丹参2-C-甲基-D-赤藓糖醇2,4-环焦磷酸合成酶(SmMCS)基因全长克隆及其生物信息学分析

Cloning and bioinformatics analysis of 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase gene in Salvia miltiorrhiza

目的:从丹参毛状根中克隆2-C-甲基-D-赤藓糖醇2,4-环焦磷酸合成酶(SmMCS)全长基因,并进行生物信息学分析。方法:根据丹参转录组数据提供的基因片段设计特异引物,采用RACE方法克隆SmMCS全长cDNA,并对SmMCS进行蛋白结构预测、序列多重比对和构建进化树等分析;同时采用实时定量PCR检测Ag+诱导子诱导丹参毛状根不同时期的SmMCS基因转录水平。结果:克隆的SmMCS全长cDNA由988个核苷酸组成,具有完整编码框,编码234个氨基酸,蛋白相对分子质量约24.6 kDa,等电点pI 8.53;实时定量PCR结果表明该基因受Ag+诱导后表达水平在12 h时急剧上升并达到最高值。结论:从丹参毛状根中克隆得到一条SmMCS全长cDNA,为进一步研究丹参酮类成分的生物合成和萜类次生代谢提供靶基因。

Objective.. To clone and analysis a 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase （SmMCS） full-length cDNA from Salvia miltiorrhiza hairy roots. Method ： A full-length cDNA of SmMCS has been cloned by designing specific primers ac- cording to the transcriptome database and using the RACE strategy. ORF Finder was used to find the open reading frame of SmMCS cDNA and ClustalW has been performed to analysis the multiple amino acid sequence alignment. Phylogenetic tree has been construc- ted using MEGA 5.1. Real-time quantitative PCR have been applied to detect the transcription leveX of SmMCS from hairy roots after elicitor Ag ~ supplied. Result ： The SmMCS cDNA sequence was obtained. The full length of SmMCS cDNA was 988 bp encoding 234 amino acids. The deduced protein had isoelectric point （pI） of 8.53 and a calculated molecular weight about 24. 6 kDa. Results of real time PCR indicated that elicitor of Ag ＋ stimulated the increase of mRNA expression of SmMCS in hairy roots, and were increased dra- matically at 12 h. Conclusion： The full-length cDNA of SmMCS was cloned from S. miltiorrhiza hairy root,which can provide a gene target for further studies of tanshinones biosynthesis and terpenoid secondary metabolites.