Molecular Identification Of The Biosynthesis Pathway Of Sesquiterpenes And Functional Analysis Of PatFPPS

Pogostemon cablin,an important Lingnan Chinese herbal medicine and industrial crop,accumulates abundant patchouli oil comprised of more than 24 unique sesquiterpene compounds,the highest content of which is the patchouli alcohol with significant biological activity.Patchouli alcohol is a tricyclic sesquiterpene compound that biosynthesis through the classical mevalonate(MVA)pathway and the methylerythritol 4-phosphate(MEP)pathway.In order to study the enzymes in the biosynthesis pathway of patchouli alcohol,we used the comprehensive analysis method to analyze the sequence structure,homology,conserved domains,subcellular localization,phylogeny,functional characterization and expression patterns of the genes.The expression pattern,subcellular localization,functional analysis,gene homologous expression and heterogenous expression of PatFPPS were researched,in order to provide more possible regulatory targets for metabolic engineering of patchouli alcohol.The main research results include the following points:1.Patchouli alcohol biosynthesis-related genes were analyzed based on transcriptome78 terpenoid biosynthesis-related genes were identified in P.cablin,which were grouped into 15 families,including two single and 13 multigene families.The transcriptional levels of these genes in flowers,stems and leaves were analyzed,and found that these genes were expressed in different tissues,among which the genes of MVA pathway were mainly expressed in flowers,the expression levels of MEP pathway genes in leaves were the highest,and the expression levels of downstream key genes in flowers and leaves were relatively higher.Based on transcriptome sequencing results,the basic sequence structures of these genes were analyzed,and found that all the genes have corresponding structural domains,which is conducive to further study on the key enzymes of terpenoid synthesis pathway.2.Cloning and characterization of genes in the sesquiterpene biosynthesis pathwayBased on transcriptome analysis,genes with relatively high expression levels and complete CDS were screened for cloning,including the MVA pathway genes(PatDXS,PatDXR,PatMCT,PatCMK,PatMDS,PatHDS and PatHDR),the MEP pathway genes(PatAACT,PatHMGS,PatHMGR,PatMVK,PatPMK and PatMVD)and the downstream pathway genes(PatIPPI and PatFPPS).Bioinformatics was used to analyze the sequence structure,homology,conserved domains,subcellular localization and phylogeny of these genes.Color complementation assay was used to verify the functional activity of the MEP pathway proteins.Quantitative reverse-transcription PCR analysis indicated that the MVA pathway genes(PatAACT,PatHMGR,PatMVD and PatFPPS)participate in patchoulol biosynthesis and are mediated by MeJA.3.Molecular identification and subcellular localization of PatFPPSPatFPPS gene was isolated from P.cablin for the first time.The total length of cDNA was 1050bp,with a typical conserved structural domain,and it was highly homologous with FPPS of other plant species.Quantitative reverse-transcription PCR analysis indicated that PatFPPS showed tissue specificity,and induced by ABA,ETH,MeJA,and SA.Subcellular localization of PatFPPS was investigated by transiently expressing N-and C-terminal fusions of FPPS to the GFP in transformed into Arabidopsis protoplasts,results suggested that PatFPPS localize in the cytoplasm and probably not in the peroxisomes,and were similar to the biological information prediction.4.PatFPPS regulates the terpene metabolism and biosynthesis of patchouli alcoholFunctional characterization of the PatFPPS was assessed by complementation of mutant S.cerevisiae strain demonstrated that PatFPPS had biological activity to mediate the biosynthesis of FPS.Transient overexpression vector was constructed to observe the function of PatFPPS in homologous plants.GUS histochemical analysis confirmed that the expression vector was successfully constructed and could be expressed in the leaves of P.cablin.GC-MS revealed the patchouli alcohol content of the leaves overexpressed PatFPPS was up to 2.16 mg/g,which were～0.5-fold higher than that of the control plants,indicating that PatFPPS could catalyzed the flow of metabolism to the direction of biosynthesis of patchouli alcohol and promote the accumulation of patchouli alcohol.To further investigate the role of PatFPPS in the terpene metabolism and influence the terpene products in vivo,26 heterologous transgenic tobacco plants of PatFPPS overexpression was obtained.We used PCR analysis with genomic DNA to confirm the integrations of the PatFPPS gene into putative transgenic lines of tobacco,and qRT-PCR were performed to verify the expression levels of PatFPPS were 5-15 folds higher than that of the control.ELISA analysis revealed that FPP enzymatic activity of the overexpression lines were significantly up-regulated compared with control.The results of the contents of volatile terpenes indicate that stigmasterol,phytol,and neophytadien of overexpression lines was significantly increase which was consistent with the gene expression and FPPS enzymatic activity changes observed in the PatFPPS overexpression plants,these results indicate that PatFPPS enhance enzymatic activity,and activate terpenes biosynthesis.5.Cloning of PatFPPS promoter and screening of interaction proteinsThe 724bp PatFPPS promoter was successfully cloned using FPNI-PCR.The promoter has the core elements necessary for eukaryotic promoter(TATA-box),and has cis-elements(ERE,ABRE,BOX-W1,AT-rich element)by bioinformatics analysis.Several proteins interacted with PatFPPS promoter were identified,which lay a foundation for further study of gene functions and regulatory mechanisms of PatFPPS.