BnTR1提高甘蓝型油菜耐盐的分子机理分析及培育耐盐油菜新材料

This project is designed to analyze the expression levels and regulation patterns of key members in response to salt stress as SOS, NHX, RD29, RD22 in BnTR1 overexpression Brassica napus lines, to study the correlation between BnTR1 and the salt tolerance of transgenic palnts, to investigate the relationship between BnTR1 and regulation of Ca2+ transport in plant. It will provide molecular evidences for the salt tolerance of BnTR1 overexpression Brassica napus lines. One of the aims of this project is to investigate the development and agronomic characters of BnTR1 overexpression Brassica napus lines cultivated under artificial salinity conditions or under saline field. It will not only present proofs for salt tolerance of transgenic Brassica, but also facilitate to evaluate effects of transgenic Brassica lines on the improvement of physicochemical properties of saline soil. Ultimate goal of the project is to provide a Brassica napus material with salt tolerance for rapeseed breeding. The significance of this project is to supply molecular evidences for the salt tolerance of BnTR1 overexpression Brassica napus lines, as well as to provide new material to exploit saline soil.

本项目拟以多个过量表达BnTR1的油菜独立株系为研究对象，分析转BnTR1基因油菜中与盐胁迫应答的关键基因SOS,NHX,RD29,RD22等基因的表达情况、调控模式，BnTR1与转基因油菜耐盐能力的相关性以及BnTR1与植物调控钙离子转运的关系，从而揭示BnTR1调控植物耐盐的分子机制；通过分析模拟盐碱条件和盐碱地实地栽培条件下转BnTR1基因油菜的生长发育和综合农性性状情况，考察转基因油菜的耐盐能力；并综合评价连续播种转基因油菜对改良盐碱地土壤理化性质的效果，培育耐盐油菜育种新材料。本项目的研究将为BnTR1提高油菜耐盐性提供分子证据，为开发利用盐碱地提供耐盐油菜新材料打下基础。

在实验室模拟干旱或盐胁迫条件下，转BnTR1基因的烟草的抗性得到了很大提高，过量表达BnTR1的油菜也表现出很强的抗性。但是，在大田盐碱地实地栽培时，非转基因油菜和转基因油菜的生长发育受到不同程度的影响。该结果说明转化单一基因的油菜对复合逆境的耐受程度有限。过量表达BnTR1基因的油菜在胁迫条件下，体内DREB、SOS1、CBF、CPK等基因的转录水平不同程度上调表达，其中，钙依赖的蛋白激酶CPK3上调表达15倍。体外实验证实TR1对CPK3、CPK6进行单泛素化修饰，而对CP10、CPK28和CPK32进行多泛素化修饰。双分子荧光互补实验证实TR1和CPK8有相互作用。质谱研究结果证明TR1对CPK3进行单泛素化修饰的位点可能CPK3的414位和117位的赖氨酸（K）。钙依赖的蛋白激酶可能是TR1发挥功能的靶蛋白之一，但是具体机制需要进一步的深入研究。通过酵母双杂交系统筛选到13个与TR1相互作用的候选蛋白质，经过进一步的验证研究，TR1与CURT1C和SWEET5有明确的相互作用，推测TR1可能通过CURT1C和SWEET5发挥抗逆功能。这些结果为进一步研究TR1的功能奠定了很好的基础。