沟叶结缕草蛋白二硫键异构酶ZmPDI调控耐盐性的分子作用机制

As a perennial warm season halophyte turfgrass, Zoysia matrella is an important plant material for efficient utilization of saline-alkali land and mechanism research of plant salt-tolerance. Protein disulfide isomerase (PDI) is an important protein involved in the protein folding pathway of endoplasmic reticulum, however, the detailed role of PDI in plant salt tolerance regulation remains unclear. In previous study, we successfully identified a candidate salt-tolerant gene ZmPDI encoding a protein disulfide isomerase by yeast expression library screening. The results indicated that expression of ZmPDI gene could be induced by salt stress, whereas the salt tolerance of ZmPDI transgenic Arabidopsis plant was significantly improved, suggesting ZmPDI might play important roles in salt tolerance regulation of Zoysia matrella. In this research, we try to elucidate the molecular mechanism of ZmPDI-mediated salt tolerance of Zoysia matrella by performing following experiments: (1) The function of ZmPDI in salt tolerance and down-stream genes and proteins regulated by ZmPDI will be revealed through over-expression and RNAi suppression of ZmPDI expression in Zoysia matrella, (2) The core salt-inducible cis-element within ZmPDI promoter will be excavated for upstream transcription factors screening by yeast one-hybrid system in order to explore the up-stream transcription regulation mechanism of ZmPDI expression. Execution of this research can not only enrich the understanding of the molecular mechanism how protein disulfide isomerase regulates salt tolerance of plants, but also provide theoretical basis for molecular breeding of other salt-tolerance turfgrass based on PDI genes.

沟叶结缕草(Zoysia matrella)是重要的盐生暖季型草坪草，是盐渍地高效利用和植物耐盐机理研究的优异植物材料。前期研究发现参与内质网蛋白正确折叠的沟叶结缕草蛋白二硫键异构酶编码基因ZmPDI表达受盐诱导上调，异源表达ZmPDI可显著提高拟南芥耐盐性，提示ZmPDI在沟叶结缕草耐盐性调控中起着重要作用。本研究拟通过以下实验进一步阐明ZmPDI调节沟叶结缕草耐盐性的分子机制：(1) 在沟叶结缕草中过量表达和RNAi抑制表达ZmPDI，明确ZmPDI的耐盐功能及其下游调控基因和蛋白网络；(2)采用启动子分段缺失突变筛选ZmPDI启动子序列中关键的盐诱导元件，通过酵母单杂交筛选与盐诱导元件结合的上游转录因子，解析ZmPDI的上游转录调控机制。本项目的成功实施不仅将深化对蛋白二硫键异构酶调控植物耐盐性分子机制的理解，也将为基于PDI基因的耐盐草坪草分子育种提供重要的理论依据。

沟叶结缕草(Zoysia matrella)是重要的盐生暖季型草坪草，是盐渍地高效利用和植物耐盐机理研究的优异植物材料。前期研究发现参与内质网蛋白正确折叠的沟叶结缕草蛋白二硫键异构酶编码基因ZmPDI表达受盐诱导上调，异源表达ZmPDI可显著提高拟南芥耐盐性，提示ZmPDI在沟叶结缕草耐盐性调控中起着重要作用。本研究通过构建了超表达载体pCAMBIA1305.2-ZmPDI，对禾本科模式作物水稻和沟叶结缕草分别进行异源转化和同源转化，并敲除水稻中PDI的同源基因，进一步验证了PDI基因的耐盐功能；之后利用转录组蛋白组测序技术，对ZmPDI转基因的水稻和结缕草进行测序，并筛选到了11个可能受到ZmPDI基因调控的差异基因和10个可能受到ZmPDI基因调控的差异蛋白。比对分析显示，被ZmPDI基因正调控的下游基因与铁、锌离子结合、蛋白质结合和氧化还原过程密切相关，被ZmPDI基因的负调控的下游基因与水解酶活性和膜紧密联系。因此推断ZmPDI基因调控植物耐盐的分子机制主要通过改善和调节与膜有关的离子和蛋白质结合以及水解酶的氧化还原过程从而提高植物的耐盐性；为了探究ZmPDI上游调控转录因子，采用启动子分段缺失突变筛选ZmPDI启动子中的关键盐诱导元件，通过酵母单杂交筛选出了5个ERF转录因子，通过酵母和烟草系统，进一步验证了ZmERF4能通过直接连接GCC-Box元件，促进ZmPDI基因表达。本项目的成功实施不仅将深化对蛋白二硫键异构酶调控植物耐盐性分子机制的理解，也将为基于PDI基因的耐盐草坪草分子育种提供重要的理论依据。