[Objective] The objective of this study is to clone gentian geraniol 10-hydroxylase gene GrG10H,to analyze its bioinformatics characters,gene structure,prokaryotic expression and tissue expression,and to provide a theoretical base for elucidation of getiopicroside biosynthesis pathway.[Method] RT-PCR and gene cloning were adopted to isolate the gene GrG10H from the leaves of Gentiana rigescens.Bioinformatics tools were used to analyze the characters of both DNA sequence and its coding protein.Clustal X2.1 software was used to make the multiple sequence alignments between the GrG10H protein and their homologous sequences,and the phylogenetic tree of homologous species was constructed by MEGA6.0.The real-time fluorescent quantitative RT-PCR was applied to analyze the expression of GrG10H in root,stem and leaf.[Result] GrG10H gene was obtained from G.rigescens leaves by RT-PCR,and was deposited into GenBank (accession number:KJ418410).The results of bioinformatics analysis showed that GrG10H gene had a length of 1834 bp containing two exons and one intron.Its ORF (open reading frame) was 1548 bp coding for 515 amino acids.Its relative molecular weight was 57.74 kD with the isoelectric point of 9.02.GrG10H protein was the member of cytochrome P450 superfamily and may localize in endoplasmic reticulum;there was a transmembrane region in N-terminal (21-43),while the first 20 amino acids resided within the membrane and that of 44-515 were out of membrane;GrG10H was a hydrophilic stable protein without signal peptide and composed of mainly α-helix (40.58％) and irregular coils (44.85％).GrG10H protein was close to SmG10H of Swertia mussotii.The results of prokaryotic expression of GrG10H gene in E.coli showed that the recombinant protein was approximately 83.74 kD (containing GST tag protein 26.00 kD),which was consistent with the theoretical size.The tissue-specific expression results indicated that GrG10H gene was primarily expressed in leaf.[Conclusion]GrG10H gene was cloned and expressed in E.coli.successfully,and it may mainly functioned in leaves.
NETL
NSTL
万方数据
维普
