal

    Obaid Ur Rehman, Muhammad Uzair, Muhammad Shahbaz Farooq, Bilal Saleem, Safira Attacha, Kotb A Attia, Umer Farooq, Sajid Fiaz, Wael H El-Kallawy, Itoh Kimiko, Muhammad Ramzan Khan

    Molecular biology reports   2023.7

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    Language:English   Publishing type:Research paper (scientific journal)  

    BACKGROUND: Alkaline-salt is one of the abiotic stresses that slows plant growth and developmental processes and threatens crop yield. Long non-coding RNAs (lncRNAs) are endogenous RNA found in plants that engage in a variety of cellular functions and stress responses. METHOD: lncRNAs act as competing endogenous RNAs (ceRNA) and constitute a new set of gene control. The precise regulatory mechanism by which lncRNAs function as ceRNAs in response to alkaline-salt stress remains unclear. We identified alkaline-salt responsive lncRNAs using transcriptome-wide analysis of two varieties including alkaline-salt tolerant [WD20342 (WD)] and alkaline-salt sensitive [Caidao (CD)] rice cultivar under control and alkaline-salt stress treated [WD20342 (WDT, and Caidao (CDT)] conditions. RESULTS: Investigating the competitive relationships between mRNAs and lncRNAs, we next built a ceRNA network involving lncRNAs based on the ceRNA hypothesis. Expression profiles revealed that a total of 65, 34, and 1549 differentially expressed (DE) lncRNAs, miRNAs, and mRNAs were identified in alkaline-salt tolerant WD (Control) vs. WDT (Treated). Similarly, 75 DE-lncRNAs, 34 DE-miRNAs, and 1725 DE-mRNAs (including up-regulated and down-regulated) were identified in alkaline-salt sensitive CD (Control) vs. CDT (Treated), respectively. An alkaline-salt stress ceRNA network discovered 321 lncRNA-miRNA-mRNA triplets in CD and CDT, with 32 lncRNAs, 121 miRNAs, and 111 mRNAs. Likewise, 217 lncRNA-miRNA-mRNA triplets in WD and WDT revealed the NONOSAT000455-osa_miR5809b-LOC_Os11g01210 triplet with the highest degree as a hub node with the most significant positive correlation in alkaline-salt stress response. CONCLUSION: The results of our investigation indicate that osa-miR5809b is dysregulated and plays a part in regulating the defense response of rice against alkaline-salt stress. Our study highlights the regulatory functions of lncRNAs acting as ceRNAs in the mechanisms underlying alkaline-salt resistance in rice.

    DOI: 10.1007/s11033-023-08648-2

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  • Evaluation of sugarcane promising clones based on the morphophysiological traits developed from fuzz. International journal

    Bilal Saleem, Muhammad Uzair, Muhammad Noman, Kotb A Attia, Muqing Zhang, Mona S Alwahaibi, Nageen Zahra, Muhammad Kashif Naeem, Arif A Mohammed, Sajid Fiaz, Itoh Kimiko, Muhammad Ramzan Khan

    PeerJ   11   e15646   2023.7

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    Sugarcane is one of the critical commercial crops and principal sources of ethanol and sugar worldwide. Unfavorable conditions and poor seed setting rates hinder variety development in sugarcane. Countries like Pakistan directly import fuzz (true seed) and other propagation material from the USA, China, Brazil, etc. In this study, we imported fuzz from China, developed 29 genotypes germinating in the glasshouse, and evaluated at field conditions along with two local checks (CPF-251 and HSF-240). Morphophysiological data were recorded, including plant height (PH), cane length (CL), internodal length (IL), tiller number (TN), brix percentage (B), cane diameter (CD), chlorophyll a (Chl. a), chlorophyll b (Chl. b), and total chlorophyll (T. Chl). Results showed highly significant (p < 0.001) differences among the sugarcane accessions for all the studied traits. High broad-sense heritability (81.89% to 99.91%) was recorded for all the studied parameters. Genetic Advance (GA) ranges from 4.6% to 65.32%. The highest GA was observed for PH (65.32%), followed by CL (63.28%). Chlorophyll leaching assay was also performed at different time points (0, 50, 100, 150, and 200 min). All the genotypes showed the same leaching trend at all times, and better performing genotypes showed less leaching compared to poor performing, indicating the high amount of cutin and wax on the leaf surface. Correlation analysis showed that PH, CL, IL, and TN had significant associations. Principal components analysis (PCA) further confirms these results. Based on PCA and correlation results, PH, CL, IL, and TN can be utilized as a selection criterion for sugarcane improvement. Genotypes such as NS-4a, NS-5, NS-6, NS-8, NS-9, and NS-15 are recommended for future breeding programs related to sugarcane variety development.

    DOI: 10.7717/peerj.15646

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  • Evaluation of functional kompetitive allele-specific PCR (KASP) markers for selection of drought-tolerant wheat (Triticum aestivum) genotypes. International journal

    Marya Rubab, Summiya Jannat, Haytham Freeg, Hina Abbas, Kotb A Attia, Sajid Fiaz, Nageen Zahra, Muhammad Uzair, Safeena Inam, Asad Hussain Shah, Itoh Kimiko, Muhammad Kashif Naeem, Muhammad Ramzan Khan

    Functional plant biology : FPB   2023.6

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    Wheat (Triticum aestivum) is a major crop around the globe and different techniques are being used for its productivity enhancement. Germplasm evaluation to improve crop productivity mainly depends on accurate phenotyping and selection of genotypes with a high frequency of superior alleles related to the trait of interest. Therefore, applying functional kompetitive allele-specific PCR (KASP) markers for drought-related genes is essential to characterise the genotypes for developing future climate-resilient wheat crop. In this study, eight functional KASP markers and nine morphological traits were employed to evaluate the 40 wheat genotypes for drought tolerance. Morphological traits showed significant variation (P≤0.05) among the genotypes, except tiller count (TC), fresh root weight (FRW) and dry root weight (DRW). PCA biplot showed that 63.3% phenotypic variation was explained by the first two PCs under control treatment, while 70.8% variation was explained under drought treatment. It also indicated that root length (RL) and primary root (PR) have considerable variations among the genotypes under both treatments and are positively associated with each other. Hence, the findings of this study suggested that both these traits could be used as a selection criterion to classify the drought-tolerant wheat genotypes. KASP genotyping accompanied by morphological data revealed that genotypes Markaz, Bhakar Star, China 2, Aas and Chakwal-50 performed better under drought stress. These outperforming genotypes could be used as parents in developing drought-tolerant wheat genotypes. Hence, KASP genotyping assay for functional genes or significant haplotypes and phenotypic evaluation are prerequisites for a modern breeding program.

    DOI: 10.1071/FP23032

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  • Genetic characterization of coarse and basmati rice (Oryza sativa L.) through microsatellite markers and morpho-agronomic traits

    Tahira Luqman, Zia-ul Qamar, Aqsa Tabasum, Wael. H. El-Kallawy, Talha Nazir, Safira Attacha, Sajid Fiaz, Muhammad Azhar Nadeem, Amjad Hameed, Zahra Maryum, Itoh Kimiko, Kotb Attia

    Genetic Resources and Crop Evolution   2023.6

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    Publishing type:Research paper (scientific journal)   Publisher:Springer Science and Business Media LLC  

    DOI: 10.1007/s10722-023-01620-w

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    Other Link: https://link.springer.com/article/10.1007/s10722-023-01620-w/fulltext.html

  • Sero-survey of bovine herpes virus-1 in dromedary camels and associated risk factors. International journal

    Abdelfattah Selim, Salma Shoulah, Roua A Alsubki, Fatima M Albohairy, Kotb A Attia, Itoh Kimiko

    BMC veterinary research   18 ( 1 )   362 - 362   2022.9

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    Infectious bovine rhinotracheitis (IBR) is a major animal health hazard in many countries throughout the world, caused by bovine herpesvirus-1 (BoHV-1). The study's goal was to evaluate the prevalence of BoHV-1 seropositivity among dromedary camels in three governorates in northern Egypt, as well as to identify risk variables related with BoHV-1 seropositivity. A total of 321 blood samples were collected randomly from dromedary camels living in the selected governorates and examined for presence of BoHV-1 antibody using ELISA test. The overall seroprevalence of BoHV-1 among examined camels was 5.92% (95%CI: 3.82-9.06). Univariable analysis confirmed that the significant association (P < 0.05) between sex, history of abortion, contact with small ruminants and herd size and BoHV-1 seropositivity. Using multiple logistic regression analysis, the following risk factors were identified to be related with the presence of BoHV-1 infection: sex (OR = 2.54, 95%CI: 0.63-10.22), history of abortion (OR = 4.16, 95%CI: 1.30-13.27), contact with small ruminants (OR = 5.61, 95%CI: 1.67-18.80) and large herd size (OR = 10.52, 95%CI: 2.46-44.91). This study estimated the disease's seroprevalence in Egyptian dromedary camels, implying that camels could act as a BoHV-1 reservoir for transmission to other species.

    DOI: 10.1186/s12917-022-03448-5

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  • A survey of bluetongue infection in one-humped camels (Camelus Dromedarius); seroprevalence and risk factors analysis. International journal

    Abdelfattah Selim, Roua A Alsubki, Fatima M Albohairy, Kotb A Attia, Itoh Kimiko

    BMC veterinary research   18 ( 1 )   322 - 322   2022.8

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    Bluetongue (BT) is an insect-borne, non-contagious viral disease which affects domestic ruminants including camels and is transmitted by Culicoides spp. Clinical symptoms of BT are typically seen in sheep, although subclinical BT infections are mostly seen in cattle, goats, and camelids. The goal of the present study was to evaluate the sero-prevalence of Bluetongue virus (BTV) in camels from some governorates in Egypt's southern and northern regions, as well as the infection's potential risk factors. During 2020-2021, a cross sectional study was conducted to screen presence of anti-BTV antibodies in 400 serum samples, which were collected randomly from camels, examined using competitive enzyme-linked immunosorbent assay (cELISA). The sera of 102 out of 400 camels tested positive for BTV, representing a frequency of 25.5%. Moreover, the odds of sero-positivity were higher among camels living in Aswan (OR = 5.33, 95%CI: 2.35-12.11), especially in females (OR = 2.63, 95%CI = 1.44-4.09) during summer season (OR = 2.40, 95%CI = 1.20-4.81). Furthermore, the probability of getting BTV infection increased when camels were exposed to the insect vectors (OR = 1.63, 95%CI = 0.87-3.09). The high prevalence of BTV in camels in several Egyptian regions highlights the need for more epidemiological investigations of BTV infection in other ruminant species in order to better control BT disease in these regions.

    DOI: 10.1186/s12917-022-03421-2

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  • Molecular epidemiological survey, genetic characterization and phylogenetic analysis of Anaplasma ovis infecting sheep in Northern Egypt. International journal

    Mourad Ben Said, Kotb A Attia, Roua A Alsubki, Arif A Mohamed, Itoh Kimiko, Abdelfattah Selim

    Acta tropica   229   106370 - 106370   2022.5

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    Anaplasma ovis is the most common etiologic agent of ovine anaplasmosis, mainly transmitted by ticks. The present study aimed to determine the molecular prevalence of A. ovis in sheep from Egypt and assessed the associated risk factors. The study was conducted, between January and December 2020, in four governorates situated in Northern Egypt. Blood samples from 355 asymptomatic sheep were collected and examined by the use of PCR specific to A. ovis. Diversity analysis and phylogenetic study based on partial msp4 gene sequence were performed on revealed A. ovis DNA. Overall, the molecular prevalence rate of A. ovis was 15.5% and the highest rate was observed in Kafr ElSheikh governorate (16.8%). Statistical analysis revealed that A. ovis infection was significantly related to sheep gender and to tick infestation. The risk factors that were found to be associated with A. ovis infection in exposed sheep were: female sex (OR=2.6, 95%CI: 1.13-6.12), and infestation with ticks (OR=2.1, 95%CI: 1.11-3.79). The analysis of A. ovis msp4 sequences revealed two different genotypes classified in the Old World sub-cluster with other Egyptian isolates. Investigation on prevalence, risk factors and genetic variability of A. ovis in sheep reported in this study is important for the implementation of control programs. Further studies are needed to determine the vectors and reservoirs of A. ovis in Egyptian small ruminants and to identify the real economic impact of A. ovis infection on the country.

    DOI: 10.1016/j.actatropica.2022.106370

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  • Gentian FLOWERING LOCUS T orthologs regulate phase transitions: floral induction and endodormancy release. International journal

    Hideyuki Takahashi, Masahiro Nishihara, Chiharu Yoshida, Kimiko Itoh

    Plant Physiology   188 ( 4 )   1887 - 1899   2022.3

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    Perennial plants undergo a dormant period in addition to the growth and flowering phases that are commonly observed in annuals and perennials. Consequently, the regulation of these phase transitions in perennials is believed to be complicated. Previous studies have proposed that orthologs of FLOWERING LOCUS T (FT) regulate not only floral initiation but also dormancy. We, therefore, investigated the involvement of FT orthologs (GtFT1 and GtFT2) during the phase transitions of the herbaceous perennial gentian (Gentiana triflora). Analysis of seasonal fluctuations in the expression of these genes revealed that GtFT1 expression increased prior to budbreak and flowering, whereas GtFT2 expression was induced by chilling temperatures with the highest expression occurring when endodormancy was released. The expression of FT-related transcription factors, reportedly involved in flowering, also fluctuated during each phase transition. These results suggested the involvement of GtFT1 in budbreak and floral induction and GtFT2 in dormancy regulation, implying that the two gentian FT orthologs activated a different set of transcription factors. Gentian ft2 mutants generated by CRISPR/Cas9-mediated genome editing had a lower frequency of budbreak and budbreak delay in overwintering buds caused by an incomplete endodormancy release. Our results highlighted that the gentian orthologs of FRUITFULL (GtFUL) and SHORT VEGETATIVE PHASE-like 1 (GtSVP-L1) act downstream of GtFT2, probably to prevent untimely budbreak during ecodormancy. These results suggest that each gentian FT ortholog regulates a different phase transition by having variable responses to endogenous or environmental cues, leading to their ability to induce the expression of distinct downstream genes.

    DOI: 10.1093/plphys/kiac007

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  • Cross-sectional survey on Mycobacterium avium Subsp. paratuberculosis in Dromedary Camels: Seroprevalence and risk factors. International journal

    Abdelfattah Selim, Kotb A Attia, Roua A Alsubki, Itoh Kimiko, Mohamed Z Sayed-Ahmed

    Acta tropica   226   106261 - 106261   2022.2

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    Johne's disease is a chronic disease with great concern in ruminants and caused by Mycobacterium avium subsp. paratuberculosis (MAP). A cross-sectional study was conducted from February 2019 to January 2020 to estimate the prevalence of MAP infection among camels which are kept in three governorates in Nile Delta of Egypt. A total of 440 serum samples were examined by ELISA for detection of MAP antibodies. The multivariable logistic regression model was performed to determine the associated risk factors for MAP infection in examined camels. Overall, the seroprevalence of MAP infection was found to be 7.5% among examined camels. The multivariable logistic regression model was performed to determine the associated risk factors for MAP infection in examined camels. The main findings revealed that the risk of getting MAP infection increased among elder camels (>10 years old) with signs of diarrhea, having communal water source and in camels grazing in the same pasture (odds ratio >1). However, geographic location, sex and contact with cattle had not significant impact regarding to seroprevalence of MAP infection in camels. The present findings confirm presence of MAP among camels which is a potential risk factor for contamination of environment and spreading of infection. Therefore, further studies for detection of infected animals in early stage are needed beside the estimated risk factors in this study to build an efficient control program.

    DOI: 10.1016/j.actatropica.2021.106261

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  • Assessment of Seroprevalence and Associated Risk Factors for Anaplasmosis in Camelus dromedarius. International journal

    Roua A Alsubki, Fatima M Albohairy, Kotb A Attia, Itoh Kimiko, Abdelfattah Selim, Mohamed Z Sayed-Ahmed

    Veterinary sciences   9 ( 2 )   2022.1

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    (1) Background: Anaplasmosis is an infectious disease in camels caused by an obligate intracellular bacterium that is transmitted by ticks. (2) Methods: A cross-sectional study was conducted during 2020 to study the seroprevalence of Anaplasma spp. among Camelus dromedarius in three governorates in Egypt and assess the associated risk factors. Serum samples from 365 camels were examined by a competitive enzyme-linked immunosorbent assay (cELISA) test. (3) Results: Overall, the seroprevalence of anaplasmosis among camels was 18.6%. Multivariable logistic regression was performed, and it was discovered that tick infestation, application of acaricides, grooming practice and body condition were potential risk factors for Anaplasma spp. infection (odds ratio > 1) in dromedary camels. In contrast, the locality in which the camels lived and their age were not significant effects with regard to the occurrence of anaplasmosis. (4) Conclusions: The current findings suggest that improvement of protective measures to limit the effects of the identified risk factors can help to reduce the spread of anaplasmosis among camels in Egypt.

    DOI: 10.3390/vetsci9020057

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  • Identification of C-T novel polymorphism in 3rd exon of OsSPL14 gene governing seed sequence in rice. International journal

    Muhammad Salah Ud Din, Xiukang Wang, Salman Alamery, Sajid Fiaz, Haroon Rasheed, Muhammad Abid Khan, Shahid Ullah Khan, Sumbul Saeed, Niaz Ali, Kalim Ullah Marwat, Kotb Attia, Itoh Kimiko, Shabir Hussain Wani

    PloS one   17 ( 3 )   e0264478   2022

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    Recently food shortage has become the major flagging scenario around the globe. To resolve this challenge, there is dire need to significantly increase crop productivity per unit area. In the present study, 24 genotypes of rice were grown in pots to assess their tillering number, number of primary and secondary branches per panicle, number of grains per panicle, number of grains per plant, and grain yield, respectively. In addition, the potential function of miR156 was analyzed, regulating seed sequence in rice. Furthermore, OsSPL14 gene for miR156 was sequenced to identify additional mutations within studied region. The results demonstrated Bas-370 and L-77 showed highest and lowest tillers, respectively. Bas-370, Rachna basmati, Bas-2000, and Kashmir Basmati showed high panicle branches whereas, L-77, L-46, Dilrosh, L-48, and L-20 displayed lowest panicle branches. Bas-370 and four other studied accessions contained C allele whereas, L-77 and 18 other investigated accessions had heterozygous (C and T) alleles in their promoter region. C-T allelic mutation was found in 3rd exon of the OsSPL14 gene. The sequence analysis of 12 accessions revealed a novel mutation (C-T) present ~2bp upstream and substitution of C-A allele. However, no significant correlation for novel mutation was found for tillering and panicle branches in studied rice accessions. Taken together present results suggested novel insight into the binding of miR156 to detected mutation found in 3rd exon of the OsSPL14 gene. Nevertheless, L-77, L-46, Dilrosh, L-48, and L-20 could be used as potential breeding resource for improving panicle architecture contributing yield improvement of rice crop.

