罗马洋甘菊HMGR基因的克隆与表达分析

Cloning and Expression Analysis of HMGR Gene from Chamaemelum nobile

3-羟基-3-甲基戊二酰-CoA还原酶(3-hydroxy-3-methyl glutaryl coenzyme A reductase,HMGR)是植物萜类化合物甲羟戊酸合成途径中的关键限速酶。为研究HMGR基因在洋甘菊萜类化合物合成代谢中的功能,根据前期罗马洋甘菊转录组注释HMGR的Unigene序列设计引物,以罗马洋甘菊c DNA为模板,采用RT-PCR方法,从罗马洋甘菊中克隆得到一个长为1 856 bp的HMGR基因,命名为CnHMGR(Gen Bank登录号为KU589282)。CnHMGR基因序列包含1个1 746 bp长的开放阅读框,编码582个氨基酸,生物信息学软件预测蛋白质分子量和等电点分别为62.5 k Da和6.80。氨基酸序列多重比对结果显示,CnHMGR蛋白质与其他植物的HMGR蛋白具有高度相似性,并包含HMGR蛋白质家族中的2个HMG-CoA结合基序:TTEGCLUA、EMPVGYVQIP以及2个NADP(H)结合基序:TVGGGT、DAMGMNM,表明CnHMGR属千罗马洋甘菊HMGR家族成员。组织表达分析显示CnHMGR在罗马洋甘菊的不同组织均有表达,在花中表达量最高,在茎中表达量最低。通过对CnHMGR基因的克隆及表达特性分析,为后续深入研究其在洋甘菊倍半萜合成途径中的功能奠定了理论基础。

3-hydroxy-3-methyl-glutaryl-CoA reductase（3-hydroxy-3-methyl glutaryl coenzyme A reductase,HMGR） is one of the key rate-limiting enzymes in mevalonic（MVA） pathway of terpenoid biosynthesis.To analysis the function of HMGR gene in terpenoid biosynthesis in Chamaemelum nobile,a HMGR gene（designated as CnHMGR,Gen Bank accession number KU589282） was cloned from C.nobile using RT-PCR method.The fulllength c DNA of CnHMGR gene was 1 856 bp and contained an open reading frame（ORF） of 1 746 bp,which encodes a 582 amino-acid protein.The theoretical molecular weight and PI of the CnHMGR were 62.5 k Da and 6.80,respectively.Multi-alignment comparison analysis showed the protein sequence of CnHMGR had high similarity with those of HMGR proteins from other plants.Furthermore,CnHMGR has two HMG-CoA-binding motifs：TTEGCLVA,EMPVGYVQIP,and two NADP（H）-binding motifs：TVGGGT,DAMGMMM,suggesting CnHMGR is one of HMGR family mumbers in C.nobile.The results of tissue expression analysis showed that CnHMGR was constituently expressed indifferent tissues of C.nobile,with highest expression level in flowers and lowest expression level in stems.The present study cloned,characterized and analyzed the tissue expression pattern of CnHMGR in C.nobile,providing basic data for further studying the function of CnHMGR in sesquiterpene biosynthesis of C.nobile.