Cloning and expression of EPSP synthase gene from Sclerotinia sclerotiorium in Escherichia coli

Arom gene without an intron of Scleortinia sclerotiorum had been amplified and sequenced. The gene codes a pentafunctional AROM protein. In order to obtain a great amount of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) of the fungi, one of AROM protein domains, the DNA fragment encoding dehydroquinate synthase (DHQS) and EPSPS of arom gene as well as the fragment encoding EPSPS were cloned into the vector pGEX-4t-2 and pET28b respectively to construct four expression vectors: pGEX-DE, pGEX-E, pET-DE and pET-E. The expression vectors were transformed into Escherichia coli DH5α, E. coli BL21 (DE3) or E. coli JM109. The results of SDS-PAGE analysis and enzyme assay showed that the above sequences were translated in E. coli, and the E. coli BL21 (DE3) cells transformed by pGEX-E, pET-DE or pET-E had EPSPS activity, which indicated that these fragments of arom gene from S. sclerotiorum could be expressed separately. The construction of the heterogeneous expression system of EPSPS from S. sclerotiorum provided a basis for inhibitor design of the enzyme.