Gene Expression Profile Analysis In Polygonatum Odoratum(Mill.)Druce And The Study Of Key Enzyme Genes Associated With Polysaccharide Synthesis

Objective: Polygonatum odoratum(Mill.)Druceis(P.odoratum)is a well-known traditional Chinese herb.Polysaccharides are major bioactive components of P.odoratum,which can be used to treat rheumatic heart disease,cardiovascular disease,diabetes and improve immunity.We have identified potential genes and transcription factors(TFs)that regulate polysaccharide synthesis in P.odoratum using RNA sequencing data from leaf,stem,and rhizome tissues.Methods:Polysaccharide content in P.odoratum samples(rhizomes,stems,and leaves)was determined using a UV-spectrophotometer.The RNA from each sample(rhizomes,stems,and leaves)was extracted,and a c DNA library was constructed.Each c DNA library was sequenced by using the BGISEQ-500 system.Unigenes were obtained by filtering and de novo assembling.To determine the functions of the unigenes,all unigenes were annotated to NCBI databases,including Nt,Nr,KEGG,KOG,Inter Pro,GO,and Swiss Prot.Key enzyme genes responsible for polysaccharide biosynthesis were identified by using KEGG annotations.The expression levels of each unigene were estimated using RSEM.Differentially expressed genes were authenticated using the Possion Dis method.Transcription factors are predicted by Plant TFDB,and transcription factors involved in polysaccharide synthesis are obtained.The expression levels of key enzyme genes involved in polysaccharides synthesis were detected by q RT-PCR.Results:Polysaccharide content was highest in rhizomes.A total of 32.75 Gb of data was obtained from transcriptome sequencing.The Q30 of each sample was greater than89.33%,and the GC content of each sample was approximately 43.57%.After thorough quality control and filtering,transcriptomes were generated,and 112,443 unigenes were assembled and clustered using Trinity software.The average unigene length was 1,240 bp and the N50 was 1,937 bp.Of these unigenes,47.35%(53,237)were longer than1,000 bp,and 66.81%(75,126)were longer than 500 bp.76,714 unigenes were annotated in public databases,with the following annotation distribution: 72,709(NR:64.66%),56,712(NT: 50.44%),55,223(Swissprot: 49.11%),56,755(KEGG: 50.47%),17,752(GO: 15.79%),58,103(KOG: 51.67%),and 53,845(Pfam: 47.89%).18 key enzymes responsible for polysaccharide biosynthesis were identified by KEGG annotations and enrichment analysis,namely,sac A,HK,scr K,MPI,PMM,GMPP,GMDS,TSTA3,GPI,pgm,UGP2,GALE,UGDH,AXS,UXE,UGE,RHM,and UER1).16,655 differentially expressed genes(DEGs)were found by the comparison of rhizome tissue with stem and leaf tissues,including 7,647 up-regulated unigenes and7,797 down-regulated unigenes.2,865 unigenes were annotated in the Plant TFDB and assigned to 58 TF families.73 TFs involved in polysaccharide synthesis were predicted.In addition,the expression level of some key enzyme genes were verified by quantitative real-time PCR.Conclusion: This study substantially enlarged the public transcriptome datasets of this species,and provided insight into detection of novel genes involved in synthesis of polysaccharides and other secondary metabolites.These data will provide a comprehensive genetic resource that will enable improvements in understanding of regulation of polysaccharide biosynthesis and accumulation at the molecular level.