细胞壁关联的类受体激酶基因CaWAKL20介导ABA信号调控辣椒耐热性的分子机制

Heat stress has become one of the most important environmental factors adversely affecting crops production; however, so far, the molecular mechanisms of plant thermotolerance development are incompletely understood. In previous research work, we found wall-associated receptor-like kinase gene CaWAKL20 negatively regulates pepper thermotolerance by repressing ABA signaling. This project will further explore the contributions of CaWAKL20 to pepper thermotolerance regulation, by analyzing the effects of fluidity modifier of cell membrane and changing of extracellular domain on CaWAKL20 expression. At the same time, the interactions between CaWAKL20 and its predicted partners in ABA signaling pathway were confirmed point-to-point by yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays. And then, taking the pepper plants with CaWAKL20 silencing and pepper or tomato lines with CaWAKL20 overexpression as materials, the heat responses of gene expression of ABA synthesis, metabolism, transport and signaling transduction, and degradation of ABA response proteins were investigated. At last, the thermotolerance-related functions of CaWAKL20 interacting partner genes and ABA signaling genes regulated by CaWAKL20 were validated by the techniques of gene silencing and overexpression. By this project, the molecular mechanisms of CaWAKL20 regulating pepper thermotolerance through mediating ABA signaling will be primarily clarified, which will then provide experimental proofs for fully understanding the molecular mechanisms of plant thermotolerance development.

热胁迫已经成为影响农作物生产的重要环境因子之一，然而目前植物耐热性形成的分子机制尚未完全阐明。我们前期发现，细胞壁关联的类受体蛋白激酶基因CaWAKL20通过抑制ABA信号而负调控辣椒耐热性。在此基础上，本项目拟首先分析细胞膜流动性改变和膜外结构域更换对CaWAKL20基因表达的影响，进一步明确其耐热调控功能，同时对ABA信号途径中与CaWAKL20有预测互作或共表达关系的蛋白成员进行点对点验证。然后以CaWAKL20基因的辣椒沉默表达植株、辣椒或番茄过表达株系为试材，分析热胁迫下ABA合成、代谢、转运以及信号转导基因表达和ABA响应蛋白降解情况。最后，将ABA信号途径中与CaWAKL20互作或受CaWAKL20调控的目标基因进行沉默表达和过表达，验证其耐热功能。通过本项目实施，有望初步明确CaWAKL20基因介导ABA信号调控辣椒耐热性的分子机制，为全面阐明植物耐热性形成的机制提供依据。

辣椒耐热性形成的分子机制尚未完全阐明。在前期研究基础上，本项目进一步明确了细胞壁关联类受体蛋白基因CaWAKL20在辣椒耐热性中的功能，分析了CaWAKL20与ABA信号途径的互作关系、细胞膜流动性改变对CaWAKL20基因表达及辣椒耐热性影响、番茄中CaWAKL20同源蛋白SlWAKL18的序列及表达特性，筛选了CaWAKL20的互作蛋白并探讨了其耐热功能。另外，鉴定了辣椒热激转录因子基因CaHsfA2的上游转录因子并分析了其耐热功能，研究了金属伴侣蛋白基因CaHPP7、糖基转移酶基因CaGnT-III、自噬相关蛋白基因CaATG16在辣椒耐热性中的功能。项目研究主要取得了以下结果。.（1）CaWAKL20基因的表达水平被热胁迫抑制而被外源ABA诱导。CaWAKL20基因沉默表达提高了辣椒的耐热性，而过表达降低了拟南芥的耐热性。CaWAKL20基因过表达降低了拟南芥对ABA的敏感性，同时抑制了ABA信号途径基因对热胁迫的响应。.（2）CaWAKL20与ABA信号转导途径相关组分的互作模式尚不明确。.（3）细胞膜流动性诱导剂苯甲醇（BA）和抑制剂二甲基亚砜（DMSO）处理均不能诱导CaWAKL20基因的表达，对辣椒耐热性的影响也不明显。.（4）番茄SlWAKL18与辣椒CaWAKL20具有相同的蛋白结构，在进化上高度保守，并且SlWAKL18能与细胞壁组分结合。SlWAKL18基因的表达水平受热胁迫调控。.（5）筛选获得了16个CaWAKL20的候选互作蛋白，其中CaDSK2a与CaWAKL20的互作关系得到验证。CaDSK2a基因沉默表达提高了辣椒的耐热性。.（6）筛选获得了热激转录因子基因CaHsfA2的上游转录因子CaBES1。CaBES1负调控CaHsfA2基因的表达，并且CaBES1基因沉默表达提高了辣椒耐热性，过表达降低了拟南芥耐热性。.（7）CaHPP7、CaGnT-III和CaATG16基因的沉默表达均降低了辣椒耐热性，而过表达均提高了拟南芥耐热性。.通过项目的实施，进一步明确了CaWAKL20对辣椒耐热性的调控作用及其作用机制，为将来深入阐明辣椒耐热分子机制奠定了基础。同时，项目实施挖掘出的辣椒耐热相关基因为采用基因工程技术创制辣椒耐热种质和选育辣椒耐热品种提供了理论依据。