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Analysis of induction mechanism of desiccationtolerance of embryos and production of osmotic tolerantplants

胚胎耐干燥诱导机制分析及耐渗透植物生产

基本信息

批准号：
10660004
负责人：
TAKAHATA Yoshihito
金额：
$ 2.18万
依托单位：
Iwate University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
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

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10660004/
关键词：
Adventitionsembryo Desiccationtolerance Absicicacid Lea gene Brassica Transformation

项目摘要

1. ME-leaN4 is late embryogenesis abundant (Lea) gene isolated from microspore-derived embryos of Brassica napus which have been induced desiccation tolerance by ABA.In situ hybridization and immunocytochemistry showed that expression of the ME-leaN4 mRNA and protein was found on desiccation tolerant embryos but not on desiccation sensitive ones.2. Scanningelectron microscopy was employed to compare desiccation-tolerant and -sensitive microspore-derived embryos. The external surface of the desiccation-tolerant embryos was uniformly shriveled by desiccation and their internal tissue system was well preserved. In contrast, in desiccation-sensitive embryos, dehydration caused tearing of the epidermis and collapse of the internal tissue system.3. T1_1 plants of transgenictobacco having ME-leaN4 which was regulated by 35SCaMV or pMAT promoter were investigated about the osmotic stress tolerance. A part of T_1 plants showed slightly increase of desiccation tolerance, but most of T_1 plants did not show. When Nail tolerance was investigated, growth of non-transgenic plants was inhibited by 1% Nail treatment, but the inhibition of growth of T1 plants was slight.4. Promoter region(323bp) of ME-leaN4 was isolated. The expression analysis using chimeric gene constructs containing ME-leaN4 promoter fragment fused to GUS gene indicated that the promoter is activated by ABA, sorbitol or Nail. Mutation analysis of the promoter ascertained the presence of two ABRE (ABA response element) in this region.

1. ME-leaN 4是从阿坝诱导的油菜小孢子胚中分离到的晚期胚胎丰富（莱亚）基因，原位杂交和免疫细胞化学分析表明，ME-leaN 4基因的mRNA和蛋白在脱水耐受胚中表达，而在脱水敏感胚中不表达.利用扫描电镜对脱水耐受性和脱水敏感性小孢子胚进行了比较。耐脱水胚的外表面因脱水而均匀皱缩，其内部组织系统保存完好。相反，在干燥敏感的胚胎中，脱水导致表皮撕裂和内部组织系统崩溃。对转35 SCaMV或pMAT启动子调控的ME-leaN 4基因烟草T1_1代植株进行了渗透胁迫抗性研究。部分T_1代植株的脱水耐性略有提高，但大部分T_1代植株的脱水耐性没有提高。在耐甲性研究中，1%Nail处理对非转基因植株的生长有抑制作用，但对T1代植株的生长抑制作用很小.分离ME-leaN 4的启动子区（323 bp）。利用含有ME-leaN 4启动子片段与GUS基因融合的嵌合基因构建体的表达分析表明，该启动子被阿坝、山梨醇或Nail激活。启动子的突变分析确定了两个ABRE（阿坝反应元件）在该区域的存在。