玉米ZmCIPK42蛋白激酶参与盐胁迫反应的调控机理研究

CBL-interacting protein kinases (CIPKs) which contain Ser/Thr kinase domain are unique protein kinase in higher plants. They especially interact with CBLs (calcineurin B-like proteins). Recent studies reveal that CBL-CIPK networks play important roles in stress signaling transduction pathways. Preliminarily, we found that ZmCIPK42 was up-regulated by high salt, osmotic and heat stress. Overexpression ZmCIPK42 in Arabidopsis rendered salt tolerant. Following we will focus four researches: First, by constructing the overexpression vector and transform the maize, analysis the phenotype of transgenic plant under different salt stress, to assess the tolerance of transgenic plants; Next, by using yeast two hybrid method to screen the CBL protein which interacted with ZmCIPK42; Then, by screening maize cDNA library of AD vector to screen target protein of ZmCIPK42; Finally, by using BiFC and Co-IP method to analyze interact mechanism and phosphorylation of downstream target protein to explicate the signaling pathway of salt tolerance. By this research we would not only provide a theoretical basis for the salt tolerance pathway of ZmCIPK42, but also provide important candidate genes for genetically modified organism of maize.

玉米类钙调磷酸酶B互作蛋白激酶(CBL-interacting protein kinases, CIPKs)是高等植物中特有的一类丝/苏氨酸蛋白激酶，它与CBL(Calcineurin B-Like protein)蛋白特异性互作后在植物逆境应答过程中起关键调控作用。在玉米的43个CIPK家族基因中，我们发现ZmCIPK42基因受高盐、渗透和高温胁迫后上调表达，在拟南芥中过表达ZmCIPK42呈现耐盐表型。因此，本项目拟通过构建该基因植物表达载体，转化玉米自交系，研究转基因植株在高盐胁迫条件下的贡献值；通过酵母双杂等方法分析该基因与玉米CBL家族基因之间的互作与定位以及发挥生物学功能的机制；通过筛选玉米cDNA文库发掘与ZmCIPK42互作的蛋白，进而通过免疫共沉淀技术或者磷酸化技术分析确定该蛋白在逆境胁迫信号传导途径中作用。本研究可为植物抗逆基因研究提供理论依据和重要的候选基因。

植物钙调神经磷酸酶B（CBL）和与其相互作用蛋白激酶（CIPK）复合物在植物生物和非生物胁迫响应中起着重要作用。在本研究中，我们发现一个参与盐胁迫响应的基因ZmCIPK42。通过遗传学、细胞生物学手段，对ZmCIPK42参与的耐盐机理进行研究，主要研究结果为：（1）在突变体库中发现一个ZmCIPK42基因突变体，该突变体编码区发生G到A突变，导致编码蛋白提前终止。突变体Zmcipk42植株雄穗分枝减少、对盐胁迫处理敏感。（2）ZmCIPK42基因在不同组织中均有转录，在NaCl胁迫下表达量显著提高。（3）在玉米和拟南芥中的过表达ZmCIPK42导致转基因植株对高盐胁迫的耐受性增强。（4）通过酵母双杂交（Y2H）筛选试验，发现ZmCIPK42能够和ZmCBL1，ZmCBL4相互作用。（5）通过酵母双杂交（Y2H）技术筛选玉米cDNA文库，鉴定了一种与ZmCIPK42互作的蛋白酶抑制剂ZmMPI，盐胁迫条件下ZmMPI呈现显著上调表达趋势。这些结果表明ZmCIPK42可能介导了玉米对盐胁迫的反应。