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Molecular biological study and breeding of cold response in wheat

小麦冷反应的分子生物学研究及育种

基本信息

批准号：
10044207
负责人：
NAKAMURA Chiharu
金额：
$ 3.33万
依托单位：
KOBE UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B).
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 2000
项目状态：
已结题

项目摘要

Japan side(Kobe University)1)Six clones of cold-responsive genes were isolated from a winter-hardy common wheat cultivar 'Mironovska 808', and their strucutre and expression were studied. a)Wcor14 and Wcs19 : genes gelonging to a Cor(cold-responsive)family. Wcor14 is homologous to cold- and light-responsive barley Bcor14b. Wcs19 has a N-terminal region homologous to that of Wcor14 and Bcor14b. WCOR14, WCS19 and BCOR14b have a 51-amino-acid N-terminal choloroplast leader peptide. b)Wdhn13 : a gene belonging to a hedydrin family. A genomic clone of Wcas19 including a putative promoter was also obtained. c) Wrab17 : a gene homologous to GA_3-responsive ES2A from a GA_3-insensitive barley mutant. d)Wrab19 : a gene belonging to a group-3 lea gene family. e)Wltr10 : a member of cereal-specific Ltr family. f)Waox1 and Waox2 : genes encoding cyanide-insensitive alternative oxidase. 2)Copy number of each gene was determined by genomic Southern blot analysis. Chromosome assignment was made using … More nulli-tetrasomic lines and D genome chromosome substiotution lines. Waox1 and Waox2 were shown to be nonhomoeologous genes. 3)Bioassy for freezing tolerance was conducted using 'Mironovska 808' and a spring wheat cv.'Chinese Spring'. Enhancement of freezing tolerance by cold acclimation and significant cultivar differences in the level of freezing tolerance were shown. 4)Gene expression was studied by Northern blot analysis. Expression of Wcor14, Wcs19 and Wltr10 was low temperature-specific. Expression of Wrab17 and Wrab19 was induced by low temperature and also modulated by GA_3 and ABA.Expression of Waox1 and Waox2 was enhanced by low temperature and KCN.A positive correlation was observed between the level of expression of these genes and the level of freezing tolerance in the two cultivars. 5)Transportation of WCOR14 and WCS19 to chloroplasts and that of WAOX1 and WAOX2 to mitonchodria was proven by the transient expression assay using GFP-fusion constracts. 6)Genomic clones of Waox1 and Waox2 were isolated and their exson-intron structures were determined. 7)WCOR14 and WDHN13 were synthesized in E.coli and their antibodies were raized using mouse and rabbit.Bulgarian side(Institute of Genetic Enginnering)1)Bioassay for freezing tolerance was conducted using a large number of tetraploid and hexaploid wheat species and cultivars. 2)Proline systhetase genes were introduced into tobacco plants and the transformants were characterized in their prolione and fructan contents. 3)Near infrared spectrophotometry was applied to monitor cellular changes during freezing/dehydration stresses. Less

1）从抗寒小麦品种Mironovska 808中分离到6个冷应答基因克隆，并对其结构和表达进行了研究。a）Wcor 14和Wcs 19：属于Cor（冷反应）家族的基因。Wcor 14与冷和光响应大麦Bcor 14 b同源。Wcs 19具有与Wcor 14和Bcor 14 b同源的N-末端区域。WCOR 14、WCS 19和BCOR 14 b具有51个氨基酸的N-末端叶绿体前导肽。B）Wdhn 13：属于hedydrin家族的基因。还获得了包括推定启动子的Wcas 19的基因组克隆。c）Wrab 17：与大麦赤霉素敏感突变体ES 2A同源的基因。d）Wrab 19：属于第3组莱亚基因家族的基因。e）Wltr 10：谷物特异性Ltr家族的成员。f）Waox 1和Waox 2：编码氰化物不敏感的交替氧化酶的基因。2)通过基因组Southern印迹分析确定每个基因的拷贝数。染色体分配使用 ...更多信息缺四体系和D染色体组染色体代换系。Waox 1和Waox 2为非同源基因。3)利用“Mironovska 808”和春小麦cv.中国之春结果表明，低温锻炼提高了品种的抗冻性，且品种间的抗冻性存在显著差异。4)通过北方印迹分析研究基因表达。Wcor 14、Wcs 19和Wltr 10的表达具有低温特异性。Wrab 17和Wrab 19基因的表达受低温诱导，GA 3和阿坝对它们的表达也有调节作用，而Waox 1和Waox 2基因的表达受低温和KCN的诱导而增强，这两个基因的表达水平与两个品种的抗冻性呈正相关。5)通过使用GFP融合质粒的瞬时表达试验证明了WCOR 14和WCS 19向叶绿体的运输以及WAOX 1和WAOX 2向线粒体的运输。6)分离了Waox 1和Waox 2的基因组克隆，并测定了它们的外显子-内含子结构。7）在大肠杆菌中合成WCOR 14和WDHN 13，并使用小鼠和兔产生它们的抗体。保加利亚方面（遗传工程研究所）1）使用大量四倍体和六倍体小麦物种和栽培种进行抗冻性的生物测定。2)将脯氨酸合成酶基因导入烟草植株中，并对转化体的脯氨酸和果聚糖含量进行了表征。3)近红外分光光度法用于监测冷冻/脱水胁迫过程中细胞的变化。少