Analysis Of Polysaccharide Biosynthesis Of Codonopsis Pilosula Under Drought Stress Based On Transcriptome Sequencing

Codonopsis pilosula(Franch.)is a perennial herb in the Campanulaceae.It has a long history as one of the ten Long Herbal.Polysaccharide is main medicinal ingredient and plays an important role in regulating immunity,scavenging free radicals and anti-aging.Codonopsis pilosula is mainly produced in the northern arid and semi-arid mountainous areas of Weiyuan and Longxi of Gansu Province,With global warming,drought occurs frequently,which affects the growth and quality formation of Codonopsis pilosula,but the influencing mechanism is not clear yet.Based on this background,in this study,the outdoor pot water control experiment was conducted to carry out water management according to 90%-85%(Normal Water: CK),70%-65%(Light Drought),55%-50%(Moderate Drought)and40%-35%(Severe Drought)of the maximum water holding capacity in the field until the vigorous growth period of Codonopsis pilosula.Sampling for polysaccharide content determination and High-throughput sequencing,screening Codonopsis pilosula polysaccharide synthesis related genes,and gene analysis under the different levels of drought differentially expressed in roots,stems,leaves,combined with the difference of polysaccharide content to illustrate the codonopsis pilosula polysaccharide synthesis mechanism under drought stress,provide reference basis for from gene level to cultivate high quality Codonopsis pilosula.Preliminary results were obtained as follows:1、Through the analysis of polysaccharide contents in the roots,stems and leaves of Codonopsis pilosula under drought treatments showed that:(1)There were significant differences in polysaccharide content in roots,stems and leaves,with a general trend of roots >stems ≈ leaves,and the polysaccharide content in roots was about 10 times that in leaves and stems,respectively.(2)Polysaccharide content in roots and leaves of Codonopsis pilosula under light drought stress was significantly and extremely significantly higher than that of other treatments,with the order of Light Drought > CK > Moderate Drought > Severe Drought;There was no significant difference in the content of polysaccharide in stem among the four treatments.The results showed that light drought was beneficial to the accumulation of Codonopsis pilosula polysaccharide.2、Bioinformatics analysis of transcriptome sequencing results found that:(1)Compared with CK,Unigene was differentially expressed of various tissues in Codonopsis pilosula under light,moderate and severe drought stress,and there were 19 303,15 142 and 7 943 differentially expressed genes in leaves,respectively,the differences of Unigene in stem were7 550,6 069 and 6 532,respectively,there were 7 297,10 169 and 9 364 Unigene differences in roots,respectively.(2)GO functional classification of differentially expressed Unigene showed that the cytological components were mainly concentrated in Cell,Cell part,Organelle and Organelle part;Biological processes mainly focus on Cellular process and Metabolic process;Molecular functions mainly focus on Binding and Catalytic activity.(3)KEGG enrichment on differentially expressed Unigene found that Carbohydrate metabolism,Energy metabolism,Amino acid metabolism,Lipid metabolism,Carbohydrate metabolism and Other amino acids metabolism were involved.3、By further analysis of differentially expressed Unigene:(1)20 enzyme genes related to the polysaccharide synthesis pathway of Codonopsis pilosula were screened,including UGP2,SPS,SS,sac A,HK,scr K,Gal E,GAE,UGDH,AXS,UXE,RHM,PGM,GPI,man A,PMM,GMPP,UXS1,UER1,GAPDH,They are derived from Starch and Sucrose metabolism(Ko00500),Amino sugar and Nucleotide sugar metabolism(Ko00520),and Glycolysis/Gluconeogenesis(Ko00010).(2)Compared with CK,polysaccharide synthase genes were differentially expressed in different tissues of Codonopsis pilosula under light,moderate and severe drought stress,among them,there were 15,10 and 5 in leaves,4,1 and 4in stems,2,7 and 6 in roots.(3)Analysis of key genes with high expression showed that UGDH,GAE and RHM genes were upregulated and overexpressed in leaves under light drought compared with CK,indicating that UGDH positively regulates UDP-glucose decomposition into UDP-glucuronic and GAE catalyzed its conversion to UDP-glucuronate ester in leaves under light drought and UDP-galactose and UDP-L arabinose were further synthesized.RHM positively regulates UDP-glucose metabolism to UDP-4-dehydro-6-deoxy-D-glucose for further UDP-rhamnose synthesis;UGDH,GAE and RHM were down-regulated under moderate and severe drought.By reason of the foregoing,Light drought stress treatment is beneficial to the synthesis of carbohydrate through photosynthesis in Codonopsis pilosula leaves.And metabolized into UDP-glucose and fructose under the catalysis of sucrose synthase and sucrose phosphate synthase.They further metabolized to UDP-L-arabinose,UDP-galactose,UDP-xylose,UDP-rhamnose,GDP-mannose and other monosaccharides under the enzymatic catalysis in the polysaccharide synthesis pathway.These substances are catalyzed by glycosyltransferase,very little of them are converted into polysaccharide storage in leaves and stems,most of them is transported through the stem to the root for conversion into polysaccharide accumulation and as the main pharmacodynamic components of Codonopsis pilosula to exert immunological competence.