花生小GTP结合蛋白基因AhRab7提高耐盐性的分子机理研究

Rab proteins form the largest family of the small GTP-binding proteins, they regulate growth, development and morphogenesis in plant. Interestingly, some of the Rab genes are found to be responsive by different environmental stresses. In our previous studies, a few mutants with salt tolerance obtained via physical and chemical mutagenesis in combination with tissue culture, then one stress-inducible Rab7 gene of Arachis hypogaea differentially expressed between the wild parent and mutant with salt resistrance was identified by SSH technology, and the open reading frames (ORF) were overexpressed in E. coli and peanut, respectively. The results showed that the Rab7 gene can help them resist salt stress. To further clarify the molecular regulation mechanism involved in salt tolerance of Rab7 gene in peanut,in this study, RNA sequencing will be carried out using transgenic plants with salt resistance as materials after salt treatment to analyze the regulation pathway on salt resistance of Rab7 gene. Moreover, we will clone its promoter and analyze the cis-regulatory elements of the promoter that mediate expression of Rab7 gene, and then construct different truncated vectors to transform peanut. After salt stress treatment, GUS staining of tissues and activity analysis will be carried out to confirm the cis-acting elements responsive to salt stress. This study will supply gene resources with independent intellectual property rights to new germplasms creation with tolerace to salinity stress via genetic engineering. Simultaneously, it will lay the foundation for clarifying molecular mechanism of Rab7 gene responsive to salinity stress and cloning regulator upstream, so this study has both great theoretical and practical significance.

Rab蛋白是小GTP结合蛋白家族最大的亚族，调控着植物的生长发育、形态建成，有些成员还受多种逆境胁迫的诱导表达。本课题组前期利用理化诱变与组培相结合筛选出一批耐盐突变体，并应用SSH技术筛选出在野生型和突变体中差异表达的1个Rab7基因，分别在大肠杆菌和花生中表达后，重组菌和转基因植株耐盐能力显著增强。为进一步探明花生Rab7基因在盐胁迫中的分子调控机理，本研究拟对盐胁迫处理后的转基因耐盐植株进行RNA-Seq，分析Rab7基因参与的耐盐调控途径。此外，克隆其启动子，分别缺失不同的区段后与GUS基因融合，转化花生，并施以盐胁迫处理，通过GUS组织化学染色和活性分析后，确定该启动子上盐胁迫应答的关键顺式调控元件。本项目的开展，旨在为创造耐盐新种质提供具有自主知识产权的基因，系统地阐明Rab7基因在花生耐盐中的分子调控机制，同时为克隆上游的调节因子等研究奠定基础，因而具有理论和实践双重意义。

Rab亚族是小GTP结合蛋白家族最大的亚族，其成员Rab7受多种逆境胁迫的诱导表达。本课题组前期在花生中筛选出1个Rab7基因，分别在大肠杆菌和花生中表达后，重组菌和转基因植株耐盐能力显著增强。. 为进一步探明花生Rab7基因在盐胁迫中的分子调控机理，本研究利用稳定遗传的过表达Rab7基因的花生植株，盐胁迫处理前后进行RNA-Seq，利用KEGG富集分析确定Rab7基因主要调控以下途径：光合作用、氧化磷酸化、AMPK信号途径、植物激素信号转导、生物钟、类黄酮合成、突触囊泡循环、植物和病原菌互作、PI3K-Akt信号途径、内质网蛋白加工、抗原加工和呈现等途径。. Rab7基因可以调控乙烯响应的AP2、MYB、RING-H2型锌指蛋白等转录因子，以及大量的耐盐相关基因：如LEA、RD22、过氧化物酶、CBL-互作蛋白激酶、钙结合蛋白等，从而提高转基因花生植株的耐盐性。. 此外，克隆了Rab7基因启动子，分别缺失不同的区段后与GUS基因融合，转化烟草，并施以盐胁迫处理，通过GUS组织化学染色和活性分析以及荧光定量PCR分析，初步筛选出了该启动子上盐胁迫应答的关键顺式调控元件。. 本项目初步阐明了Rab7基因在花生中的调控途径和转基因植株耐盐的分子调控机制，同时初步确定了盐胁迫相关的重要顺式调控元件，因而具有重要的理论价值。