地黄RgCDPK基因的克隆与表达分析

Cloning and expression analysis of calcium-dependent

关键词:  地黄, 钙依赖蛋白激酶, 生物信息学, 组织特异性

Key words:  Rehmannia glutinosa, calcium-dependent protein kinase, bioinformatics, tissue specificity

摘要:

钙依赖型蛋白激酶(calcium-dependent protein kinases, CDPKs)是高等植物细胞中重要的钙离子信号受体,在植物抵御逆境胁迫过程中发挥着重要作用。该研究以地黄为材料,设计特异引物,克隆地黄RgCDPK基因全长序列,并使用在线软件进行生物信息学分析,采用荧光定量PCR技术进行组织特异性分析。结果表明:(1)克隆得到的地黄CDPK基因长度为1 770 bp,编码589个氨基酸;(2)多序列比对和结构分析显示,该蛋白含有钙依赖蛋白激酶典型结构域丝氨酸/苏氨酸蛋白激酶区及EF-手性区。系统进化分析表明其与拟南芥 AtCDPK28 的同源关系最近,因此命名为RgCDPK(Genbank登录号为MT024235);(3)组织特异性分析得出RgCDPK在地黄叶中表达量最高。该研究成功克隆出地黄CDPK基因,且发现该基因在不同组织中的表达存在差异,为以后深入研究CDPK在地黄连作障碍等生物及非生物胁迫中的分子机制提供理论基础。

Abstract:

Calcium-dependent protein kinases(CDPKs)are important calcium sensors in higher plants, which play an important role in plant resistance to envirenment stress. In this study, a full-length cDNA of RgCDPK gene was cloned from Rehmannia glutinosa. Meanwhile, the bioinformatics analysis was used the online software, quantitative real-time PCR technique was used to detect RgCDPK expression level in different tissues. The results were as follows:(1)The full-length cDNA sequence of RgCDPK gene was 1 770 bp and encoded 589 amino acid residues;(2)Multiple sequence alignments and structural analysis revealed that its protein contained serine/threonine protein kinase region and EF-hand region, which were typical domains of calcium-dependent protein kinase. Phylogenetic analysis showed the highest similarity with Arabidopsis AtCDPK28. Thus, the gene was defined as RgCDPK(Genbank accession No. MT024235);(3)The expression analysis of RgCDPK in different tissues revealed that high transcript levels occurred at leaves. In this study, the CDPK gene of R. glutinosa was successfully cloned, and the expression of the gene in different tissues was found to be different. The results of this study provide theoretical basis for further study on the function of CDPK in biotic, abiotic stresses and continuous cropping obstacles.