Gene Cloning And Expression And Single Nucleotide Polymorphisms Analysis Of Caffeoyl-coenzyme A-O-methyltransferase In Camellia Sinensis

It is well known that caffeoyl-coenzyme A O-methyltransferase(CCoAOMT)is a key enzyme in the biosynthesis of lignin and catalyzes the methylation of caffeoyl-CoA to feruloyl-CoA in guaiacyl lignin biosynthesis.As an S-adenosyl-L-methionine(SAM)-dependent O-methyltransferase,CCoAOMT transfers the methyl groups of SAM to the aromatic ring of monolignols.The CCoAOMT gene in Camellia sinensis was also catalyzed the formations of O-methylated EGCGs[(-)-epigallocatechin-3-O-(3-O-methyl)gallate(EGCG3"Me),(-)-epigallocatechin-3-O-(4-O-methyl)gallate(EGCG4"Me)and(-)-3'-O-methyl-epigallocatechin-3-O-gallate(EGCG3'Me)].In this research,CCoAOMT gene were cloned,the inducing conditions for producing recombinant protein were optimized and the enzymatic properties were investigated.The single nucleotide polymorphisms(SNP)diversity of CCoAOMT gene in different tea cultivars was analyzed by PCR amplification combined with Sanger sequencing.The main results are as follows:1.RNA was extracted from Jinmudan tea leaves and cDNA was obtained by reverse transcription.CCoAOMT gene DNA code region was amplified by RT-PCR.The DNA fragment was cloned into expression vector pET-28a and recombinant pET-CCoAOMT.Eexpression of the plasmid in Escherichia coli BL21(DE3)resulted in the production of recombinant protein(approximately 28 KDa).The optimum conditions for producing recombinant protein were as follows.The optimum induced concentration of IPTG was 0.05 mmol/L,the induction temperature was 37 °C and the induction time was 4h.2.EGCG3"Me could be synthesised from EGCG catalyzed by CCoAOMT recombinant protein in vitro.The enzyme exhibited the highest activity at 37 °C and pH 4.0 and the optimal substrate concentration was 0.05mmol/L EGCG.3.In this study,the relative expression of caffeyl coenzyme A-O-methyltransferase in different tea cultivars was analyzed by using GAPDH as the internal reference gene.The results showed that the expression of CCoAOMT in Rougui cultivar leaves was the highest.The expression of CCoAOMT in Rougui,Yingchun,Chiyeqilan,Maoxie cultivar leaf was highly expressed than in other tea cultivar leaves.4.A single nucleotide polymorphism(SNP)diversity of caffeyl coenzyme A-O-methyltransferase(CCoAOMT)gene in 25 tea cultivars was analysised.A total of 16 SNPs was detected and 1 SNP was found on average every 46 bases.The frequency of SNP from the first to the fourth amplification zones is 1.24%,3.61%,0.54%,and 1.53%,respectively.The results show that the frequency of occurrence of SNP in the second amplification zone is the highest.There were 11 transition(68.8%)and 5 transversion(31.2%)of mutation types.The ransversion of mutation types was A(?)G,C(?)T and the transversion of mutation types was A?C,A?T and G(?)C.Among the SNPs,2 were synonymous mutations and 14 were non synonymous mutations which altered the amino acid.