Cloning And Functional Confirmation Of CsECGT Gene In Camellia Sinensis

Galloylated catechins are the products of non-galloylated catechins with three hydroxy of its C ring gallic acylation.Existing data showed:the acylation of plants metobolic substances needed activity of acyl donor which are abundance in plants,like CoA thioesters, 5-O-caffeoylquinate,1-O-galloy1-β-D-glucose.Galloylated catechins were produced by epicatechin:1-O-galloy1-β-D-glucose-O-galloyltransferase(ECGT) catalysis from β-glucogallin and non-galloylated catechins.But,it was not easy to find clear evidence which could show the reaction above in tea plants(Camellia sinensis (L.) O. Ktunze).There was hydrolytic enzyme in tea plants(Camellia sinensis (L.) O. Ktzeis),which hydrolyze galloylated catechins into non-galloylated catechins,thus,disturbing the synthesis of galloylated catechins through ECGT’s biosynthesis.This paper researched series experiments like CsECGT’s prokyriotic expression CsECGT’s sequence analysis,over expression vector construction,ECGT’s expression and extraction and functional verification of ECGT.The main results of this paper were listed as follow:1.Having built two CsECGT prokaryotic expression systems, such as PET28-ECGT/ PET32-ECGT recombinant Escherichia coli. In a series of optimization expression condition exploration,like conventional SDS-PAGE protein assay, enzyme purification means, biotransformation, HPLC, etc.,there was no outstanding experimental results.2.Analyzing the sequence of CsECGT gene:its total length was 1726bp,including a complete coding region 1443bp,5’non translation region(UTR) 72bp,3’non translation region(UTR) 180bp and polyA 31bp.The GC content was 51.83% in its coding region which coded a protein constituted by 480 amino acids(ECGT).Comparative analyzing by NCBI blast tool,finding that the protein coded by CsECGT had high identity with Serine acyltransferase of other species,especially Serine acyltransferase of Zea mays,Solanum pennellii,Clitoria ternatea with 40% identity alike.3.Successfully constructed over-expression vector PCXSN-ECGT of ECGT/empty vector PCDG-CCDB,and transformed those vectors into Agrobacterium tumefaciens GV3101.4.Mediated by Agrobacterium tumefaciens GV3101,successfully transformed over-expression vector PCXSN-ECGT, empty vector PCDG-CCDB into the fresh leaves of tobacco(Nicotiana alata).Through induced differentiation, rooting of these leaves,positive tobacco plants were obtained.5.According to the sequence features of expression vector PCXSN,designed primers HybF/HybR.And PCR results with these primers successfully turned out the feasibility of induced systems(resistance screening).Taking it into account the expression direction tendency of over-expression vector PCXSN-ECGT and the sequence characteristics of the ECGT gene,designed CsECGTOF/CXSN-Seq F primers to further verify the positive tabacco plants.6.Extracting the enzymes of tabacco’s fresh leaves,then verified by SDS-PAGE electrophoresis, the results showed, CsECGT gene with fragment size 55KD was successfully expressed in tobacco(Nicotiana alata).7.Using methanol and formic acid directly extracted the fresh leaves of tobacco(Nicotiana alata), the HPLC analyzing showed:no catechins were detected under the extract of fresh leaves quantity. Extracted enzymes of tobacco fresh leaves with 50mM Phosphate buffer, establishing enzyme reaction system in vitro.And preliminary got a conclude that enzymes encoded by CsECGT genes had substance specificity among EC/ EGC.