玉米ZmCIPK6基因的克隆及植物表达载体的构建

Cloning of ZmCIPK6 Gene and Construction of Its Plant Expression Vector

根据MaizeDGB数据库中ZmCIPK6基因序列设计引物,采用RT-PCR技术从玉米中克隆了1个ZmCIPK6基因。ZmCIPK6基因c DNA全长1 739 bp,5′-非编码区长281 bp,3′-非编码区长111 bp,编码区长1 347 bp,编码448个氨基酸,预测分子量为48.8 KDa,等电点为9.14。氨基酸同源性分析发现,ZmCIPK6与高粱SbCIPK6同源性较高。根据ZmCIPK6的基因序列和植物表达载体pCAMBIA3301的多克隆位点设计带有限制性内切酶位点的特异性引物,以质粒pMD18-T-ZmCIPK6为模板,PCR扩增ZmCIPK6基因片段,双酶切目的片段及载体,回收后连接,构建成该基因的植物表达载体。经PCR检测及测序鉴定,表明成功构建了ZmCIPK6基因的植物表达载体,为进一步遗传转化研究基因的功能奠定基础。

According to the sequence of ZmCIPK6 in MaizeDGB database,primers were designed. RT-PCR method was used to clone ZmCIPK6 gene. A gene coding for ZmCIPK6 was isolated from maize （Zea mays）.The full length ZmCIPK6 cDNA was 1 739 bp,including a 281 bp 5’-UTR,an ORF of 1 347 bp,and a 111 bp 3’-UTR.This cDNA sequence encoded a polypepi-de of 448 amino acid residues with a predicted molecular mass of 48.8 kDa and a basic isoelectric point of 9.14. The deduced amino acid sequence had a high homology with SbCIPK6 from Sorghum bicolor.According to the restriction enzyme sites of expression vector pCAMBIA3301 and sequence of ZmCIPK6 gene,one pair of primers containing restriction enzyme sites were designed. The ZmCIPK6 gene was obtained from pMD18-T-ZmCIPK6 by PCR. PCR product and the plasmid pCAMBIA3301 were digested by the corresponding restricted enzymes respectively,and then the ZmCIPK6 gene was cloned into pCAMBI-A3301 vector.PCR and sequencing results suggest that plant expression vector of ZmCIPK6 gene was constructed successfully.