Cloning, Expression Of HMGR, G10H And IS And Correlation With Gentiopicroside Accumulation In Gentiana Rigescens

Objective In order to obtain the three potential key enzymes in the biosynthetic pathway of gentiopicroside in Gentiana rigescens Franch.ex Hemsl.It includes3-hydroxy-3-methylglutaryl coenzyme A reductase,geraniol 10-hydroxylase and iridoid synthase genes.To express the above three genes in the way of prokaryotic expression and analyze the relationships between the expressions and the synthesis of gentiopicroside.Methods The c DNA sequence of HMGR ? G10 H and IS was obtained from G.rigescens by RT-PCR technique.It based on the transcript from the transcrptome.The prokaryotic expression of three genes was carried out by using the p EASY-E1/BL21 expression system.The physical and chemical properties,secondary structure and tertiary structure of three genes protein were forecasted and analyzed by using related Software.Adopt the real-time fluorescence quantification PCR to detect the three genes in G.rigescens roots,stems and leaves of different growth stages.The contents of gentiopicroside in different parts of different growth stages in G.rigescens Franch.were determined by ultra performance liquid chromatography.SPSS 20 software was used to analyze the correlation between the relative expression of three genes and the content of gentiopicroside.Results Two HMGR genes were cloned and named GRHMGR-1 and GRHMGR-2.GRHMGR-1 has a full length of 1747 bp and an ORF of 1605 bp,which encodes 534 amino acids.GRHMGR-2 has a full length of 1731 bp and an ORF of 1572 bp,which encodes 523 amino acids.Both proteins encoded 3-hydroxy-3-Coenzyme A reductase possess a typical polypeptide site.One G10 H sequence of full-length 1400 bp,with a1248 bp ORF,encoding 415 amino acids,named GRG10 H.The conserved region analysis shows that it belongs to the cytochrome P450 family;A 1746 bp genome sequence of G10 H was predicted to contain three exons and two introns.One IS gene of full-length 1388 bp,with a 1176 bp ORF,encoding 391 amino acids was identified as GRIS.Conserved region analysis showed that it was a member of the NADB-Rossmann muperfamily.GRHMGR-1,GRHMGR-2,GRG10 H and GRIS were separately successfully expressed as 58 KDa,56 KDa,48 KDa and 44 KDa target proteins in Escherichia coli.GRHMGR and GRG10 H were all expressed in the roots,stems and leaves of G.rigescens at different growth stages.GRIS was detected only in stems and leaves of different growth stages of G.rigescens.The results showed that the contents of gentiopicroside in G.rigescens root increased with the increase of the growth years,while the stem and leaf decreased gradually with the growth period.Conclusions The possible key genes of HMGR,G10 H and IS were obtained and the prokaryotic expression system was constructed,which provided a basis for the establishment of eukaryotic expression system.The expression levels of GRHMGR,GRG10 H and GRIS were not significantly correlated with the accumulation of gentiopicroside.