苏云金芽胞杆菌鲇泽亚种HD-133 cry1D的表达调控

Regulation of Expression of the cry1D in Bacillus thuringiensis subsp.aizawai HD-133

利用PCR扩增基因cry1D启动子及上游区片段 ,在测序的基础上构建含cry1D lacZ融合基因穿梭质粒 ,导入不同遗传背景的苏云金芽胞杆菌菌株中 ,并以cry1Ab lacZ融合基因为对照测定 β 半乳糖苷酶活性 ,检测启动子上游区的作用。结果表明 ,cry1D lacZ和cry1Ab lacZ融合基因在不同遗传背景的菌株中表达完全不同 ,也许一些宿主专一性的因子参与了转录调控 ;而在同一菌株中Ccry1D lacZ和cry1Ab lacZ的表达差异是由于上游区的不同以及竞争有限的σ因子所致。利用PCR定点诱变技术突变其SD序列GGGGA为GGAGG后 ,cry1D lacZ融合基因的表达提高了 1 0～ 1 6倍。表明GGAGG是苏云金芽胞杆菌合适的SD序列 。

Influence of the promoter and upstream region on expression of cry1D was tested. Firstly, the PCR products of cry1D promoter and its upstream were obtained followed by the construction of shuttle plastid containing cry1D-lacZ. Then the cry1D-lacZ was introduced into different hosts. Analysis of activity of β-galactosidase showed that the cry1D and cry1Ab expressed differently in different hosts. Comparison of expression of cry1D with cry1Ab in the same host revealed that different upstream and competition for σ-factor were involved in the difference of cry1D and cry1Ab expression. Influence of transnational initiation efficiency on expression of cry1D was studied here. Analysis of SD sequence of cry1D showed that it was obviously different from that of other cry genes published. After site-mutation using PCR was introduced into cry1D SD sequence (GGGGA→GGAGG),cry1D-lacZ expressed higher 1.0～1.6 times than that before mutation, suggesting that GGAGG might be most effective SD sequence for Bacillus thuringiensis, meanwhile, low transnational initiation efficiency caused by improper SD sequence might lead to low expression of cry1D in HD133.