蛋白激酶GsCBRLK在大豆盐胁迫信号转导途径中的调控机理研究

Elucidation of the regulatory mechanisms of key salt tolerant genes is the premise of crops’ molecular breeding. GsCBRLK was isolated from wild soybean during our previous study. This gene was reported as being a putative novel calmodulin-binding membrane receptor-like protein kinase whose activity was up-regulated in a calcium-dependent manner. It was found that over-expression of GsCBRLK in Arabidopsis, alfalfa and soybean plants resulted in enhanced salt tolerance. However, the regulatory mechanism of GsCBRLK on plant salinity stress tolerance is yet to be characterized. The hypothesis is that GsCBRLK will be activated by the salt signal and transform the signal by phosphorylating substrates, which results in enhanced salt tolerance in plants.In order to verify this hypothesis, the substrates of GsCBRLK will be identified at first. The approach inculdes an in vitro kinase assay-based substrate screening and endogenous kinase dependent phosphorylation profilling. The phophorylated substrates identified by phosphoproteomics will be analysed using bioinformatic methods. The interaction between GsCBRLK and candidate substrates will be tested by BiFC, GST-pull down and other methods. And the direct targets will be identified by in vitro kinase assay between GsCBRLK and substrates using proteins purified from E.coli expression. In-gel kinase assay will be performed with protein isolated from wild soybean plants treated with different concentration and different time point of NaCl using the bona fide substrates embeded in SDS-PAGE gel.The result will verify the hypothesis that GsCBRLK phosphorylation status will be changed under salt treatment. Taken together, the aim of this project is to elucidate the mechanism of enhanced salt tolerance in wild soybean regulated by GsCBRLK, and the ultimate goal is to provide the theoretical foundation, technical assitance and plant material to molecular breeding of salt tolerant crops.

阐明耐盐关键基因的调控机理是进行耐盐分子育种的重要前提。前期研究已获得野生大豆蛋白激酶基因GsCBRLK，体外实验表明其具有Ca2+依赖CaM调控的自磷酸化和底物磷酸化活性，过量表达该基因可显著提高植物的耐盐性，但作用机理尚不明确。我们推测盐胁迫下GsCBRLK以激活状态通过磷酸化未知底物蛋白将胁迫信号向下传递。为证实这一假设，本项目拟采用基于体外磷酸化的蛋白质组学方法在多肽水平筛选GsCBRLK的底物蛋白，分析其磷酸化位点；进行GsCBRLK与候选底物的体内、体外相互作用验证及体外磷酸化验证，明确蛋白激酶和底物的相互作用模式；利用筛选出的特异性底物进行体内磷酸化分析验证高盐胁迫对GsCBRLK激酶活性的影响；最终阐明GsCBRLK提高大豆耐盐性的分子调控机理。为通过遗传工程途径改良大豆耐盐性提供理论依据和材料储备。

高盐等逆境环境条件严重制约农业生产，是我国区域农业发展所面临的重要问题，提高作物的耐逆性是开发利用盐碱地的前提，而对候选基因进行详细的抗逆功能分析并弄清关键基因所参与的分子调控机理是转基因育种研究和产业化的重要前提。本研究利用农杆菌介导的遗传转化将GsCBRLK基因转入大豆植株，经分子生物学检测获得GsCBRLK基因超表达且稳定遗传的转基因大豆株系；经过耐盐性检测，筛选耐盐转基因大豆株系。利用iTRAQ定量蛋白质组学手段分析盐胁迫处理前后GsCBRLK基因超表达的转基因大豆株系和未转基因对照植株蛋白质组的差异，原始数据已提交ProteomeXchange Consortium，接收号为PXD002851。数据分析得到以下结果：.1. 根据iTRAQ结果，盐胁迫处理后在大豆叶片中得到278个差异表达蛋白，其中237个表达量增加，41个表达量降低；同时在大豆根中得到440个差异表达蛋白，其中354 个表达量增加，86个表达量降低。通过对差异表达蛋白的功能分析，得出大豆幼苗在高盐胁迫下的早期应答机制：首先，胁迫信号转导和膜蛋白在胁迫处理早期被激活，调控下游蛋白的表达；第二，活性氧清除系统启动并与其它非生物胁迫响应蛋白协同互作；第三，新陈代谢途径被重构以适应盐胁迫。通过构建蛋白-蛋白相互作用网络，发现蛋白质代谢，能量供应及光合作用协同互作，共同重新建立了盐胁迫下的细胞稳态。.2. 根据iTRAQ结果，GsCBRLK调控了大豆叶片中574个蛋白、大豆根中1176个蛋白的表达，这些蛋白参与信号转导、转录过程、能量代谢、蛋白折叠和降解以及胁迫响应过程。GsCBRLK参与了盐胁迫下大豆幼苗的活性氧清除、光合作用等生理过程；尤其重要的是GsCBRLK重构了盐胁迫下大豆植株的Ca2+/CaM信号转导途径，调控了一系列信号分子的转录水平及蛋白表达。.研究结果从新的视角进一步揭示了GsCBRLK调控大豆耐盐性的分子机理，补充完善盐胁迫下的信号转导途径，为关键调控基因的利用提供理论基础。