Study On The Function Of Polyphenol Oxidase In Clematis Terniflora DC.& Cloning And Expression Analysis Of Prenyltransferases In Coumarin Biosynthesis

Clematis terniflora DC.is a traditional Chinese medicine which is commonly used for anti.tumor and chronic prostatitis treatment.The Clematisterniflora DC.Has well therapeutie effect,however the content of active ingredient is low and that seriously affected the promotion and application of it.The growth and development of plants are often affected by environment,and the inappropriate environment will put stress on them.Ultraviolet light,as a comnon physical stress factor,has an important impact on the growth and development of plants.In the early researches,we conducted an in-depth study on the response of Clematis ternifora to ultraviolet induction and metabolic disturbance in dark culture(HUV-B+D),and found that double stress had an obvious up-regulation trend on the effective components.On the related study of the influenced secondary metabolic pathways found that coumarins,as a secondary metabolite with abundant pharnacological activities,showed a significant increase in expression during the cultivation process.In the further study,the biosynthetic pathways of coumarins was deduced and the key enzyme polyphenol oxidase was cloning and eukaryotic expression.Based on previous results,we make a further study on the function of the polyphenol oxidase in Clematia terniflora,moreover,the discovery,identification,cloning and expression of prenyltransferases in the coumarin biosynthesis pathway was also conducted.The main contents and results of this topic are as follows:(1)Study on the function of polyphenol oxidase in Clematis terniflora.The combination of qRT-PCR and enzymatic techniques can prove that the successful establishment of PPO silence modle by Virus Induced Gene Silencing(VIGS)in Clematis temoflora.Proteomics analysis showed that,compared with plants with VIGS-vector(VV),proteins in PPO silent plants(VC)showed significant changes,among which proteins related to secondary metabolism,protein metabolism,REDOX,photosynthesis and glyeolysis pathway were up-regulated.Differential proteomics analysis combined with HUV-B-+D further showed that proteins related to photosynthesis,glycolysis,stress,REDOX and protein metabolism were still higher in VC plants than in VV plants.Mapman analysis showed that the differentially expressed proteins were mainly involved in light reaction and ealvin cycle in photosynthesis.Further analysis illustrated that the expression level of ATP synthase,the content of chlorophyll,and the photosynthesis rate were increased in VC plants compared to VV plants pre-and post HUV-B+D.These results indicate that the silence of PPO elevated the plant photosynthesis by activating glycolysis process,regulating Calvin cycle and providing ATP for energy metabolism.This study provides a prospective approach for increasing crop yield in agricultural production.(2)Cloning and expression analysis of prenyltransferases in coumarin biosynthesis.In combination with TBLASTN database search,4 coumarin prenyltransferase sequences in Clematis ternflora with high scores were obtained,and three of them were obtained by PCR with complete open reading frames.Compared with PcPT for searching database,it was found that the similarity of the three sequences was 35%,41%and 42%,respectively,and both of them contain the characteristic sequences common to prenyltransferases:NQxDxxD and KD(I/L)×D×(E/D)GD(× represents any amino acid),then,phylogenetic analysis showed that two of them were located in the prenyltransferase branch of coumarin and one was located in the homogentisic branch.The results of qRT-PCR showed that all three sequences were up-regulated after the culture of HUV-B+D,but only the change trend of CtPT1 in the culture process was consistent with that of CtPPO and coumarin.The most likely coumarin prenyltransferase gene CtPT1,with 400 amino acids,molecular weight of 44.1kDa and isoelectric point of 9.32,was screened out from the binding prediction results.It was an unstable hydrophobic protein with 8 transmembrane regions and was a membrane binding protein.PESC-CtPT1-GFP recombinant vector was constructed and the target protein was induced and expressed in yeast.For the high protein concentration of pESC-vector,the SDS-PAGE analysis did not observe obvious bands,but the westen bloting results proved that CtPT1 was successfully expressed in yeast.By constructing pMDC83-OtPT1 recombinant vector,agrobacterium-mediated infection of tobacco leaves was completed.And confocal observation showed that COPT1-GFP was located on chloroplasts.These results showed that CtPT1 is a chloroplast membrane binding protein.At last,the tissue specificity analysis showed that it was distributed in root,stem,leaf and flower of Clematis terniflora,but the content in root and leaf was relatively high,and the content in leaf was twice as high as that in root.