Molecular biological study and breeding of cold response in wheat

1)从冬小麦品种‘Mironovska 808’中分离到6个冷响应基因克隆，并对其结构和表达进行了研究。a)Wcor14和Wcs19：属于Cor（冷反应）家族的基因。Wcor14与冷响应和光响应的大麦Bcor14b同源。Wcs19具有与Wcor14和Bcor14b同源的n端区域。WCOR14、WCS19和BCOR14b具有51个氨基酸的n端叶绿体先导肽。b)Wdhn13：一个属于hedydrin家族的基因。还获得了一个包含假定启动子的Wcas19基因组克隆。c) Wrab17：大麦ga_3不敏感突变体中ga_3敏感的ES2A同源基因。d)Wrab19：属于group-3 lea基因家族的基因。e)Wltr10：谷类特异性Ltr家族成员。f)Waox1和Waox2：编码氰化物不敏感替代氧化酶的基因。2)通过基因组Southern blot分析确定每个基因的拷贝数。采用多非四染色体系和D基因组染色体替代系进行染色体配位。Waox1和Waox2为非同源基因。3)以“Mironovska 808”和一个春小麦cv为材料，进行抗冻性生物测定。“中国春”。结果表明，冷驯化提高了小麦的抗冻能力，品种间抗冻能力差异显著。4) Northern blot检测基因表达。Wcor14、Wcs19和Wltr10的表达具有低温特异性。Wrab17和Wrab19的表达受低温诱导，并受GA_3和ABA的调控。低温和KCN增强了Waox1和Waox2的表达。在两个品种中，这些基因的表达量与抗冻能力呈正相关。5)利用gfp融合构建物进行瞬时表达实验，证实WCOR14和WCS19向叶绿体转运，WAOX1和WAOX2向线粒体转运。6)分离了Waox1和Waox2的基因组克隆，确定了它们的外显子内含子结构。7)在大肠杆菌中合成了WCOR14和WDHN13，并分别在小鼠和家兔身上扩增了抗体。保加利亚方面（遗传工程研究所）1)利用大量四倍体和六倍体小麦品种和栽培品种进行了抗冻性生物测定。2)将脯氨酸合成酶基因导入烟草植株，对其转化体进行了脯氨酸和果聚糖含量鉴定。3)采用近红外分光光度法监测冷冻/脱水胁迫下细胞的变化。少

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