Cloning and expression of EPSP synthase gene from Sclerotinia sclerotiorium in Escherichia coli
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摘要: 不含有内含子的核盘菌arom基因已经被扩增、测序.该基因编码五功能的AROM蛋白.为了大量获得核盘菌AROM蛋白的结构域之一5-烯醇丙酮酰莽草酸-3-磷酸合酶(EPSPS),将该菌arom基因编码脱氢奎尼酸合酶(DHQS)和EPSPS两结构域的DNA序列和只编码EPSPS结构域的DNA序列分别克隆入载体pGEX-4t-2和载体pET28b中,构建了4个表达载体pGEX-DE、pGEX-E、pET-DE和pET-E,并将其转入大肠杆菌DH5α、大肠杆菌BL21(DE3)或大肠杆菌JM109中表达.酶活测定和SDS-PAGE分析结果显示,上述编码序列在大肠杆菌细胞内获得了表达,含有表达载体pGEX-E、pET-DE和pET-E的大肠杆菌BL21(DE3)转化子具有EPSPS的催化活性,说明核盘菌arom基因的这些DNA片段可以被单独表达.核盘菌EPSPS异源表达系统的建立为该酶的抑制剂设计奠定了基础.Abstract: 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.
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Keywords:
- gene expression /
- Sclerotinia sclerotiorum /
- arom gene /
- Escherichia coli
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