关键词: 薤白')">薤白, EPSP合成酶, cDNA克隆, 原核表达

Abstract: 【Objective】 Allium macrostemon Bunge is a weed plant and it is very hard to control in cotton field because of its glyphosate resistance. To reveal its mechanism of glyphosate resistance can lead to a molecular understanding and utilization. 【Method】 The cDNA of EPSPs gene that is responsible to glyphosate resistance were amplified and cloned by RT-PCR and RACE. The glyphosate resistance of the protein is tested by expression of the EPSPs cDNA in E. coli. 【Result】 The cloned full-length cDNA is 1 821 bp with an open reading frame of 1 569 bp. The cDNA encodes a putative 522 amino acids protein. BLAST and protein structure estimation analysis revealed that it is homologous to EPSP synthase of many other plants but with same characterized sites. The cDNA is designed of EPSPsA. The PCR products of the cDNA coding region were recombined into the expression vector pRSET-A and transformed into bacterium E.coli BL21 (DE3) for expression. The target protein with a molecular mass of 55 kD was identified in PAGE after inducing with IPTG. The bacteria show an increased resistance ability when treated with glyphosate. Conclusion】 It is concluded that the EPSPs of Allium macrostemon Bunge has the feature and trait of glyphosate resistance.

Key words: Allium macrostemon Bunge')">Allium macrostemon Bunge, EPSP synthase, cDNA cloning, prokaryotic expression