盐胁迫下棉花根源油菜素内酯基因的筛选及功能分析研究

Soil salinity is a serious threat to global agriculture, and even more prevalent as the intensity of land use increases. In arid and semiarid regions of the world, limited rainfall, high temperature and evapotranspiration, as well as inadequate freshwater management have contributed to an increase in soil salinity. Although cotton is considered as an salt-tolerant crop, its salt tolerance is still limited. Breeders have sought to make cotton more tolerant to salt through various methods including traditional plant breeding and biotechnological approaches, and some progresses were promised but still no value in practical performance. So it is necessary to find new ways to understand and improve cotton salt tolerance. Brassinosteroid as a plant hormone, it can regulate plant salt tolerance, and it do not undergo long-distance transport in plant and play a role in situ. Roots first "feel" the stress when plants exposed to exceesive salt, so this research would study the influence of brassinosteroid in roots on cotton salt tolerance and the identification of new genes in the pathway of brassinosteroid biosynthesis, degradation and signal transduction which related to salt tolerance. .First, a hydroponic culture experiment using NaCl to induce salt stresses and adding brassinosteriod to solution is carried out to evaluate the effects of brassinosteriod on cotton growth and development and physiology and gene expression, to analyse the expression levels of brassinosteroid metabolic genes in roots when cotton subjected to salt stress. And then, the relationship is established between cotton salt resistance and the expression of brassinosteroid metabolic genes, and the key genes are found during the pathway of brassinosteroid biosynthesis, degradation and signal transduction which related to cotton salt tolerance. Finally, the key genes in brassinosteroid metabolic pathways are cloned, and then they are constructed into root expression vector with root promoter and transformed into plant to testing plant tolerance to salt stress. The results would valuable to understand the role of brassinosteroid metabolic genes in cotton roots sujected to salt stress, and it could provide new ways and new genes for innovating the cotton germplasm resources.

棉花是盐碱地先锋作物，发展盐碱地植棉对于缓解粮棉争地矛盾极具意义，改良棉花耐盐性已成为科研主攻方向之一。油菜素内酯作为一种植物内源激素具有原位作用特性，可调控植物耐盐性。作物根系是盐胁迫作用的首要器官，因此本项目拟开展根源油菜素内酯调控棉花耐盐性的机理及关键基因的功能分析研究。首先从根系外用油菜素内酯对棉花耐盐性的调控入手，研究外源油菜素内酯对盐胁迫下棉花形态、生理功能、下游基因表达的影响。并重点分析盐胁迫下根系油菜素内酯合成、降解、转导途径相关基因的表达情况，建立油菜素内酯代谢相关基因的表达与棉花耐盐性间的关系，进而确定棉花根系中与耐盐性相关的油菜素内酯基因。在此基础上，通过转基因技术利用根系特异启动子使油菜素内酯基因在根系定向表达，验证作物根源油菜素内酯基因在盐胁迫下的功能。研究结果不仅可以进一步明确棉花根源油菜素内酯基因对耐盐性的调控作用，还可为棉花种质资源的创新提供新途径、新基因。

植物耐盐性是复杂的数量性状，近年来利用常规技术在一定程度上提高了棉花耐盐性，但离生产应用的要求还有一定距离，因此有必要寻找新的途径、新的基因来创制新的耐盐资源。油菜素内酯作为一种植物内源激素，可调控植物的耐盐性，但对盐胁迫棉花的调控作用尚未明确。为此本项目首先研究了油菜素内酯对棉花耐盐性的影响。结果表明，外源油菜素内酯对盐胁迫下棉花的形态、生理功能、基因表达水平均有明显影响。数字表达谱结果显示，盐胁迫下棉花根系蛋白质合成相关基因和叶片光合相关基因及逆境响应基因的表达水平受到油菜素内酯调控。此外，外源油菜素内酯增强盐胁迫下棉花的渗透调节能力、降低离子毒害，从而导致盐胁迫下棉花叶片叶绿素含量上升、根系活力增强、最终生物量提高。在此基础上，分析了盐胁迫下根系油菜素内酯合成、代谢、信号转导途径相关基因的表达情况，发现盐胁迫下棉花中油菜素内酯相关基因DWF4、DET2、BES1、UGT73C5等的表达受到外源油菜素内酯的显著调控。结合前人研究结果选择油菜素内酯信号转导途径基因BES1、代谢途径基因UGT73C5作为候选基因进行基因功能鉴定研究。通过转基因技术将上述基因在拟南芥中过量表达，发现过量表达BES1、UGT73C5基因改变拟南芥耐盐性。项目的开展明确了外源油菜素内酯对棉花耐盐性的影响，筛选出与棉花耐盐性相关的油菜素内酯基因。