    DOI: 10.1371/journal.pone.0264478

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  • Molecular Characterization and Functional Localization of a Novel SUMOylation Gene in Oryza sativa. International journal

    Eid I Ibrahim, Kotb A Attia, Abdelhalim I Ghazy, Kimiko Itoh, Fahad N Almajhdi, Abdullah A Al-Doss

    Biology   11 ( 1 )   2021.12

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    Small ubiquitin-related modifier (SUMO) regulates the cellular function of diverse proteins through post-translational modifications. The current study defined a new homolog of SUMO genes in the rice genome and named it OsSUMO7. Putative protein analysis of OsSUMO7 detected SUMOylation features, including di-glycine (GG) and consensus motifs (ΨKXE/D) for the SUMOylation site. Phylogenetic analysis demonstrated the high homology of OsSUMO7 with identified rice SUMO genes, which indicates that the OsSUMO7 gene is an evolutionarily conserved SUMO member. RT-PCR analysis revealed that OsSUMO7 was constitutively expressed in all plant organs. Bioinformatic analysis defined the physicochemical properties and structural model prediction of OsSUMO7 proteins. A red fluorescent protein (DsRed), fused with the OsSUMO7 protein, was expressed and localized mainly in the nucleus and formed nuclear subdomain structures. The fusion proteins of SUMO-conjugating enzymes with the OsSUMO7 protein were co-expressed and co-localized in the nucleus and formed nuclear subdomains. This indicated that the OsSUMO7 precursor is processed, activated, and transported to the nucleus through the SUMOylation system of the plant cell.

    DOI: 10.3390/biology11010053

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  • Adduct Formation of Delamanid with NAD in Mycobacteria Reviewed

    Mikayo Hayashi, Akihito Nishiyama, Ryuki Kitamoto, Yoshitaka Tateishi, Mayuko Osada-Oka, Yukiko Nishiuchi, Shaban A. Kaboso, Xiuhao Chen, Mamoru Fujiwara, Yusuke Inoue, Yoshikazu Kawano, Masanori Kawasaki, Tohru Abe, Tsutomu Sato, Kentaro Kaneko, Kimiko Itoh, Sohkichi Matsumoto, Makoto Matsumoto

    Antimicrobial Agents and Chemotherapy   64 ( 5 )   2020.4

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    Publishing type:Research paper (scientific journal)   Publisher:American Society for Microbiology  

    <title>ABSTRACT</title>
    Delamanid (DLM), a nitro-dihydroimidazooxazole derivative currently approved for pulmonary multidrug-resistant tuberculosis (TB) therapy, is a prodrug activated by mycobacterial 7,8-didemethyl-8-hydroxy 5-deazaflavin electron transfer coenzyme (F<sub>420</sub>)-dependent nitroreductase (Ddn). Despite inhibiting the biosynthesis of a subclass of mycolic acids, the active DLM metabolite remained unknown. Comparative liquid chromatography-mass spectrometry (LC-MS) analysis of DLM metabolites revealed covalent binding of reduced DLM with a nicotinamide ring of NAD derivatives (oxidized form) in DLM-treated <named-content content-type="genus-species">Mycobacterium tuberculosis</named-content> var. Bacille de Calmette et Guérin. Isoniazid-resistant mutations in the type II NADH dehydrogenase gene (<italic>ndh</italic>) showed a higher intracellular NADH/NAD ratio and cross-resistance to DLM, which were restored by complementation of the mutants with wild-type <italic>ndh</italic>. Our data demonstrated for the first time the adduct formation of reduced DLM with NAD in mycobacterial cells and its importance in the action of DLM.

    File: Antimicrobial Agents and Chemotherapy-2020-Hayashi-e01755-19.full.pdf

    DOI: 10.1128/aac.01755-19

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  • Adduct Formation of Delamanid with NAD in Mycobacteria Reviewed

    Mikayo Hayashi, Akihito Nishiyama, Ryuki Kitamoto, Yoshitaka Tateishi, Mayuko Osada-Oka, Yukiko Nishiuchi, Shaban A. Kaboso, Xiuhao Chen, Mamoru Fujiwara, Yusuke Inoue, Yoshikazu Kawano, Masanori Kawasaki, Tohru Abe, Tsutomu Sato, Kentaro Kaneko, Kimiko Itoh, Sohkichi Matsumoto, Makoto Matsumoto

    Antimicrobial Agents and Chemotherapy   64 ( 5 )   2020.4

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    Publishing type:Research paper (scientific journal)   Publisher:American Society for Microbiology  

    Delamanid (DLM), a nitro-dihydroimidazooxazole derivative currently approved for pulmonary multidrug-resistant tuberculosis (TB) therapy, is a prodrug activated by mycobacterial 7,8-didemethyl-8-hydroxy 5-deazaflavin electron transfer coenzyme (F
    <sub>420</sub>
    )-dependent nitroreductase (Ddn). Despite inhibiting the biosynthesis of a subclass of mycolic acids, the active DLM metabolite remained unknown. Comparative liquid chromatography-mass spectrometry (LC-MS) analysis of DLM metabolites revealed covalent binding of reduced DLM with a nicotinamide ring of NAD derivatives (oxidized form) in DLM-treated
    <named-content content-type="genus-species">Mycobacterium tuberculosis</named-content>
    var.

    DOI: 10.1128/aac.01755-19

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  • Increased CD5+ B-cells are associated with autoimmune phenomena in lepromatous leprosy patients. Reviewed

    Kotb A, Ismail S, Kimiko I, Mohamed W, Salman A, Mohammed AA

    Journal of infection and public health   12 ( 5 )   656 - 659   2019.9

  • Optimized Nuclear Pellet Method for Extracting Next-Generation Sequencing Quality Genomic DNA from Fresh Leaf Tissue. Reviewed

    Rana MM, Aycan M, Takamatsu T, Kaneko K, Mitsui T, Itoh K

    Methods and protocols   2 ( 2 )   54 - 54   2019.6

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    Next-generation sequencing (NGS) is a revolutionary advancement allowing large-scale discovery of functional molecular markers that has many applications, including plant breeding. High-quality genomic DNA (gDNA) is a prerequisite for successful NGS library preparation and sequencing; however, few reliable protocols to obtain such plant gDNA exist. A previously reported nuclear pellet (NP) method enables extraction of high-yielding gDNA from fresh leaf tissue of maize (Zea mays L.), but the quality does not meet the stringent requirements of NGS. In this study, we optimized the NP method for whole-genome sequencing of rice (Oryza sativa L.) through the integration of simple purification steps. The optimized NP method relied on initial nucleus enrichment, cell lysis, extraction, and subsequent gDNA purification buffers. The purification steps used proteinase K, RNase A, phenol/chloroform/isoamyl alcohol (25:24:1), and chloroform/isoamyl alcohol (24:1) treatments for protein digestion and RNA, protein, and phenol removal, respectively. Our data suggest that this optimized NP method allowed extraction of consistently high-yielding and high-quality undegraded gDNA without contamination by protein and RNA. Moreover, the extracted gDNA fulfilled the quality metrics of NGS library preparation for the Illumina HiSeq X Ten platform by the TruSeq DNA PCR-Free Library Prep Kit (Illumina). We provide a reliable step-by-step guide to the extraction of high-quality gDNA from fresh leaf tissues of rice for molecular biologists with limited resources.

    File: 20190625_Optimized Nuclear Pellet Method for Extracting Next-Generation Sequencing Quality Genomic DNA from Fresh Leaf Tissue, Md Masud Rana at al..pdf

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  • Salt Tolerance Improvement in Rice through Efficient SNP Marker-Assisted Selection Coupled with Speed-Breeding. Reviewed International journal

    Md Masud Rana, Takeshi Takamatsu, Marouane Baslam, Kentaro Kaneko, Kimiko Itoh, Naoki Harada, Toshie Sugiyama, Takayuki Ohnishi, Tetsu Kinoshita, Hiroki Takagi, Toshiaki Mitsui

    International journal of molecular sciences   20 ( 10 )   2585   2019.5

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    Salinity critically limits rice metabolism, growth, and productivity worldwide. Improvement of the salt resistance of locally grown high-yielding cultivars is a slow process. The objective of this study was to develop a new salt-tolerant rice germplasm using speed-breeding. Here, we precisely introgressed the hst1 gene, transferring salinity tolerance from "Kaijin" into high-yielding "Yukinko-mai" (WT) rice through single nucleotide polymorphism (SNP) marker-assisted selection. Using a biotron speed-breeding technique, we developed a BC3F3 population, named "YNU31-2-4", in six generations and 17 months. High-resolution genotyping by whole-genome sequencing revealed that the BC3F2 genome had 93.5% similarity to the WT and fixed only 2.7% of donor parent alleles. Functional annotation of BC3F2 variants along with field assessment data indicated that "YNU31-2-4" plants carrying the hst1 gene had similar agronomic traits to the WT under normal growth condition. "YNU31-2-4" seedlings subjected to salt stress (125 mM NaCl) had a significantly higher survival rate and increased shoot and root biomasses than the WT. At the tissue level, quantitative and electron probe microanalyzer studies indicated that "YNU31-2-4" seedlings avoided Na+ accumulation in shoots under salt stress. The "YNU31-2-4" plants showed an improved phenotype with significantly higher net CO2 assimilation and lower yield decline than WT under salt stress at the reproductive stage. "YNU31-2-4" is a potential candidate for a new rice cultivar that is highly tolerant to salt stress at the seedling and reproductive stages, and which might maintain yields under a changing global climate.

    File: 20190526_Salt Tolerance Improvement in Rice through Efficient, Md Masud Rana at al..pdf

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  • Optimized method of extracting rice chloroplast DNA for high-quality plastome resequencing and de Novo assembly Reviewed

    Takeshi Takamatsu, Marouane Baslam, Takuya Inomata, Kazusato Oikawa, Kimiko Itoh, Takayuki Ohnishi, Tetsu Kinoshita, Toshiaki Mitsui

    Frontiers in Plant Science   9 ( 266 )   1 - 13   2018.2

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    Chloroplasts, which perform photosynthesis, are one of the most important organelles in green plants and algae. Chloroplasts maintain an independent genome that includes important genes encoding their photosynthetic machinery and various housekeeping functions. Owing to its non-recombinant nature, low mutation rates, and uniparental inheritance, the chloroplast genome (plastome) can give insights into plant evolution and ecology and in the development of biotechnological and breeding applications. However, efficient methods to obtain high-quality chloroplast DNA (cpDNA) are currently not available, impeding powerful sequencing and further functional genomics research. To investigate effects on rice chloroplast genome quality, we compared cpDNA extraction by three extraction protocols: liquid nitrogen coupled with sucrose density gradient centrifugation, high-salt buffer, and Percoll gradient centrifugation. The liquid nitrogen–sucrose gradient method gave a high yield of high-quality cpDNA with reliable purity. The cpDNA isolated by this technique was evaluated, resequenced, and assembled de novo to build a robust framework for genomic and genetic studies. Comparison of this high-purity cpDNA with total DNAs revealed the read coverage of the sequenced regions
    next-generation sequencing data showed that the high-quality cpDNA eliminated noise derived from contamination by nuclear and mitochondrial DNA, which frequently occurs in total DNA. The assembly process produced highly accurate, long contigs. We summarize the extent to which this improved method of isolating cpDNA from rice can provide practical progress in overcoming challenges related to chloroplast genomes and in further exploring the development of new sequencing technologies.

    File: 20180228_Optimized Method of Extracting Rice Chloroplast DNA for High-Quality Plastome Resequencing and de Novo Assembly., Takeshi Takamatsu at al..pdf

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  • Proteomic and Glycomic Characterization of Rice Chalky Grains Produced Under Moderate and High-temperature Conditions in Field System

    Kentaro Kaneko, Maiko Sasaki, Nanako Kuribayashi, Hiromu Suzuki, Yukiko Sasuga, Takeshi Shiraya, Takuya Inomata, Kimiko Itoh, Marouane Baslam, Toshiaki Mitsui

    Rice   9 ( 1 )   2016.12

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  • N-glycomic and microscopic subcellular localization analyses of NPP1, 2 and 6 strongly indicate that trans-golgi compartments participate in the golgi to plastid traffic of nucleotide pyrophosphatase/phosphodiesterases in rice Reviewed

    Kentaro Kaneko, Takeshi Takamatsu, Takuya Inomata, Kazusato Oikawa, Kimiko Itoh, Kazuko Hirose, Maho Amano, Shin-Ichiro Nishimura, Kiminori Toyooka, Ken Matsuoka, Javier Pozueta-Romero, Toshiaki Mitsui

    Plant and Cell Physiology   57 ( 8 )   1610 - 1628   2016.8

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    Nucleotide pyrophosphatase/phosphodiesterases (NPPs) are widely distributed N-glycosylated enzymes that catalyze the hydrolytic breakdown of numerous nucleotides and nucleotide sugars. In many plant species, NPPs are encoded by a small multigene family, which in rice are referred to NPP1-NPP6. Although recent investigations showed that N-glycosylated NPP1 is transported from the endoplasmic reticulum (ER)-Golgi system to the chloroplast through the secretory pathway in rice cells, information on N-glycan composition and subcellular localization of other NPPs is still lacking. Computer-assisted analyses of the amino acid sequences deduced from different Oryza sativa NPP-encoding cDNAs predicted all NPPs to be secretory glycoproteins. Confocal fluorescence microscopy observation of cells expressing NPP2 and NPP6 fused with green fluorescent protein (GFP) revealed that NPP2 and NPP6 are plastidial proteins. Plastid targeting of NPP2-GFP and NPP6-GFP was prevented by brefeldin A and by the expression of ARF1(Q71L), a dominant negative mutant of ADP-ribosylation factor 1 that arrests the ER to Golgi traffic, indicating that NPP2 and NPP6 are transported from the ER-Golgi to the plastidial compartment. Confocal laser scanning microscopy and high-pressure frozen/freeze-substituted electron microscopy analyses of transgenic rice cells ectopically expressing the trans-Golgi marker sialyltransferase fused with GFP showed the occurrence of contact of Golgi-derived membrane vesicles with cargo and subsequent absorption into plastids. Sensitive and high-throughput glycoblotting/ mass spectrometric analyses showed that complex-type and paucimannosidic-type glycans with fucose and xylose residues occupy approximately 80% of total glycans of NPP1, NPP2 and NPP6. The overall data strongly indicate that the trans-Golgi compartments participate in the Golgi to plastid trafficking and targeting mechanism of NPPs.

    File: 20160506_N-Glycomic and Microscopic Subcellular Localization Analyses., K. Kaneko et al..pdf

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  • Golgi/plastid-type manganese superoxide dismutase involved in heat-stress tolerance during grain filling of rice Reviewed

    Takeshi Shiraya, Taiki Mori, Tatsuya Maruyama, Maiko Sasaki, Takeshi Takamatsu, Kazusato Oikawa, Kimiko Itoh, Kentaro Kaneko, Hiroaki Ichikawa, Toshiaki Mitsui

    PLANT BIOTECHNOLOGY JOURNAL   13 ( 9 )   1251 - 1263   2015.1

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    Superoxide dismutase (SOD) is widely assumed to play a role in the detoxification of reactive oxygen species caused by environmental stresses. We found a characteristic expression of manganese SOD 1 (MSD1) in a heat-stress-tolerant cultivar of rice (Oryza sativa). The deduced amino acid sequence contains a signal sequence and an N-glycosylation site. Confocal imaging analysis of rice and onion cells transiently expressing MSD1-YFP showed MSD1-YFP in the Golgi apparatus and plastids, indicating that MSD1 is a unique Golgi/plastid-type SOD. To evaluate the involvement of MSD1 in heat-stress tolerance, we generated transgenic rice plants with either constitutive high expression or suppression of MSD1. The grain quality of rice with constitutive high expression of MSD1 grown at 33/28 degrees C, 12/12 h, was significantly better than that of the wild type. In contrast, MSD1-knock-down rice was markedly susceptible to heat stress. Quantitative shotgun proteomic analysis indicated that the overexpression of MSD1 up-regulated reactive oxygen scavenging, chaperone and quality control systems in rice grains under heat stress. We propose that the Golgi/plastid MSD1 plays an important role in adaptation to heat stress.

    File: 201512_Golgiplastid-type manganese superoxide dismutase involved in heat-stress tolerance during grain filling of rice., Shiraya_et_al_Plant_Biotechnology_Journal.pdf

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  • Determination of genomic location and structure of the transgenes in marker-free rice-based cholera vaccine by using whole genome resequencing approach Reviewed

    Mio Mejima, Koji Kashima, Masaharu Kuroda, Natsumi Takeyama, Shiho Kurokawa, Yoshiko Fukuyama, Hiroshi Kiyono, Kimiko Itoh, Toshiaki Mitsui, Yoshikazu Yuki

    PLANT CELL TISSUE AND ORGAN CULTURE   120 ( 1 )   35 - 48   2015.1

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    We previously developed a molecularly uniform rice-based oral cholera vaccine (MucoRice-CTB) by using an overexpression system for modified cholera toxin B-subunit, CTB (N4Q) with RNAi to suppress production of the major rice endogenous storage proteins. To establish MucoRice-CTB for human use, here we developed hygromycin phosphotransferase selection marker-free MucoRice-CTB by using two different Agrobacterium tumefaciens, each carrying a distinct T-DNA for co-transformation. In the marker-free candidates from co-transformants by segregation in the seed progeny, we selected a line with high CTB expression, line 51A, which we advanced to the T6 generation by self-pollination to obtain a homozygous line without down-regulation of CTB expression. Southern blot analyses showed that three copies of the CTB gene, but not the backbone of the T-DNA binary vector, were inserted into the rice genome of MucoRice-CTB line 51A. By whole genome resequencing, we showed that the transgenes in this line were inserted into intergenic regions in chromosome 3 and chromosome 12. We determined that two full-length copies, each containing the CTB and RNAi expression cassettes, were inserted in a tandem reverted orientation into chromosome 3. An additional copy of the CTB over-expression cassette with a truncated RNAi cassette was inserted into chromosome 12. These findings provide useful information for the establishment of a seed bank of marker-free MucoRice-CTB for human use.

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  • Nucleotide Pyrophosphatase/Phosphodiesterase 1 Exerts a Negative Effect on Starch Accumulation and Growth in Rice Seedlings under High Temperature and CO2 Concentration Conditions Reviewed

    Kentaro Kaneko, Takuya Inomata, Takahiro Masui, Tsutomu Koshu, Yukiho Umezawa, Kimiko Itoh, Javier Pozueta-Romero, Toshiaki Mitsui

    PLANT AND CELL PHYSIOLOGY   55 ( 2 )   320 - 332   2014.2

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    Nucleotide pyrophosphatase/phosphodiesterase (NPP) is a widely distributed enzymatic activity occurring in both plants and mammals that catalyzes the hydrolytic breakdown of the pyrophosphate and phosphodiester bonds of a number of nucleotides. Unlike mammalian NPPs, the physiological function of plant NPPs remains largely unknown. Using a complete rice NPP1-encoding cDNA as a probe, in this work we have screened a rice shoot cDNA library and obtained complete cDNAs corresponding to six NPP genes (NPP1-NPP6). As a first step to clarify the role of NPPs, recombinant NPP1, NPP2 and NPP6 were purified from transgenic rice cells constitutively expressing NPP1, NPP2 and NPP6, respectively, and their enzymatic properties were characterized. NPP1 and NPP6 exhibited hydrolytic activities toward ATP, UDP-glucose and the starch precursor molecule, ADP-glucose, whereas NPP2 did not recognize nucleotide sugars as substrates, but hydrolyzed UDP, ADP and adenosine 5'-phosphosulfate. To gain insight into the physiological function of rice NPP1, an npp1 knockout mutant was characterized. The ADP-glucose hydrolytic activities in shoots of npp1 rice seedlings were 8% of those of the wild type (WT), thus indicating that NPP1 is a major determinant of ADP-glucose hydrolytic activity in rice shoots. Importantly, when seedlings were cultured at 160 Pa CO2 under a 28 degrees C/23 degrees C (12h light/12 h dark) regime, npp1 shoots and roots were larger than those of wild-type (WT) seedlings. Furthermore, the starch content in the npp1 shoots was higher than that of WT shoots. Growth and starch accumulation were also enhanced under an atmospheric CO2 concentration (40 Pa) when plants were cultured under a 33 degrees C/28 degrees C regime. The overall data strongly indicate that NPP1 exerts a negative effect on plant growth and starch accumulation in shoots, especially under high CO2 concentration and high temperature conditions.

