大豆耐盐相关基因GmNACx在大豆耐盐性中的作用机制研究

Soybean (Giycine max L.) is one of the most important food and economic crops and has not high salinity tolerance. So far, it is hard to perform the soybean transgene and was not reported about the funtional analysis of salt tolerance related soybean genes in soybean itself. An efficient soybean transgenic technique has been established in our lab and a sanility tolerant soybean cultivar SHENGDOU No.9 was selected from 179 cultivars. A transcriptomic comparison was performed in SHENGDOU No.9 between salinity-stressed and non-stressed treatments and several salt responsive genes were selected as candidate salinity tolerance related genes.GmNACx,one of the NAC transcription factors,was further cloned and over expressed in soybean and the T3 transgenic seed have been obtained. Based on these working foundations, this project is to study the function and molecular mechanism of GmNACx in salinity tolerance in soybean. The contents of the project include: protein sub-cellular localization, trancativation, gene expression patterns in different tissues and in response to abiotic stresses and plant hormores; the obtainment of GmNACx over-expression and RNAi transgenic soybean seedlings and.their validation with Southern-blot and qRT-PCR, the salinity tolerance and physiological index of the transgenic seedlings, to make sure the gene function in salinity tolerance in soybean; analysis of gene networks those were transcriptionally regulated by GmNACx with the transcriptomic comparison between the transgenic and control soybeans, screen of GmNACx's direct target genes with ChIP-sequence, the confirmation of the direct regulation of GmNACx on its potential targets with EMSA, ChIP etc.. These findings will reveal the molecular mechnism of how GmNACx function in salinity tolerance in soybean and lay foundation of the aqquirement of new salinity tolerant soybean germplasm.

大豆（Glycine max L.）是重要的粮食及经济作物，耐盐性较差。大豆基因转化较难，有关耐盐基因在大豆耐盐中的作用研究未见报道。本实验室建立了一种高效的大豆转化技术，筛选出耐盐大豆- - 圣豆9号，建立了其在盐及非盐胁迫下的表达谱，比较了它们的表达差异，筛选出NAC转录因子基因GmNACx，并获得其过表达转基因大豆。在此基础上，本项目拟开展GmNACx的耐盐作用机制研究。包括亚细胞定位、转录激活活性、组织特异性表达及其在盐和激素处理下的表达变化；GmNACx RNAi转基因大豆的获得及分子鉴定，两种转基因大豆耐盐性、生理指标及耐盐相关基因表达分析，确定该基因耐盐功能；分析转基因大豆及对照转录组差异，结合ChIP-sequence，筛选其下游靶基因，进行EMSA、ChIP等验证，以期揭示该基因调控大豆耐盐的分子机制，为创建转基因耐盐大豆新种质奠定基础。

大豆是重要农作物，耐盐性较差，其耐盐性由多基因控制，因此，大豆耐盐基因发掘和功能鉴定是实现其耐盐性改良的前提。目前，有关大豆耐盐功能基因的发掘和功能鉴定虽有一些报道，但它们的耐盐功能鉴定大多数是来自于模式生物拟南芥等，加之大豆遗传转化尚存在转化效率低等问题，从而制约了耐盐基因在大豆中的应用。因此，创制具有自主知识产权的转基因大豆新品种是当务之急。.本研究获得了一个大豆盐胁迫相关的转录因子基因GmNACx，进行了以下研究：.1、进行了大豆耐盐基因GmNACx在大豆耐盐中的作用的研究：.1）完成了GmNACx表达模式、亚细胞定位与转录活性分析；.2）利用Southern Blot和qRT-PCR对转基因大豆进行了分子生物学鉴定，筛选出3个单拷贝、高表达的GmNACx大豆纯系以及GmNACx-RNAi转基因大豆T1代；.3）完成了35S::GmNACx大豆耐盐性鉴定，发现35S::GmNACx转基因大豆在不同浓度 NaCl处理后，主根长度及苗生长势明显高于对照，而GmNACx-RNAi转基因大豆与之相反；.4）检测了转基因大豆和对照在盐和非盐处理下生理生化变化，发现在盐下，35S::GmNACx植株根茎叶中H2O2、Na+和K+含量高于野生型；脯氨酸含量、SOD和POD活性亦高于对照，表明GmNACx过表达可提高大豆的耐盐性；.5）分析了在0.4%的盐碱地下35S::GmNACx生长情况，发现35S::GmNACx大豆在株高、单节最多荚数、单株结荚总数和单株粒数上均显著高于野生型大豆。 .6）检测了35S::GmNACx和对照在盐处理6h下的转录组变化。发现在NaCl处理下，一些ABA、ROS合成关键酶基因表达均上调表达；利用EMSA实验，证明了GmNACx能直接结合ROS合成酶编码基因GmRBOHB-1的启动子，表明GmNACx可能通过直接调控上述基因表达来提高大豆的耐盐性，解析了该基因在耐盐中的作用机制。.2、进行了耐盐碱转GmNACx大豆耐盐新种质选育研究：.1）35S:: GmNACx大豆连续3年种植0.3-0.4%的盐碱地上，发现其在株高、单节最多荚数、单株结荚总数和单株粒数上显著高于对照，证明了该基因的过表达促进了大豆产量和耐盐碱能力。.2）完成了35S:: GmNACx的农业转基因生物中间试验，选育出抗盐、高产的转基因大豆新品系3个，可望用于大规模盐碱地的生