薤白EPSP合成酶基因cDNA的克隆与原核表达分析

 【目的】揭示棉田杂草薤白（Allium macrostemon Bunge）抗草甘膦的分子机理，开发利用其草甘膦抗性特性。【方法】运用RT-PCR结合RACE技术,分离克隆了薤白中草甘膦作用的靶酶EPSP合成酶（EPSPs）基因cDNA，并将其构建成原核表达载体在大肠杆菌中诱导表达和鉴定其抗性。【结果】薤白EPSP合成酶基因cDNA序列全长1 821 bp，编码一段522个氨基酸的推导蛋白质。经BLAST及蛋白质结构预测，蛋白具有EPSPs的特征序列，并与已报道的EPSPs序列有高同源性，确认克隆的cDNA序列即是薤白EPSPs基因序列，命名为EPSPsA。将该cDNA与原核表达载体pRSET-A重组后，构建成重组表达质粒pRSET-A-EPSPsA，并转化至大肠杆菌BL21（DE3），用IPTG诱导了目标蛋白的高效表达。经SDS-PAGE电泳分析显示，该蛋白的分子量约为55 kD，与预期大小一致。通过草甘膦对表达细菌处理，表达菌对草甘膦的抗性显著提高。【结论】薤白EPSPs对草甘膦具有一定抗性。

Cloning and Prokaryotic Expression of EPSP Synthase Gene CDNA of Allium macrostemon Bunge

【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.