Cloning and expression of Fusarium moniliforme CGMCC 0536 D-lactonohydrolase gene in Escherichia coli / 生物工程学报

ABSTRACT

The total cDNA obtained through reverse transcription of F. oxysporum CGMCC 0536 mRNA used as template, a fragment about 1.5kb was amplied with oligo(dT)15 primer and a gene specific primer designed on the base of the sequence of both NH2-terminus and the cDNA sequence encoding D-lactonohydrolase of Fusarium oxysporum reported on the NCBI, then the fragment was cloned to the pMD18-T vector and sequenced. The sequence encoding D-lactonohydrolase of F. moniliforme CGMCC 0536 shows a high homology of 90.06% with that of F. oxysporum indicating that the gene encoding D-lactonohydrolase is highly conservative. Two specific primers were designed according to the sequence result, and a fragment, 1146bp, was amplied using hot start PCR with these two specific primers. Subsequently, the resulting products were digested with EcoR I and Sal I and ligated to the pTrc99a vector digested with the same enzymes using T4 DNA ligase. the recombinant plasmid, pTrc99a-LAC, was transformed into Escherichia coli JM109. The two positive clones were induced with IPTG, and enzymes expressed in Escherichia coli JM109, the enzyme activity was about 37U and 41U respectively. The expression products were analyzed by SDS-polyacrylamide gel electrophoresis indicating that about 40kD protein was obtained.