小麦TaNRX-B1基因调控抗旱性的功能解析

Drought has become one of the most serious stress factors affecting wheat production in China. It is one of the most economical and efficient means of ensuring wheat high and stable yield to breed and plant cultivars with drought resistance. It is very important for breeding drought resistant cultivars to interpret the function of drought resistant genes. Our previous studies showed that gene TaNRX-B1 in wheat was related to drought resistance using a population of recombinant inbred lines, natural population with a large number of wheat cultivars, near-isogenic wheat lines differing at the TaNRX-B1 locus, and its Arabidopsis overexpression transgenic lines. The mechnism of the gene under drought stress will be dissected in this project. In order to further prove the drought resistance of the gene TaNRX-B1, wheat transformation lines with overexpression or suppressing (RNA interference) expression of the gene will be obtained by transformation technology, respectively. Morphological, physiological and biochemical properties related to drought resistances, and spatio-temporal expression of the gene TaNRX-B1 in the seedlings of wheat transformation lines and their wild type under different water treatments will be analyzed. To determine the action site of TaNRX-B1, a subcellular localization assay will be conducted by transiently expressing TaNRX-B1-GFP fusion protein in onion epidermal cells and Arabidopsis protoplasts. Various plasmid vectors with 5’-truncated TaNRX-B1 promoters fused with the reporter gene GUS will be constructed and transformed into wheat callus, respectively. Activity of the gene TaNRX-B1 promoter under different water treatments will be analyzed. RNA-Seq will be used for gene expression analysis in response to water stress treatments in the seedlings of wheat transformation lines and their wild type. Differentially expressed genes related to plant stress response were validated by qPCR. Downstream target genes of the gene TaNRX-B1 will be screened out. Interaction proteins of TaNRX-B1 will be screened out using yeast two-hybrid and bimolecular fluorescence complementation. Expression of reporter genes GUS and LUC in wheat callus will be detected. Regulation of downstream target genes of gene TaNRX-B1 will be illustrated.

干旱已成为影响我国小麦生产最严重胁迫因素之一，选育和推广抗旱品种是确保小麦高产稳产最经济最有效途径之一，解析抗旱基因的功能可为抗旱新品种的选育提供理论依据。课题组前期利用小麦自然群体、重组自交系群体、近等基因系和转拟南芥研究表明，小麦的TaNRX-B1基因与抗旱性相关。本项目拟对其功能进行解析。通过分析过表达和RNAi沉默小麦转基因后代，进一步鉴定该基因的抗旱性功能；通过研究TaNRX-B1基因在拟南芥和洋葱瞬时表达，以及启动子5’系列缺失片段驱动GUS基因在小麦响应水分胁迫瞬时表达，明确TaNRX-B1基因表达蛋白的细胞部位并初步揭示启动子响应水分胁迫的机制；通过小麦转基因后代和野生型的转录组测序和分析，筛选与抗旱相关的下游靶基因；用酵母双杂交和双分子荧光互补技术，鉴定互作蛋白；通过小麦原生质体培养和转化技术并结合GUS和LUC报告基因研究，揭示TaNRX-B1基因调控下游靶基因的机理。

小麦是我国最重要的粮食作物之一。干旱已成为影响我国小麦生产最严重非生物胁迫因素之一，严重影响了我国的粮食安全。选育和推广抗旱性品种是确保小麦高产稳产最经济最有效的途径之一，挖掘抗旱相关基因并解析作用机制是小麦抗旱性新品种选育的基础。本项目以小麦的TaNRX1基因为研究对象，通过转基因小麦鉴定了其抗旱性，定位了该基因表达蛋白的亚细胞位置，分析了启动子响应干旱胁迫的分子机制，筛选了该基因调控抗旱性的下游靶基因，获得了与TaNRX1互作的蛋白，解析了其调控小麦抗旱性的机理。研究结果表明，TaNRX1基因受干旱胁迫的诱导表达，过表达能明显增强小麦的抗旱性，而干扰表达能明显降低小麦的抗旱性，该基因正调控小麦的抗旱性；与野生型相比，干旱胁迫下过表达TaNRX1小麦的根系较发达，叶片持水保水性较强，体内活性氧积累量和膜质氧化程度较低，而干扰表达小麦正好相反，初步揭示了调控抗旱性的生理机理；TaNRX1蛋白被定位于细胞核、细胞质和细胞膜；启动子的193bp区段是干旱胁迫诱导表达的最小基本功能区段，可作为单子叶和双子叶植物转基因的诱导型启动子，其中的36bp是干旱诱导表达的关键区段，含有响应干旱的元件；TaNRX1蛋白含有3个TRX的结构域，均具有还原二硫键的活性，第3个结构域的活性最强，第2个结构域最弱，为改良该蛋白的活性提供了靶位点；TaNRX1能与TaVDAC1、TaPDI、TaPP2Ac、TaTRX-h蛋白及自身之间相互作用；互作蛋白的基因过表达，能明显提高拟南芥的抗旱性或抗盐性；通过调节物质代谢与转运、信号传导等途径之中相关蛋白的氧化还原状态的平衡，维持干旱胁迫下靶蛋白正常的生物功能，增强小麦抗氧化能力，提高小麦的抗旱性。本项目研究的结果，为小麦抗旱育种提供了优异基因资源和理论指导，为深入研究小麦TaNRX1基因抗旱性的分子机制奠定了理论基础，研究结果具有重要的理论和实践意义。