调控TaGST1基因表达的转录因子的分离及其功能研究

Glutathione-S-transferases (GSTs)，one key enzyme of the ascorbate- glutathione（ASA-GSH） cycle, play major roles in the response of plants to abiotic stresses metabolism. In previous studies, we isolated the full-length cDNA sequences of the glutathione-S-transferase 1 (GST1) and 2 (GST2) genes in wheat and measured the transcription levels of eight ASA-GSH cycle-related genes from wheat plants under abiotic stress. These indicated that the transcription levels of GST1/2 genes was significantly enhanced after drought stress. Using proteomic method, moreover, a set of abiotic stresses-responsive protein species with significantly altered abundance were identified in the wheat plant suffered from abiotic stresses, such as drought, salt, copper, and etc. Pathway Studio software was used to construct the abiotic stresses-responsive protein species interaction network, and showed that this interaction network including some of putative key stresses-responsive protein species. TaGST1, one of these identified protein species, was significantly upregulated, and participated in the above interaction network by interacting with different abiotic stresses-responsive protein species during abiotic stress. These demonstrated that TaGST1 plays an important role in abiotic tolerance. Accordingly, the promoter of TaGST1, including many abiotic stresses-responsive elements, has been isolated in our lab. And the expression of GUS was higher in salinity stress than that of the control plants, suggesting that this promoter can be used for both constitutive as well as stress inducible expression of gene(s). .Based on these data, to further explore the molecular mechanism of TaGST1 gene, in the present study, the transcription factors, which combine with the promoter of TaGST1, are further identified using yeast one-hybrid method. The function of the isolated transcription factors have been identified by transgenic wheat. And then, the transcription levels of TaGST1 gene and the parameters of growth will be measured in the transgenic wheat plants in order to explore the function of the isolated transcription factors in abiotic tolerance. These results will help to further explore the molecular mechanism of TaGST1 gene under abiotic stress.

谷胱甘肽转移酶（GST）是抗坏血酸酸（ASA）和谷胱甘肽（GSH）循环中活性氧清除的关键酶。在前期研究中，申请人辨析出小麦基因组内ASA-GSH循环的8个关键酶基因，转录水平测定了逆境胁迫小麦植株8个ASA-GSH相关酶基因的表达，发现其在不同胁迫时间点的表达量显著上升，其中TaGST1/2在胁迫处理中的转录水平表达均上调，表明TaGST在小麦幼苗响应逆境胁迫中发挥了重要作用。进而采用双向电泳技术，在翻译水平上研究了小麦幼苗对盐、干旱等非生物胁迫的响应机制，辨析出一批小麦逆境胁迫响应蛋白，其中TaGST1蛋白的表达丰度显著上升，从而在翻译水平证明了TaGST1在小麦逆境胁迫响应中的重要功能。本项目拟采用酵母单杂交、EMSA、ChIP-qPCR等方法，分离出调控TaGST1表达的转录因子；进而通过转基因的方法验证转录因子在逆境胁迫下的功能，从而揭示TaGST1在响应逆境胁迫中的分子机制。

谷胱甘肽转移酶（GST）在抗坏血酸酸和谷胱甘肽循环中是活性氧清除的关键酶。项目负责人前期研究发现小麦GST1在转录和蛋白的表达丰度被干旱等逆境胁迫显著诱导表达，并参与多种小麦逆境胁迫的蛋白通路，说明其在小麦响应逆境胁迫中发挥了重要作用。在此基础上，为进一步解析TaGST1在逆境胁迫中的分子调控作用，项目负责人分离了该基因上游启动子序列，利用酵母单杂交方法将其与小麦逆境胁迫酵母cDNA文库进行杂交，筛选出调控TaGST1表达的调控因子；并利用另外的酵母单杂试验验证了转录因子TaWRKY74和TaERFL1a与TaGST1启动子存在互作，利用双荧光素酶实验验证了TaCPK34与TaGST1的互作；进而利用BSMV-VIGS沉默法分别获得了上述调控因子的沉默植株，并在逆境胁迫条件下验证了其作用效果以及与TaGST1的关系，从而进一步解析了TaGST1在逆境胁迫条件下的分子调控机制。