| Paris polyphylla var.Chinensis,a plant belongs to the genus Paris in the Liliaceae family,its rhizome is one of the official sources of Paris material used in a medicinal extract of the Chinese medicine Chonglou.The rhizome of Paris polyphylla var.Chinensis contains variety of active components,and the most important medicinal ingredient are polyphyllins.Due to a variety of wild resources in the genus Paris,including Paris polyphylla var.Chinensis,are on the verge of exhaustion,and their artificial breeding period are long,there is a large gap between the supply and demand of polyphyllin in the market for present Chinese pharmaceutical industry.It had become a current research hotspot that regulate artificially the synthesis of polyphyllins to alleviate the resource crisis of Paris polyphylla var.Chinensis and meet the demand of the polyphyllins in the pharmaceutical industry by using the synthetic biology methods.However,it is still not fully resolved as for the biosynthetic pathway of polyphyllin.To deeply understand the information of the polyphyllin biosynthesis,the candidate genes related to saponin synthesis were screened and their expression characteristics were studied in this paper through the analysis of pacbio transcriptome sequencing performed on a mixture sample from the multiple tissues.By combineding with phylogenetic tree analysis,two candidate genes that may be related to the synthesis of polyphyllins were further screened out.And on the base of obtaining the full length c DNAs of the two candidate genes,the prokaryotic expression of the candidate genes was performed.The main research are as follow:(1)The rhizomes,stems,leaves,flowers,fruit pods and seeds of Paris polyphylla var.Chinensis were taken as one mixed sample to perform the PacBio sequencing.Fifty-two thousand five hundred and thirty-seven high-quality Isoforms were obtained with an average length of 2607 bp.The obtained sequences were annotated by NR,NT,KEGG,SWISSPROT and GO databases,and 40725 isoforms were obtained which accounted for77.52% of all identified isoforms.A total of 39342 CDS were obtained by CDS prediction on all high-quality ISOFORMS,with an average length of 759 bp.In addition,SSR analysis showed that the number of dinucleotide repeats were highest in above isoforms which reached 9925.(2)According to the KEGG analysis of full-length transcriptome data,12 key candidate genes in the saponin synthesis pathway were selected.And then the RNA samples were extracted from roots,rhizomes,stems,leaves,flowers and seeds of Paris polyphylla var.Chinensis,and the expression levels of 12 candidate genes were analyzed by q RT-PCR.The results showed that among the 12 candidate genes,2 genes were expressed at low in all tissues,3 genes were mainly expressed in rhizomes,2 genes were expressed highly in flowers,3 genes were expressed highly in both stems and leaves,and the other 2 genes were expressed highly in leaves.(3)Furthermore,bioinformatics method was used to analyze the glycotransferase genes in the downstream pathway of polyphyllins,and 45 candidate genes of uridine diphosphate glycotransferase(UGT)were screened out,among which 31 UGTs had PSPG boxes at their C-terminals.The physical and chemical properties of the protein products of the 45 UGT genes were analyzed and the secondary structures were predicted.The results showed that the 45 UGT proteins were mostly acidic proteins with the length of 46～479 amino acids.The phylogenetic tree analysis showed that the 45 UGT genes were mainly distributed in groups A,D,G,L and K,among which 15 genes were distributed in group D related to saponin synthesis,and 1 gene was distributed in subgroup UGT80.Expression analysis showed that 7 genes were up-regulated significantly in rhizomes,6 genes were up-regulated significantly in leaf,10 genes were up-regulated significantly in stem,5 genes were upregulated significantly in seed,4 genes were up-regulated significantly in flower and 13 genes were up-regulated significantly in root.(4)The full lengths of two UGTs(PpUGT01 and PpUGT42),which were more than1000 bp in length and clustered in group D,were spliced by electronic cloning technique.Bioinformatics analysis showed that the full length of PpUGT01 c DNA was 1485 bp and its encoding protein length was 492 aa,the relative molecular weight of the protein was53836.69 Da with the 5.73 isoelectric point,the instability index and lipid index of PpUGT01 were 34.22 and 90.22,respectively.The PpUGT01 was hydrophilic protein,and there was no transmembrane domain and signal peptide in protein.The full length of PpUGT42 c DNA was 1446 bp and the length of the encoding protein was 482 aa.The relative molecular weight of the encoding product was 53241.54 Da which isoelectric point was 5.53,its instability index was 47.38,and the fat index was 90.15.The PpUGT42 was hydrophilic protein and there was no transmembrane domain and signal peptide.(5)The prokaryotic expression vectors of PpUGT01 and PpUGT42 were constructed respectively,and their prokaryotic expression were analyzed in E.coli.The results showed the soluble expression product of PpUGT01 could be obtained when PpUGT01 was induced at 16℃ with 1 m M IPTG for 16 h.And the soluble expression product was detected 4 h later,when PpUGT42 was induced by 1 m M IPTG at 37℃.Taken together,PpUGT01 and PpUGT42 were expressed successfully in E.coli and presented as soluble proteins.(6)Diosgenin and pianogenin were used as substrates,and uridine diphosphate glucose,uridine diphosphate glucuronic acid and uridine diphosphate galactose were used as glycosyl donors,the crude enzyme solution of PpUGT42 was incubated at 30 ℃ for 12 h.The reaction products were detected by HPLC,and the results showed that the crude enzyme solution of PpUGT42 could not catalyze the glycosylation of diosgenin and pianogenin under this condition. |
