Functional Studies About Polygala Tenuifolia CYP714E38 (C-23) In Saccharomyces Cerevisiae And CYP716A249 (C-28) In Arabidopsis Thaliana

Rationales:Polygala tenuifolia is the dried roots of a plant of the Polygalaceae family which comes from P.tenuifolia Willd or P.sibirica L.It has the effects of calming the nerves and benefiting the mind,communicating the heart and kidneys,eliminating phlegm,and reducing swelling.The triterpenoid saponins,a secondary metabolite from P.tenuifolia,have anti-inflammatory and antiviral effects.However,the traditional methods for obtaining triterpenoid saponins have low levels of medicinal active ingredients,high costs,and few resources,which cannot meet existing needs.The way of synthetic biology can avoid the shortcomings of the traditional way,and can obtain a large number of Triterpenoid saponin with high quality,high efficiency and safety.β-amyrin of the synthetic pathway of triterpenoid saponins in P.tenuifolia is the node,and its previous mechanism of action has been basically clarified.The downstream pathway of this pathway remains to be resolved.In the early stage,the research team screened cytochrome P450 monooxygenase(CYP450/P450s)at the C-23 position that is closely related to the synthesis of presenegenin through high throughput sequencing technology combined with second and third generation transcriptome technology,phylogenetic analysis,gene co-expression analysis,and other methods.It is predicted that CYP714E38 can catalyze oleanolic acid to produce presenegenin.Functional studies on CYP714E38 have been carried out in prokaryotic expression vectors,but the results are not ideal.Eukaryotic expression systems such as Saccharomyces cerevisiae can express introns and transmembrane regions,and have a post-translational protein modification processing system,which is more suitable for the functional research of CYP450 in eukaryotic organisms.Therefore,in this study,CYP714E38 was transformed into the S.cerevisia eukaryotic expression system to continue its vivo or vitro functional research,and several other candidate CYP450 were rescreened through bioinformatics analysis.In the future,other students in the research group will continue to study these CYP450.In addition,the research team previously verified the catalytic activity of CYP716A249 which can catalyze β-amyrin to oleanolic acid through heterologous expression in S.cerevisiae.In order to increase the functional research methods of CYP450.With short growth cycle,rapid development,small size,easy planting,easy cultivation,simple genome,and simple genetic manipulation of Arabidopsis thaliana.we attempt to establish a functional study of CYP450 in Arabidopsis in vivo.The progress of CYP716A249 can catalyze β-amyrin to oleanolic acid was converted to theresearch platform of Arabidopsis,and the function of CYP716A249 was validated again.Objrctive : 1.To screen the candidate CYP450 of P.tenuifolia at the C-23 position,using bioinformatics methods to predict the structure and function of the candidate CYP450,providing a new idea for the subsequent functional research of P.tenuifolia CYP450 and the exploration of the synthesis pathway of Polygala triterpenoid saponins.2.To carry out functional research on CYP714E38 in the heterologous expression system of S.cerevisiae.3.To replicate the Arabidopsis functional research platform of Arabidopsis thaliana.and convert the process of CYP716A249 catalysising β-amyrin to oleanolic acid to the Arabidopsis research platform,we conduct the functional validation of CYP716A249 again,to lay a foundation for the subsequent study of Polygala tenuifolia CYP450.Methods: 1.Based on the previous screening of CYP450 candidate genes at the C-23 position by the research team,amino acid sequence similarity and homology modeling and molecular docking technology were added to further screen the CYP450 at the C-23 position.Existing bioinformatics methods were used to predict and analyze the function of the three CYP450 candidate genes at the C-23 position.2.We construct an ADH1p-CYP714E38-ADH1 t gene expression cassette,transform the CYP714E38 gene into a delta DNA specific site in S.cerevisiae using lithium acetate transformation method,perform functional validation in vivo or vitro of S.cerevisiae.Positive strains were identified using Polymerase Chain Reaction(PCR)technology,and metabolites obtained from in vitro and in vivo reactions were analyzed using Ultra high performance liquid chromatography mass spectrometry(UHPLC-MS).3.We use seamless cloning technology to construct a recombinant expression vector pCAMBIA1300-35S-CYP716A249,carry out genetic transformation of Arabidopsis,and obtain transgenic Arabidopsis Quantitative Real-time PCR(qRT-PCR)and UHPLC-MS techniques were used to verify whether the CYP716A249 gene was successfully transformed into Arabidopsis at the gene and metabolite levels.Results : 1.A new screening method was used to screen out three candidate CYP450 genes at the C-23 position.By predicting and analyzing the sequence characteristics of the three candidate CYP450 at the C-23 position,a new direction was opened for the subsequent screening of CYP450 in P.tenuifolia,as well as providing a new idea for the subsequent functional research of CYP450 in P.tenuifolia and the exploration of triterpenoid saponin synthesis pathways in P.tenuifolia.2.The results of functional validation in vitro and enzyme activity reaction in yeast indicate that CYP714E38 can catalyze oleanolic acid to produce hederagenin,which provids experience for the subsequent analysis of triterpenoid saponin synthesis pathways in eukaryotic heterologous expression systems.3.qRT-PCR results shows that the CYP716A249 gene was successfully transformed into Arabidopsis,and the results of UHPLC-MS shows that the content of oleanolic acid did increase in Arabidopsis after the transformation of the CYP716A249.Both results shows that the CYP716A249 had been successfully transformed into Arabidopsis and played a role which provided a new way for the subsequent research of P.tenuifolia CYP450 in the plant platform.Conciusion:1.CYP714E38 can catalyze oleanolic acid to produce presenegenin in vivo or vitro of S.cerevisiae.2.CYP716A249 was successfully transferred into Arabidopsis,increasing the content of oleanolic acid in Arabidopsis.