Gene Mining And Function Study Of Key Enzymes Involved In Polyphyllins Of Paris Polyphylla Smith Var.Chinensis(Franch.)Hara Based On Modern Omics Techniques

P.polyphylla var chinensis is a perennial medicinal plant in the genus Paris L.of the Liliaceae family.It uses rhizomes for medicinal purposes and has excellent medicinal value.It has great economic value and development potential in the traditional Chinese medicine market.The characteristic product of Aesculus aesculus is steroidal saponins,and its pharmacological effects in anti-tumor and antibacterial aspects have been confirmed in recent years.Although there are a lot of mature researches on the separation and identification of chemical components of Aesculus aesculus,there are relatively few reports on its biosynthetic pathway and key enzyme genes.Based on this,the work carried out on this subject is as follows:1.Using HPLC-Q-TOF-MS/MS to analyze the chemical components in P.polyphylla var chinensis,through database search and comparison with standard products,infer 35 chemical components.Furthermore,the HPLC method was used to determine the content of steroidal saponins in the rhizome of P.polyphylla var chinensis at different growth years,and the accumulation of steroidal saponins in P.polyphylla var chinensis was obtained.The test found that the total saponins content of P.polyphylla var chinensis was the highest in the first-year rhizomes(0.9639%),and its content was about 4 times that of the 4-year-old rhizomes(0.2307%).There were significant differences in different periods.Considering the combined content and growth years,4 key periods of 1a,4a,7a and 10 a were selected for transcriptome and proteome research.2.Using iTRAQ proteomics technology to qualitatively and quantitatively analyze the differential proteins of P.polyphylla var chinensis in 4 key periods(1a,4a,7a,10a),and obtain the changes of differential proteins of P.polyphylla var chinensis in different growth years.law.The total protein number of P.polyphylla var chinensis was identified as 2637,and the number containing at least 2 Unique peptides was 1963.Through further bioinformatics analysis,it is found that there are 426 proteins related to the secondary metabolism of P.polyphylla var chinensis of which 13 are involved in the synthesis of terpenoid backbone proteins,and 5 are steroidal saponins biosynthetic pathways,which are preliminary analyzed from the protein level.Biosynthetic pathway of polyphyllin.3.The second-generation high-throughput sequencing technology was used to sequence the high-throughput transcriptome and gene expression profile of the four key stages of P.polyphylla var chinensis.A total of 20.32 Gb Clean Data was obtained.After assembly,129085 Unigenes were obtained.After database comparison,62420 Unigenes can be matched with public databases such as Nr,Nt,Swissprot,KEGG,KOG,Pfam,GO,etc.,with an annotation rate of 48.36%,and a transcriptome library of P.polyphylla var chinensis was successfully constructed.Through mining the transcriptome data of P.polyphylla var chinensis,systematically analyzed Unigenes that may be involved in the biosynthesis of steroidal saponins of C.odorifera,and obtained 594 candidate gene sequences of 20 types of key enzymes that may be involved in the synthesis of steroidal saponins of polyphyllin.Among them,the number of CYP450 and GT genes accounted for a relatively large number,reaching 483.These candidate sequences covered all the genes in the biosynthetic pathway of polyphyllin,and greatly enriched the genetic resources related to the effective components of P.polyphylla var chinensis.4.Using gene expression profiling and sequencing combined with DESeq software and Benjamini-Hochberg method,we analyzed and obtained 4 differentially expressed genes(DEG Unigene)in the key periods of P.polyphylla var chinensis.In 1a-VS-10 a and7a-VS-10 a,the number of differential genes was the largest,30909 and 32860,respectively,of which 12723 and 14181 were up-regulated genes;in 4a-VS-7a,The number of up-regulated genes reached 15,823.Through "content + transcriptome + expression profile" co-expression pattern analysis to find out 15 differential genes,respectively encoding the 14 enzyme genes in the steroidal saponins synthesis pathway of P.polyphylla var chinensis:DXS,DXR,MCT,IDS,AACT,HMGR,MK,MVD,GPS,FPS,SS,SE,CYP450 and GT.The expression level(FPKM)and content of these candidate genes in the samples of the four periods showed consistent changes.It is speculated that these enzyme genes may play a key role in the biosynthesis of steroidal saponins.5.Design primers for 15 key candidate genes for PCR amplification and full-length cloning,of which 13 candidate genes can amplify bands,the size of which is consistent with the expected amplified fragment length.Through ligation,transformation,and sequencing,7full-length genes of P.polyphylla var chinensis were finally obtained and analyzed by bioinformatics.The real-time fluorescent quantitative PCR technology was used to analyze the spatial and temporal expression of Glycosyltransferase(PpGT),the key enzyme in the synthesis of saponins from polyphyllin,and its expression level was consistent with the trend of gene expression profile data.These full-length genes laid the foundation for the elucidation of the biosynthetic pathway of polyphyllin and provided genetic resources for the next step of molecular regulation and functional verification.