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Bulletin of Botanical Research ›› 2016, Vol. 36 ›› Issue (2): 258-265.doi: 10.7525/j.issn.1673-5102.2016.02.015

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Cloning and expression analysis of GrCMS gene in Gentiana rigescens

ZHANG Xiao-Dong1;LI Cai-Xia1;WANG Yuan-Zhong2*   

  1. 1.College of Resources and Environment,Yuxi Normal University,Yuxi 653100;
    2.Institute of Medicinal Plants,Yunnan Academy of Agricultural Sciences,Kunming 650223
  • Online:2016-03-15 Published:2016-05-20

Abstract: 2-C-methyl-D-erythritol 4-phosphatecytidyltransferase(CMS, EC is the third enzyme in methylerythritol phosphate(MEP) pathway. The Open Reading Frame(ORF) of GrCMS gene was cloned by RT-PCR technology from young leaves of Gentiana rigescens based on the transcriptome of G.rigescens and its prokaryotic and the tissue specific expression analysis were performed. The ORF of GrCMS gene(accession number: KJ917164) was 933 bp long coding for a protein of 310 amino acids, and the predicted relative molecular weight of GrCMS was 34.23 kD with its theoretical pI of 7.68. The results of GrCMS protein analysis showed that GrCMS which possessed the conserved domains of CMS proteins and may localize in chloroplast was a hydrophilic stable protein without signal peptide, and it was composed of mainly α-helix(26.45%) and random coils(54.84%). By phylogenetic analysis, GrCMS was close to CrCMS of Catharanthus roseus. By prokaryotic analysis, the recombinant protein of GrCMS gene in E.coli was approximately 60.23 kD(containing GST tag protein 26 kD), which was consistent with the anticipated size. By real-time PCR analysis, GrCMS gene was primarily expressed in leaf. Our results will provide reference for further functional researches of GrCMS gene and the biosynthetic pathway of gentiopicroside.

Key words: Gentiana rigescens, 2-C-methyl-D-erythritol 4-phosphatecytidyltransferase, gene cloning, expression analysis