Construction Of Expression Plasmid Of Streptococcus Mutans CAT-pacA-CTB-DOCK8/DHR Fusion Gene

Objective: The aim of the study to construct recombinant plasmid with the encodinggene of pacA of S.mutans, catalytic region in glucosyltransferase, cholera toxin B subnnit,and DOCK8/DHR. Construct Stable and efficient expressing tomato fruit plasmid andfusion protein using fruit-specific promoter E8in tomato was expressed with flexiblepeptide, which could provides an important basis for further study of transformation oftomato.Methods:The experiment was divided into two partsPart one: Construction of the recombinant plasmid cat-pacA-ctxB-DOCK8/DHR1.Design specific primers and obtain target genes cat, pacA, ctxB and DOCK8/DHR withPCR amplification.2.Construction of intermediate vectors pMD-cat,pMD-pacA-ctxB and pMD-DOCK8/DHRthrough T-A cloning.3.pMD-cat,pMD-pacA-ctxB and pMD-DOCK8/DHR were digested with restrictionenzymes to construct the recombinant vector pET-cat-pacA-ctxB-DOCK8/DHR.Part two: Construction of the plant expression plasmid p2301-E8-cat-pacA-ctxB-DOCK8/DHR.pET-cat-pacA-ctxB-DOCK8/DHR were removed by restriction enzymes to get the fusiongene cat-pacA-ctxB-DOCK8/DHR, which recombined with the directional cloning andplant transformation carrier (pCAMBIA2301) to obtain the plant expression plasmidp2301-cat-pacA-ctxB-DOCK8/DHR. This process also needed to join the fruit cell-specificpromoter E8extracted from the tomato genome.Results:1.The recombinant plasmid pET-cat-pacA-ctxB-DOCK8/DHR which were successfully introduced through the tests of restriction enzyme digestion the4.9kb size fragme nts and were consistent with the prediction and the sites inserted in the right direction.2.The recombinant plasmid p2301-cat-pacA-ctxB-DOCK8/DHR,which obtained11.6kband4.9kb gene DNA fragment was digested by restriction enzyme KpnI and SalI.Conclusion:A recombinant plasmid called pET-cat-pacA-ctxB-DOCK8/DHR was successful-y constructed；Successfully construct the plant expression plasmid p2301-E8-cat-pacA-ctxB-DOCK8/DHR.