南方型紫花苜蓿盐诱导蛋白基因克隆及功能分析

It was the first to solve planting problems of alfalfa in the southern region due to introduction of foreign high fall dormancy of alfalfa varieties,but alfalfa was not very tolerant to salts and was limited large-scale cultivation in the south Yangtze river seabeach regions.Traditional breeding methods due to by the Southern hot and humid environmental conditions, cultivation of ew varieties of salt-tolerant alfalfa was difficult, low efficiency, long time.Genetic engineering could be directed breeding and speeding the breeding process,but its core of the problem was first obtained a good salt tolerance gene and was clear to key molecular mechanisms and regulation mechanism of he target traits of alfalfa salt. The project was intended to take advantage of Southern type Millenium afalfa salt-tolerant mutant materials by comprehensive application of theory and method of comparative genomics，differential proteomics etc, seperated and Identificated alfalfa salt-induced specific protein , cloned alfalfa salt key new 2 or 3 genes by basing on N_terminal sequence of the purified protein to degenerate oligonucleotide primers and verifed the new gene functions. Completion of this project was contributed to the salt tolerance of alfalfa protein and gene level deeper reveals the molecular mechanism of the formation of proprietary alfalfa salt-tolerant 2 or 3 new genes and laied a solid theoretical foundation for the cultivation of suitable alfalfa varieties planting in the south Yangtze river seabeach regions.

国外高秋眠级紫花苜蓿品种引进，率先解决了紫花苜蓿在江南地区的种植难题，但紫花苜蓿耐盐能力有限，限制了在江南海涂区大面积种植。传统育种方法受到南方高温高湿环境条件限制，培育耐盐紫花苜蓿新品种难度大、效率低、时间长。基因工程可以实现定向育种，加快育种过程，但其问题的核心首先要获得优良的耐盐相关基因，明确紫花苜蓿耐盐目标性状的关键分子机制和调控机理。本项目拟利用南方型"盛世"紫花苜蓿耐盐突变体为材料，综合应用比较基因组学、差异蛋白组学和生物信息学等理论和方法，分离和鉴定紫花苜蓿盐诱导"特异蛋白"，依据目标特异蛋白N端部分氨基酸序列设计简并引物，克隆紫花苜蓿耐盐关键新基因2-3个并验证新基因功能。本项目的完成有助于从蛋白和基因水平上更深层次揭示紫花苜蓿的耐盐分子机制，形成具有自主知识产权的紫花苜蓿耐盐的关键新基因2-3个，为培育适合江南海涂种植南方型紫花苜蓿新品种奠定坚实的应用理论基础。

国外高秋眠级紫花苜蓿品种引进，率先解决了紫花苜蓿在江南地区的种植难题，但紫花苜蓿耐盐能力有限，限制了在江南海涂区大面积种植。该项目研究结果建立了南方型紫花苜蓿Millennium品种高效再生体系，完成再生体系仅需60-70d。获得了一份耐1.2% NaCl可以稳定遗传的紫花苜蓿耐盐突变体。发现AsA-GSH循环效率是紫花苜蓿耐盐机制的重要生理基础。采用双向电泳法成功鉴定出紫花苜蓿耐盐突变体盐胁迫差异蛋白，叶片中有26 个差异蛋白，15个上调，11个下调。这些差异蛋白质主要参与膜转运相关蛋白、蛋白代谢、光合代谢、细胞壁和细胞骨架、碳水化合物和能量、信号转导和应激反应等途径；根中18个差异蛋白， 8个上调，10个下调。参与膜转运相关蛋白、蛋白代谢，核苷酸代谢、细胞壁和细胞骨架、碳水化合物和能量等途径。采用iTRAQ定量蛋白质组学技术，高通量地筛选与紫花苜蓿盐胁迫相关的差异蛋白，在蛋白质组水平上明确根和叶片响应适应盐胁迫机制不同，发现紫花苜蓿耐盐突变体和野生型可能运用不同的防御策略来响应盐胁迫。利用转录组和蛋白质组的关联研究技术可以有效地发现紫花苜蓿耐盐相关候选蛋白基因。克隆了一批差异蛋白基因。构建了MsMSR、MsP450超表达载体，获得超表达MSR、P450烟草转基因植株。发现MsTPS 作为一个类受体蛋白激酶受盐逆境胁迫诱导，该基因的过量表达提高了拟南芥的抗盐性。研究结果发表相关文章9篇，其中一级5篇，中文核心4篇。另有两篇英文已投稿，培养4名研究生。该研究为深入认识紫花苜蓿盐胁迫的应答分子调控机制提供理论依据。