    File: 201402_Nucleotide Pyrophosphatase Phosphodiesterase 1 Exerts a Negative Effect on Starch Accumulation and Growth in Rice Seedlings under High Temperature and , K. Kaneko et al..pdf

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  • Lack of starch synthase IIIa and high expression of granule-bound starch synthase I synergistically increase the apparent amylose content in rice endosperm Reviewed

    Naoko Crofts, Katsumi Abe, Satomi Aihara, Rumiko Itoh, Yasunori Nakamura, Kimiko Itoh, Naoko Fujita

    PLANT SCIENCE   193   62 - 69   2012.9

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    Rice endosperm starch is composed of 0-30% linear amylose, which is entirely synthesized by granule-bound starch synthase I (GBSSI: encoded by Waxy, Wx). The remainder consists of branched amylopectin and is elongated by multiple starch synthases (SS) including SSI, IIa and Ilia. Typical japonica rice lacks active SSIIa and contains a low expressing Wx(b) causing a low amylose content (ca. 20%).
    WAB2-3 (SS3a/Wx(a)) lines generated by the introduction of a dominant indica Wx(a) into a japonica waxy mutant (SS3a/wx) exhibit elevated GBSSI and amylose content (ca. 25%). The japonica ss3a mutant (ss3a/Wx(b)) shows a high amylose content (ca. 30%), decreased long chains of amylopectin and increased GBSSI levels. To investigate the functional relationship between the ss3a and Wx(a) genes, the ss3a/Wx(a) line was generated by crossing ss3a/Wx(b) with SS3a/Wx(a), and the starch properties of this line were examined. The results show that the apparent amylose content of the ss3a/Wx(a) line was increased (41.3%) compared to the parental lines. However, the GBSSI quantity did not increase compared to the SS3a/Wx(a) line. The amylopectin branch structures were similar to the ss3a/Wx(b) mutant. Therefore, Wx(a) and ss3a synergistically increase the apparent amylose content in rice endosperm, and the possible reasons for this increase are discussed. (C) 2012 Elsevier Ireland Ltd. All rights reserved.

    File: 201209_Lack of starch synthase IIIa and high expression of granule-bound starch synthase I synergistically increase the apparent amylose content in rice endosperm., Crofts N at al..pdf

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  • Differential localizations and functions of rice nucleotide pyrophosphatase/phosphodiesterase isozymes 1 and 3 Reviewed

    Kentaro Kaneko, Chie Yamada, Ai Yanagida, Tsutomu Koshu, Yukiho Umezawa, Kimiko Itoh, Hidetaka Hori, Toshiaki Mitsui

    PLANT BIOTECHNOLOGY   28 ( 1 )   69 - 76   2011

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    Our previous investigation demonstrated that ADP-glucose hydrolytic nucleotide pyrophosphatase/phosphodiesterase (NPP) 1 is transported from the endoplasmic reticulum (ER)-Golgi system to the plastids via a secretory pathway in rice cells [Nanjo et al. (2006) Plant Cell 18: 2582-2592]. In this study, we analyzed the enzymatic characteristics and subcellular localization of its isozyme, NPP3. Unlike NPP1, NPP3 exhibited no hydrolytic activity toward ADP-glucose and no plastid-targeting ability. Furthermore, there was a clear difference between their N-terminal proteolytic processing schemes to form mature enzyme proteins. NPP1 is matured to a 70-kDa protein by two-step proteolytic processing. We detected the 72 kDa form of NPP1 in the microsomes of rice cells in addition to the 70 kDa mature protein, strongly suggesting that proprotein processing occurs post-translationally in the ER-Golgi system. To clarify the existence of the plastid-targeting signal of NPP1, the plastid localization of a series of carboxy-terminal truncated NPP1 proteins fused with green fluorescence protein was tested in rice cells. The results showed that NPP1 cannot be delivered to the plastid by the N-terminal region, including the ER signal sequence and the proprotein processing site, and that the peptide region, from 308 to 478 amino acid residues, is probably important for the transport of NPP1 into plastids in rice cells.

    File: 2011_Differential localizations and functions of rice nucleotide pyrophosphatase phosphodiesterase isozymes 1 and 3.pdf

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  • Isoform-Specific Localization of Brassica rapa Nitrilases in Root Infected with Plasmodiophora brassicae Revealed Using In Situ Hybridization Probes Improved with Locked Nucleic Acids Reviewed

    Toshiki Ishikawa, Keiichi Okazaki, Tomohiko Nagaoka, Kimiko Itoh, Toshiaki Mitsui, Hidetaka Hori

    JOURNAL OF PLANT GROWTH REGULATION   29 ( 2 )   210 - 222   2010.6

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    We established an in situ hybridization (ISH) technique by modification of hybridization probes with locked nucleic acids (LNAs) and demonstrated isoform-specific localization of transcripts of Brassica rapa nitrilase (BrNIT-T) genes in clubroot tissue infected with Plasmodiophora brassicae. Chimeric oligo DNA probes containing LNAs demonstrated highly improved specificities and could discriminate between BrNIT-T1 and BrNIT-T2. These LNA-containing probes were applied to ISH. BrNIT-T1 was strongly expressed in cells containing expanding secondary plasmodia of P. brassicae, but not in cells containing resting spores. On the other hand, BrNIT-T2 transcripts were localized in noninfected cells rather than infected cells during the clubroot growth phase but coexisted with mature resting spores at a later phase of clubroot development. Immunostaining for indole-3-acetic acid (IAA) revealed IAA accumulation in cells containing growing plasmodia. IAA immunostaining in infected cells was reduced as the pathogen formed resting spores, but the signal was again enhanced in cells containing mature resting spores at a later phase of infection, suggesting that IAA is involved in both the early growth and the latest maturation phase of clubroot development. Expression of BrNIT-T1 and BrNIT-T2 in turnip roots was upregulated by exogenous treatment with cytokinin and jasmonic acid, respectively. Thus, these two phytohormones are possible triggers of abnormal IAA production in clubroot tissue via induction of the respective nitrilase. Given these results, we propose a model for isoform-specific roles of B. rapa nitrilases in auxin biosynthesis involved in phytohormone crosstalk during development of clubroot disease.

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  • Structures of endosperm starch from a rice wx cultivar expressing Wxa transgene.

    I Hanashiro, T. Wakayama, M. Hasegawa, T. Higuchi, K. Itoh, T. Fukuyama, Y. Takeda

    J Applied Glycoscience   56 ( 2 )   65 - 70   2009.7

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    Structural changes caused by the introduction of the <i>Wx<sup>α</sup></i> transgene, which encodes granule-bound starch synthase I (GBSSI), into the non-transgenic <i>wx</i> cultivar Musashimochi were examined and compared with the previous results (<i>Plant Cell Physiol</i>., 49, 925-933 (2008), using a transgenic line (WAB3-5) established independently from those used previously. Increases of amylose content of starch and extra-long chain (ELC) content of amylopectin were observed in the line WAB3-5 (actual amylose, 15.2%; ELC, 11.6% by weight). An HPSEC profile (size distribution) of WAB3-5 amylose showed characteristics in-between of those of <i>Wx<sup>α</sup></i> and <i>Wx<sup>b</sup></i> cultivars, and WAB3-5 amylose had a comparably high degree of branching, which was indicated by the molar fraction of branched molecules (MF<sub>B</sub>, 0.38) and number of chains of branched molecules (NC<sub>B</sub>, 10.5). Amylose from cv. Yumetoiro (<i>Wx<sup>α</sup></i>) had a comparable degree of branching (MF<sub>B</sub>, 0.35; NC<sub>B</sub>, 13.7) with the WAB3-5 amylose. Chain-length distribution of WAB3-5 amylopectin showed a slightly higher amount of B<sub>2</sub>+B<sub>3</sub> chains than cv. Musashimochi. These results are consistent with those of the previous study, providing additional evidence for the proposed role of GBSSI in both amylose and ELC synthesis in rice endosperm. ELCs of amylopectins from line WAB3-5 and cvs. IR36 and Yumetoiro showed size distributions that were basically similar to each other but distinct from those of their amylose counterpart. ELCs were hydrolyzed by β-amylolysis of amylopectins with different extents of trimming, 66.3, 92.0 and 77.0% for line WAB3-5, cv. IR36 and cv. Yumetoiro, respectively, suggesting the branched structure of the ELCs is different among the three amylopectins.

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  • Granule-bound starch synthase I is responsible for biosynthesis of extra-long unit chains of amylopectin in rice Reviewed

    Isao Hanashiro, Kimiko Itoh, Yuki Kuratomi, Mina Yamazaki, Toshinari Igarashi, Jun-ichi Matsugasako, Yasuhito Takeda

    PLANT AND CELL PHYSIOLOGY   49 ( 6 )   925 - 933   2008.6

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    A rice Wx gene encoding a granule-bound starch synthase I (GBSSI) was introduced into the null-mutant waxy (wx) rice, and its effect on endosperm starches was examined. The apparent amylose content was increased from undetectable amounts for the non-transgenic wx cultivars to 21.6-22.2% of starch weight for the transgenic lines. The increase was in part due to a significant amount of extra-long unit chains (ELCs) of amylopectin (7.5-8.4% of amylopectin weight), that were absent in the non-transgenic wx cultivars. Thus, actual amylose content was calculated to be 14.9-16.0% for the transgenic lines. Only slight differences were found in chain-length distribution for the chains other than ELCs, indicating that the major effect of the Wx transgene on amylopectin structure was ELC formation. ELCs isolated from debranched amylopectin exhibited structures distinct from amylose. Structures of amylose from the transgenic lines were slightly different from those of cv. Labelle (Wx(a)) in terms of a higher degree of branching and size distribution. The amylose and ELC content of starches of the transgenic lines resulted in the elevation of pasting temperature, a 50% decrease in peak viscosity, a large decrease in breakdown and an increase in setback. As yet undetermined factors other than the GBSSI activity are thought to be involved in the control of formation and/or the amount of ELCs. Structural analysis of the Wx gene suggested that the presence of a tyrosine residue at position 224 of GBSSI correlates with the formation of large amounts of ELCs in cultivars carrying Wx(a).

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  • Establishment of High amylose Type Japonica rice by Agrobacterium-mediated gene transfer

    Mari Hasegawa, Akiko Sekikawa, Fuminori Tanno, Kimiko Itoh

    Bull Facul. Agric Niigata Univ.   61 ( 1 )   41 - 45   2008.3

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  • Molecular cloning of Brassica rapa nitrilases and their expression during clubroot development Reviewed

    Toshiki Ishikawa, Keiichi Okazaki, Haruka Kuroda, Kimiko Itoh, Toshiaki Mitsui, Hidetaka Hori

    MOLECULAR PLANT PATHOLOGY   8 ( 5 )   623 - 637   2007.9

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    Three isoforms of nitrilase were cloned from turnip, Brassica rapa L., and their expression during clubroot development caused by Plasmodiophora brassicae was investigated. The isoforms were designated BrNIT-T1, BrNIT-T2 and BrNIT-T4 based on homology to known nitrilases. BrNIT-T1 and BrNIT-T2 have 80% homology to three nitrilases from Arabidopsis thaliana (AtNIT1, AtNIT2 and AtNIT3). BrNIT-T4 showed 90% homology to AtNIT4. To confirm their enzyme activity, the recombinant proteins were expressed in Escherichia coli. The recombinant BrNIT-T1 and BrNIT-T2 but not BrNIT-T4 converted indole-3-acetonitrile to indole-3-acetic acid, an endogenous plant auxin, although kinetic analysis showed that indole-3-acetonitrile is a poor substrate compared with various aliphatic and aromatic nitriles. By contrast, the recombinant BrNIT-T4 specifically converted beta-cyano-L-alanine to aspartic acid and asparagine and these findings agree with the idea that it is involved in the cyanide detoxification pathway. Real-time PCR analysis clearly showed that these isoforms were differentially expressed during clubroot development. BrNIT-T1 transcripts were very low in non-infected roots but were enhanced up to 100-fold in infected roots exhibiting club growth. By contrast, BrNIT-T2 transcripts remained at a very low level during clubroot formation. All these results clearly indicate the specific involvement of BrNIT-T1 in clubroot formation. The BrNIT-T4 transcripts were substantially reduced in the clubroot-growing phase, but thereafter they increased rapidly to a level found in non-infected roots as the clubroot growth reached a plateau. These findings suggest the specific involvement of BrNIT-T4 in clubroot maturation. In fully developed clubs, the BrNIT-T1 and BrNIT-T2 transcripts also increased. Free indole-3-acetic acid (IAA) content increased in the early and the latest phase of infected roots compared with noninfected roots, but decreased substantially at the middle phase. Thus, free IAA may play a role in the initiation and maturation of clubroot. Total IAA content was significantly higher in infected roots than in non-infected roots throughout clubroot development and IAA conjugation/conjugate hydrolysis system as well as BrNIT-Ts appear to be involved in clubroot development.

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  • Proteomic characterization of tissue expansion of rice scutellum stimulated by abscisic acid Reviewed

    Tsuyoshi Asakura, Shota Hirose, Satoru Asatsuma, Yohei Nanjo, Tetsuyo Nakaizumi, Kimiko Itoh, Hidetaka Hori, Setsuko Komatsu, Toshiaki Mitsui

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   71 ( 5 )   1260 - 1268   2007.5

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    We found that appropriate treatment with a highly potent and long-lasting abscisic acid analog enhanced the tissue expansion of scutellum during early seedling development of rice, accompanied by increases of protein and starch accumulation in the tissue. A comparative display of the protein expression patterns in the abscisic acid analog-treated and non-treated tissues on two dimensional gel electrophoretogram indicated that approximately 30% of the scutellar proteins were induced by abscisic acid. The abscisic acid-induced proteins included sucrose metabolizing, glycolytic, and ATP-producing enzymes. Most of these enzyme proteins also increased during the seedling growth. In addition, the expression of some isoforms of UDP-glucose pyrophosphorylase, 3-phosphoglycerate kinase, and mitochondrial ATP synthase beta chain was stimulated in the scutellum, with suppressed expression of a-amylase. We concluded that abscisic acid directly and indirectly stimulates the expression of numerous proteins, including carbohydrate metabolic enzymes, in scutellar tissues.

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  • Loading of Fura-2 intoliquid organ cultured adventitious root of turnip (Brassica rapa L.) resistant to clubroot pathogenPlasmodiophora brassicae and determination of [Ca2+]cyt.

    Takahashi H, Ishikawa T, Hayakawa T, Itoh K, Mitsui T, Hori H

    Bull. Facul. Agric. Niigata Univ.   60   61 - 66   2007.3

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  • Histochemical analysis of Bacillus thuringiensis Cry1A toxin binding to midgut epithelial cells of Bombyx mori Reviewed

    Delwar M. Hossain, Tohru Hayakawa, Yasuyuki Shitomi, Kimiko Itoh, Toshiaki Mitsui, Ryoichi Sato, Hidetaka Hori

    PESTICIDE BIOCHEMISTRY AND PHYSIOLOGY   87 ( 1 )   30 - 38   2007.1

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    We analyzed the binding of the Bacillus thuringiensis insecticidal toxins, CrylAa, CrylAb and CrylAc, to midgut tissue of the silkworm, Bombyx mori with ligand blot analysis and histochemical observations. CrylAa, CrylAb and CrylAc bound to unique sets of proteins in various subcellular fractions prepared by centrifugation. CrylAa bound to various proteins in all subcellular fractions, whereas CrylAb bound to a single protein of similar to 180 kDa in all fractions as shown by Western blot analysis. CrylAc bound to proteins which were primarily similar to 100-120 kDa in all fractions. CrylA toxins were labeled with fluorescent dye and Cy3-labeled CrylAa, CrylAb and CrylAc were shown to localize primarily to the apical membrane region. However, they also localized to basement or basolateral membranes. The distribution of a 252-kDa membrane protein (P252) of the B. mori midgut, which was recently identified as a plausible candidate for receptor of CrylA toxins were also examined with histochemical methods. Substantial signals of FITC-labeled antibody against P252, even though not all, were evident in the apical cells, and these were coincident with Cy3-CrylAa and Cy3-CrylAc signals. (c) 2006 Elsevier Inc. All rights reserved.

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  • Isolation and Characterization of GAMYB mutant rice Reviewed

    Y Tanaka, K Nakano, T Mitsui, H Hori, K Itoh

    Bull Facul Agric Niigata Univ   58 ( 2 )   103 - 108   2006.12

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  • Flowering induced by 5-azacytidine, a DNA demethylating reagent in a short-day plant, Perilla frutescens var. crispa Reviewed

    H Kondo, H Ozaki, K Itoh, A Kato, K Takeno

    PHYSIOLOGIA PLANTARUM   127 ( 1 )   130 - 137   2006.5

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    Treatment with 5-azacytidine, a DNA demethylating reagent, induced flowering in Perilla frutescens (L.) Britton var. crispa (Thunb. ex Murray) Decne. ex L. H. Bailey, an absolute short-day plant under long days. The 5-azacytidine treatment induced slight suppression of vegetative growth but had no obvious effect on any other phenotypes. The Southern hybridization analysis of the genomic DNA isolated from the leaves of 5-azacytidine-treated plants and digested with restriction enzyme, methylation-insensitive Msp I or methylation-sensitive Hpa II with P. frutescens 25S-18S rDNA intergenic spacer probe indicated that the 5-azacytidine treatment caused demethylation of the genomic DNA. The 5-azacytidine-induced flowering was delayed as compared with the short day-induced flowering. Flowers were formed even at the lower nodes which had not been directly treated with 5-azacytidine. The results suggest that DNA demethylation induced flowering by inducing the production of a transmissible flowering stimulus in P. frutescens.

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  • Plasmodiophora brassicae-induced cell death and medium alkalization in clubroot-resistant cultured roots of Brassica rapa Invited Reviewed

    H Takahashi, T Ishikawa, M Kaido, K Takita, T Hayakawa, K Okazaki, K Itoh, T Mitsui, H Hori

    JOURNAL OF PHYTOPATHOLOGY   154 ( 3 )   156 - 162   2006.3

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    Plasmodiophora brassicae causes clubroot in the turnip, Brassica rapa L. We used organ cultures of adventitious roots from B. rapa seedlings to investigate the initial response of resistant and susceptible cultivars to P. brassicae infection. Primary plasmodia of P. brassicae were observed in root hairs of both susceptible and resistant cultured roots. On the other hand, secondary plasmodia were able to proliferate only in the susceptible root culture but not in the resistant one. Root cultures from the susceptible cultivar all developed clubroot 4 weeks after treatment with 10(4), 10(5) or 10(6) spores/ml, but roots from the resistant cultivar did not develop clubroot under the same conditions. Cell death, as measured by Evans blue and TTC dye methods, was observed in cultured roots from the resistant cultivar but did not occur in roots from the susceptible cultivar after exposure to P. brassicae spores. Cell death was inhibited almost completely by EGTA and verapamil but not by the calmodulin antagonist W7. These results suggest the involvement of Ca2+ in P. brassicae-induced cell death. Alkalization of the root culture medium of the resistant cultivar was observed 2 days after treatment with P. brassicae spores but was not observed in root culture medium from the susceptible strain. We conclude that our root culture system must be a useful tool for further studies of the molecular mechanism of clubroot resistance.

