Molecular Cloning, Expression And Protein Purification Of Monoterpene Synthase Gene In Cymbopogon Citratus

The comprehensive utilization of plant resources has always been the focus on research and the development of plant products with economic value and urgent need of the market.The secondary metabolites of most plants include terpenoids,alkaloids and phenylpropane,etc.,which play extremely important roles in plant defense and human health.For plants,generating metabolite is a means of protecting themselves against harsh conditions;for human being,many plant secondary metabolites have important functions in the treatment of diseases and are also important sources of medicines and cosmetics.Terpenoids are the most diverse natural products among the countless compounds produced by plants,and have important pharmacological effects and a wide range of biological functions.Therefore,it is of great significance to elucidate the synthetic pathways of terpenoids and study their molecular regulation mechanisms.C.citratus has short growth cycle to develop large-scale cultivation with small investment and quick effect.The essential oil extracted from its stems and leaves has a wide range of applications in food industry and medicine,etc.The research shows that terpenoids are the main components in the essential oil of C.citratus.Previous studies on C.citratus mainly focused on the extraction and composition analysis of its essential oils,but there were relatively few studies on the biosynthesis and metabolism regulation of its important compounds,and the functions of most genes in the genome were not clear,it is impossible to regulate its unique synthetic pathway to greatly increase its content.In this study,the monoterpene synthase gene was cloned from C.citratus,full length cDNA sequence of PTS gene was cloned from C.citratus after bioinformatics analysis.Then prokaryotic expression vector were constructed,the optimal conditions for prokaryotic expression were explored,and the enzyme purification were performed.This study is of great significance to elucidate the metabolic pathways and functions of the key enzymes of citronella terpenes,and also to provide reference and modification targets for the industrial fermentation of terpenes.In details,full length cDNA sequence of monoterpene synthase gene was cloned from C.citratus for the first time,containing a1185 bp open reading frame(ORF)coding for 394 amino acids.The pGEX and pET28a(+)was chosen as the expression vector of target gene to construct recombinant plasmid and expressed in Escherichia coli.Optimized expression conditions in terms of the effects of induction time,induction temperature,expression vector and host bacteria for the prokaryotic expression were investigated,and the optimal induction expression conditions were found with successfully purified the protein.In summary,the cloning of the monoterpene synthase gene of citronella with successful expression and purification in prokaryotic expression system are conducive to the research and discovery of other monoterpene synthetase genes,and provide a potential guide in further investigation on mechanism of monoterpene biotechnology as well as producing highly value monoterpene at large scale by biotechnology.