山茱萸萜类合成途径关键酶HMGR1基因的克隆与分析

Cloning and analysis of a key enzyme of HMGR1 gene involved in terpene biosynthesis of Cornus officinalis

目的克隆山茱萸Cornus officinalis HMGR1的cDNA序列并进行生物信息学分析。方法以山茱萸转录组数据中unigenec100572_g1序列为参考,在其开放阅读框两端设计特异引物,经实时荧光定量PCR(RT-PCR)扩增、连接pTOPO-T载体克隆并测序,获得CoHMGR1基因的cDNA序列,并利用生物信息学方法对该基因及其编码蛋白进行相关分析。结果CoHMGR1基因长2116 bp,含长度为1338 bp的完整开放阅读框(ORF),编码445个氨基酸,为疏水性蛋白。多序列比对和亲缘关系分析显示,CoHMGR1基因编码的氨基酸序列与喜树的序列相似性较高,达79.56%。结论首次对山茱萸叶片和果实进行比较转录组测序的基础上,成功克隆并分析了CoHMGR1基因,为进一步研究CoHMGR1蛋白的功能及山茱萸萜类合成途径的分子机制奠定基础。

Objective To clone and analyze the cDNA sequence of 3-Hydroxy-3-methylglutaryl coenzyme-a reductase(HMGR1) of Cornus officinalis. Methods In this study, specific primers were designed at both ends of the open reading frame(ORF) based on the unigene(c100572_g1) in the transcriptome data from C. officinalis. Subsequently, the c DNA sequence of Co HMGR1 gene was amplified by RT-PCR, cloned into the pTOPO-T vector and sequenced. This gene and its encoded protein were analyzed using bioinformatics methods. Results The results suggested that CoHMGR1 was 2 116 bp in length andthe ORF was 1 338 bp in length, which encodes 445 amino acids and is a hydrophobic protein. Multiple sequence alignment and phylogenetic analysis showed that the amino acid sequence encoded by this gene has a high similarity with that of Camptotheca acuminate, reaching 79.56%. Based on the first comparison of transcriptome sequencing from leaves and fruits of C. officinalis, the Co HMGR1 gene was successfully cloned and analyzed. Conclusion This study laid a foundation for studying the function of CoHMGR1 protein and the molecular mechanism of terpene biosynthesis pathway of C. officinalis.