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  • Involvement of alpha-amylase I-1 in starch degradation in rice chloroplasts Reviewed

    S Asatsuma, C Sawada, K Itoh, M Okito, A Kitajima, T Mitsui

    PLANT AND CELL PHYSIOLOGY   46 ( 6 )   858 - 869   2005.6

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    To determine the role of alpha-amylase isoform I-1 in the degradation of starch in rice leaf chloroplasts, we generated a series of transgenic rice plants with suppressed expression or overexpression of alpha-amylase I-1. In the lines with suppressed expression of alpha-amylase I-1 at both the mRNA and protein levels, seed germination and seedling growth were markedly delayed in comparison with those in the wild-type plants. However, the growth retardation was overcome by supplementation of sugars. Interestingly, a significant increase of starch accumulation in the young leaf tissues was observed under a sugar-supplemented condition. In contrast, the starch content of leaves was reduced in the plants overexpressing alpha-amylase I-1. In immunocytochemical analysis with specific anti-alpha-amylase I-1 antiserum, immuno-gold particles deposited in the chloroplasts and extracellular space in young leaf cells. We further examined the expression and targeting of alpha-amylase I-1 fused with the green fluorescent protein in re-differentiated green cells, and showed that the fluorescence of the expressed fusion protein co-localized with the chlorophyll autofluorescence in the transgenic cells. In addition, mature protein species of alpha-amylase I-1 bearing an oligosaccharide side chain were detected in the isolated chloroplasts. Based on these results, we concluded that alpha-amylase I-1 targets the chloroplasts through the endoplasmic reticulum-Golgi system and plays a significant role in the starch degradation in rice leaves.

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  • Dissection of gene function by RNA silencing Reviewed

    S. M. Shahinul Islam, Takahiro Miyazaki, Fuminori Tanno, Kimiko Itoh

    Plant Biotechnology   22 ( 5 )   443 - 446   2005

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    The RNA silencing technique is an effective tool to examine the biological function of the target mRNA in plants. The recent development of versatile-type RNAi vectors, which are driven by constitutive promoters, and GATEWAY™ cloning technology makes it easy to construct the RNAi vectors with trigger sequences and to analyze the function of a target gene. Although these vectors are highly useful, constitutive defects of the target mRNA expression sometimes result in lethality or seed abortion. Here, we summarize recent approaches to RNA silencing research designed to overcome these difficulties and to dissect gene expression.

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  • Intron loop Wx 5'UTR dsRNA vectors mediated endosperm specific silencing of Wx gene of rice Reviewed

    F Tanno, H Ozaki, K Itoh

    Bull Facul Agric Niigata Univ   57 ( 2 )   121 - 128   2004.12

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  • Posttranscriptional regulation of alpha-amylase II-4 expression by gibberellin in germinating rice seeds Reviewed

    Y Nanjo, S Asatsuma, K Itoh, H Hori, T Mitsui, Y Fujisawa

    PLANT PHYSIOLOGY AND BIOCHEMISTRY   42 ( 6 )   477 - 484   2004.6

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    Hormonal regulation of expression of alpha-amylase II-4 that lacks the gibberellin-response cis-element (GARE) in the promoter region of the gene was studied in germinating rice (Oryza sativa L.) seeds. Temporal and spatial expression of alpha-amylase II-4 in the aleurone layer were essentially identical to those of alpha-amylase I-1 whose gene contains GARE, although these were distinguishable in the embryo tissues at the early stage of germination. The gibberellin-responsible expression of a-amylase II-4 was also similar to that of alpha-amylase I-1. However, the level of a-amylase II-4 mRNA was not increased by gibberellin, indicating that the transcriptional enhancement of a-amylase II-4 expression did not occur in the aleurone. Gibberellin stimulated the accumulation of Ca-45(2+) into the intracellular secretory membrane system. In addition, several inhibitors for Ca2+ signaling, such as EGTA, neomycin, ruthenium red (RuR), and W-7 prevented the gibberellin-induced expression of a-amylase II-4 effectively. While the gibberellin-induced expression of a-amylase II-4 occurred normally in the aleurone layer of a rice dwarf mutant d1 which is defective in the alpha subunit of the heterotrimeric G protein. Based on these results, it was concluded that the posttranscriptional regulation of a-amylase II-4 expression by gibberellin operates in the aleurone layer of germinating rice seed, which is mediated by Ca2+ but not the G protein. (C) 2004 Elsevier SAS. All rights reserved.

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  • Proteomic identification of alpha-amylase isoforms encoded by RAmy3B/3C from germinating rice seeds Reviewed

    Y Nanjo, S Asatsuma, K Itoh, H Hori, T Mitsui

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   68 ( 1 )   112 - 118   2004.1

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    We isolated and identified 10 alpha-amylase isoforms by using beta-cyclodextrin Sepharose affinity column chromatography and two-dimensional polyacrylamide gel electrophoresis from germinating rice (Oryza sativa L.) seeds. Immunoblots with anti-alpha-amylase I-1 and II-4 antibodies indicated that 8 isoforms in 10 are distinguishable from alpha-amylase I-1 and II-4. Peptide mass fingerprinting analysis showed that there exist novel isoforms encoded by RAmy3B and RAmy3C genes. The optimum temperature for enzyme reaction of the RAmy3B and RAmy3C coding isoforms resembled that of alpha-amylase isoform II-4 (RAmy3D). Furthermore, complex protein polymorphism resulted from a single a-amylase gene was found to occur not only in RAmy3D, but also in RAmy3B.

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  • Introduction of Wx transgene into rice wx mutants leads to both high- and low-amylose rice Reviewed

    K Itoh, H Ozaki, K Okada, H Hori, Y Takeda, T Mitsui

    PLANT AND CELL PHYSIOLOGY   44 ( 5 )   473 - 480   2003.5

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    The Waxy (Wx) gene encodes a granule-bound starch synthase (GBSS) that plays a key role in the amylose synthesis of rice and other plant species. Two functional Wx alleles of rice exist: Wx(a), which produces a large amount of amylose, and Wx(b), which produces a smaller amount of amylose because of the mutation at the 5' splice site of intron 1. Wx(b) is largely distributed in Japonica cultivars, and high amylose cultivars do not exist in Japonica cultivars. We introduced the cloned Wx(a) cDNA into null-mutant Japonica rice (wx). The amylose contents of these transgenic plants were 6-11% higher than that of the original cultivar, Labelle, which carries the Wx(a) allele, although the levels of the Wx protein in the transgenic rice were equal to those of cv. Labelle. We also observed a gene-dosage effect of the Wx(a) transgene on Wx protein expression, but a smaller dosage effect was observed in amylose production with over 40% of amylose content in transgenic rice. Moreover, one transgenic line carrying eleven copies of the transgene showed low levels of Wx expression and amylose in the endosperm. This suggested that the integration of excessive copies of the transgene might lead to gene silencing.

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  • Phosphate Modulates Ca2+ Uptake and ± Amylase Secretion in Suspension-Cultured Cells of Rice Reviewed

    Satoru Asatsuma, Yohei Nanjo, Mohammad A. Kashem, Kimiko Itoh, Hidetaka Hori, Masahiro Ohshima, Toshiaki Mitsui

    Plant Biotechnology   20 ( 3 )   187 - 194   2003

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    Effects of phosphate on the Ca2 uptake and the sucrose-controlled secretion of ±-amylase molecules in cultured rice cells were investigated. Phosphate markedly stimulated Ca2, uptake into rice cells, particularly at the outer cell layer of the cell cluster. Phosphate also increased the synthesis and extracellular liberation of a - amylase II-4 in sucrose-supplemented cells. The distribution pattern of enzyme in rice cell clusters induced by phosphate was similar to that of Ca2+ uptake. Phosphate did not increase the level of mRNA of ±-amylase II-4, indicating that phosphate stimulates the translation and posttranslational secretory processes of ±-amylase II-4 in the presence of sucrose. Furthermore, phosphate enhanced both the Ca2r uptake and α-amylase II-4 synthesis in the microsomes. These results strongly suggested that the ratio of phosphate to sugar is important for regulating the Ca2+ uptake, and that phosphate and sugar precisely coordinate the Ca2- mediated synthesis and extracellular liberation of α-amylase II-4. © 2003, Japanese Society for Plant Cell and Molecular Biology. All rights reserved.

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  • [Morphological mutagenesis of rice inflorescence by insertion of Ac transposon]. Reviewed

    Itoh K, Tanaka Y, Nakano K

    Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme   47 ( 12 Suppl )   1658 - 1659   2002.9

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  • Posttranscriptional regulation of alpha-amylase expression by gibberellin in germinating rice seeds Reviewed

    Y Nanjo, S Asatsuma, S Mikami, H Hori, K Itoh, T Mitsui

    PLANT AND CELL PHYSIOLOGY   43   S133 - S133   2002

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  • Resting Spore of Plasmodiophora brassicae Proliferates only in the Callus of Clubroot Disease-Susceptible Turnip but Increases the PAL Activity in the Callus of Clubroot Disease-Resistant Turnip Reviewed

    Hideyuki Takahashi, Satoko Muraoka, I. T.O. Kimiko, Toshiaki Mitsui, Hidetaka Hori, Akira Kiso

    Plant Biotechnology   18 ( 4 )   267 - 274   2001.3

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    The calluses were induced from turnips, Brassicae campestris L. which were susceptible and resistant to Plasmodiophora brassicae in Murashige-Skoog's agar medium supplemented with 1.0 ppm 6-benzylaminopurine and 0.5 ppm α - naphthalene acetic acid. When a 10 μ1 water suspension containing 104 resting spores of P. brassicae was placed on the surface of the calluses, about 3 × 105 zoosporangium-like spheroids (SLS) were recovered from the susceptible calluses after 24 h of the treatment but no SLS was found in the resistant callus. On 6th day after the treatment, the SLS increased to about 4 × 106 in the susceptible callus. Upon inoculation of resistant callus with 104 resting spores, the phenylalanine ammonia lyase (PAL) activity increased about 4-fold after 20 h, however, such increase was not observed in the susceptible callus during the same period. The constitutive PAL activity of susceptible callus was roughly one 6th of that of resistant callus.

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  • Enhancement of Germination of Spores from Obligatory Plant Pathogen, Plasmodiophora brassicae causing clubroot disease. Reviewed

    Satoshi Ogawa, Hideyuki Takahashi, Tohru Hayakawa, Kimiko Itoh, Toshiaki Mitsui, Hidetaka Hori, Akira Kiso

    Bull. Facul. Agric. Niigata Univ.   54 ( 1 )   35 - 43   2001

  • Possible involvement of phosphoinositide-Ca2+ signaling in the regulation of alpha-amylase expression and germination of rice seed (Oryza sativa L.). Reviewed

    MA Kashem, K Itoh, S Iwabuchi, H Hori, T Mitsui

    PLANT AND CELL PHYSIOLOGY   41 ( 4 )   399 - 407   2000.4

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    We have studied the effects of neomycin, a potent inhibitor of inositol phospholipid-specific phospholipase C (PLC), on the germination of rice seed and the gibberellin-induced expression of alpha-amylase in the aleurone layer and the scutellar tissues. It was shown that, in the absence of exogenous Ca2+, neomycin markedly reduced the germination speed and seedling growth of rice seeds and inhibited the gibberellin-induced expression of alpha-amylase in both secretory tissues. In addition, neomycin decreased the formation of inositol 1,4,5-trisphosphate (IP3) in the gibberellin-treated aleurone layer and the scutellar tissues. However, the inhibitory effects on the germination speed and the expression of alpha-amylase activity were overcome by supplementation of Ca2+. In addition, gibberellin elevated the level of IP3, and ABA prevented the gibberellin-induced formation of IP3, although ABA alone did not alter the IP3 level. The expression of a membrane-bound PLC molecule in rice aleurone layer was shown to be induced by gibberellin, and the gibberellin-induced expression of PLC was markedly delayed by treatment with ABA, These results strongly suggest that the phosphoinositide-Ca2+ signal transduction pathway may play an important role in the gibberellin-induced expression of alpha-amylase molecules closely related to the germination processes of rice seed.

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  • Sucrose-controlled transport and turnover of alpha-amylase in rice (Oryza sativa L.) cells Reviewed

    T Mitsui, T Loboda, Kamimura, I, H Hori, K Itoh, SI Mitsunaga

    PLANT AND CELL PHYSIOLOGY   40 ( 8 )   773 - 783   1999.8

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    We studied the sucrose-controlled intracellular transport and turnover of alpha-amylase molecules in suspension cultured cells of rice (Oryza saliva L.) employing pulse labeling techniques with [S-35] amino acids. The secretion of two classes of a-amylase isoforms, alpha-amylase I-1 (encoded by RAmy1A) and II-4 (encoded by RAmy3D) was differentially controlled by sucrose. In rice cells labeled with [S-35] amino acids under different sucrose-supplemented conditions, sucrose preferentially prevented the extracellular liberation of [S-35]-labeled alpha-amylase II-4 molecules from rice cells at around 2 mM, whereas the de novo protein synthesis still occurred at this concentration. Pulse-chase experiments showed that sucrose regulates the intracellular transport of [S-35]alpha-amylase II-4 molecules and stimulates the protein turnover. However, cycloheximide, a protein synthesis inhibitor, was induced the extracellular liberation and reduced the turnover of [S-35]alpha-amylase II-4 molecules in the presence of sucrose. These results strongly suggested that newly synthesized sucrose-induced proteins are involved in the posttranslational regulation on sucrose-controlled alpha-amylase secretion in rice cells.

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  • Two distinct types of silencing show different tissue specificity and stability in Wx gene silencing(proceedings) Reviewed

    K. Itoh, H. Ozaki, T. Mitsui, K. Shimamoto

    Agriculture Biotechnology: Laboratory, Field and Market   221 - 222   1998.12

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  • Effects of (+)-8 ',8 ',8 '-trifluoroabscisic acid on alpha-amylase expression and sugar accumulation in rice cells Reviewed

    MA Kashem, H Hori, K Itoh, T Hayakawa, Y Todoroki, N Hirai, H Ohigashi, T Mitsui

    Planta   205   319 - 326   1998.5

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    The effects of (+)-8',8',8'-trifluoroabscisic acid (trifluoro-ABA) on alpha-amylase expression were studied in rice embryoless half-seeds, scutella, and suspension-cultured cells derived from the embryo, and the effects of the analog on sugar accumulation were also studied in scutella and suspension-cultured cells. Treatment with (+)-trifluoro-ABA strongly inhibited the gibberellic acid-inducible expression of alpha-amylase I-1 encoded by RAmy1A in the aleurone layers of embryoless half-seeds at the levels of transcription, protein synthesis, and enzyme activity. It was also found that (+)-trifluoro-ABA stimulated (i) the uptake of glucose from the incubation medium and (ii) the synthesis of sucrose in scutellar tissues and suspension-cultured cells of rice. The biological activity of(+)-trifluoro-ABA was found to be more potent and persistent than that of natural ABA. We further examined the effects of trifluoro-ABA on the expression of alpha-amylase I-1 in scutellar tissues and suspension-cultured cells. It was found that (+)-trifluoro-ABA did not inhibit the formation of alpha-amylase I-1 in the absence of external glucose. However, glucose and (+)-trifluoro-ABA cooperatively suppressed the formation of alpha-amylase I-1. Judging from these results, we conclude that the regulatory mechanism for the expression of alpha-amylase I-1 in the scutellar epithelium is distinguishable from that operating in the aleurone layer.

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  • Transposon tagging in rice Reviewed

    Takeshi Izawa, Toru Onishi, Toshitugu Nakano, Nobuhiro Ishida, Hiromasa Enoki, Hisako Hashimoto, Kimiko Itoh, Rie Terada, Denxing Wu, Chikara Miyazaki, Tomoko Endo, Shigeru Iida, Ko Shimamoto

    Plant Molecular Biology   35 ( 1-2 )   219 - 229   1997.9

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    To develop an efficient gene isolation method for rice we introduced the maize Ac/Ds system into rice. Extensive analysis of their behavior in rice for several generations indicated that Ac and Ds in the presence of Ac transposase gene actively transpose in rice. A wide spectrum of mutations affecting growth, morphogenesis, flowering time and disease resistance have been obtained in the population carrying Ac/Ds and some of them were genetically analyzed. Main efforts are currently being made to isolate genes responsible these mutations. In addition, a number of Ac/Ds were mapped on chromosomes and mapped elements will be used in the future for directed tagging of genes with known chromosomal positions.

    File: 199709_TransposonTaggingInRice.pdf

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  • Silencing of waxy genes in rice containing Wx transgenes Reviewed

    Kimiko Itoh, Midori Nakajima, Ko Shimamoto

    Molecular genetics and genomics   255 ( 4 )   351 - 358   1997.7

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    In order to study gene silencing in a monocot system we introduced a waxy (Wx) gene into rice. In the pollen grain of the transgenic wild-type plants, two types of Wx gene silencing were observed: Type I in which all the pollen grains showed the mutant (wx) phenotype, and Type II in which 50% of the pollen grains showed the wx phenotype. Analysis of Wx gene expression in the progeny of selfing and outcrossing indicated that Wx gene silencing was meiotically transmitted to the offspring. The number of transgene copies and transgene loci was determined by Southern blot analysis and suggested that the Wx transgene may have a paramutagenic effect on the endogenous Wx genes. In contrast to the pollen grain, the wx phenotype was not observed in the endosperm. However, the level of WAXY (WX) protein in the endosperm of Type I lines was similar to that in non-transgenic seed, while in Type II lines two classes of seeds, showing high and low levels of the protein segregated. When the same transgene was introduced into wx mutants in which no Wx transcript was detectable, the transgene behaved as a dominant Mendelian factor and no silencing was found, suggesting that the activity of the endogenous Wx gene influences the silencing phenomenon. Our study of Wx gene silencing in rice extends the well-known phenomenon of gene silencing, so far observed mainly in dicots, to a cereal.

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  • The alpha-amylase multigene family Reviewed

    Toshiaki Mitsui, Kimiko Itoh

    Trends Plant Science   2 ( 7 )   255 - 261   1997.7

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    Many alpha-amylase genes have been cloned from various cereals, and sequence analysis reveals that these fall into two major classes and three subfamilies. In parallel studies, many alpha-amylase isoforms have been characterized and their corresponding genes identified. Understanding of the regulatory mechanisms that operate to control expression of the alpha-amylase multigene family has also advanced in recent years. In the light of the improved understanding of alpha-amylase activity, we have addressed a classical argument over the 'scutellum versus aleurone' concept. This disagreement concerns the site of synthesis and secretion of alpha-amylase. We have focused our attention on this by comparing gene expression at the transcriptional and post-transcriptional level.

