Study On The Enzyme Gene Related To The Synthesis Pathway Of Saponins Of Polygonatum Sinensis

Polygonatum sibiricum Red.(Liliaceae)is a traditional medicinal plant in China,whichhas the characteristics of medical and edible.And it's aslo has high economic value and development potential in medicine,health care and food.Steroidal saponins is a main product in P.sibiricum,which had been proved to have good pharmacological effects of anti-tumor and anti-bacterial and so on.At present,the research on steroidal saponins is mainly focused on the extraction and identification of chemical constituents.The steroidal saponins biosynthetic pathway and key enzyme genes are still not clear.Based on these problems,we performed our work as followings.1.14 P.sibiricum samples were collected and the different habitats were determined.The results showed that there were significant differences in the contents of steroidal saponins of P.sibiricum between Henan,luoyang(3.14 mg/g)and Shaanxi,Lueyang(9.58 mg/g).We used the two samples to construction the transcriptome libarary by RNA-Seq..We received 72.06 Gb Clean Data after data quality control and trimming with Q30 at 93.31%.A total of 74,730 Unigene were assembled and blasted with the database NR,Swiss-Prot,GO,COG,KOG,KEGG,Pfam.Among them,35,793 Unigene were annotated to at least one database,accounting to 48.28%of the total Unigene.The transcriptome libraries of P.sibiricum we constructed were successful.2.In transcriptome database,we found 146 Unigene in total,which encodes 32 enzymes related to biosynthesis of steroidal saponins,covering almost all the nodes in steroidal pathway.It enriched enzyme genes information of steroidal saponins biosynthesis pathway in P.sibiricum.3.Using the DESeq software and Beniamini-Hochberg methods to analyze gene differentially expression quantity between two samples,we obtained 7,016 DEG Unigenes,including 4,396 up regulated and 2,657 down regulated genes.Through the DEG gene function annotation and enrichment analysis in KEGG pathway,we fonud 10 differential genes related to steroidal saponins biosynthetic pathway,These genes was identified as HMGS,HMGR,DXS,HDS,FPPS,SQLE,CAS,SMT1,SC5DL and DHCR24.The FPKM values of these differential genes were significantly increased in samples of Shannxi,Lueyang,supposing that these enzyme genes play a key role in steroidal pathway.4.A total of eight housekeeping genes(18S,ACT,TUA,TUB,CYP,GAPDH,EF-1? and UBQ)were screened by qRT-PCR.According to GeNorm,NormFinder and BestKeeper software,the results showed that TUB gene was the most stable in different tissues of P.sibiricum,which was the best reference gene.5.The expression parterrn of enzyme genes was analyzed by qRT-PCR.The results showed that the expression of eight genes(HMGS,HMGR,HDS,FPPS,SQLE,CAS,SMTI and DHCR24)was consistent with the trend of1 DGE gene.And we found that the changes of expression of these genes were similar to the accumulation of steroidal saponins.Terefore,these genes may play an important regulation role in steroidal pathway.6.One gene related to biosynthesis of steroidal saponins,PsCAS,wascloned and the protein structure,physicochemical properties,and phylogenetic tree were analyzed.The results showed that the open reading frame(ORF)of PsCAS was 2,268 bp,encoding 761 amino acid residues in the protein,with conservative structure of OSCs protein family.The r calculated molecular weight of PsCAS was 86.5 kDa and the isoelectric point was 6.14.The PsCAS may have the function of catalyzing 2,3-oxysalane and has a close relationship with the CAS protein of Dioscorea zingiberensis.The cloning of CAS gene provides a stable foundation for its effect on saponins amount of P.sibiricum s and expression regulation.