Excavation And Expression Analysis Of Genes Related To The CYP And UGT Enzymes Of Triterpenoid Saponin Biosynthesis In Gynostemma Pentaphyllum

Objective: At present,the transcriptome sequencing of Gynostemma pentaphyllum has been analyzed,while the proteome sequencing has not been carried out to analyze the protein expression level of Gynostemma pentaphyllum,and there are many studies on the genes of skeletinase in the biosynthesis of gypenosides,such as squalene synthase and squalene epoxidase.However,there is a lack of understanding of the post modifying enzyme genes: cytochrome P450 and UDP-dependent glycosyltransferase genes or overall analysis of their differential expression in different tissues.In this study,we analyzed the dynamic changes of protein composition and activity in Gynostemma pentaphyllum from the overall point of view.The study aim focuses on the underlining for the target gene expression regulation mechanism of genes related to biosynthesis of triterpernoid saponins in Gynostemma pentaphyllum through the excavation of postmodifying enzyme genes and their tissue differenctial expression analysis.Methods: Illumina Nextseq 500 and i TRAQ / TMT technology were used to obtain the transcriptome database and the results of protein quantitative sequencing of Gynostemma pentaphyllum in the study.Through conjoint analysis of the above two datas,some key enzyme genes that may be involved in the biosynthesis of gypenosides were excavated,and their differential expression in the roots,stems and leaves of Gynostemma pentaphyllum was analyzed.Meanwhile,CYP or UGT genes with significant differences in expression were screened and their c DNAs were cloned by RT-PCR.The functions of the coding proteins were analyzed and compared by bioinformatics analysis.Finally,RTq PCR was used to verify the tissue differential expression of the post-modification enzyme genes at the transcription level to elucidate the mechanism of triterpenoid saponin biosynthesis.Results: A total of 3925 proteins were identified by proteomic sequencing,of which 2537 were quantified in this study.Combined with the results of pre transcriptome sequencing,a total of 21 triterpene saponin skeleton and side chain post-modification enzyme genes were excavated.The correlationt of significant difference genes in proteomic sequencing and transcriptome sequencing is different in roots,stems,and leaves of Gynostemma pentaphyllum.Among them,GPJ vs GPG has good academic consistency in the two sequencing analyses,but GPY vs GPG and GPY vs GPJ show poor correlation.There are 641 putative cytochrome P450(CYP450)and 178 putative UDP –glycosyltransferase(UGT)in the transcriptome database served as candidate genes of triterpenoid saponin post-modification enzymes,meanwhile at least,18 CYPs and 11 UGTs with significant different expression were uncovered respectively,while only 7 CYPs and 7 UGTs were found through proteomic quantitative sequencing analysis.The8 c DNAs of CYP and 5 c DNAs of UGT with complete ORF regions and partial unigenes of 2 CYP and 2 UGT were cloned successfully.By bioinformatics analyses related to the cloned CYPs or UGTs,the conserved domains of CYP or UGT have been found in all CYP or UGT proteins with full-length ORF regions,respectively.Through phylogenetic tree analysis,we found that they are closely related to the corresponding genes of Cucurbitaceae plants.Finally,the results showed that most of post-modification enzymes were significantly higher in expression in Gynostemma pentaphyllum leaves and stems compared with roots,and some CYP and UGT such as UGT91A1,UGT76B1 were expressed differently through RT-q PCR,but the results all showed obvious tissue specificity.Conclusion: Conjointing transcriptome data with proteomic sequencing data for the integrated analysis is a good way to quickly excavate the post-modification enzyme genes involved in the biosynthesis of Gynostemma pentaphyllum triterpene saponin.Eight CYP c DNAs and five UGT c DNAs of the Gynostemma pentaphyllum with complete ORF were successfully cloned.Bioinformatics predictions were carried out on the physicochemical properties,structural characteristics,functions,system evolution relationship and active site analysis of the post-modification enzymes in the Gynostemma pentaphyllum triterpene saponin synthesis pathway.Transcription level analysis showed that the gene expression of the post-modification enzymes in the Gynostemma pentaphyllum triterpenoid saponin synthesis pathway exhibits tissue characteristics,which can provide important information for the targeted expression regulation of triterpenoid saponin biosynthesis in Gynostemma pentaphyllum in the future.