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  • Characteristics of genetic variation in the progenies of protoplast-derived plants of rice, Oryza sativa cv Nipponbare Reviewed

    M Yamagishi, K Itoh, T Koba, Y Sukekiyo, K Shimamoto, T Shimada

    THEORETICAL AND APPLIED GENETICS   94 ( 1 )   1 - 7   1997.1

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    Genetic variation in protoplast-derived rice (Oryza sativa L.) plants was characterized using first and second generation selfed progenies. A total of 133 regenerated plants were obtained from ten protoplasts of the japonica rice cultivar Nipponbare. Sixty two regenerated plants which set enough seeds for the subsequent held tests at the next generation and were derived from five protoplasts were selected, and their selfed seeds were used as the first selfed-seed progeny (Pt-1 generation). Fifteen plants were selected from each of the 15 Pt-1 lines, and their selfed seeds were used for tests at the Pt-2 generation. Thirty seven Pt-1 lines (60%) segregated plants with detrimental mutant characters of yellow-green phenotype, dwarf stature, dense and short panicle, or low seed fertility. According to the segregation patterns in the lines having mutated plants among those originated from the same protoplasts, the stages of mutation induction were estimated. Additionally, five quantitative traits were changed in almost all Pt-1 and Pt-2 lines. Varied quantitative traits of heading date, number of spikelets per panicle, and seed fertility, were in a heterozygous state. However, culm and panicle lengths showed high uniformity, whereas reduced culm and panicle lengths were caused by mutational changes in polygenes and/or multiple genes.

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  • Effects of 8′,8′,8′-trifluoroabscisic acid on α-amylase secretion and sugar accumulation in rice cells Reviewed

    M. A. Kashem, K. Itoh, T. Hayakawa, T. Mitsui, Y. Todorold, N. Hirai, H. Ohigashi

    Nippon Nogeikagaku Kaishi   71   1 - 5   1997

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    DOI: 10.1271/nogeikagaku1924.71.sup_1

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  • DEVELOPMENTAL AND HORMONAL-REGULATION OF RICE ALPHA-AMYLASE(RAMY1A)-GUSA FUSION GENES IN TRANSGENIC RICE SEEDS Reviewed

    K ITOH, J YAMAGUCHI, N HUANG, RL RODRIGUEZ, T AKAZAWA, K SHIMAMOTO

    PLANT PHYSIOLOGY   107 ( 1 )   25 - 31   1995.1

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    Transgenic seeds of rice (Oryza sativa L.) were used to investigate temporal, spatial, and hormonal regulation of a rice Lu-amylase gene, RAmy1A. Two overlapping segments of the RAmy1A promoter were fused to the coding region of the bacterial reporter gene, gusA. The resulting promoter-gusA fusions, pE4/GUS (-232 to +31) and pH4/GUS (-748 to +31), were used separately to transform rice protoplasts. beta-Glucuronidase (GUS) activity was detected in germinated transgenic seeds, although the two constructs showed no significant difference in timing or location of GUS expression. Both constructs first expressed GUS in the scutellar epithelium and then in the aleurone layer. Aleurone expression of GUS activity was strongly induced when embryoless half-seeds were treated with gibberellic acid. GUS expression in the aleurone layer was also suppressed by abscisic acid. These results indicate that the 5' regulatory region from -232 to +31 is sufficient for temporal, spatial, and hormonal regulation of RAmy1A gene expression.

    File: 199501_Developmental and Hormonal Regulation of Rice.pdf

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  • Molecular analysis of genetically modified Brassica and rice Reviewed

    Kimiko ITOH

    1994.8

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    Authorship:Lead author, Corresponding author   Language:English   Publishing type:Doctoral thesis  

    遺伝的に修飾された植物ゲノムの解析により,細胞工学的手法や遺伝子導入法等の実験的アプローチの有効性を論じ,これらの手法により明らかにされた未知の遺伝現象の解析によって新しい研究分野の創出の可能性を提示した。

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  • Insect resistant rice generated by introduction of a modified delta-endotoxin gene of Bacillus thuringensis. Reviewed

    Hideya Fujimoto, Kimiko Itoh, Mikihiro Yamamoto, Junko Kyozuka, Ko Shimamoto

    BIO-TECHNOLOGY   11 ( 10 )   1151 - 1155   1993.10

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    As a first step towards development of insect resistant rice we have introduced a truncated delta-endotoxin gene, cryIA(b) of Bacillus thuringiensis (B.t.) which has specific biological activity against lepidopteran insects into a japonica rice. To highly express the cryIA(b) gene in rice the coding sequence was extensively modified based on the codon usage of rice genes. Transgenic plants efficiently expressed the modified cryIA(b) gene at both mRNA and protein levels. Bioassays using R2 generation plants with two major rice insect pests, striped stemborer (Chilo suppressalis) and leaffolder (Cnaphalocrosis medinalis), indicated that transgenic rice plants expressing the CryIA(b) protein are more resistant to these pests than untransformed control plants. Our results suggest that the B.t. endotoxin genes will be useful for the rational development of new rice varieties resistant to major insect pests.

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  • Trans-activation and stable integration of the maize transposable element Ds cotransfected with the Ac transposase gene in transgenic rice plants. Reviewed

    Ko Shimamoto, Chikara Miyazaki, Hisako Hashimoto, Takeshi Izawa, Kimiko Itoh, Rie Terada, Yosisige Inagaki, Shigeru Iida

    MOLECULAR & GENERAL GENETICS   239 ( 3 )   354 - 360   1993.6

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    To develop an efficient gene tagging system in rice, a plasmid was constructed carrying a non-autonomous maize Ds element in the untranslated leader sequence of a hygromycin B resistance gene fused with the 35S promoter of cauliflower mosaic virus. This plasmid was cotransfected by electroporation into rice protoplasts together with a plasmid containing the maize Ac transposase gene transcribed from the 35S promoter. Five lines of evidence obtained from the analyses of hygromycin B-resistant calli, regenerated plants and their progeny showed that the introduced Ds was trans-activated by the Ac transposase gene in rice. (1) Cotransfection of the two plasmids is necessary for generation of hygromycin B resistant transformants. (2) Ds excision sites are detected by Southern blot hybridization. (3) Characteristic sequence alterations are found at Ds excision sites. (4) Newly integrated Ds is detected in the rice genome. (5) Generation of 8 bp target duplications is observed at the Ds integration sites on the rice chromosomes. Our results also show that Ds can be trans-activated by the transiently expressed Ac transposase at early stages of protoplast culture and integrated stably into the rice genome, while the cotransfected Ac transposase gene is not integrated. Segregation data from such a transgenic rice plant carrying no Ac transposase gene showed that four Ds copies were stably integrated into three different chromosomes, one of which also contained the functional hph gene restored by Ds excision. The results indicate that a dispersed distribution of Ds throughout genomes not bearing the active Ac transposase gene can be achieved by simultaneous transfection with Ds and the Ac transposase gene.

    File: 199306_Shimamoto1993_Article_Trans-activationAndStableInteg.pdf

    DOI: 10.1007/bf00276933

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  • GENETICALLY ENGINEERED RICE RESISTANT TO RICE STRIPE VIRUS, AN INSECT-TRANSMITTED VIRUS Reviewed

    T HAYAKAWA, YF ZHU, K ITOH, Y KIMURA, T IZAWA, K SHIMAMOTO, S TORIYAMA

    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA   89 ( 20 )   9865 - 9869   1992.10

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    The coat protein (CP) gene of rice stripe virus was introduced into two japonica varieties of rice by electroporation of protoplasts. The resultant transgenic plants expressed the CP at high levels (up to 0.5% of total soluble protein) and exhibited a significant level of resistance to virus infection. Plants derived from selfed progeny of the primary transformants also expressed the CP and showed viral resistance, indicating stable transmission of the CP gene and the trait of resistance to the next generation. Moreover, the virally encoded stripe disease-specific protein was not detected in transgenic plants expressing CP 8 weeks after inoculation, indicating protection before viral multiplication. These studies demonstrated that CP-mediated resistance to virus infection can be extended to cereals and to the viruses transmitted by an insect vector (planthopper).

    DOI: 10.1073/pnas.89.20.9865

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  • INSITU HYBRIDIZATION WITH SPECIES-SPECIFIC DNA PROBES GIVES EVIDENCE FOR ASYMMETRIC NATURE OF BRASSICA HYBRIDS OBTAINED BY X-RAY FUSION Reviewed

    K ITOH, M IWABUCHI, K SHIMAMOTO

    THEORETICAL AND APPLIED GENETICS   81 ( 3 )   356 - 362   1991

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    We have previously reported production of somatic hybrids between B. oleracea and B. campestris by fusion of B. oleracea protoplasts with X-irradiated B. campestris protoplasts, in order to transfer a part of the B. campestris genome into B. oleracea. Our previous analysis of morphology, chromosome number, and isozyme patterns of the hybrids suggested that they are asymmetric in nature. To obtain further evidence for the asymmetric nature of the hybrids, we isolated B. campestris-specific repetitive sequences and used them for in situ hybridization of the chromosomes of the hybrids. The repetitive DNA probes could specifically identify 8 out of 20 chromosomes of the B. campestris genome, and analysis of the hybrids indicates that 1-3 chromosomes of B. campestris are lacking in all five hybrids examined, giving clear evidence for the asymmetric nature of the hybrids. Furthermore, in situ hybridization revealed that some of the abnormal chromosomes observed in the hybrids are generated by rearrangements of B. campestris chromosomes caused by X-irradiation. Altogether, our study indicates that in situ hybridization using species-specific repetitive sequences is a useful tool to analyze chromosomal compositions of various types of hybrids obtained by cell fusion or conventional methods.

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  • MOLECULAR AND CYTOLOGICAL CHARACTERIZATION OF REPETITIVE DNA-SEQUENCES IN BRASSICA Reviewed

    M IWABUCHI, K ITOH, K SHIMAMOTO

    THEORETICAL AND APPLIED GENETICS   81 ( 3 )   349 - 355   1991

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    We isolated three different repetitive DNA sequences from B. campestris and determined their nucleotide sequences. In order to analyze organization of these repetitive sequences in Brassica, Southern blot hybridization and in situ hybridization with metaphase chromosomes were performed. The sequence cloned in the plasmid pCS1 represented a middle repetitive sequence present only in B. campestris and not detected in closely related B. oleracea. This sequence was localized at centromeric regions of six specific chromosomes of B. campestris. The second plasmid, pBT4, contained a part of the 25S ribosomal RNA gene, and its copy number was estimated to be 1,590 and 1,300 per haploid genome for B. campestris and B. oleracea, respectively. In situ hybridization with this sequence showed a clear signal at the NOR region found in the second largest chromosome of B. campestris. The third plasmid, pBT11, contained a 175-bp insert that belongs to a major family of tandem repeats found in all the Brassica species. This sequence was detected at centromeric regions of all the B. campestris chromosomes. Our study indicates that in situ hybridization with various types of repetitive sequences should give important information on the evolution of repetitive DNA in Brassica species.

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  • Computer simulation for microscopic images of barley chromosomes. Reviewed

    Kiichi Fukui, Kimiko Itoh

    Bulletin of the National Institute of Agrobiological Resources   5 ( 5 )   21 - 36   1989.12

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    種々の光学系を用いたオオムギ染色体の顕微鏡画像を数量的に捉えられる事を明らかにした。染色体画像解析システム構築に伴う,システム性能の追求の一環として行った研究である。共同研究に付き分担部分の抽出不可能。

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    Other Link: http://agriknowledge.affrc.go.jp/RN/2010451355

  • Demonstration of the contour lines in barley chromosomes. Reviewed

    Kiichi Fukui, Katsuyuki Kakeda, Kimiko Itoh

    Chromosome Information Service (現 Chromosome Science)   41   19 - 21   1986.4

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    オオムギ染色体の顕微鏡画像を用いて, 画像解析により様々な処理効果のシミュレーションを行った。共同研究につき分担部分の抽出不可能。

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Books

  • Biostimulants in Alleviation of Metal Toxicity in Plants

    S.M. Shahinul Islam, Anwar Hossain, Mahmodol Hasan, Kimiko Itoh, Narendra Tuteja( Role: Contributor ,  Application of Trichoderma spp. as biostimulants to improve soil fertility for enhancing crop yield in wheat and other crops)

    Academic Press is an imprint of Elsevier  2023  ( ISBN:9780323996006

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  • 植物化学調節実験実験マニュアル

    山口淳二, 伊藤紀美子, 島本功( Role: Contributor)

    植物化学調節学会  1997.10 

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  • 細胞工学別冊 植物細胞工学シリーズ4. モデル植物の実験プロトコールーイネ・シロイヌナズナ編

    伊藤紀美子( Role: Contributor)

    秀潤社  1996.4 

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MISC

  • グリコーゲン代謝酵素の欠損及びイネデンプン合成酵素の発現が大腸菌のグリコーゲン構造と代謝に及ぼす影響の研究

    伊藤可那, 伊藤可那, 福島真美, 松木順子, 花城勲, ZABALZA Goizeder Almagro, 高橋秀行, 金古堅太郎, 三ツ井敏明, POZUETA=ROMERO Javier, 伊藤紀美子

    応用糖質科学   10 ( 4 )   2020

  • OsSUMO3,4,5の細胞内局在性の比較解析

    伊藤菜々美, 野口夏希, 及川和聡, 伊藤紀美子, 伊藤紀美子

    日本植物細胞分子生物学会大会・シンポジウム講演要旨集   36th   2018

  • Effect of Adding Appropriate Mixture of NPK and Chicken Manure on Growth and Yield of TR-9 Paddy Variety on Beach Ridges Interspersed with Swales (BRIS) Soil

    Awang Azwan, Mohamadu Boyie Jalloh, Pang Su Kuan, Itoh Kimiko, Mitsui Toshiaki, Mohd. Dandan Alidin

    新潟大学農学部研究報告 = Bulletin of the Faculty of Agriculture, Niigata University   68   43 - 48   2016.2

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    Conversion of fertile lands to other uses has been one of limiting factors for paddy production in Malaysia. Infertile soil such as Beach Ridges Interspersed with Swales (BRIS) soil has a potential for paddy production if it&#039;s fertility has been improved. We, therefore, conducted a study to determine the optimum rate of NPK and chicken manure for growth and yield of TR-9 paddy variety grown on BRIS soil and also to determine the nutrient content of the BRIS soil after different high application rates of NPK and chicken manure. Treatments were three NPK rates at 60:30:30 kg ha^&lt;-1&gt;, 100:60:60 kg ha^&lt;-1&gt; and 150:90:90 kg ha^&lt;-1&gt;, and three chicken manure rates at 20 t ha^&lt;-1&gt;, 40 t ha^&lt;-1&gt; and 60 t ha^&lt;-1&gt;, arranged in a complete randomized design. Our results showed that TR-9 paddy variety growth and yield were not significantly affected by combination of NPK and chicken manure when grown on BRIS soil. NPK rate of 60:30:30 kg ha^&lt;-1&gt; produced the highest percentage of productive tillers (71.62 %) and 1000 grains weight (24.73 g). Chicken manure rate of 20 t ha^&lt;-1&gt; produced the highest plant height (127.75 cm), culm height (85.58 cm), percentage of filled grains (83.73 %), 1000 grains weight (25.81 g), extrapolated yield (11.16 tons ha^&lt;-1&gt;) and dry weight (464.44 g). Furthermore, application of high NPK and chicken manure to BRIS soil have significantly increased soil nutrients and organic carbon content which in turn can promote better growth and yield.マレーシアでは肥沃な土地が非農業的な用途に使用され、水稲生産の制限要因の一つとなっている。しかし、湿地に散在する浜堤(BRIS)土壌の様な痩せ地でも施肥で改良すれば水稲生産に使用可能である。そこで、我々はBRIS 土壌においてイネ品種TR-9の成長及び収量に最適な化成肥料組成及び鶏糞の施肥量を決定し、種々の組成の化成肥料または異なる量の鶏糞を施肥した後のBRIS 土壌の栄養素含有量の違いを調査する事を目的として研究を行った。3種の化成肥料組成、60:30:30 kg ha^&lt;-1&gt;、100:60:60 kg ha^&lt;-1&gt;、150:90:90 kg ha^&lt;-1&gt;または3種の鶏糞量、20 t ha^&lt;-1&gt;、40 t ha^&lt;-1&gt;、60 t ha^&lt;-1&gt; を施肥し、完全無作為化法を用いて実験を行った。その結果、 BRIS 土壌で成長させたイネ品種TR-9の成長及び収量に対して化成肥料の組成や鶏糞施肥量の違いがもたらす影響には統計学的に有意な違いはなかった。60:30:30 kg ha^&lt;-1&gt; の化成肥料の施肥区では、円錐花序の付いた稈の産生能(71.62 %)と 1000 粒重(24.73 g)において、最も高い値が示された。また、20 t ha^&lt;-1&gt; の鶏糞施肥区では草高(127.75 cm)、稈の高さ(85.58cm)、籾の充満率(83.73 %)、1000 粒重(25.81 g)、補外収量(11.16 tons ha^&lt;-1&gt;)、乾燥重量(464.44 g)において、最も高い値が示された。さらに、化成肥料または鶏糞を施肥したBRIS 土壌では、有意に土壌養分と有機炭素含有量が増加しており、イネの成長と収量の改善を促進できる事が示された。

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  • Ca-5 質量分析器を用いた澱粉物性の異なるイネ品種間のプロテオーム解析(澱粉の生合成,一般講演,一般社団法人日本応用糖質科学会平成27年度大会(第64回))

    伊藤 紀美子, 渡邊 茉莉, 金古 堅太郎, 二瓶 正崇, 三ツ井 敏明

    応用糖質科学 : 日本応用糖質科学会誌   5 ( 3 )   B47   2015.8

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  • Cloning, expression, and intracellular localization of rice SUMO genes

    Ikarashi Yuta, Noguchi Natsuki, Attia Kotb, Kitajima-Koga Aya, Mitsui Toshiaki, Itoh Kimiko

    新潟大学農学部研究報告   65 ( 1 )   77 - 83   2012.9

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    Small ubiquitin-related modifier (SUMO) is a type of ubiquitin-like proteins and regulates various protein functions through post-translational modification. The SUMO precursor proteins are processed by C-terminal cleavage reaction at the end of the di-glycine(GG) motif, and then activated and bind to substrate proteins by a series of enzymatic reaction. We cloned SUMO1-3 genes in rice, and analyzed the transient expression and intracellular localization of the DsRed fusion proteins, DsRed:SUMO1, 2, and 3 in onion epidermal cells by using confocal laser scanning microscopy. The DsRed signals from DsRed:SUMO1 and DsRed:SUMO2 were detected both in nuclei and cytoplasmic location, but not in nucleoli. In the case of DsRed:SUMO3, the DsRed signal was detected mainly in nucleus, and formed sub-nuclear domain like structure. We also tested effect of the GG motif on intracellular localization of SUMO proteins. The GG deletion mutant vectors, pDsRed:SUMO1ΔGG, pDsRed:SUMO2ΔGG, and pDsRed:SUMO3ΔGG were constructed and transiently expressed in onion epidermal cells. Result showed that the deletion mutation of GG motif suppressed the accumulation of the DsRed:SUMOΔGG proteins in the nucleus. These results indicated that the C-terminal processing of OsSUMO precursor proteins are necessary for OsSUMO localization to nucleus in onion epidermal cells.Small-ubiquitin related modifier (SUMO)はユビキチン様タンパク質の一種であり、翻訳後修飾により多様なタンパク質の機能を調節する。SUMO 前駆体タンパク質はC末側に存在するジグリシン(GG)モチーフの末端でプロセシングされ、一連の酵素反応により活性化され基質タンパク質に結合する。私たちはイネSUMO1-3遺伝子を単離し、DsRed 融合タンパク質であるDsRed:SUMO1, 2、および3をタマネギ表皮細胞において一過的に発現させ、その細胞局在性を共焦点レーザー顕微鏡により解析した。DsRed:SUMO1およびDsRed:SUMO2のシグナルは核と細胞質にも分布したが、核小体には観察されなかった。DsRed:SUMO3の場合は、主に核に局在し、そのシグナルは細胞核ドメイン様構造を形成した。私たちはまた、GGモチーフ欠失変異細胞内局在性に関わる影響をテストした。GG モチーフ欠失変異を持つベクター、pDsRed:SUMO1ΔGG、pDsRed:SUMO2Δ GG、およびpDsRed:SUMO3ΔGG を構築し、タマネギ表皮細胞において発現させた。GG モチーフの欠失変異はDsRed:SUMO Δ GG タンパク質の核への集積を抑制した。これらの結果から、OsSUMO 前駆体タンパク質のC末端のプロセシングがタマネギ表皮細胞におけるOsSUMO タンパク質の核への局在性に必要であることが示唆された。

