Study On The Identification And Expression Of Functional Genes Related To Ginsenoside Biosynthesis In Panax Quinquefolius L.

Panax quinquefolius L. is one of the precious perennial herbs and well known forits outstanding effectiveness of anti-fatigue, anti-aging, beauty maintenance and so on.Ginsenosides are thought to be the main metabolites of Panax quinquefolius L. for itspharmacological activity. Recent studies show that dammarane-type ginsenosides areregarded as the major pharmaceutical ginsenoside constituents in Panax quinquefoliusL.. Dammarenediol synthase (DS), protopanaxadiol synthase (D12H) andprotopanaxatriol synthase (P6H) of cytochrome oxidase P450are considered as thekey enzymes in dammarane-type ginsenosides biosynthesis pathway. Precursors2,3-oxidosqualene can be converted into dammarenediol-II with the catalysis of DS,then the dammarenediol-II is converted into protopanaxadiol under the hydroxylationof D12H at C-12position, follow with P6H involves in hydroxylation at C-6position,the protopanaxadiol could be converted into protopanaxatrioL. Subsequently,protopanaxadiol and protopanaxatriol can synthesize monomer ginsenoside under thecatalysis of glycosyltransferase. Our group had already finished the identification andanalysis of protopanaxadiol synthase gene (PqD12H) in Panax quinquefolius L.previously. However, the gene of dammarenediol synthase and protopanaxatriolsynthase in Panax quinquefolius L. had not been identified.This study started with the isolation and characterization of dammarenediolsynthase gene and protopanaxatriol synthase gene from Panax quinquefolius L., fromthe beginning to end, we did the gene cloning, sequence analysis, heterologousexpression identification, heterologous co-expression and over-expression regulationsuccessively. Overall, this study could improve the ginsenoside biosynthesis pathwayin Panax quinquefolius L., laid a foundation for the heterologous expression ofginsenosides, also had a certain reference value to the regulation of ginsenosidebiosynthesis and germplasm innovation in Panax quinquefolius L.. The main resultsof our study are as follows:1. Primers were designed according to the published conserved sequences ofdammarenediol synthase gene and protopanaxatriol synthase gene between Panaxginseng and Panax notoginseng respectively, then the full-length gene ofdammarenediol synthase and protopanaxatriol synthase in Panax quinquefolius L.were amplified by RACE successfully. The sequences were named PqDS and CYP6Hwith the bioinformatics analysis, GenBank accession No. were KC316048and KC190491.2. After we got the open reading frame region of PqDS and CYP6H, heterologousexpression vectors were constructed through enzyme digestion and ligationtechnology respectively, then transformed into saccharomyces cerevisiae competentcell for heterologous expression identification. Collected the yeast extract anddetected by High Performance Liquid Chromatography (HPLC), the results indicatedthat both two genes could be expressed into protease with active function successfully.3. We transformed all the heterologous expression vectors pAUR123-PqDS,pAUR123-PqD12H and pAUR123-CYP6H into a single saccharomyces cerevisiaecell according to the order of phases, then the co-expression engineering bacteriayeast-pAUR123-PqDS-PqD12H-CYP6H was constructed. HPLC analysis of the yeastextract showed that genes PqDS, PqD12H and CYP6H could be co-expressed insaccharomyces cerevisiae to produce the protopanaxatriol, and the yield wasapproximately13.85μg/g FW.4. Additionally, we also constructed over-expression vectors pBI121-PqDS andpBI121-CYP6H, then transformed into agrobacterium rhizogenes A4and obtained theover-expression engineering bacteria A4-pBI121-PqDS and A4-pBI121-CYP6Hrespectively. Through the infection of Panax quinquefolius L. root, we got theover-expression hairy root, then detected the content changes of total ginsenosidesand monomer ginsenoside Rb1, Rb2, Rc, Rd, Re and Rg1to analyzeexpression regulation effect of ginsenoside response to gene PqDS and CYP6Hover-expression. The results indicated that over-expression of gene PqDS couldimprove the prodution of monomer ginsenoside by15.44%-46.55%, and the amountof total ginsenosides also increased by24.56%; what is more, the over-expression ofCYP6H could increase the protopanaxatriol-type monomer ginsenoside by17.80%-19.9%, while the protopanaxadiol-type monomer ginsenosides decreased by4.46%-11.26%, and the content of total ginsenosides still grew by9.24%.