Molecular Regulation Mechanism Of Taxol Biosynthesis In Taxus Media Cv.Hicksii Cell Line

In vitro culture of plant materials often led to genetic variation,described assomaclonal variation,including morphology,fertility,growth potentiality,resistibility andsecondary metabolism aspects.Genetic variation can result in unstable and uncontrollablesecondary metabolite production in large-scale plant cell culture:firstly,in differentgrowing periods and culture environments,cell morphology,growth rate and ability ofsecondary metabolite production have changed;secondly,in the same passage times andculture environments,different small calli demonstrated as different morphology,color,growth rate,asynchronization of cell mitosis cycle and ability of secondary metaboliteproduction.For these reasons,the high-yield germplasm cell can not be applied insecondary metabolite production by large-scale plant cell culture efficiently.Thus,study onimpacting factors and molecular mechanism of genetic variation during the process of invitro plant cell culture is the foundation of plant cell industrial application.In this study,wetake Taxus media cv.Hicksii as research object and try to discover molecular regulationmechanism of taxol biosynthesis in Taxus media cv.Hicksii cell line at chromatosome,genetranscript level,genetic level and epigenetic level.Detailed study as followed:Genetic variation of Taxus during long-term in vitro taxus cell culture:Gradual loss ofpaclitaxel yield in taxus cell line during 5 years in vitro cell culture was observed,withdecreasing cell browning,increasing growth rate and chromatosome variation.Transcriptlevel of four genes encoding key enzymes in the paclitaxel biosynthesis pathway wereheavily lower and little DNA sequence polymorphism were found by AFLP assayed.While,both HPLC and MSAP methods displayed variation of DNA methylation pattern ofdifferent samples.Effect of environment on genetic stability of Taxus media cv.Hicksii cell duringlong-term in vitro taxus cell culture:compared with environment factors,as culture method,light condition,condition medium,plant auxin play an important role in taxol contentvariation.Auxin content impacted not only taxol production,but also cell phenotype,transcript level of four genes encoding key enzymes in the paclitaxel biosynthesis pathway,DNA methylation level and pattern.Taxus cell line genetic stability during process of low temperature preservation:taxus cell line was cultured in low temperature for 30 d,60 d,180 d and 1 a,and recovered tonormal temperature for 30 d,60 d,180 d,separately.Along with taxol and 10-DAB contentvariation,cell phenotype,transcript level of four genes encoding key enzymes in thepaclitaxel biosynthesis pathway,DNA methylation level and pattern happened variation.Repeated low temperature preservation could maintain taxus cell line genetic stability inlong-term.Correlation analysis between taxol content of Taxus media cv.Hicksii and cellbiomass,key enzyme expression,DNA methylation suggested that DNA methylationshowed a significant correlation with taxol content of Taxus media cv.Hicksii.Taxus cellline treated with 5-Aza-CdR could inhibit CMT activity and lower DNA methylationlevel.De-methylation progress maybe stimulate the expression of serial genes related withtaxol biosynthesis.Therefore,DNA methylation maybe regulate taxol production in Taxuscell line.In addition,molecular regulation mechanism of cold synchronization treatment wasstudied.The expression levels of DXR,HMGR,GGPPS and DBAT decreased rapidly after24 h induced by MJ in cells untreated with cold.On the contrary,expression level of DBATwas improved and expression level of those genes decreased slowly in cells with coldtreatment,and the genes expression maintained higher levels after 60 h induced by MJ.Incells with cold synchronization treatment and MJ induction,the high and steady expressionof taxol biosynthetic pathway key genes maybe one reason for the steady improvement oftaxol content.