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  • Aa1-3 Starch synthaseIIIa(SSIIIa)欠損とインディカ型Wx遺伝子(GBSSI)を組み合わせた高アミロース米創出の試み(澱粉の構造・物性・利用,一般講演,日本応用糖質科学会平成24年度大会(第61回))

    クロフツ 尚子, 阿部 克, 相原 里美, 伊藤 るみ子, 中村 保典, 伊藤 紀美子, 藤田 直子

    応用糖質科学 : 日本応用糖質科学会誌   2 ( 3 )   B28   2012.8

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  • イネヌクレオチドピロホスファターゼ/ホスホジエステラーゼのプラスチド局在に関する研究

    梅澤幸歩, 金古堅太郎, 甲州努, 石本卓也, 伊藤紀美子, 林八寿子, 豊岡公徳, 三ツ井敏明

    日本農芸化学会大会講演要旨集(Web)   2012   2012

  • Interaction between Bacillus thuringiensis Cyt2Aa toxin and Phosphatidylcholine-liposome

    小山 尚生, 萩野谷 功輔, 田中 未希, Promodonkoy B, Angsuthanasombat C, 三ツ井 敏明, 谷口 正之, 高屋 朋彰, 伊藤 紀美子, 堀 秀隆

    Bulletin of the Faculty of Agriculture, Niigata University   63 ( 2 )   69 - 76   2011.3

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    Bacillus thuringiensisが生産するCyt2Aaは、双翅目昆虫を殺虫する細胞毒性殺虫タンパク質で、その殺虫メカニズムを詳細に解明することは持続可能な農業を実現するために重要である。活性型Cyt2Aaを得るために、プロテネースKにより部分分解を行ったところ、反応時間が120分を超えると過度な分解が進行し、活性化断片と考えられる23kDaの断片が消失した。60分間の反応で得られた23kDaの消化断片はアミノ酸配列解析の結果、33番目のリシンと34番目のスレオニンの間で切断されていた。一方、90分間の消化処理で得られたペプチドは、さらに38番目のセリンまで4残基が消化切断されていたが、これを最小活性断片であるとし、蛍光物質カルセインを内部に取り込んだ人工脂質膜リポソームを作製し小孔形成活性を測定した。精製したCyt2Aaを、ホスファチジルコリンリポソーム(PC-Lipo)と反応させ、その小孔形成活性を放出されるカルセインの蛍光量から評価した。反応開始30分で小孔形成活性は最大となった。3時間かけて小孔形成活性が最大値に達するCry1Aとの反応に比べて、Cyt2AaがPC-Lipoに対して早い破壊活性を持つことが示唆され、Cyt2AとCry1Aは異なる反応様式でPC-Lipoに小孔を形成し、Cyt2Aは脂質膜とより早く反応すると考えられた。

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  • Changes in rice Golgi apparatus in response to sugar starvation

    Mitsui Toshiaki, Asakura Tsuyoshi, Takayama Kazutoshi, Nakayama Yuki, Ishiyama Ryuichi, Hirose Shota, Katamine Hiroki, Aoyama Makoto, Kaneko Kentaro, Itoh Kimiko, Hori Hidetaka

    Bulletin of the Faculty of Agriculture, Niigata University   63 ( 2 )   63 - 68   2011.3

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    我々は糖欠乏に応答したイネゴルジ体の変化を見出した。イネ懸濁培養細胞は糖欠乏条件下で培養された時、加水分解酵素、特にα-アミラーゼを活発に分泌する。正常および糖欠乏条件下で培養した細胞より調整した、NDPase結合ゴルジ膜をショ糖密度勾配遠心分離により分析したところ、糖欠乏条件下では正常条件下より高密度にシフトすることが分かった。このことは環境要因に対してゴルジ膜構成成分が動的に再編成されることを示唆する。

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  • Ethylene production during clubroot development in turnip

    Ishikawa Toshiki, Okazaki Keiichi, Itoh Kimiko, Mitsui Toshiaki, Hori Hidetaka

    Bulletin of the Faculty of Agriculture, Niigata University   63 ( 2 )   83 - 87   2011.3

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    Auxin and cytokinin play a crucial role for initiation stage of clubroot development in Plasmodiophora brassicaeinfectedcruciferous plants. On the other hand, the roles of phytohormones in a later maturation stage of clubroot remainunclear, regardless of significant accumulation of endogenous auxin indole-3-acetic acid (IAA) in fully developed clubrootgetting deteriorated. Here we analyzed the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) contents andACC oxidase (ACO) activity during clubroot development in turnip (Brassica rapa) to evaluate involvement of ethylene inclubroot development. Ethylene biosynthesis capacity estimated from endogenous ACC contents and ACO activity wasnot significantly affected in early growing clubroot but was elevated in later maturating tissues. This result suggests thatethylene biosynthesis is affected by P. brassicae infection and its level was coordinated with IAA during the maturationphase. Real-time PCR analysis of nitrilase, an IAA biosynthetic enzyme, demonstrated that treatment of turnip roots withACC inhibits nitrilase expression, whereas ACC-dependent ethylene biosynthesis is well-known to be upregulated by IAA.These insights imply that IAA biosynthesis via nitrilase precedes and induces ethylene production in the maturatingclubroot. This is the first report that ethylene could be involved in the maturation and deterioration of clubroot.Plasmodiophora brassicae 感染による根こぶ組織形成の初期段階におけるオーキシンやサイトカイニンといった植物ホルモンの関与がよく知られる一方で、根こぶ組織の成熟、腐熟段階における植物ホルモンの関与はほとんどわかっていない。本研究では、根こぶ組織成熟過程へのエチレンの関与に着目し、根こぶ病の進行に伴うエチレン前駆体1- アミノシクロプロパン-1- カルボン酸(ACC)含量とACC 酸化酵素(ACO)活性の変動を解析した。ACC 含量とACO 活性から推定されるエチレン生合成活性は、肥大初期の根こぶ組織で大きな変化は無かったが、肥大後期から腐熟期にある根こぶ組織では大きく増加していることが示された。リアルタイム定量PCR 解析の結果、ACC はオーキシン合成酵素ニトリラーゼのmRNA 発現を強く抑制することが明らかとなり、根こぶ成熟期におけるオーキシンの増加はエチレンの蓄積に先立って起こることが示唆された。

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  • イネヌクレオチドピロホスファターゼ/ホスホジエステラーゼのプラスチド局在化機構に関する研究

    梅澤幸歩, 金古堅太郎, 甲州努, 石本卓也, 伊藤紀美子, 林八寿子, 豊岡公徳, 三ツ井敏明

    生化学   2011

  • Rice Golgi proteome: identification and characterization of N-acetylglucosaminyltransferase-I-like protein

    Mitsui Toshiaki, Asakura Tsuyoshi, Nakayama Yuki, Takayama Kazutoshi, Hirose Shota, Katamine Hiroki, Aoyama Makoto, Karahashi Ayumi, Kaneko Kentaro, Ishiyama Ryuichi, Itoh Kimiko, Hori Hidetaka, Kajiura Hiroyuki, Fujiyama Kazuhito

    Bulletin of the Faculty of Agriculture, Niigata University   63 ( 1 )   19 - 27   2010.8

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    A shotgun proteomic analysis of the nucleoside diphosphatase (NDPase)-associated Golgi membranes isolated from ricesuspension-cultured cells was performed by multi-dimensional liquid chromatography-tandem mass spectrometry. The Golgimembranes contained an N-acetylglucosaminyltransferase-I-like protein, in addition to reversibly glycosylated polypeptide,putative xylosyltransferase and vacuolar sorting receptor. N-acetylglucosaminyltransferase-I-like protein (GNTI-LP)synthesized by a cell-free transcription/translation system from the cDNA clone catalyzed the reaction forming (GlcNAcβ1-2)(Manα1-6) (Manα1-3) (Man)3- (GlcNAc)2- PA from (Manα1-6) (Manα1-3) (Man)3- (GlcNAc)2- PA and UDP-GlcNAc, indicatingthat the protein is an N-acetylglucosaminyltransferase-Ⅰ. The optimal pH for the enzyme reaction was 8.0, but the enzymeactivity was expressed at a broad pH range between 5 and 8. The optimal temperature was 30℃. The enzyme requiredMn2+ and Mg2+, and the activity was inhibited with EDTA. The Km value for UDP-GlcNAc was determined to be 0.58 μM.Furthermore, the N-terminal region including a transmembrane domain (M1 to Q120) was not essential for the enzymeactivity. Western blot analyses with anti-GNTI-LP revealed that the Golgi membranes actually accumulate a protein thatmigrates as a 51 kDa band in SDS gels.イネ培養細胞から単離したNDPase- 結合ゴルジ体膜のショットガンプロテオミック解析を多次元液体クロマトグラフタンデム質量分析装置を用いて行った。ゴルジ体膜には可逆性グリコシル化ポリペプチド、キシロシルトランスフェラーゼ、液胞選別レセプターに加えて、N-アセチルグルコサミニルトランスフェラーゼI 様タンパク質(GNTI-LP)が含まれていた。無細胞転写・翻訳系を用いてcDNA クローンから合成したGNTI-LP は、(Manα1-6)(Manα1-3)(Man)3(- GlcNAc)2-PA とUDP-GlcNAc から(GlcNAcβ1-2()Manα1-6()Manα1-3()Man)3(- GlcNAc)2-PA を形成する反応を触媒し、N-アセチルグルコサミニルトランスフェラーゼⅠであることが明らかになった。酵素反応の至適pH は8.0であるが、酵素活性はpH 5からpH 8の広い範囲で維持された。また、至適温度は30℃であった。酵素はMn2+ とMg2+ を要求し、EDTA により活性が阻害された。UDP-GlcNAc に対するKm 値は0.58 μM を示した。さらに、膜貫通ドメインを含むN 末端領域(M1からQ120)は酵素活性に必須ではないことが分かった。抗GNTI-LP 抗体を用いたウエスタンブロット解析によりゴルジ膜は確かにSDS ゲル中で51 kDa のバンドとして泳動されるタンパク質を蓄積することが明らかになった。

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  • Synthesis of high quality probe using locked nucleic acid (LNA) and exploitation for ISH

    Ishikawa Toshiki, Okazaki Keiichi, Itoh Kimiko, Mitsui Toshiaki, Hori Hidetaka

    Bulletin of the Faculty of Agriculture, Niigata University   63 ( 1 )   35 - 39   2010.8

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    高度に保存された塩基配列を有するmRNA のin situ hybridization(ISH)による特異的検出を目的として、locked nucleicacid(LNA)を利用したキメラオリゴヌクレオチドプローブを作製し、その実用性を評価した。塩基配列で90%以上の相同性を示すカブのニトリラーゼアイソフォームBrNIT-T1及びBrNIT-T2との間で相同性の低い24~25塩基の領域に相補的なオリゴヌクレオチドを設計し、両者間で異なる塩基(9~10塩基)をLNA に置換したLNA-DNA キメラオリゴヌクレオチドを作製した。配列中にLNA を部分的に導入することにより、DNA のみでは55~57℃であったオリゴヌクレオチドのTm 値は78~82℃に上昇し、ISH 解析に十分な値を示した。BrNIT-T1及びBrNIT-T2の全長転写産物に対する結合性をドットブロットノザンハイブリダイゼーションにより調査したところ、BrNIT-T1及びBrNIT-T2のそれぞれの配列に対応するLNA-DNA キメラプローブは互いの転写産物に非特異的に結合することなく、目的の転写産物のみを検出できることが確認された。さらにこれらのキメラプローブをカブ根こぶ病組織のISH 解析に応用し、従来のcRNA プローブを用いた解析では得られなかった、BrNIT-T1とBrNIT-T2の時空間的な発現特異性を明らかにすることに成功した。To achieve specific detection of highly-conserved sequences by in situ hybridization (ISH), locked nucleic acid(LNA)-containing oligonucleotide probes were designed and assessed. Two isoforms of turnip nitrilases, BrNIT-T1 andBrNIT-T2, that possess more than 90% homology at the nucleotide level, were targeted in this study. Oligonucleotidescorresponding to a relatively different region between BrNIT-T1 and BrNIT-T2 were generated to contain LNAs in placeof DNA bases different between the two sequences. The partial incorporation of LNA into DNA oligonucleotides providedsufficient Tm values for ISH analysis. Dot-blot hybridization assay confirmed that the two LNA-modified oligonucleotideprobes corresponding to sequences of BrNIT-T1 and BrNIT-T2 specifically hybridized with the desired RNA sequencebut not with another one. Advantages of the LNA-modified probes in ISH analysis were also observed as specific andsensitive detection of BrNIT-T localization. Based on these results, we here report that partial incorporation of LNA intooligonucleotide probe is useful for highly specific detection of similar genes in ISH analysis.

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  • Biosynthesis and degradation of starch

    Mitsui Toshiaki, Itoh Kimiko, Hori Hidetaka, Ito Hiroyuki

    Bulletin of the Faculty of Agriculture, Niigata University   62 ( 2 )   49 - 73   2010.3

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    Higher plants accumulate transitory and storage starches in chloroplasts of illuminated photosynthetic organs and amyloplasts of heterotrophic organs, respectively. In addition to the plastids, the cytosol is also involved in the control of starch biosynthesis. These compartments are metabolically interconnected by means of carriers localized in the envelope membrane of the plastid. Multiple enzymes involved in starch biosynthesis have been identified from the genetic and biochemical analyses of various plant mutants exhibiting phenotypic changes in starch accumulation. Especially, genetic mutants of maize have offered valuable information to elucidate the role and function of various enzymes in starch biosynthesis. With the involvement of genetic manipulation techniques in the last decade, it has became theoretically possible to manipulate and create plants with novel, useful, and powerful property of starch. We summarize the knowledge on plant starch metabolism and introduce the developments on the procedure and regulation of starch biosynthesis and on the mechanism of energy recovery through degradation of starch.高等植物は、光合成器官である葉緑体および従属栄養器官であるアミロプラストにそれぞれ一過性または貯蔵性デンプンを蓄積する。デンプン生合成にはプラスチドに加え細胞質もまた制御に関与している。これらのコンパートメントはプラスチド膜に局在するキャリアーによって代謝的につながっている。デンプン蓄積の表現型が異なるさまざまな変異体植物の遺伝学的および生化学的解析により、デンプン生合成に関与する複数の酵素が同定されてきた。特に、トウモロコシの変異体はデンプン生合成における様々な酵素の機能と役割を決める情報を提供してきた。さらに、近年の遺伝子操作技術にともない、新規かつ有用なデンプンの性質を持つ植物体の作製および操作が可能になっている。我々は植物のデンプン代謝についての知見を概説するとともに、デンプン生合成の制御とその手法の発達およびデンプン分解によるエネルギー回収のメカニズムを紹介する。

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  • イネにおけるプラスチド局在糖タンパク質ヌクレオチドピロホスファターゼの糖鎖構造解析

    金古堅太郎, 甲州努, 梅澤幸歩, 北嶋(古賀)彩, 天野麻穂, 西村紳一郎, 豊岡公徳, 伊藤紀美子, 三ツ井敏明

    生化学   82 ( 9 )   2010

  • Studies on plastid targeting signal of rice α-amylase I-1

    Mizutani Rie, Okada Hisao, Hamada Yuki, Okubo Ena, Itoh Taeko, Itoh Kimiko, Mitsui Tosiaki

    Journal of Applied Glycoscience Supplement   2010   33 - 33   2010

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    DOI: 10.11541/jsag.2010.0.33.0

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  • Changes in amylose contents and molecular structures of rice endosperm starch with decreased starch branching enzyme IIb

    Idichi Kazuhisa, Hanashiro Isao, Abe Katsumi, Ito Kimiko

    Journal of Applied Glycoscience Supplement   2010   12 - 12   2010

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    DOI: 10.11541/jsag.2010.0.12.0

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  • Glycoproteomic analysis of chloroplasts isolated from rice mature leaves

    浜田 侑紀, 北嶋 彩, 岡田 久夫, 大久保 英奈, Awang A, 金古 堅太郎, 高山 和俊, 堀 秀隆, 伊藤 紀美子, 三ツ井 敏明

    Bulletin of the Faculty of Agriculture, Niigata University   62 ( 1 )   25 - 30   2009.9

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    イネ葉緑体タンパク質のグライコプロテオーム解析を行った。イネ成熟葉から不連続パーコール密度勾配遠心分離法を用いて無傷の葉緑体を単離した。葉緑体タンパク質を調製し、SDS-ポリアクリルアミドゲル電気泳動の後、ペルオキシダーゼ標識Concanavalin Aを用いたレクチンブロッティングを行った。ペルオキシダーゼ標識Concanavalin Aにより検出されたいくつかのタンパクバンドを質量分析装置を用いて解析した結果、ERシグナルペプチドとN結合型糖鎖結合部位を持つホスホグリセリン酸キナーゼ様タンパク質がイネ葉緑体に局在していることが示唆された。

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  • Structures of Endosperm Starch from a Rice wx Cultivar Expressing Wx^a Transgene

    HANASHIRO Isao, WAKAYAMA Takuji, HASEGAWA Mari, HIGUCHI Toshiyuki, ITOH Kimiko, FUKUYAMA Toshinori, TAKEDA Yasuhito

    Journal of Applied Glycoscience   56 ( 2 )   65 - 70   2009.4

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    Structural changes caused by the introduction of the &lt;i&gt;Wx&lt;sup&gt;&amp;alpha;&lt;/sup&gt;&lt;/i&gt; transgene, which encodes granule-bound starch synthase I (GBSSI), into the non-transgenic &lt;i&gt;wx&lt;/i&gt; cultivar Musashimochi were examined and compared with the previous results (&lt;i&gt;Plant Cell Physiol&lt;/i&gt;., 49, 925-933 (2008), using a transgenic line (WAB3-5) established independently from those used previously. Increases of amylose content of starch and extra-long chain (ELC) content of amylopectin were observed in the line WAB3-5 (actual amylose, 15.2%; ELC, 11.6% by weight). An HPSEC profile (size distribution) of WAB3-5 amylose showed characteristics in-between of those of &lt;i&gt;Wx&lt;sup&gt;&amp;alpha;&lt;/sup&gt;&lt;/i&gt; and &lt;i&gt;Wx&lt;sup&gt;b&lt;/sup&gt;&lt;/i&gt; cultivars, and WAB3-5 amylose had a comparably high degree of branching, which was indicated by the molar fraction of branched molecules (MF&lt;sub&gt;B&lt;/sub&gt;, 0.38) and number of chains of branched molecules (NC&lt;sub&gt;B&lt;/sub&gt;, 10.5). Amylose from cv. Yumetoiro (&lt;i&gt;Wx&lt;sup&gt;&amp;alpha;&lt;/sup&gt;&lt;/i&gt;) had a comparable degree of branching (MF&lt;sub&gt;B&lt;/sub&gt;, 0.35; NC&lt;sub&gt;B&lt;/sub&gt;, 13.7) with the WAB3-5 amylose. Chain-length distribution of WAB3-5 amylopectin showed a slightly higher amount of B&lt;sub&gt;2&lt;/sub&gt;+B&lt;sub&gt;3&lt;/sub&gt; chains than cv. Musashimochi. These results are consistent with those of the previous study, providing additional evidence for the proposed role of GBSSI in both amylose and ELC synthesis in rice endosperm. ELCs of amylopectins from line WAB3-5 and cvs. IR36 and Yumetoiro showed size distributions that were basically similar to each other but distinct from those of their amylose counterpart. ELCs were hydrolyzed by &amp;beta;-amylolysis of amylopectins with different extents of trimming, 66.3, 92.0 and 77.0% for line WAB3-5, cv. IR36 and cv. Yumetoiro, respectively, suggesting the branched structure of the ELCs is different among the three amylopectins.

