Cloning And Analysis Of 4CL Genes In Tea Plant [Camellia Sinensis (L.)]

Phenylpropanoid metabolic pathway is an important secondary metabolic pathway in plant,which involved in the biosynthesis of lignin,flavonoids and anthocyanins.P-coumaric acid coenzyme A ligase(4CL)is the last enzyme of phenylpropanoid pathway which located in the branch of the phenylpropanoid pathway,lignin production and flavonoids pathway.It plays an important role in the secondary metabolites pathway in plant.In this paper,we cloned two 4CL genes in tea plant(Cs4CL);through prokaryotic expression technique,HPLC technology,UPLC-MS technology and spectrophotometric assays,we identified the function of HfusionH HproteinH of these two 4CL.Cs4 CL eukaryotic expression vectors were also constructed and transformed into tobacco and Arabidopsis.The results are as following:1.Frist,we screened two EST sequences of Cs4 CL gene from NCBI and Anhui Agriculture University tea transcriptome database.The full-length cDNA of two Cs4 CL genes were obtained by RACE technology.Cs4CL1 and Cs4CL2 showed 62.63% identity in amino acid by using software DANMAN 8.0.They both have the Box I and Box II motifs which are highly conserved in 4CL proteins in plant.Phylogenetic analysis showed that the two Cs4 CLs belong to two different subgroups.2.Through prokaryotic expression system,Cs4CL1 and Cs4CL2 were cloned into the petSUMO vector respectively,and then transformed into Escherichia coli.BL21.After induced,Cs4CL1 and Cs4CL2 fusion protein were separated and purified by using Cobalt ion affinity resin.Using HPLC and UPLC-MS detection technology,we detected the enzyme activity and verified the reaction products of fusion protein,then identified the catalytic of these two fusion proteins.3.Using spectrophotometric assay,we studied the optimum reaction temperature,pH and stable characteristics of these two enzymes,as well as the substrate specificity of the Cs4CL1 and Cs4CL2 recombinant protein were detected.The optimal reaction pH of Cs4CL1 was 6.5,and the optimal reaction temperature of Cs4CL1 was 50 ℃;the optimum stable temperature of Cs4CL1 was 0 ℃,when the optimum stable pH of Cs4CL1 was 7.0;the optimum substrate was coffee acid.The optimal reaction pH of Cs4CL2 was 7.5,and the optimal reaction temperature of Cs4CL2 was 50 ℃;The optimum stable temperature of Cs4CL2 was-20℃,when the optimum stable pH of Cs4CL1 was 6.0;the optimum substrate was p-coumric cid.4.Over expression plasmids of pCB2004-Cs4CL1 and pCB2004-Cs4CL2 were constructed,and transformation of tobacco was performed.We also constructed the eukaryotic expression plasmids pGWB5-Cs4CL1 and p GWB5-Cs4CL2,genetic transformation of HarabidopsisH was performed.The results of PCR showed that the target genes Cs4CL1 and Cs4CL2 had been transformed into tobacco and Arabidopsis thaliana successfully...