Transcriptome Analysis Of Polygonatum Cyrtonema Hua And Study Of Key Enzyme Genes Involved In Polysaccharide Biosynthesis

Objective: Polygonatum cyrtonema Hua(P.cyrtonema)is one of the most important herbs used in traditional Chinese medicine.This plant contains polysaccharides that exhibit a broad range of pharmacological functions including immuno-enhancement,antiviral,anti-tumor,anti-aging,and immunomodulatory activities.We therefore carried out RNA sequencing on P.cyrtonema leaf,root,and rhizome tissues in order to identify the potential genes that are involved in polysaccharides synthesis.The expression profile of polysaccharides in different P.cyrtonema tissues was analyzed to provide experimental evidence for the molecular biology level of the tubers as medicinal parts in this plant.Methods:The fresh P.cyrtonema samples were picked and the total RNA of tubers,leaves and roots tissues were extracted for establishing the c DNA libraries of these tissues.Then,the c DNA libraries were performed on Illumina Hi Seq 4000 platform.After filtering,the clean reads were obtained and assembled into transcripts using Trinity software,clustering into unigene using the software TGICL.By Blastx software,unigene was mapped to the known databases NT,NR,GO,COG,KEGG,Swiss Prot and Inter Pro,obtaining functional annotations.The polysaccharide relevant pathways,including amino sugar and nucleotide sugar metabolism,starch and sucrose metabolism,glycolysis / gluconeogenesis,interconversion of pentose and glucuronic acid,galactose metabolism,the pentose phosphate pathway,fructose and mannose metabolism,were searched and annotated according to the Ko number of the KEGG pathway.Numerous genes that encodes enzymes participated in the polysaccharide relevant pathways were therefore identified and analysed.The total polysaccharide content of all the three different tissues in P.cyrtonema was detected by UV-spectrophotometry.While a few key enzyme genes related to the biosynthesis of polysaccharides were experimentally validated using quantitative real-time PCR(q RT-PCR).Results : Illumina high-throughput sequencing of the P.cyrtonema transcriptome generated approximately 31.66 Gb clean reads.A total of 164,573 Unigenes were obtained with a mean length of 734 bp,an N50 value of 1,234 bp,and a GC value of44.23%;86,063 of them were annotated in public databases,consisting of 75,427(45.83%)by NR,47,418(28.81%)by NT,47,723(29.00%)by Swiss Prot,58,627(35.62%)by KOG,54,881(33.35%)by KEGG,22,649(13.76%)by GO,and 58,525(35.56%)by Inter Pro.Within KEGG pathway annotation,3,753 Unigenes were mapped to amino sugar and nucleotide sugar metabolism and 1,038 genes were annotated in starch and sucrose metabolism,of which,360 Unigenes encoding sac A,HK,scr K,MPI,PMM,GMPP,GMDS,TSTA3,GPI,pgm,UGP2,GALE,UGE,UGDH,AXS,UXE,RHM,UER1,and UGT enzymes,were mapped to polysaccharides biosynthesis.Multiple genes encoding transcription factors(TFs)invovled in polysaccharides synthesis were identified and analysed.Differential expression analysis showed that the expression level of the Unigenes that encodes sac A,RHM,MPI,GMDS,TSTA3,UER1 and UGE enzymes were higher in rhizomes than leaves or roots.Total polysaccharide content was 4.76% in rhizomes,3.06% in roots,and 1.62% in leaves.The m RNA unigene expression levels encoding HK,scrk,and sac A were also experimentally examined using q RT-PCR,which confirmed the reliability of our transcriptional data.Conclusion:1.We conducted a comprehensive transcriptome analysis of P.cyrtonema plants and identified 86,063 functional genes of P.cyrtonema.The RNA-Seq data sets from the three tissues of P.cyrtonema were deposited in the NCBI Sequence Read Archive(SRA)database under the accession number SRP166090.2.The biosynthesis pathway and key enzymes of P.cyrtonema polysaccharide were systematically analyzed.3.Genes encoding key enzyme that involved in the synthesis of polysaccharides from P.cyrtonema was identified,such as sac A,RHM,MPI,GMDS,TSTA3,UER1 and UGE.The m RNA expression level in rhizomes is higher than roots and leaves,which is consistent with the highest content of polysaccharide in rhizomes,indicating that the expression levels of these enzyme genes may be positively correlated with the yield of polysaccharides.