    DOI: 10.5458/jag.56.65

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  • Establishment of high-amylose type Japonica rice by Agrobacterium-mediated gene transfer

    Hasegawa Mari, Sekikawa Akiko, Tanno Fuminori, Itoh Kimiko

    Bulletin of the Faculty of Agriculture, Niigata University   61 ( 1 )   41 - 45   2008.9

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    デンプン顆粒結合型デンプン合成酵素をコードするWx遺伝子はアミロース合成を司る鍵酵素である。これまでの研究から、直接導入法によりwx欠損変異体に外来Wx遺伝子を導入したイネ(WxR/wx)では、高アミロース型と低アミロース型双方の表現型を示した。そのうち複数の系統の後代において、不安定かつ不活性化された表現型が現れた。この原因は、外来遺伝子が多コピー導入されたことにより、反復配列誘導性ジーンサイレンシングが起きたためである。この不安定な表現型の出現を解決するために、バイナリーベクターpWABを構築し、アグロバクテリウム法によりwx欠損変異体に導入した。得られた形質転換イネ、WAB/wx系統では低コピー数の外来遺伝子が確認され、胚乳において高アミロース型であり、その胚乳形質は安定に次世代に遺伝した。

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  • GAMYB遺伝子の転写後・翻訳後調節の解析

    五十嵐雄太, 宮嵜敬弘, 近藤尚, 永井理子, 南條洋平, 三ツ井敏明, 伊藤紀美子

    生化学   4P-0786   2008

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  • Regulation of photoperiodic flowering related gene expression by DNA methylation

    KONDO Hiroshi, KATO Akira, ITOH Kimiko, OKAZAKI Keiichi, TAKENO Kiyotoshi

    42   47 - 47   2007.10

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    We found that the treatment with a DNA demethylating reagent, 5-azacytidine (azaC) induced flowering in Perillafrutescens, suggesting that the flowering of P. frutescens was epigenetically regulated. The progeny of the plants flowered by azaC treatment did not flower under non-inductive photoperiod. The flowering-related genes activated by azaC treatment may be deactivated by remethylation during the generation change. Thus, we hypothesized that the expression of flowering-related genes is regulated by DNA methylation. Accordingly, we compared the genomic DNA extracted from photoperiodically induced P. frutescens and that from non-induced control plants by MS-AFLP technique, and detected some fragments specific to the DNA sample of photoinduced plants. This suggests that the inductive photoperiod changed DNA methylation status. Some flowering-specific fragments were cloned, and some of them contained CpG island-like regions which are known to play an important role in epigenetical regulation in mammals. We found a fragment homologous to the gene encoding Zn-finger domain-containing protein of rice. Zn-finger domain is contained in the protein important to vernalization, suggesting a relationship to epigenetics. Interestingly, this fragment had a CpG island-like region. These results suggest that the photoperiodic flowering of P.frutescens may be regulated by DNA methylation in CpG islands and the genes for Zn-finger containing proteins.

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  • Evaluation of roles amidase which converts indole-3-acetamide to indole-3-acetic acid, in formation of clubroot in turnip

    Ishikawa Toshiki, Kuroda Haruka, Okazaki Keiichi, Itoh Kimiko, Mitsui Toshiaki, Hori Hidetaka

    Bulletin of the Faculty of Agriculture,Niigata University   60 ( 1 )   53 - 60   2007.8

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    Amidase was investigated if it was one of auxin-producing enzymes of Brassica rapa. We found amidase activity to convert indole-3-acetamide to indole-3-acetic acid in soluble protein extracts from B. rapa. Optimum condition for the enzyme activity was searched and two optimum temperatures were obtained at 45 and 55℃. Search for the heat stability of the enzyme strongly suggested that the two summits of the optimum temperatures were resulted from occurrence of variant amidases showing different temperature stabilities. The possibility of the presence of several amidase isoforms was supported by the following two observations, i.e., firstly, amidase activities at 45 and 55℃ had different pH optimums, 8.5 and 7.5, respectively. Secondly, the enzyme activities at the two temperatures were differentially fluctuated during the vegetative growth of turnip and clubroot development. Fluctuation of the amidase activity and IAA contents observed in various turnip tissues such as healthy turnip leaf, hypocotyl and roots suggested that the enzyme had a constitutive role for keeping IAA homeostasis in those tissues. Interestingly, the amidase in turnip was shown to have high activity in hypocotyl and root rather than leaf, unlike Arabidopsis one. Changes of the enzyme activity during development of B. rapa was analyzed using the tissues of clubrootdiseased turnips. The activities fluctuated differently from each other in the temperature at 45 and 55℃ in infected tissues. In addition, activity at 45℃ was specifically enhanced in a later phase of clubroot development, whereas one at 55℃ increased only in an early phase in the infected root tissues. These results indicated that the amidase played an important role in turnip growth and clubroot development.

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  • Loading of fura-2 into liquid organ cultured adventitious root of turnip (Brassica rapa L.) resistant to clubroot pathogen Plasmodiophora brassicae and determination of <Ca[2+]>cyt

    Takahashi Hideyuki, Ishikawa Toshiki, Hayakawa Tohru, Itoh Kimiko, Mitsui Toshiaki, Hori Hidetaka

    Bulletin of the Faculty of Agriculture,Niigata University   60 ( 1 )   61 - 66   2007.8

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    Our previous study using liquid organ cultured adventitious roots from turnip (Brassica rapa L.) showed that Ca2+ is required for induction of defense responses in the cultured roots on the treatment with Plasmodiophora brassicae resting spores (Takahashi et al., 2006). To evaluate change in [Ca2+]cyt in cultured root cells on contact with the spores, acetoxymethyl ester derivative of Fura-2 (Fura-2/AM) was loaded into the roots. When ionophore A23187 was treated with Ca2+ simultaneously, the Fura-2 fluorescence ratio that represents relative [Ca2+]cyt increased promptly, showing that the Fura-2/AM system is suitable to evaluate [Ca2+]cyt change in cultured root cells in a second range. Applicability of this method for studies on Ca2+ fluctuation against various extracellular stimuli was supported by observations that treatment with mannitol or NaCl also immediately increased the Fura-2 ratio. When the Fura-2/AM loaded roots were treated with resting spores, no [Ca2+]cyt change was observed during 500 second but when treated with spores pre-incubated with germination-enhancing suspension (GES), a slight but reproducible increase in [Ca2+]cyt was observed. We conclude that although further analysis is needed, the Fura-2/AM system will contribute to revealing the Ca2+ involvement in clubroot-resistance response.

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  • RNAiから見えてきた植物の細胞特異的な 発現制御

    伊藤紀美子, 関公二, 丹野史典

    化学と生物   43 ( 5 )   329 - 335   2005.12

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    DOI: 10.1271/kagakutoseibutsu1962.43.329

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  • RNAiから見えてきた植物の細胞特異的な発現制御

    伊藤 紀美子, 関 公二, 丹野 史典

    化学と生物   43 ( 5 )   329 - 335   2005.5

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    DOI: 10.1271/kagakutoseibutsu1962.43.329

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  • Intron Loop Wx 5'UTR dsRNA Vectors Mediated Endosperm Specific Silencing of Wx Gene of Rice

    TANNO Fuminori, OZAKI Hiroko, ITOH Kimiko

    Bulletin of the Faculty of Agriculture,Niigata University   57 ( 2 )   121 - 128   2005.3

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    To evoke endosperm specific disruption of gene function and quantitatively regulated RNAi, ihp dsRNA vectors, pWRI-A and pWRI-B, were constructed by using endosperm specific promoter and 1st intron of Wx gene. pWRI-B carries a single mutation at the 5&#039; splicing site of 1st intron and the mutation reduces the mature transcript of the dsRNA gene. Both vectors efficiently disrupted Wx mRNA function among 96-100% of transgenic rice, and the degrees of Wx gene suppression at WRI-B/Wxb endosperms were weaker than that of WRI-A/Wxb endosperms. These results clearly showed the Wx promoter regulates endosperm specific RNAi. Moreover, the controlling the splicing efficiency can quantitatively regulates the suppression effect on RNAi.胚乳特異的な遺伝子機能の破壊及びRNAiの量的調節について解明するため、胚乳特異的プロモーターで誘導され、第一イントロンをループに持つRNAiベクター、pWRI-A、pWRI-Bを構築した。pWRI-Bは第一イントロンの5&#039;側スプライシング部位に塩基置換があり、RNAi特異的二本鎖RNAの蓄積量が減少する。両方のRNAiベクターとも96~100%の形質転換体で内在Wx mRNAの発現抑制が観察された。さらに、WRI-B/Wxbの抑制効果はWRI-A/Wxbよりも弱かった。これらの結果により、Wxプロモーターが胚乳特異的なRNAiを誘導させることが明らかとなった。さらに、スプライス効率の調節がRNAiの抑制効果を制御できる可能性が示唆された。

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  • The permanent flowering state and flowering induced by DNA demethylation.

    H Kondo, T Miura, K Itoh, A Kato, K Takeno

    PLANT AND CELL PHYSIOLOGY   46   S132 - S132   2005

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  • Quest of florigen by physiological methods - the case studies in Pharbitis nil and Perilla frutescens

    H Kondo, K Itoh, A Kato, K Takeno

    PLANT AND CELL PHYSIOLOGY   46   S10 - S10   2005

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  • Involvement of alpha-amylase I-1 for starch metabolism in rice chloroplasts

    C Sawada, S Asatsuma, A Kitajima, K Itoh, T Mitsui

    PLANT AND CELL PHYSIOLOGY   46   S106 - S106   2005

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  • Functional analysis of rice nucleotide pyrophosphatase

    Y Nanjo, N Ikarashi, H Oka, K Itoh, J Pozueta-Romero, T Mitsui

    PLANT AND CELL PHYSIOLOGY   46   S106 - S106   2005

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  • Research for inactivation of gene function at Wx locus of rice

    K Seki, F Tanno, H Ozaki, K Itoh

    PLANT AND CELL PHYSIOLOGY   46   S148 - S148   2005

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  • Research and development of RNAi vectors for tissue-specific disruption of gene function.

    F Tanno, K Seki, K Itoh

    PLANT AND CELL PHYSIOLOGY   46   S7 - S7   2005

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  • Characterization of rice nucleotide pyrophosphatase isoform

    Y Kondo, Y Nanjo, K Itoh, T Mitsui

    PLANT AND CELL PHYSIOLOGY   46   S106 - S106   2005

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  • イネの花成におけるジベレリンシグナルの関与

    西村貫, 宮崎敬弘, ISLAM S. M. Shahinul, 三ツ井敏明, 三ツ井敏明, 横井修司, 島本功, 伊藤紀美子, 伊藤紀美子

    日本分子生物学会年会講演要旨集   28th   2005

  • Utilization of published databases as a supplementary material for the genomics lecture.

    Itoh Kimiko

    大学教育研究年報   9 ( 9 )   41 - 45   2004.3

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    Language:Japanese   Publisher:Niigata University  

    In field of genomics, many genome-related on-line databases are published and are available. In this trial, to assist students in a better understanding of concept of the genomics, information of the published databases was utilized for a supplementary material of genomics lecture. Students can learn the scheme of genome information and concrete example for the description of the gene information through the database operation. Demonstration of the database operation and hard copy of the web-based materials assisted in downloading precise gene information from the published databases. The trial helped students their understanding of the lecture and increasing their interest in various genome information from the published databases. According to the survey, 73.7% of the respondents answered the trial was effective in deepening a understanding of the lecture.

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  • Flowering induced by DNA demethylation in Perilla frutescens var. crispa

    H Kondo, H Ozaki, K Itoh, A Kato, K Takeno

    PLANT AND CELL PHYSIOLOGY   45   S72 - S72   2004

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  • Analysis of alpha I-1 co-suppression line in rice

    S Asatsuma, C Sawada, M Ohshima, K Itoh, H Hori, T Mitsui

    PLANT AND CELL PHYSIOLOGY   45   S105 - S105   2004

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  • アミロライスの開発

    伊藤 紀美子

    年報   2004   211 - 215   2004

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    Language:Japanese   Publisher:飯島記念食品科学振興財団  

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  • Suppression of Wx gene expression by stable transformation of RNAi vectors.

    F Tanno, K Seki, H Ozaki, K Itoh

    PLANT AND CELL PHYSIOLOGY   45   S36 - S36   2004

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  • Purification and cDNA cloning of rice nucleotide pyrophosphatase

    Y Nanjo, S Kurokawa, Y Kondo, K Itoh, J Potueta-Romero, T Mitsui

    PLANT AND CELL PHYSIOLOGY   45   S139 - S139   2004

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  • Acトランスポゾンの挿入によって生じた イネ花序の形態変異

    伊藤紀美子, 田中義人, 中野浩一

    蛋白質 核酸 酵素   47 ( 12 )   1658 - 1559   2002.12

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  • Paramutation at waxy locus of rice

    OZAKI H, TANNO F, SAWAZAKI E, WADA H, ITOH K

    育種学研究 = Breeding research   4 ( 1 )   33 - 33   2002.3

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  • Characterization of Inositol 1,4,5-Trisphosphate-Receptor Like Glycoprotein in Rice Cells :

    Iwabuchi Satoru, Watanabe Satoko, Ito Kimiko, Hori Hidetaka, Mitsui Toshiaki

    Plant and cell physiology   41   s65   2000

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    Other Link: https://projects.repo.nii.ac.jp/?action=repository_uri&item_id=183786

  • PHOSPHOINOSITIDE-SIGNALING PATHWAY IS INVOLVED FOR GA INDUCED α-AMYLASE EXPRESSION IN GERMINATING RICE SEEDS.

    KASHEM M. A., ITOH K., HORI H., MITSUI T

    40   s161 - s161   1999.3

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  • Gene silencing in transgenic plants

    SHIMAMOTO Ko, MORINO Kazuko, KANNO Tatsuo, KYOZUKA Junko, NAITO Satoshi, ITOH Kimiko

    育種学最近の進歩   39   18 - 21   1997.10

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  • Homology-dependent Gene Silencing in Plants

    ITOH Kimiko

    Kagaku To Seibutsu   34 ( 8 )   503 - 509   1996.8

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    Language:Japanese   Publisher:Japan Society for Bioscience, Biotechnology, and Agrochemistry  

    DOI: 10.1271/kagakutoseibutsu1962.34.503

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  • GENE SILENCING AT THE WAXY LOCUS OF RICE:COSUPPRESSION AND PARAMUTATION

    SHIMAMOTO Ko, ITOH Kimiko

    36   S3   1995.3

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  • CP-Mediated Resistance against RSV of Transgenic Rice Plants by Expressing with Tissue Specific Promoter

    HAYAKAWA T, KIMURA Y, ITOH K, McELROY D, WU R

    Annals of the Phytopathological Society of Japan   60 ( 3 )   378 - 378   1994.6

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  • INSECT-RESISTANT RICE GENERATED BY INTRODUCTION OF A MODIFIED DELTA-ENDOTOXIN GENE OF BACILLUS-THURINGIENSIS

    A TANAKA, H FUJIMOTO, K ITOH, M YAMAMOTO, J KYOZUKA, K SHIMAMOTO

    BIOCONTROL SCIENCE AND TECHNOLOGY   4 ( 4 )   485 - 485   1994

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  • Toward Molecular Cloning of Blast Resistance Genes by Maize Transposable Elements, Ac/Ds, in Rice

    HASHIMOTO H, ITOH K, IZAWA T, TERADA R, MIYAZAKI C, IIDA S, SHIMAMOTO K

    Annals of the Phytopathological Society of Japan   59 ( 3 )   273 - 273   1993.6

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  • (188) 遺伝子導入によるイネ縞葉枯ウイルス抵抗性イネの作出 (日本植物病理学会大会)

    早川 孝彦, 朱 亜峰, 木村 雄輔, 伊藤 紀美子, 島本 功, 鳥山 重光

    日本植物病理學會報   57 ( 3 )   441 - 441   1991.7

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Presentations

  • グリコーゲン代謝酵素の欠損及びイネデンプン合成酵素の発現が大腸菌のグリコーゲン構造と代謝に及ぼす影響の研究

    伊藤可那, 福島真美子, 松木順子, 花城勲, ゴイゼデール ザバルザ, 高橋秀行, 金古堅太郎, 三ツ井敏明, ハビア ポズエタ ロメロ, 伊藤紀美子

    第69回日本応用糖質科学会2020年度大会(Web開催)  2020.9 

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    Event date: 2020.9

    Language:Japanese   Presentation type:Poster presentation  

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  • Development of Super-high amylose starch Invited International conference

    Kimiko ITOH, Daichi GOTO, Katsumi ABE, Isao HANASHIRO, Yasunori NAKAMURA, Ryuichi SHIRATORI

    The 5th International conference on Agricultural and Biological Science (ABS 2019)  2019.7 

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  • Rice GBSSI affects glycogen structure and metabolome in glycogen metabolic enzyme deficient- and non-deficient Escherichia coli. International conference

    ITOH Kimiko

    Fisiología Vegetal  2019.6 

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    File: アブスト集のコピー.pdf

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  • Comprehensive identification of OsSUMO binding proteins in rice exposed to high temperature stress condition. International conference

    T. KUSHIOKA, H. ATAKA, A. KOTB, K. KANEKO, T. MITSUI, K. ITOH

    Human Proteome Organization 12th Annual World Congress  2013.9  HUPO

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    Venue:YOKOHAMA  

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  • 高温ストレス条件下におけるOsSUMO結合タンパク質の網羅的同定

    串岡拓也, 金古堅太郎, KOTB ATTA, 一色正之, 三ツ井敏明, 伊藤紀美子

    第54回日本植物生理学会年会  2013.3  日本植物生理学会

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    Language:English   Presentation type:Oral presentation (general)  

    Venue:岡山大学  

    PF125(0600)
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  • イネSUMO/ E2パラログによる細胞内局在性制御の研究

    野口夏希, 三ツ井敏明, 一色正之, 伊藤紀美子

    第54回日本植物生理学会年会  2013.3  日本植物生理学会

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    Language:Japanese   Presentation type:Poster presentation  

    Venue:岡山大学  

    PF124(0599)
    要旨集 p259

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Industrial property rights

  • Wx遺伝子発現抑制方法および該方法に用いられる遺伝子

    佐藤 文彦, 伊藤 紀美子, 丹野 史典, 尾崎 寛子

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    Application no:特願2003-146245  Date applied:2003.5

    Patent/Registration no:特許第3855033号  Date issued:2006.12

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Research Projects

  • オイルボディを介した植物のレジリエンス獲得の分子機構の研究

    Grant number:19K06039

    2019.4 - 2023.3

    System name:科学研究費助成事業

    Research category:基盤研究(C)

    Awarding organization:日本学術振興会

    高橋 秀行, 伊藤 紀美子

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    Grant amount:\4290000 ( Direct Cost: \3300000 、 Indirect Cost:\990000 )

    多年生植物は,冬芽のなどの休眠器官を形成し,休眠することで耐冷性・耐凍性を獲得し越冬する.その後,春になりストレスが解除されると萌芽し成長を開始する.前年度までの研究から,貯蔵脂質であるトリアシルグリセロール(TAG)を蓄積するオイルボディが,成長回復(レジリエンス)を調節することで萌芽の成立に関わる可能性が見出されている.
    本年度は,レジリエンスとの関連性が推測されている脂質について,野生型とobap1ゲノム編集個体で,低温ストレス処理時からストレス解除までの挙動を経時的に調査した.低温ストレス処理時には,これら脂質の蓄積が観察されたが,野生型とobap1ゲノム編集個体で顕著な差は検出されなかった.一方,低温ストレス解除後の脂質を定量したところ,野生型では脂質量が減少したがobap1ゲノム編集個体では蓄積したままであった.特にTAGにその傾向が強く,検出された殆どのTAGが野生型に比べ優位に蓄積していることが判明した.そこで,TAGの分解産物である脂肪酸を定量すると,低温ストレス解除後のobap1ゲノム編集個体では脂肪酸量が減少していることが明らかとなった.これらの結果から,低温ストレスによって蓄積したTAGは低温ストレス解除後の成長回復のエネルギー源であり,OBAP1がTAGの分解に関与している可能性が示唆された.
    TAGから遊離した脂肪酸は,ペルオキシソームにおけるβ酸化を経てATP合成に用いられる.本研究で,β酸化に関与する酵素遺伝子の発現量を調査したところ,低温ストレス処理時からストレス解除までに明らかな変動は確認されず,野生型とobap1ゲノム編集個体で差は確認されなかった.この結果は,β酸化の機構は低温ストレスには影響されず,OBAP1とも関与していないことを示している.即ち,β酸化に供される脂肪酸量の多少がレジリエンスに強く影響する可能性が示された.

