Molecular Cloning, Expression And Functional Characterization Of Squalene Epoxidase And Dammarenediol Synthase From Panax Ginseng

Ginseng is the precious traditional Chinese medicine in a long history, and has a important medicinal and commercial value. The main effective components of ginseng is ginseng saponins. At present, it is at least60ginseng saponins that be separated from ginseng. Ginseng is harvested in a long period and the yield and quality of Ginseng has been affected by other conditions, such as climate environment, regional factors, plant diseases and insect pests. Therefore, it is not easily to get a lot of ginseng saponins. If we are further understand the biosynthetic pathway and the molecular mechanism of specific regulation in course of saponins synthesis. The yield of ginsengoside should be improved.This experiment continued research group project, using a four-year old ginseng root tissue cDNA as a template. To clone squalene epoxidase(SQE) and dammarenediol synthase(DS) which are key enzyme in the biosynthetic pathway of ginseng saponins. The full-length of open reading frame of SQE is161lbp, coding536amino acid. Comparing on the Genbank, the nucleotide sequence is identity of99.75%, the amino acid sequence identity of99.81%.The nucleotide sequence and amino acid sequence of DS gene are99.87%, comparing on the Genbank. In this study, the mRNA expression levels of SQE and DS of ginseng fibrous roots were detected in the different year (1year,3year,4years,5year) by quantitative PCR technique. The results showed that the transcription of SQE and DS is up with the parameters age, the ginsenoside accumulation has the same result. SQE and DS gene have great relation with the production of ginsenosides, providing a reliable basis reference data for future research.On the basis of the cloned gene, we have successfully built the SQE and DS prokaryotic expression vector pET-30a-the SQE and pET-30a-the DS. The recombinant proteins of SQE and DS were expressed in the recipient strain of E.coli Rosetta and detected by SDS-PAGE electrophoresis. The result showed the recombinant protein has high purity. By adding different amounts of enzyme is reaction and the same amount of squalene is as a substrate. The different enzyme content of system is produce dammarenediol which were detected by LC-MS. The results showed that in vitro SQE and DS recombinant protein has a certain activity. In the case of a certain substrate, the Dharma ene diol formation increases with the amount of enzyme. The experiments show that the accumulation intermediates of ginsenoside biosynthetic pathway has a significant effect by adding SQE and DS, and has effect on the yield of ginsenoside.