Molecular biological study and breeding of cold response in wheat

日方(神户大学)1)从耐寒普通小麦品种“Mironovska 808”中分离得到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 与冷光响应大麦 Bcor14b 同源。 Wcs19 具有与 Wcor14 和 Bcor14b 同源的 N 末端区域。 WCOR14、WCS19 和 BCOR14b 具有 51 个氨基酸的 N 端叶绿体前导肽。 b)Wdhn13：属于 Hedydrin 家族的基因。还获得了包含推定启动子的 Wcas19 基因组克隆。 c)Wrab17：与来自GA_3不敏感大麦突变体的GA_3响应性ES2A同源的基因。 d)Wrab19：属于3组lea基因家族的基因。 e)Wltr10：谷物专用 Ltr 家族的成员。 f)Waox1 和 Waox2：编码对氰化物不敏感的替代氧化酶的基因。 2)通过基因组Southern印迹分析确定每个基因的拷贝数。使用无效四体系和 D 基因组染色体取代系进行染色体分配。 Waox1和Waox2被证明是非同源基因。 3)使用“Mironovska 808”和春小麦品种“中国春”进行耐冻性生物测定。冷驯化增强了耐冻性，并且耐冻性水平存在显着的品种差异。 4)通过Northern印迹分析研究基因表达。 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，并受到GA_3和ABA的调节。低温和KCN增强Waox1和Waox2的表达。这些基因的表达水平与两个品种的耐冻性水平呈正相关。 5)通过使用GFP融合合约的瞬时表达测定证明了WCOR14和WCS19向叶绿体的转运以及WAOX1和WAOX2向线粒体的转运。 6)分离了Waox1和Waox2的基因组克隆并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，并用小鼠和兔产生了其抗体。保加利亚方面(遗传工程研究所)1)利用大量的四倍体和六倍体小麦品种和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物中，并对转化子的脯氨酸和果聚糖含量进行表征。 3）近红外分光光度法用于监测冷冻/脱水应激期间的细胞变化。较少的

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