Transformation Screening And Identification Of Ginseng CYP716A47 Gene In Physcomitrella Patens

Physcomitrella patens is a new model organism developed in recent years in foreign countries,because it has a high efficiency of homologous recombination,and its growth model is relatively simple and easy to mass culture.At present,the scientists have successfully used transgenic P.patens produce a variety of human proteins.Moreover,currently,the scientists have used P.patens to express terpene synthase genes,which provided as a basis for the feasibility of combinatorial biosynthesis of ginsenoside in P.patens.Ginsenoside is one of the main active ingredients of ginseng,however,it is difficult to obtain ginsenosides,which has been a bottleneck problem in drug development and production.Currently the acquisition of ginsenosides is mainly from extracting ginseng,this method is of low resource utilization and of high cost.Since ginsenoside biosynthetic pathway has been revealed,the combinatorial biosynthesis woulde be the ideal way to obtain ginsenosides.In recent years,attempts have been made to introduce some ginsenosides biosynthesis enzymes into E.coli or yeast for combinatorial biosynthresis and have made some achievements.P.patens has some unique advantages over E.coli and yeast as a host,for example,photoautotrophic culture,low cost,already industrialized culture,high homologous recombination rate,easy genetic manipulation and itself has terpenoid metabolic pathways,great transformation potential,terpene product can be secreted into the medium,easy purification and so on.Ginseng CYP716A47 gene encodes an enzyme capable of catalysing the dammarenediol to become protopanoxadiol,it is an important gene in the synthesis of protopanoxadiol type ginsenosides.Objective:This topic will try to transfer ginsenosides synthase CYP716A47 into P.patens for exogenous expression,through constructing an expression vector of ginsenosides synthase CYP716A47 and transforming P.patens to accomplish the homologous recombination,and through resistance screening of antibiotic and identification in molecular level and protein level to acquire transgenic P.patens,which has a stable expression of CYP716A47 gene.Methods:Design the histidine-tagged primers to amplified CYP716A47 gene and do a double digestion together with Phsp-HB-NPT,which is an expression vector and contains homologous fragments of P.patens,after connection and transformation,ultimately acquire the Phsp-CYP-NPT,which is an expression vector of CYP716A47 gene.This paper uses both BCD and PPNH4 medium to culture P.patens,and by addition 2 g/L of the ammonium tartrate and glucose to induce the physcomitrella patens’ protonema.Using driselase to prepare P.protoplasts,and by using two types of PEG-mediated transformation methods to transform the protoplasts.Regenerative P.patens will be screened by antibiotic G418(4 mg/mL).After two circles of antibiotic screening,extract P.patens genomic DNA and RNA,which survived after antibiotic selection and then do the identification of PCR and RT-PCR,then do the identification of Western Blot to acquire transgenic P.patens,which has a stable expression of CYP716A47 gene.Results:Successfully added histidine tag at the C-terminus of the CYP716A47 gene,and connected it with expression vectors,finally acquired the expression vector:Phsp-CYP-NPT.P.patens protonema using of two medium growth very well,and the activities of these prepared protoplasts are good too,but in the end,only the protoplasts using the first method,have regenerated to P.patens plants,regeneration rate is 0.037%.After two rounds of the G418 antibiotic selection,only part of them survived,survival rate is 63.8%.After PCR,RT-PCR and Western Blot identification,we acquired transgenic P.patens,which has a stable expression of CYP716A47 gene.