Cloning And Tissue Expression Of Functional Genes In Steroidal Saponins Biosynthesis From Paris Fargesii Franch

Steroidal saponins is a kind of secondary metabolites with high medicinal value, to explore and study its synthetic pathway and realize its metabolic regulation has a great significance for the modernization of Chinese medicine.Paris fargesii Franch is a genus of Paris Liliaceae, which has the effects of heat-clearing and detoxifying. And steroidal saponins is the major medicinal effective ingredient of it. Steroidal saponins is synthesized by the mevalonate pathway and 3-hydroxy-3-methyl coenzyme A reductase (HMGR) is just the first rate-limiting enzyme.The total RNA was extracted from Paris fargesii Franch then the conserved sequence,3'cDNA and 5'cDNA of gene HMGR were amplified to make up of a full-length cDNA encoding HMGGR(designated as PfHMGR). PfHMGR is 1,973 bp encoding 578 amino acids, its bioinformatics analysis showed that the protein molecular weight is about 61kDa, theoretical isoelectric point is 6.64 and it has high homology with other plant HMGRs and contains two transmembrane domains and tree catalytic domains. It has two HMG-CoA binding sites and two NADP (H) binding sites as well.The E. coli expression vector for PfHMGR was constructed for the first time. PfHMGR was transformed into E. coli and the identification showed that PfHMGR can replace AtIPI to promote the accumulation of carotenoids, which demonstrated the expression of PfHMGR in E. coli.Real time quantitative PCR was performed to make a research on the differential expression in different plant tissue, respectively on IPPI, FPS and SS, which are functional genes in the biosynthetic pathway of steroidal saponins, to make a conclusion that all three genes are expressed in root, stem and leaf. IPPI was expressed most in stem, least in root; FPS most in stem, least in leaf; SS most in root, least in leaf. Primers were designed to perform amplifications of coding sequences of PfHMGR in root, stem and leaf, take housekeeping gene 18S as control, the result detected by electrophoresis revealed that PfHMGR was expressed most in root, least in leaf.