Study Of Regulation Mechanism For Secondary Metabolites Disturbance In Clematis Terniflora DC.under UVB Irradiation&Cloning And Expression Analysis Of CtPPO In Coumarin Biosynthesis

The low content of traditional Chinese medicine and natural medicine effective ingredient is the key factor that limits the development of traditional Chinese medicine.In recent years,the quality of Chinese herbal medicines has been increasing by improving the quality and the cultivation environment of authentic medicinal herbs.UV-induction significantly increases active ingredients content of medicinal plants and provides a new method to improve the quality of medicinal herbs.The response of plants to UV irradation is mainly reflected in the formation and accumulation of UV-absorbing and photoprotective secondary metabolites.Most of these secondary metabolites are the active ingredients of traditional Chinese medicine,including flavonoids,coumarins,anthraquinones,terpenes,alkaloids and so on.Clematis terniflora DC.is commonly used for anti-tumor and chronic prostatitis treatment in northern Zhejiang Province.The therapeutic effect of Clematis terniflora DC.is good,but the active ingredient content is low which seriously affected the promotion and application of it.Our group conducted an in-depth research on the responses of C.terniflora after UVB irradiation and dark treatment(HUVB + D),and found that the physiological status and secondary metabolism of C.terniflora under stress were significantly changed.Analysis showed that the active ingredient content was increased significantly,and coumarin was a major component of changed secondary metabolites,including 10-demethyl-luvangeti,4,6,7-trimethoxy-5-methyl-2H-chromen-2-one and luvangetin.Our group further proved that polyphenol oxidase(PPO)is the key enzyme in the biosynthesis of esculetin,the basic structure of coumarin,which mainly catalyzes the lactonization of the side chain of caffeic acid to esculetin.Based on previous results,the mechanism of the disturbance of secondary metabolite of Clematis terniflora DC.after HUVB + D was further studied,moreover,the cloning and expression PPO of C.terniflora was also conducted,which is a key enzyme in coumarin biosynthesis pathway.The main contents and results of this topic are as follows:(1)The mechanism of secondary metabolic disturbance in Clematis terniflora DC.under HUVB + D.Combination of metabolomics,qRT-PCR,enzymatic techniques,and biochemical analysis were used to study the mechanism of secondary metabolic disturbance under HUVB + D.In this study,we proved that UVR8(UV resistance locus 8)specific pathway and JA/SA signaling pathway were activated in C.terniflora under HUVB +D,GC-MS based metabolomic analysis demonstrated that the amino acids,carbohydrates,lipids,and organic acids metabolism were increased after HUVB + D.Cluster and KEGG pathway analysis showed that the accumulation of caffeic acid and dihydrocoumarin were higher in JG than that in CG and IG.And the accumulation of proline were higher in IG than that in CG and JG.It was revealed that JA signaling may enhance the transformation from nitrogen to carbon,which increased the reserve of carbon resource for production of secondary metabolites.In all,UVR8 specific pathway and JA/SA signaling pathway were activated in C.terniflora under HUVB + D and co-increase of JA and SA reconstructed the dynamic stability of transformation from nitrogen to carbon,which effectively enhanced the oxidative defense and tolerance to HUVB + D in C.terniflora by the increased secondary metabolites and proline.(2)Cloning and expression of PPO in Clematis terniflora DC(CtPPO).A total of 1975 bp,including an ORF 590 amino acids,was abtained by EST database and RACE technique combined with genome walking technique.CtPPO was predicated as an unstable and hydrophilic protein with the molecular weight of 66.32 kD and had the closest genetic relationship with lotus PPO by physical and chemical properties prediction and phylogenetic tree analysis.The target protein was obtained through recombinant of pET-28a(+)-CtPPO and expression in Escherichia coli by IPTG induction.The result of SDS-PAGE analysis showed that the target protein was optimally expressed at 28?,180 rpm and 0.8 mM IPTG for 6 h,and expressed in both supernatant and pellet,with more fractions in the form of inclusion bodies.Through the optimization of the induction conditions,it was found that the proportion of soluble protein,induced by 0.8 mM IPTG at 16?,180 rpm,was enhanced.The supernatant of expressed recombinant protein was purified by Ni-NTA column,and a higher purity CtPPO protein was obtained.Through the above studies,the signal transduction and regulation mechanism of secondary metabolic perturbation in Clematis terniflora DC.in response to HUVB + D were basically elucidated.CtPPO,a key enzyme catalyzes the synthesis of esculetin,was cloned and prokaryotic expressed.The research enriched the mechanism of plant secondary metabolites responding to stress and provided new ideas for biosynthesis of active ingredients of medicinal plants and promoted the rational utilization of traditional Chinese medicine resources.