| Objective:Paris polyphylla is a traditional Chinese medicine.The Chinese Pharmacopoeia 2020 edition takes the dried rhizome of P.polyphylla var.yunnanensis as one of the sources of P.polyphylla.The wild resources of P.polyphylla var.yunnanensis are scarce due to the sharp increase in the market demand for medicinal materials of P.polyphylla var.yunnanensis and the unreasonable excavation of wild resources by drug farmers.The artificial cultivation of P.polyphylla var.yunnanensis is affected by factors such as provenance and planting years,and the quality is uneven.Because of the high content of saponins,the chemical composition of P.forrestii is similar to that of P.polyphylla var.yunnanensis,and the pharmacological effect is equivalent.It has the characteristics of easy cultivation and high yield in the model cultivation area,and is a potential resource of P.polyphylla.The main active ingredients of P.polyphylla are steroidal saponins.However,there is no report on the difference of steroidal saponins between P.forrestii and P.polyphylla var.yunnanensis and the molecular mechanism of the difference.This study intends to analyze the difference of steroidal saponins in the rhizomes of P.forrestii and P.polyphylla var.yunnanensis by high performance liquid chromatography.At the same time,the key genes in the steroidal saponin synthesis pathway were excavated by high-throughput transcriptome sequencing,and the differentially expressed genes in the steroidal saponin synthesis pathway of P.forrestii and P.polyphylla var.yunnanensis were screened.The expression of differential genes was analyzed by real-time fluorescence quantitative PCR,and the regulatory genes and molecular mechanisms that caused the difference in the composition and content of steroidal saponins between P.forrestii and P.polyphylla var.yunnanensis were preliminarily revealed in combination with saponin content.It lays a foundation for the development and utilization of the resources of P.forrestii,and provides a scientific basis for further study on the biosynthetic pathway of steroidal saponins in P.polyphylla.Method:(1)To establish a high performance liquid chromatography(HPLC)method for the determination of polyphyllin I,II,III,VI,VII,V,H and gracillin in rhizome of P.forrestii and P.polyphylla var.yunnanensis.The contents of rhizomes of P.polyphylla from different provenances and P.forrestii and P.polyphylla var.yunnanensis.from the same provenance were determined,and the quality of P.polyphylla was evaluated.The composition and content of 8 steroidal saponins in the rhizomes of P.forrestii and P.polyphylla var.yunnanensis were discussed by variance analysis and principal component analysis.The contents of rhizomes of P.forrestii and P.polyphylla var.yunnanensis were determined to evaluate the quality of P.forrestii.The composition and content differences of 8 steroidal saponins in rhizomes of P.forrestii and P.polyphylla var.yunnanensis were discussed by variance analysis and principal component analysis.(2)Through transcriptome sequencing,the related enzymes and genes in the steroidal saponin synthesis pathway of P.polyphylla were excavated,and the differentially expressed genes in the steroidal saponin synthesis pathway of P.forrestii and P.polyphylla var.yunnanensis were screened by KEGG MAP combined with saponin metabolism pathway.(3)The commonly used housekeeping genes were screened from the transcriptome data as candidate reference genes.The expression levels of candidate reference genes in rhizomes,stems and leaves of P.forrestii and P.polyphylla var.yunnanensis were analyzed by real-time fluorescence quantitative PCR.The stability of each candidate reference gene was comprehensively analyzed by ge Norm,Normfinder,Best Keeper and Ref Finder website,and the stability of the selected reference genes was verified.(4)Twenty significantly up-regulated differentially expressed genes in the steroidal saponin synthesis pathway of P.forrestii and P.polyphylla var.yunnanensis were selected.The expression levels of 20 differentially expressed genes in P.forrestii and P.polyphylla var.yunnanensis were quantified by real-time fluorescence quantitative PCR,and the correlation with saponin content was analyzed.Result:(1)The established HPLC content determination method can better separate the eight kinds of polyphyllin in the rhizome of P.forrestii and P.polyphylla var.yunnanensis,with small error,good accuracy,repeatability and recovery rate.The contents of polyphyllin in rhizomes of P.forrestii from four provenances were higher.There was no significant difference in the average total content of eight polyphyllin(p>0.05),and the overall quality was stable.There was no significant difference in the average total content of polyphyllin I,II and VII in the samples of P.forrestii and P.polyphylla var.yunnanensis in Tengchong Baoshan(p>0.05),but the average total content of eight kinds of polyphyllin was significantly different(p<0.01).Polyphyllin VI and polyphyllin V were different in the rhizomes of P.forrestii and P.polyphylla var.yunnanensis,and the components affecting the total content of the eight polyphyllin were mainly gracillin,polyphyllin V and polyphyllin III.(2)Through the high-throughput transcriptome sequencing of 8 samples of P.forrestii and P.polyphylla var.yunnanensis.A total of 201571 transcripts and 126111 Unigenes were obtained.A total of 593 Unigenes of 36 enzymes in the steroidal saponin biosynthetic pathway including upstream,midstream and downstream pathways were excavated.Through the KEGG MAP metabolic pathway enrichment analysis,it was found that the Unigene encoding 27 enzymes such as HMGS in the Ko00100 and Ko00900 pathways related to the synthesis of steroidal saponins was differentially expressed in the samples of P.forrestii and P.polyphylla var.yunnanensis,which may be the regulatory genes that cause the differences in the composition and content of steroidal saponins in the rhizomes of P.forrestii and P.polyphylla var.yunnanensis.(3)Through transcriptome data,8 candidate reference genes(GAPDH,ACT,α-TUB,UBC,UBQ,PP2 A,β-TUB,EF-1α)were screened out.Comprehensive analysis showed that ACT had the best comprehensive stability,followed by UBQ;GAPDH had the best stability,followed by PP2 A.Through verification,it was found that ACT and UBQ,GAPDH and PP2 A were suitable reference genes for the study of gene expression in rhizome,stem and leaf of P.forrestii and P.polyphylla var.yunnanensis,respectively,while the reference genes with poor stability could not effectively standardize the expression of target genes.(4)Through q RT-PCR expression quantification of 20 significantly up-regulated differentially expressed genes,the expression of 20 differentially expressed genes in the rhizome of P.forrestii is higher than that in the rhizome of P.polyphylla var.yunnanensis,which is consistent with the transcriptome sequencing results.The expression of HMGS was significantly positively correlated with the total content of eight steroidal saponins(p<0.05),while the expression of SQLE,CAS,CYP90 B,CYP724,UGTX1 and GT14A1 was significantly positively correlated with the total content of eight steroidal saponins(p<0.01).Conclusion:The content of steroidal saponins in rhizome of P.forrestii is high,and the overall quality is stable.he main components that cause the difference of steroidal saponins in the rhizomes of P.forrestii and P.polyphylla var.yunnanensis are gracillin,polyphyllin V,polyphyllin VI and polyphyllin III.The biosynthetic pathway of steroidal saponins is long and complex,involving 593 Unigenes of 36 enzymes.The mechanism of the difference in the content of steroidal saponins in the rhizomes of P.forrestii and P.polyphylla var.yunnanensis may be due to the up-regulation of HMGS,SQLE,CAS,CYP90 B,CYP724,UGTX1 and GT14A1 in the rhizomes of P.forrestii. |
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