Screening Of CYP450s And UGTs In The Biosynthesis Of Triterpenoid Saponin And The Preliminary Study Of Inhibitory Effect On HDDC Of Polygala Tenuifolia

Rationales:As one of the most important secondary metabolites of Polygala tenuifolia,the oleanane type of triterpenoid saponin has the pharmacological efficacy of tranquilization,eliminating sputum,detumescence,anti-depressants,anti-aging,and anti-senile.Because of the diversity of pharmacological efficacy,triterpenoid saponin of P.tenuifolia is especially suitable for developing as a lead compound of new drugs with anti-aging and neuroprotective effects.However,triterpenoid saponin is difficult for chemical synthesis and extracted from P.tenuifolia directly.Therefore,the illumination of the biosynthesis pathway of triterpenoid saponin will contribute to the further synthesis of a large number of triterpenoid saponin of P.tenuifolia in vivo.Our previous studies have indicated that the upstream biosynthesis pathway of triterpenoid saponin of P.tenuifolia was accomplished through the mevalonate(MVA)pathway,SQS and SQE were found in the midstream pathway.However,only P-AS was found in the the downstream pathway,which synthesized triterpenoid saponin precursor.CYP450s and UGTs are large family of enzymes,so screening CYP450s and UGTs is so important to the the illumination of the biosynthesis pathway of triterpenoid saponin in P.tenuifoliaThe previous studies based on in vivo and cell level have demonstrated that P.tenuifolia have therapeutic effect for Parkinson’ disease(PD).However,these studies lack the relevant research at the protein level,and with the disadvantages of longer molding cycle,more influence factors,and higher cost.Therefore,to establish a screening hDDC specific inhibitor model in vitro will contribute to the in-depth study on anti-PD effects of P.tenuifolia.Objective:1.Clarify the downstream biosynthetic pathway of triterpenoid saponin and to rich the genetic resources of P.tenuifolia,and screening the key enzymes in the downstream biosynthesis pathway of triterpenoid saponin of P.tenuifolia—CYP450s and UGTs.2.To establish a screening hDDC specific inhibitor model in vitro for the study on anti-PD effects of P.tenuifolia.Method:1.Application of digital gene expression(DGE)sequencing technologies on roots and leaves of P.tenuifolia parallel with three times,which the difference contents of triterpenoid saponin,respectively.Because of the difference contents of triterpenoid saponin in roots and leaves,screening the differentially expressed unigenes relevant to biosynthetic pathway of triterpenoid saponin,which are up-regulated in roots of P.tenuifolia.2.Based on the DGE data,using RT-qPCR to detect the expression level of the 22 unigenes(17 CYP450s and 5 UGTs)involved in triterpenoid saponin biosynthesis pathway of P.tenuifolia.Then screening the key CYP450s and UGTs through the correlation analysis.3.To establish a screening hDDC specific inhibitor model in vitro through the hDDC and PEPC and enzyme activity assay.And screening the inhibitory activity on hDDC from the different extracts(95%ethanol extract,water extract)and fractions(petroleum ether extract,ethyl acetate extract,chloroform extract,n-butyl alcohol extract,extraction water extracts)of P.tenuifolia.Results:1.In this study,we obtained the transcription database of on roots and leaves of P.tenuifolia,respectively.A total of 60,437,865 reads in roots and 61,926,272 reads in leaves were found in the transcription database,and 494,113 contigs,298,586 transcripts,and 64,171unigene were assembled,which have perfected the early sequencing data.2.There were 6943(10.88%)differentially expressed uingenes between the transcription database of on roots and leaves of P.tenuifolia with the good reproducibility.The up-regulated unigenes were annotated to the KEGG database,including PMK,FPPS,and β-AS.A total of 22 unigenes were screened relevant to biosynthetic pathway of triterpenoid saponin,including 17 CYP450s and 5 UGTs.3.CYP71A1,CYP94A2,CYP714C2,CYP78A9,CYP734A6(CYP734A1),and UGT73C4 were closely related to the downstream biosynthetic pathway of triterpenoid saponin of P.tenuifolia through the correlation analysis by RT-qPCR DGE combined with RT-qPCR can effectively screen key enzymes and the discover new genes in the biosynthetic pathway of triterpenoid saponin in P.tenuifolia4.Different extracts and extraction parts of P.tenuifolia were screened the inhibitory activity through a screening hDDC specific inhibitor model in vitro.The results showed that chloroform extract part of P.tenuifolia have inhibiting effect on hDDC,which thechloroform extract part of P.tenuifolia may have therapeutic effect for PD.The screening hDDC specific inhibitor model in vitro can be used for subsequent high throughput screening.Conclusion:The results of CYP71A1,CYP94A2,CYP714C2,CYP78A9,CYP734A6(CYP734A1),and UGT73C4 were closely related to the downstream biosynthetic pathway of triterpenoid saponin of P.tenuifolia provide scientific basis for the further illumination of the biosynthesis pathway.In addition,the results of 95%ethanol extract and chloroform extract part of P.tenuifolia may have therapeutic effect for PD,which are not only lay a scientific foundation for the future isolation and structure identification of the effective chemical constituents from the above parts,but also provide a new thought for the study of therapeutic effect and mechanisms of P.tenuifolia for PD.