大豆NAC膜结合转录因子GmNTLx在大豆耐盐中的作用机制研究

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.

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。

本项目开展了GmNTLx蛋白“膜联-释放”特性及其耐盐作用机制研究，筛选了水解GmNTLx的蛋白酶；分析了非生物胁迫因子及激素对蛋白释放的影响；对GmNTLx进行了亚细胞定位、转录激活活性、组织特异性表达及其对盐和激素的响应研究；分析了过表达和抑制表达转基因大豆耐盐性及生理指标，确定了其耐盐功能，获得了大豆耐盐新种质；比较了174个大豆品种中的该基因序列差异，发现了与耐盐性相关的变异位点；分析了转基因大豆及对照在盐及非盐下的转录组差异，初步揭示了其调控的基因网络；获得了GmNTLx直接调控的靶基因GmDREBa和GmNDR1；通过酵母双杂交，筛选出了2个与其互作的蛋白GmNHXx和GmARDx，利用BiFC验证及大豆转基因过表达植株，确定其与靶基因互作关系，验证了靶基因调控大豆耐盐性的作用，从而揭示了其调控大豆耐盐的分子机制。