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  • Development of ultra-high-amylose rice starch of BEs triple mutant and study of its properties for utilization

    Grant number:15K00779

    2015.4 - 2018.3

    System name:Grants-in-Aid for Scientific Research

    Research category:Grant-in-Aid for Scientific Research (C)

    Awarding organization:Japan Society for the Promotion of Science

    Itoh Kimiko

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    Grant amount:\4680000 ( Direct Cost: \3600000 、 Indirect Cost:\1080000 )

    To develop starch with a novel property, a ΔBEs/Wxa line with the reduced expression of three starch branching enzymes was developed. The appearance of trait of the mature seeds was white and opaque, and floury. Round and small starch grain was observed in the cross section of the seeds. The X-ray diffraction pattern of the ΔBEs/Wxa starch showed B type pattern. The unit chain length distribution of the starch showed that short- and long- chain fractions were greatly reduced. The apparent amylose content was up to 65% maximum, calculated from weight distribution of the unit chains. The true amylose content was up to 47.7 % maximum. Since the ΔBEs/Wxa starch was fine grain, round, and floury, it is expected to be used as an ultrafine grain powder.

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  • イネの温度ストレス応答に関わる翻訳後修飾因子SUMOの細胞生物学的研究

    2010.9 - 2011.3

    System name:受託研究(一般受託研究)

    Awarding organization:財団法人佐々木環境技術振興財団

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    Grant type:Competitive

    Grant amount:\1000000

    生物における温度ストレスに関わる制御システムとして、タンパク質翻訳後修飾の一つ、SUMO化が知られている。SUM0(Small Ubiquitin-like modifier) は低分子のタンパク質修飾因子であり、標的タンパク質に結合することをSUMO化と呼ぶ。本研究以前に、イネにおいて、既に報告されたものも含めて5つのSUMO配列を見いだし、遺伝子を単離した。本研究ではこれらを用いて温度ストレス環境下におけるSUMOの標的タンパク質の同定および、細胞学的な研究を進めた。

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  • イネにおける高温ストレス応答に対する植物SUMO修飾機構の細胞機能研究

    2010.8 - 2012.8

    System name:科学研究費助成事業

    Research category:特別研究員奨励費

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    Grant type:Competitive

    Grant amount:\2400000 ( Direct Cost: \2400000 )

    SUMO化はタンパク質の翻訳後修飾のひとつであり、タンパク質間相互作用/活性調節/細胞内移行に関わっており、ユビキチン化と同様なシステムによって行われる。
    申請者らは、イネにおいて5つのSUMO類似配列およびSUMO化に関わる遺伝子の単離を行った。(1)免疫沈降・TOF-MS/LC-MS/MSによるタンパク質同定により、高温、強光ストレス環境下におけるSUMOの標的タンパク質の同定を行う事で、温度ストレス耐性の分子メカニズムを明らかにする。また、(2)SUMOの細胞学的な研究は植物においてほとんど報告されていないが、申請者らは活性化により核に局在化し、核ドメインと見られる構造体として観察されることを明らかにした。これら核ドメインの機能・温度ストレスとの関係を明らかにする。

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  • イネデンプン代謝に関わる糖タンパク質の新奇プラスチド局在化機構の解明

    2010.4 - 2014.3

    System name:科学研究費助成事業

    Research category:基盤研究(B)

    Awarding organization:日本学術振興会

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    Grant amount:\655000 ( Direct Cost: \655000 )

    イネデンプン代謝に関わる糖タンパク質の新奇プラスチド局在化機構の解明

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  • イネスターチシンターゼアイソザイムを用いたテーラーメイドデンプンの創製

    2007.4 - 2010.3

    System name:科学研究費助成事業

    Research category:基盤研究(B)

    Awarding organization:日本学術振興会

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    Grant type:Competitive

    Grant amount:\1440000 ( Direct Cost: \1344000 、 Indirect Cost:\96000 )

    デンプン生合成には多数の酵素、アイソザイムが関与しており、これらの欠損変異体は、独特のデンプンを蓄積する。中でもデンプンの直鎖を伸長するスターチシンターゼ(SS)は、最も多くのアイソザイムをもち、それらによって伸長する長さが異なり、機能分担している。本研究では、SSアイソザイムの変異体を片親あるいは宿主にして、二重変異体および組換体を作出することで、産業利用可能なものを含むユニークな米デンプンの生産に成功した。

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  • デンプン顆粒結合型アミロース合成酵素によるアミロペクチン構造の変換と物性の解析

    2004.4 - 2007.3

    System name:科学研究費助成事業

    Research category:基盤研究(B)

    Awarding organization:日本学術振興会

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    Grant amount:\15800000 ( Direct Cost: \15800000 )

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  • OsGAMyb遺伝子の改変による多収性米の分子育種

    2003.10 - 2005.9

    System name:科学研究費助成事業

    Research category:特別研究員奨励費

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    Grant type:Competitive

    Grant amount:\2400000 ( Direct Cost: \2400000 )

    OsGAMyb遺伝子の過剰発現により穂の形態、特に一次枝梗の形成が促進されることを明らかにした。

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  • 植物遺伝子/植物におけるRNA機能の不活化と遺伝子機能解析/RNAiを利用した組織特異的な遺伝子機能の不活性化の研究

    2000.12 - 2004.3

    System name:科学研究費助成事業

    Research category:未来開拓学術事業

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    Grant type:Competitive

    Grant amount:\55652040 ( Direct Cost: \55652040 )

    RNAiを利用した組織特異的な遺伝子機能の不活性化の研究

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  • カルシウムイオン濃度の変化によって発現が制御される遺伝子群の解析

    2000.4 - 2002.3

    System name:受託研究

    Awarding organization:独立行政法人農業生物資源研究所

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    Grant type:Competitive

    Grant amount:\10119000

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  • イネWx座における不活性化の分子機構の解明

    1998.7 - 2001.3

    System name:受託研究(一般受託研究)

    Awarding organization:独立行政法人農業生物資源研究所

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    Grant type:Competitive

    Grant amount:\11433000

    イネゲノムプロジェクトMP2202
    イネWx座における不活性化の分子機構の解明

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  • トランスポゾン・タギングによる発芽関連遺伝子の探索と解析

    Grant number:10182211

    1998

    System name:科学研究費助成事業

    Research category:特定領域研究(A)

    Awarding organization:日本学術振興会

    伊藤 紀美子

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    Grant amount:\3000000 ( Direct Cost: \3000000 )

    発芽は栄養成長期の器官形成における重要なステップである。発芽時に、イネでは植物ホルモン・ジベレリン(GA)が重要な役割を担い、胚乳貯蔵物質の分解酵素の合成・分泌、芽・根の伸長および葉の展開を促進する。本研究では、トランスポゾン・タギングによりイネにおけるGA情報伝達系の調節因子を含む発芽関連遺伝子の変異体をスクリーニングした。
    (1) アラビドプシス等で知られるGA情報伝達系に関わる調節遺伝因子の既知配列に基づき、イネにおける相同配列またはイネの遺伝子配列をデータベースから得てプライマーを設計した。これらのプライマーを用いて、Acの高頻度転移系統から得た9594個体のプールDNAをテンプレートにPCRを行い、遺伝子の破壊株を探索したが、現在のところまだ見つかっていない。
    (2) Acが転移あるいは分離によって除かれてもGA応答性の変異を示す変異体を選抜した。これらの変異体ではRAmylA/GUS遺伝子の発現はほぼ正常であったが、胚乳の糖化が全く起こらないか、遅延していた。これらの変異体の胚から誘導したカルスを用いて、RAmylAがコードするaアミラーゼアイソフォームA+Bの翻訳、分泌制御に関わる生化学的な解析を行った。aアミラーゼはGAのシグナルにより細胞内に流入したCa^<2+>によって、翻訳・分泌レベルにおいて制御が行われる事が知られている。そこで、Ca^<2+>ポンプ、Ca^<2+>チャネル、PLC、ER-ATPase、CaM等の阻害剤を用いてaアミラーゼの翻訳、分泌量の増減を調べた。その結果、これらの変異体ではCa^<2+>ポンプ、PLC、ER-ATPaseは正常に働いているが、Ca^<2+>チャネルかまたはCaMについて、異常が起きている可能性が示唆された。

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  • トランスポゾン・タッギングによるジベレリン情報伝達系に関わる調節遺伝子の探索

    Grant number:09251208

    1997

    System name:科学研究費助成事業

    Research category:重点領域研究

    Awarding organization:日本学術振興会

    伊藤 紀美子

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    Grant amount:\2000000 ( Direct Cost: \2000000 )

    発芽時に、イネでは植物ホルモン・ジベレリン(GA)が主要な役割を担う。この際発現する、様々なGA誘導型の遺伝子のひとつである、α-アミラーゼ遺伝子の発現を指標として、トランスポゾン・タッギングによるGA情報伝達系の調節遺伝子の探索を行った。
    [変異体の検出]Ac系統と、RAmy1A/GUS系統を交配した無胚半切種子をGAで処理し、胚乳デンプンの糖化の程度とGUSの発現レベルが正常より遅れる固体を検出した。残りの胚切片は、1/2MS、スクロース培地上で生育させ、発芽の進行度と幼植物の形態を観察した。15固体の独立のF1植物から得たF2種子のうち、約800粒のスクリーニングを行い、GA応答や発芽時の発生・成長に関与すると考えられる変異体を検出した。これらはa)RAmy1A/GUSの誘導が遅延する変異、b)乳胚の糖化が起こらない変異、c)発芽の遅延、あるいは発芽しない変異であり、全体の27.5%でいずれかの変異が観察された。
    [変異の遺伝とAcとの連鎖]変異系統(F2世代)で観察された表現型は約60系統のF2植物より得られた自殖F3種子1000粒のうち、ほぼ全てのF3種子において、遺伝していた。これら顕著な表現型を持つ17系統を選び、サザン法によりAcとの連鎖を調べた。合計270固体のF3植物を解析したうち、表現型の分離と一致するAcは観察されなかった。これらはAcの新たな挿入によるものではなく、過去にAcの活発な挿入、再転移により蓄積された変異と考えられた。
    [変異の固定]胚乳の糖化が遅延する変異を持つ系統の一部では、Acはすでに存在しないが、変異がほぼ固定し、生化学的解析に供し得る系統が得られた。また、遺伝解析を行うために、戻し交配を行い、F1種子を得た。この変異はGAの刺激により、Ca^<2+>を介してアミラーゼの翻訳・分泌を制御する経路に関わる遺伝子である可能性が高い。

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  • イネαアミラーゼ遺伝子のホルモンによる発現調節に関わる調節遺伝子の探索

    Grant number:08740613

    1996

    System name:科学研究費助成事業

    Research category:奨励研究(A)

    Awarding organization:日本学術振興会

    伊藤 紀美子

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    Grant amount:\900000 ( Direct Cost: \900000 )

    イネにおいては植物ホルモン、ジベリン(GA)が発芽初期の発生制御の主要な役割を担う。そこでGA誘導型αアミラーゼ遺伝子RAmylAをターゲット遺伝子として、トランスポゾン・タッギングによるGA情報伝達系に関与する調節遺伝子の探索を行った。RAmylA/GUS遺伝子とトウモロコシ由来のトランスポゾンA_Cを持つトランスジェニック・イネ系統を交配し、本年度、両遺伝子を持つF2植物体を選抜した。まず無胚半切種子をジベレリン処理後、GUS活性の有無を調べ、胚側は生育させて自殖F3種子を得た。また、葉からDNAを抽出し、A_Cの存在をPCR法によって調べた。このスクリーニング法では、3つの制御系に関わる変異の検出が可能である。1)GA生合成の変異では、発芽のみが抑制され、GA処理した無胚半切種子でのGUS活性は正常、2)GAシグナルをうけてから、RAmylA転写までに関わる変異の場合、無胚半切種子の糖化遅延、GUS活性の低下、3)RAmylA産物の翻訳や分泌にのみ関わる変異の場合、無胚半切種子のGUS活性は正常であるが胚乳糖化が起きない。
    本研究では、上記のようなこれまでの生理学、生化学的知見から予想されるGA誘導型α-アミラーゼ遺伝子の制御機構を裏付ける変異を多数検出した。まず、不発芽、GUSネガティブであり、胚乳が糖化しない個体が検出された。次に、発芽の遅延を示すものがF2集団のうち約20%に観察された。そのなかでも、GUS活性は正常であるが胚乳糖化が遅延したもの、胚乳糖化は正常だがGUS活性がやや弱いもの、GUS活性が無く糖化も遅延したものなど、表現型は様々であった。また、発芽が正常な個体にもGUS活性が弱い、胚乳糖化の遅延、糊粉層ではなく胚乳でGUS活性があるもの等も観察された。変異の出現頻度の高さから、当該変異の内には、遺伝的ではないものが含まれていると考えられる。これらが遺伝的な変異であるのか、またはエピジエネティックなものなのか、現在F3集団でのA_Cとの連鎖を含め、解析を進めている。

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Teaching Experience (researchmap)

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Teaching Experience

  • 基礎化学

    2024
    Institution name:新潟大学

  • 酵素化学

    2024
    Institution name:新潟大学

  • 生物化学I

    2024
    Institution name:新潟大学

  • 微生物学実験

    2023
    Institution name:新潟大学

  • Topics in Biotechnology and Biochemistry

    2022
    Institution name:新潟大学

  • スタディ・スキルズAIIc

    2022
    Institution name:新潟大学

  • 農学入門II

    2022
    Institution name:新潟大学

  • 応用生命・食品科学セミナーII

    2022
    Institution name:新潟大学

  • スタディ・スキルズAIc

    2022
    Institution name:新潟大学

  • 農学入門I

    2022
    Institution name:新潟大学

  • 食品科学概論

    2020
    -
    2022
    Institution name:新潟大学

  • スタディ・スキルズA c

    2020
    Institution name:新潟大学

  • 生物化学Ⅱ

    2018
    Institution name:新潟大学

  • 生命を知る

    2018
    Institution name:新潟大学

  • Topics in Biotechnology and Biochemistry

    2018
    -
    2022
    Institution name:新潟大学

  • 農学入門Ⅱ

    2017
    -
    2019
    Institution name:新潟大学

  • 農学入門Ⅰ

    2017
    -
    2019
    Institution name:新潟大学

  • スタディ・スキルズA a

    2017
    Institution name:新潟大学

  • 応用生命・食品科学概論

    2015
    -
    2020
    Institution name:新潟大学

  • 応用生命・食品科学特論

    2015
    -
    2020
    Institution name:新潟大学

  • スタディ・スキルズA2

    2013
    -
    2014
    Institution name:新潟大学

  • 植物分子生命科学概論

    2012
    -
    2020
    Institution name:新潟大学

  • 機器分析化学 Ⅰ

    2012
    -
    2016
    Institution name:新潟大学

  • 研究発表演習(中間発表)

    2012
    -
    2015
    Institution name:新潟大学

  • 応用生命・食品科学演習(学会発表)

    2012
    -
    2015
    Institution name:新潟大学

  • 生命・食料科学セミナーBⅠ

    2012
    -
    2015
    Institution name:新潟大学

  • 文献詳読Ⅰ

    2012
    -
    2015
    Institution name:新潟大学

  • 生命・食料科学特定研究BⅠ

    2012
    -
    2015
    Institution name:新潟大学

  • 生命・食料科学セミナーBⅡ

    2012
    -
    2014
    Institution name:新潟大学

  • 生命・食料科学特定研究BⅡ

    2012
    -
    2014
    Institution name:新潟大学

  • 文献詳読Ⅱ

    2012
    -
    2014
    Institution name:新潟大学

  • 分子生命科学演習Ⅱ

    2011
    -
    2020
    Institution name:新潟大学

  • 分子生命科学演習Ⅰ

    2011
    -
    2019
    Institution name:新潟大学

  • 細胞分子生物学

    2010
    Institution name:新潟大学

  • 分子生命科学実験

    2010
    -
    2020
    Institution name:新潟大学

  • 機器分析化学Ⅰ

    2009
    -
    2018
    Institution name:新潟大学

  • 生物学

    2009
    -
    2015
    Institution name:新潟大学

  • 機器分析化学

    2009
    -
    2010
    Institution name:新潟大学

  • 生体物質構造解析学

    2009
    -
    2010
    Institution name:新潟大学

  • 植物分子生物学

    2007
    Institution name:新潟大学

  • 生物化学実験

    2007
    Institution name:新潟大学

  • エピジェネティクス特論

    2007
    -
    2022
    Institution name:新潟大学

  • ゲノム科学

    2007
    -
    2020
    Institution name:新潟大学

  • 科学英語演習

    2007
    -
    2020
    Institution name:新潟大学

  • 放射線化学

    2007
    -
    2009
    Institution name:新潟大学

  • 生命を知る

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