野大麦转录因子HbERF6调控蛋白激酶基因HbCIPK2响应高盐干旱胁迫的分子机制研究

Wild barley (Hordeum brevisubulatum) is the relative of wheat and barley and one of important halophytic grasses in genera Hordeum. It has evolved special mechanisms to adapt salt and drought stresses. Under the foundation of previous projects, a novel protein kinase gene HbCIPK2 was identified from halophytic grass wild barley, which was induced by abiotic stress and regulated plant salt and drought tolerance. The promoter of HbCIPK2 was cloned by genome walking, and showed inducible activities under abiotic stresses. The transcriptional factor HbERF6 was screened out from wild barley cDNA library treated with salinity and drought using the HbCIPK2 promoter as bait, and the promoter sequence just contained cis-elements to which ERF can specifically bind. This proposal will aim to identify how HbERF6 regulate HbCIPK2 in vitro and vivo, analyze the biological function of HbERF6 by its overexpression in Arabidopsis and gene editing of CRISPR/Cas9 in wild barley plants and confirm the regulatory relationships between HbERF6 and HbCIPK2 through RNA sequencing. At last, the HbERF6-HbCIPK2 regulatory pathway will be dissected to mediate in response to high salt and drought stresses, which will provide not only the genetic information for the study of plant abiotic resistance and gene regulation, but also new strategies and methods for the genetic improvement of plant resistance.

野大麦是小麦、大麦的野生近缘种，也是大麦属重要的盐生植物，在长期进化中演化形成特殊的耐盐抗旱机制。课题组在前期项目资助下从盐生野大麦中挖掘出一个新的蛋白激酶编码基因HbCIPK2，它受逆境诱导表达，具有调控植物耐盐、抗旱性的功能；通过染色体步移克隆到HbCIPK2启动子，其具有逆境诱导表达活性；并以其为诱饵通过酵母单杂筛选高盐、干旱胁迫野大麦cDNA文库，获得转录因子HbERF6；HbCIPK2启动子序列含有ERF特异结合的顺式作用元件。但是关于二者之间的调控关系，目前还不清楚。在此基础上本项目拟通过体外和体内实验鉴定HbERF6对HbCIPK2转录水平的调控作用；通过模式植物拟南芥过表达和基因组编辑创制野大麦突变体分析HbERF6的生物学功能；通过转录组测序进一步确定二者的调控关系，最终解析HbERF6-HbCIPK2介导的调控途径在植物高盐、干旱胁迫响应中的作用和机制。

乙烯信号通路的识别和功能鉴定对于理解盐生植物对自然盐碱环境的适应具有重要意义。在本研究中，利用野生大麦（Hordeum brevisubulatum）CBL相互作用蛋白激酶HbCIPK2启动子（PHbCIPK2）为诱饵筛选酵母单杂交文库，鉴定了一个由盐、干旱和ABA诱导的乙烯反应转录因子（ERF）HbERF6。亚细胞定位分析显示HbERF6定位于细胞核内。酵母单杂交分析和凝胶迁移实验表明，HbERF6可直接与HbCIPK2启动子结合。双荧光素酶实验证实HbERF6能够反式激活HbCIPK2启动子。HbERF6异位表达拟南芥增强植物对盐胁迫的耐受性。通过分析转基因植物中下游基因的表达，发现HbERF6显著增强了胁迫响应基因的转录水平，如AtCIPK6、AtP5CS和AtGSTF10等。综上所述，这些结果表明HbERF6通过增强HbCIPK2的表达在植物耐盐性中发挥积极作用。