大豆NAC膜结合转录因子GmNTLx在大豆耐盐中的作用机制研究

项目名称： 大豆NAC膜结合转录因子GmNTLx在大豆耐盐中的作用机制研究

项目编号： No.31471515

项目类型： 面上项目

立项/批准年度： 2015

项目学科： 食品科学、农学基础与作物学

项目作者： 向凤宁

作者单位： 山东大学

项目金额： 90万元

中文摘要： 大豆是重要农作物，耐盐性较差。利用耐盐大豆在盐及非盐胁迫下的基因表达谱，筛选出NAC膜结合转录因子基因GmNTLx，并获得其过表达转基因大豆。在此基础上，本项目拟开展GmNTLx蛋白膜联-释放特性及其耐盐作用机制研究，包括水解GmNTLx的蛋白酶筛选；非生物胁迫因子及激素对蛋白释放的影响；亚细胞定位、转录激活活性、组织特异性表达及其对盐和激素的响应；获得GmNTLx RNAi转基因大豆，分析过表达和抑制表达转基因大豆耐盐性及生理指标，确定其耐盐功能，获得大豆耐盐新种质；比较325个大豆品种该基因序列差异，并分型，明确各基因型与耐盐性关系；分析转基因大豆及对照转录组差异，结合ChIP-seq，筛选其下游靶基因，进行EMSA、ChIP等验证；通过酵母双杂交，筛选其互作蛋白，利用BiFC、Co-IP验证，利用植物杂交技术，确定其与靶基因及互作蛋白基因的遗传关系，揭示其调控大豆耐盐的分子机制。

中文关键词： 大豆；NAC膜结合转录因子GmNTLx；耐盐性；转基因大豆；分子机制

英文摘要： Soybean (Giycine max L.) is one of the most important crops, and has lower salt tolerance. A transcriptomic comparison was performed in salt tolerant soybean SHENGDOU No.9 between salinity-stressed and non-stressed treatments and a NAC membrane-bound transcription factor GmNTLx was selected as candidate salinity tolerance gene. The GmNTLx-overexpressing transgenic soybean lines were obtained. Based on these working foundations, this project is to study the mechanism of membrane-release and the regulation function of GmNTLx in salinity tolerance in soybean. The contents of the project include: protease screening, protein sub-cellular localization, trancativation, release and gene expression patterns in different tissues and in response to abiotic stresses and plant hormones; the obtainment of GmNTLx RNAi transgenic soybean seedlings and their validation with Southern-blot and qRT-PCR, the salinity tolerance and physiological index of two kinds of transgenic soybean lines, to confirm the function of GmNTLx in salinity tolerance in soybean,to obtain the new salinity tolerant soybean germplasm; the correlation analysis of the GmNTLx gene sequence difference and salinity tolerance in different soybean species; obtainment of gene networks base on the transcriptomic comparison between the transgenic and control soybeans, screen of GmNTLx's direct target genes with ChIP-sequence, the confirmation of the direct regulation of GmNTLx on its potential targets with EMSA, ChIP etc. screen of GmNTLx's interaction protein with yeast two hybrid, and the verification with BiFC and Co-IP. Determination of genetic relationship of GmNTLx and its target genes or interaction protein genes with cross; These findings will reveal the molecular mechnism of how GmNTLx function in salinity tolerance in soybean.

英文关键词： soybean;NAC membrane-bound transcription factor GmNTLx;salt tolerance;transgenic soybean;molecular mechanism