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

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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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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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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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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(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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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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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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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
₄₂₀
)-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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<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 (F₄₂₀)-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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File： 20190303_Increased CD5+ B-cells are associated with autoimmune phenomenain lepromatous leprosy patients., Attia Kotba,b,d,∗, Samia Ismailc, Itoh Kimitoa, Waghi Mohamedb, Alamery Salmand,Arif A. MohammeddaGenomic.pdf

DOI： 10.1016/j.jiph.2019.03.001

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Publishing type：Research paper (scientific journal)   Publisher：MDPI} {AG  

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

DOI： 10.3390/mps2020054

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Other Link： http://orcid.org/0000-0002-5341-8086

Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI} {AG  

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

DOI： 10.3390/ijms20102585

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Other Link： http://orcid.org/0000-0002-5341-8086

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Frontiers Media S.A.  

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

DOI： 10.3389/fpls.2018.00266

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

DOI： 10.1186/s12284-016-0100-y

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Other Link： http://link.springer.com/article/10.1186/s12284-016-0100-y/fulltext.html

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Oxford University Press  

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

DOI： 10.1093/pcp/pcw089

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

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

DOI： 10.1111/pbi.12314

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

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.

DOI： 10.1007/s11240-014-0575-4

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

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

DOI： 10.1093/pcp/pct139

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER IRELAND LTD  

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

DOI： 10.1016/j.plantsci.2012.05.006

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE SOC PLANT CELL & MOLECULAR BIOL  

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

DOI： 10.5511/plantbiotechnology.10.1228a

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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.

DOI： 10.1007/s00344-009-9131-6

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Language：Japanese   Publisher：The Japanese Society of Applied Glycoscience  

DOI： 10.11541/jsag.2010.0.33.0

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Language：Japanese   Publisher：The Japanese Society of Applied Glycoscience  

DOI： 10.11541/jsag.2010.0.12.0

CiNii Article

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：The Japanese Society of Applied Glycoscience  

Structural changes caused by the introduction of the <i>Wx^α</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^α</i> and <i>Wx^b</i> cultivars, and WAB3-5 amylose had a comparably high degree of branching, which was indicated by the molar fraction of branched molecules (MF_B, 0.38) and number of chains of branched molecules (NC_B, 10.5). Amylose from cv. Yumetoiro (<i>Wx^α</i>) had a comparable degree of branching (MF_B, 0.35; NC_B, 13.7) with the WAB3-5 amylose. Chain-length distribution of WAB3-5 amylopectin showed a slightly higher amount of B₂+B₃ 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.

DOI： 10.5458/jag.56.65

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Other Link： http://agriknowledge.affrc.go.jp/RN/2010780954

Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

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).

DOI： 10.1093/pcp/pcn066

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

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Language：Japanese   Publisher：The Japanese Society of Applied Glycoscience  

DOI： 10.11541/jsag.2008.0.87.0

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL PUBLISHING, INC  

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.

DOI： 10.1111/J.1364-3703.2007.00414.X

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

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.

DOI： 10.1271/bbb.60675

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

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

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.

DOI： 10.1016/j.pestbp.2006.01.011

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Language：English   Publishing type：Research paper (other academic)  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL PUBLISHING  

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.

DOI： 10.1111/j.1399-3054.2005.00635.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL PUBLISHING  

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.

DOI： 10.1111/j.1439-0434.2006.01076.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

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.

DOI： 10.1093/pcp/pci091

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japanese Society for Plant Cell and Molecular Biology  

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.

DOI： 10.5511/plantbiotechnology.22.443

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Language：English   Publishing type：Research paper (other academic)  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：EDITIONS SCIENTIFIQUES MEDICALES ELSEVIER  

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.

DOI： 10.1016/j.plaphy.2004.04.005

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

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.

DOI： 10.1271/bbb.68.112

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

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.

DOI： 10.1093/pcp/pcg068

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

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.

DOI： 10.5511/plantbiotechnology.20.187

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Language：English   Publisher：OXFORD UNIV PRESS  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japanese Society for Plant Cell and Molecular Biology  

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.

