小麦TaBI-1.1的耐热功能解析

High temperature has become one of the most serious stress factors affecting wheat.production in China. Breeding new wheat cultivars with enhanced heat tolerance is an effective way to solve this problem. Discovering and using the novel heat stress tolerance gene is the key for this strategy..Our previous studies showed that TaBI-1.1 was related to heat tolerance using the transcriptome and ectopic overexpression it in Arabidopsis. The transgenic wheat lines of TaBI-1.1 with overexpression or suppressing (RNA interference) expression have been obtained and will be used to further affirm the heat tolerance of the TaBI-1.1 in this project. The subcellular localization assay will be conducted to determine the action site of TaBI-1.1 by transiently expressing TaBI-1.1-GFP fusion protein in wheat protoplasts. A series of plasmid vectors with 5ˈ-truncated TaBI-1.1 promoters fused with the reporter gene GUS will be constructed and transformed into wheat callus, respectively. Activity of the gene TaBI-1.1 promoter under different heat treatments will be analyzed. Relative and absolute quantification will be used for protein expression analysis in response to heat stress treatments in the seedlings of wheat transformation lines and their wild type. Differentially expressed proteins related to plant heat stress response were validated by parallel reaction monitoring. Downstream target proteins of TaBI-1.1 will be screened out. Interaction proteins of TaBI-1.1 will be screened out using yeast two-hybrid, bimolecular fluorescence complementation and GST pull down assay. The mechanism of TaBI-1.1 and its interaction protein to regulate the downstream target protein will be illustrated by co-transfecting protoplasts and GUS or LUC reporter system. The results will deepen the understanding of the mechanism of how plant adapt to the heat stress and supply the theoretical fundamentality for breeding heat stress tolerant cultivars.

高温已成为影响我国小麦生产的主要逆境胁迫因素之一，选育和推广耐热品种是确保小麦高产稳产的有效途径之一，发掘耐热基因和解析耐热基因的功能可为耐热新品种的选育提供种质材料和理论依据。课题组前期通过转录组分析和转拟南芥研究表明，小麦的TaBI-1.1基因与耐热性相关。本项目拟通过分析过表达和RNAi沉默小麦转基因后代，进一步鉴定该基因的耐热性功能；通过研究TaBI-1.1基因在小麦原生质体的瞬时表达，以及启动子5ˈ系列缺失片段驱动GUS基因在小麦响应高温胁迫瞬时表达，明确TaBI-1.1基因的亚细胞定位信息并初步揭示启动子响应高温胁迫的机制；通过对小麦转基因后代和野生型分别进行定量蛋白组学分析，筛选与耐热相关的下游靶蛋白；用酵母双杂交系统筛选，并结合双分子荧光互补技术和蛋白质体外结合实验鉴定与TaBI-1.1互作蛋白。本项目对小麦抗逆分子育种有重要的理论和实际意义。

高温已成为影响我国小麦生产的主要逆境胁迫因素之一，选育和推广耐热品种是确保小麦高产稳产的有效途径之一，发掘耐热基因和解析耐热基因的功能可为耐热新品种的选育提供种质材料和理论依据。在本项目的资助下，利用TaBI-1.1的小麦的过表达和RNAi沉默植株对TaBI-1.1在小麦中的耐热功能机进行了验证，结果表明在正常条件下的苗期和灌浆期，转基因植株和受体没有表型差异。在高温胁迫下，苗期和灌浆期的对照和沉默株系迅速萎蔫，过表达植株只发生部分黄化；过表达植株的叶绿素含量显著高于对照植株，沉默植株的叶绿素含量显著低于对照植株；活性氧清除相关酶SOD、CAT、POD的酶活性在过表达植株中显著高于对照植株，在沉默植株中显著低于对照植株。高温胁迫下的千粒重测定表明，过表达植株的千粒重显著高于对照植株，沉默植株的千粒重显著低于对照植株。因此，TaBI-1.1具有耐热功能，可以提高小麦的苗期和灌浆期的耐热性。选择最耐热的过表达植株和最不耐热沉默植株进行转录组和蛋白组分析，结果发现分别有四个基因和11个蛋白在过表达植株中上调表达，在沉默植株中下调表达。GO和KEGG分析发现差异表达蛋白被富集到内质网蛋白加工通路，暗示TaBI-1.1参与内质网合成相关生物学过程。酵母双杂交验证表明TaBI-1.1和两个小热激蛋白TasHSP17.8和TasHSP24.1存在互作关系。利用酵母单杂技术，筛选到TaBI-1.1的上游调控蛋白JAZ，推测JAZ可以同时调控TaBI-1.1、TasHSP-17.8和TasHSP-24.1的表达，从而提高小麦的耐热性。本研究为深入理解小麦的耐热机理提供有益信息。