摘要
为了研究稻瘟病菌效应蛋白基因功能,本研究构建了稻瘟病菌效应蛋白基因BAS1和BAS4与3×FLAG标签融合的原核表达载体及其与GFP融合的瞬时表达载体。以持有BAS1和BAS4基因的稻瘟病菌株的cDNA为模板,RT-PCR扩增目的基因BAS1和BAS4编码区,选用p3×FLAG-CMV-10为模板扩增3×FLAG基因,分别将BAS1和BAS4与3×FLAG连接到原核表达载体p GEX-4T-1中,获得原核表达载体p GEX-BAS1-3×FLAG和p GEX-BAS4-3×FLAG,测序结果表明克隆到的BAS1、BAS4和3×FLAG序列及连接方向正确。将构建好的原核表达载体转化到表达感受态细胞Transetta(DE3)中得到转化子,利用IPTG诱导表达原核表达产物,SDS-PAGE电泳结果表明,目的蛋白约为40 kD,与理论值相符。为了进一步研究BAS1和BAS4的亚细胞定位,利用同源重组方法构建了pBWA(V)HS-BAS1-GLosgfph和pBWA(V)HS-BAS4-GLosgfp瞬时表达载体。本研究所构建的BAS1和BAS4与3×FLAG融合的原核表达载体及瞬时表达载体将为进一步研究BAS1和BAS4功能及定位提供一定的实验基础。
To investigate the function of effec tor protein genes in Magnaporthe oryzae, we constructed the prokaryotic expression vectors and transient expression vectors with GFP fusion in the combination of BAS1 and BAS4 with 3×FLAG in this study. We used the cDNA of Magnaporthe oryzae strains which held BAS1 and BAS4 genes as templates, and the coding region of target genes BAS1 and BAS4 was amplified by RT-PCR. We selected the plasmid p3×FLAG-CMV-10 as a template to amplify the 3×FLAG gene, and connected the three genes BAS1,BAS4 and 3×FLAG into p GEX-4 T-1, respectively. Then, the prokaryotic expression vectors p GEX-BAS1-3×FLAG and p GEX-BAS4-3 ×FLAG were obtained. We transformed the prokaryotic expression vectors p GEX-BAS1-3 ×FLAG and p GEX-BAS4-3×FLAG into Escherichia coli Transetta(DE3) competent cells to obtain transformant,and used IPTG to induce prokaryotic expression products. The results of SDS-PAGE electrophoresis showed that the size of expressed proteins was approximately 40 kD, corresponding to their theoretical size. To further study the subcellular localization of BAS1 and BAS4, we used homologous recombination to construct transient expression vectors pBWA(V)HS-BAS1-GLosgfph and pBWA(V)HS-BAS1-GLosgfp. The prokaryotic expression vectors and transient expression vectors of BAS1 and BAS4 fused to 3 ×FLAG constructed in this study might provide an experimental basis for further study of the function and subcellular localization of BAS1 and BAS4.
引文
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