Effect Of MeJA On DS Gene Expression In Panax Ginseng And Study On Saccharomyces Cerevisiae Expressing DS Gene

Panax ginseng is one of the most famous medicinal plants and used intensively because of its potent pharmaceutical activities. Ginsenosides are the major active constituents including dammarene-type and oleanane-type saponins. So far, more than 60 kinds of ginsenosides were found. All the ginsenosides except ginsenoside R0 belong to dammarene-type saponins. Dammarene-type saponins have many physiological and pharmaceutical effects, including anti-fatigue, antihyperglycemic, anti-aging and anti-cancer effects. Dammarane-type ginsenosides are biosynthesized from dammarenediol by a sequence of two steps, namely hydroxylation and glycosidation. Dammarane type ginsenosides are biosynthesized from dammarenediol by a sequence of two steps, namely hydroxylation and glycosidation. RNA interference of DS in transgenic P. ginseng resulted in silencing of DS expression, which leads to a reduction of ginsenoside production. These results indicate that DS is a key enzyme involved in ginsenoside biosynthesis. However, mechanism of regulation in ginsenoside synthesis remains to be answered.In this study, methyl jasmonate induced effect on the growth, content of total saponins and individual ginsenoside content of Panax ginseng hairy root and DS gene expression were investigated by HPLC and semi-quantitative reverse transcription and polymerase Chain reaction. Meantime, a recombinant S.cerevisiaes strain which can produce dammarenediol-Ⅱwere constructed as yeast-pAUR123-DS. The main contents and conclusions of this study are as follows:1. First, we choosed the time of MeJA elicitation for 4 weeks from growth curve of Panax ginseng hairy root. Different concentration of MeJA elicitation could remarkably improve the growth of Panax ginseng hairy root and increase level of total ginsenoside and Rb1, Rd, Re, Rg2 ginsenoside by HPLC and colorimetry of vanillina. In the case of Rgl ginsenoside after MJ elicitation, the content was affected negatively in ginseng hairy root cultures. But the effects were weakened with increase of concentration. When the concentration of MeJA elicitation was 0.01mmol/L, growth, content of total saponins and Rb1,Rd, Re, Rg2, Rgl ginsenoside was 41.59g/L,24.1mg/g,2.4 mg/g,1.96 mg/g,5.82 mg/g,0.53mg/g,3.87 mg/g respectively.2. In order to analyze the change of DS gene expression, concentration of Mg2+, annealing temperature and cycle number of PCR system were optimized analyzed, and then a stable and convenient semi-quantitative RT-PCR system which amplified DS andβ-actin gene at the same time in different tubes was established. The PCR condition for DS andβ-actin gene were as follows:concentration of Mg2+ was 1.5 mmol/L, annealing temperature was 55℃, the primes of P-actin gene was added after 6 cycles of DS gene amplication. When the 30th cycles, they arrived the exponential period at the same time.3. The transcription of DS genes in hairy root cultures elicited by 0.1mm MeJA treatment remarkably increased as compared with the control. Compared with 24h,48h of MeJA elicitation, DS gene expression is the maximum at 12h. The difference is insignificant, so the upregulation of DS gene expression do not depend on time.4. Total RNA was isolated from P.ginseng hairy roots. Total RNA were reverse-transcribed to produce cDNA. PCRs were performed with F and MR primer. After tailing and purifaction, the PCR product was subcloned to the plasmid vector (pMD-19T), namely pMD-19T-DS. Compared with sequence of DS gene in Genebank, the positive vector has 6 basic group changed by sequence analysis of TaKaRa. However, the mutated basic group did not mutate encode sequence of amino acids. After enzyme digestion, the DS fragment was ligated into the multi-cloning site of yeast expression vector pAUR123, namely pAUR123-DS. The plasmid pAUR123-DS was transferred to S. cerevisiae strain 31476. After screening on medium and enzyme digestion, the transformant had object,namely yeast-pAUR123-DS.5. S. cerevisiae extract from transformed cells were applied onto SDS-PAGE, and then target protein of molecular weight 85.5kDa was found, whereas target protein was not found in the control. Meanwhile, dammarenediol-Ⅱwas found in secondary metabolite of S. cerevisiae by HPLC, and the content of dammarenediol-Ⅱwas 1.85mg/100g.6. Plasmid stability of transgenic engineering bacteria was detected. The consequence showed that plasmid of recombinant yeast was not stable under no selective pressure. After 168h of culture, plasmid stability of recombinant yeast was 67% under no selective pressure, and plasmid stability of recombinant yeast was above 90% under selective pressure.Analysis and prospect based on above result:First, DS is the key enzyme in biosynthensis pathway of ginsenoside, and upregulation of gene expression could improve level of dammarenediol-Ⅱand promote saponin accumulation ginsenoside. Second, MeJA elicitation could change glycosyltransferase gene expression, so the ratio of ginsenoside Rx was changed. Last, because the yield of dammarenediol-Ⅱof transgenic engineering bacteria is very low, we should knockout unwanted gene and optimize fermentation condition to improve level of dammarenediol-Ⅱ. This study is significant for further investigation of ginsenoside biosynthesis pathway and for the large-scale production of dammarenediol-Ⅱ.