File： 200103_Resting spore of Plasmodiophora brassicae proliferates only in the callus of club root disease-susceptible turnip but increases the PAL activity in the callus of club root disease-resistant turnip.pdf

DOI： 10.5511/plantbiotechnology.18.267

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

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.

File： 200004_Possible involvement of phosphoinositide-Ca2+ signaling in the regulation of alpha-amylase expression and germination of rice seed (Oryza sativa L.)..pdf

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE SOC PLANT PHYSIOLOGISTS  

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.

File： 1999_Sucrose-controlled transport and turnover of alpha-amylase in rice (Oryza sativa L.) cells..pdf

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Language：English   Publishing type：Research paper (international conference proceedings)  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER VERLAG  

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.

File： 199805_Effects of (+)-8’8’8’-trifluoroabscisic acid on alpha-amylase expression and sugar accumulation in rice cells..pdf

DOI： 10.1007/s004250050326

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：KLUWER ACADEMIC PUBL  

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

DOI： 10.1023/a:1005769605026

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER VERLAG  

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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Language：English   Publisher：ELSEVIER SCI LTD  

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.

File： 199707_The α-amylase multigene family.pdf

DOI： 10.1016/s1360-1385(97)86347-9

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER VERLAG  

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.

File： 1997_Charasteristics of genetic variation in the progenies of protoplast-derived plants of rice, Oryza sativa cv. Nipponbare..pdf

DOI： 10.1007/s001220050374

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DOI： 10.1271/nogeikagaku1924.71.sup_1

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC PLANT PHYSIOLOGISTS  

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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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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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING CO  

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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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER VERLAG  

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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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATL ACAD PRESS  

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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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER VERLAG  

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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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER VERLAG  

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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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

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

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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Language：Japanese   Publisher：日本応用糖質科学会  

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Language：English   Publisher：新潟大学農学部  

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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Language：English   Publisher：新潟大学農学部  

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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Language：English   Publisher：新潟大学農学部  

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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Language：English   Publisher：新潟大学農学部  

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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Language：Japanese   Publisher：新潟大学農学部  

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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Language：English   Publisher：新潟大学農学部  

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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Language：English   Publisher：新潟大学農学部  

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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Language：English   Publisher：The Japanese Society of Applied Glycoscience  

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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Language：English   Publisher：新潟大学農学部  

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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Language：Japanese  

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Language：Japanese   Publisher：The Japanese Society for Chemical Regulation of Plants  

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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Language：English   Publisher：Niigata University  

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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Language：English   Publisher：Niigata University  

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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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)   Publisher：編集・発行者 社団法人 日本農芸科学会  

DOI： 10.1271/kagakutoseibutsu1962.43.329

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Other Link： https://jlc.jst.go.jp/DN/JALC/00250988400?from=CiNii

Language：Japanese   Publisher：公益社団法人 日本農芸化学会  

DOI： 10.1271/kagakutoseibutsu1962.43.329

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Language：English   Publisher：Niigata University  

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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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：OXFORD UNIV PRESS  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：OXFORD UNIV PRESS  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：OXFORD UNIV PRESS  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：OXFORD UNIV PRESS  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：OXFORD UNIV PRESS  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：OXFORD UNIV PRESS  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：OXFORD UNIV PRESS  

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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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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：OXFORD UNIV PRESS  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：OXFORD UNIV PRESS  

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

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：OXFORD UNIV PRESS  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：OXFORD UNIV PRESS  

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (other)   Publisher：共立出版  

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Language：Japanese  

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Language：Japanese   Publisher：全国農業改良普及協会  

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Language：English   Publisher：Japanese Society of Plant Physiologists  

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

Language：English  

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Language：Japanese  

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

DOI： 10.1271/kagakutoseibutsu1962.34.503

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Language：English  

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Language：Japanese   Publisher：The Phytopathological Society of Japan (PSJ)  

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Language：English   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)   Publisher：CARFAX PUBL CO  

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Language：Japanese   Publisher：The Phytopathological Society of Japan (PSJ)  

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Language：Japanese   Publisher：日本植物病理学会  

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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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