紫花苜蓿MsCIPK2的克隆及功能分析

Cloning and Functional Analyses of MsCIPK2 in Medicago sativa

【目的】CIPK是植物响应逆境胁迫信号通路中一类重要的蛋白激酶,可与CBL形成CBL-CIPK复合物,启动细胞内相关应答基因的表达而应对各种非生物胁迫。发掘并研究紫花苜蓿MsCIPK基因响应非生物胁迫的分子机理,有助于揭示紫花苜蓿抗逆生物学基础,为紫花苜蓿抗逆育种提供新的基因资源。【方法】通过PCR技术克隆MsCIPK2,使用生物信息学工具分析基因序列,利用qRT-PCR技术分析MsCIPK2,以及与其互作的4个CBL基因(MsCBL2、MsCBL6、MsCBL7和MsCBL10)在紫花苜蓿各组织中的表达水平,在烟草叶片表皮细胞中,瞬时表达pCAMBIA1302-GFP-MsCIPK2融合表达载体,通过激光共聚焦显微镜观察进行亚细胞定位,利用酵母双杂交技术分析MsCIPK2与4个MsCBLs蛋白互作情况,利用发根农杆菌诱导紫花苜蓿产生过量表达MsCIPK2的毛状根,利用qRT-PCR技术分析转基因毛状根株系中相关基因的表达水平。【结果】通过PCR扩增获得MsCIPK2片段,该基因CDS为1 230 bp,编码409个氨基酸,具有典型的CIPK家族的ATP结合位点、激活环、NAF motif和PPI motif等结构域。MsCIPK2在紫花苜蓿根中表达量最高,在花中表达量最低。亚细胞定位结果显示,MsCIPK2蛋白定位于内质网。酵母双杂交试验结果显示,MsCIPK2蛋白与MsCBL2、MsCBL6、MsCBL7和MsCBL10蛋白具有相互作用,且与MsCBL10蛋白相互作用较强。MsCBL2、MsCBL6和MsCBL10在紫花苜蓿根中表达量最高,MsCBL7在荚中表达量最高。qRT-PCR结果表明,过量表达MsCIPK2的毛状根中响应非生物胁迫基因ATPase、P5CS、CYP705A5、COR47、HAK5和RD2的表达量均明显上调。在200 mmol·L^(-1)NaCl和20%PEG处理条件下,与对照相比,过量表达MsCIPK2毛状根的丙二醛含量降低,SOD活性、脯氨酸含量和可溶性糖含量增高。【结论】紫花苜蓿MsCIPK2与MsCBLs蛋白互作,主要在根中表达并响应盐和干旱胁迫,过量表达MsCIPK2可以提高紫花苜蓿的耐盐性和耐旱性,MsCIPK2可作为提高紫花苜蓿抗逆育种的候选基因。

【Objective】CIPKs are a group of important protein kinase involved in signaling pathway of plant in response to stress.They can form CBL-CIPK complex with CBL, to activate the expression of related responsive genes to cope with various abiotic stresses in cells. Exploration and study on the molecular mechanism of MsCIPK genes in alfalfa in response to abiotic stress will help to reveal the biological basis of stress resistance in alfalfa, and to provide new gene resources for alfalfa breeding with enhanced stress resistance. 【Method】The MsCIPK2 gene was cloned by using PCR, the sequence was analyzed by bioinformatics tools, and the expression level of Ms CIPK2, Ms CBL2, Ms CBL6, Ms CBL7 and MsCBL10 genes in various tissues were analyzed by using qRT-PCR. The pCAMBIA1302-GFP-MsCIPK2 vector was transiently expressed in tobacco leaf epidermal cells, and the subcellular localization was observed under laser confocal microscope. Yeast two-hybrid assay was used to analyze interaction between Ms CIPK2 and four MsCBLs proteins. Agrobacterium rhizogenes was used to generate alfalfa hairy roots over-expressing MsCIPK2.qRT-PCRs were used to analyze the expression levels of related genes in transgenic hairy root lines. 【Result】The coding sequence of MsCIPK2 gene was obtained by using PCR, and it is 1 230 bp in length, encoding 409 amino acids. The deduced MsCIPK2protein contained typical ATP binding site, activation loop, NAF motif and PPI motif as for the CIPK family genes. The expression level of MsCIPK2 gene was the highest in roots, and the lowest in the flowers of alfalfa. Subcellular localization results showed that MsCIPK2 protein was localized in the endoplasmic reticulum. Yeast two-hybrid assays showed that MsCIPK2 protein interacted with MsCBL2, MsCBL6, MsCBL7 and MsCBL10 proteins, showing stronger interaction with MsCBL10 than with other MsCBLs. The expression levels of MsCBL2, MsCBL6, and MsCBL10 were the highest in roots of alfalfa, and the expression level of MsCBL7 was the highest in pods. qRT-PCR results showed that the expression levels of abiotic stress-associated genes ATPase,P5CS, CYP705A5, COR47, HAK5 and RD2 were significantly up-regulated in hairy roots over-expressing MsCIPK2. Under the treatment of 200 mmol·L^(-1)NaCl and 20% PEG, when compared with the control hairy root line, hairy roots over-expressing MsCIPK2 had lower MDA content, and higher POD activity, proline content and soluble sugar content. 【Conclusion】MsCIPK2can interact with CBL protein, and responded to salt and drought stress in roots of alfalfa. Over-expression of MsCIPK2 can improve salt and drought stress resistance in alfalfa, and MsCIPK2 can be used as candidate gene for alfalfa breeding with improved abiotic stress resistance.