Bt群体信号应答因子nprR基因的缺失对cry1Ac基因表达的影响

Effect of Quorum Sensing response regulator nprR deletion on expression of Cry protein in Bacillus thuringiensis

【目的】研究群体信号应答蛋白编码基因nprR在苏云金芽胞杆菌(Bacillus thuringiensis,Bt)HD-73菌株晶体蛋白形成过程中的作用。【方法】通过同源重组,构建了HD-73nprR基因缺失突变菌株HD73(ΔnprR)。利用启动子-lacZ融合、SDS-PAGE方法,测定不同培养基中nprR基因转录活性及nprR基因缺失对cry1Ac转录及表达的影响。【结果】启动子转录活性分析表明,在LB和SSM培养基中nprR基因从对数期结束(T0)开始表达,稳定期持续表达。在LB培养基中,nprR基因的缺失使cry1Ac基因在生长过渡期和稳定期前期转录活性显著提高,同时HD73(ΔnprR)菌株Cry蛋白生成量也明显高于出发菌株HD-73,但是在芽胞形成释放后,Cry蛋白的表达没有明显的区别。【结论】在丰富培养基中苏云金芽胞杆菌nprR基因的缺失在生长过渡期和稳定期前期能够提高cry1Ac基因转录和表达,从而缩短了cry基因表达时间,并且Cry蛋白总产量与出发菌株相当。

[ Objective] The role of Quroum Sensing response regulator nprR on the expression of Cry protein in B. thuringiensis HD-73 was studied. [ Methods] The nprR gene deletion mutant HD73 （△nprR） was constructed by using of homologous recombination. Beta-galactosidase assay of crylAc＇-lacZ gene fusion and SDS-PAGE in both HD-73 and HD73 （△nprR） strains were performed to analyze the effect of nprR gene deletion on expression of crylAc gene. [ Resultsl Beta- galactosidase assay of nprR＇-lacZ in both LB and Schaeffer＇ s sporulation medium showed nprR gene in B. thuringiensis was initially transcripted at TO （end of Logarithmic growth phase） and keeping expression in stationary phase. Betagalactosidase assay of crylAc＇-lacZ and SDS-PAGE indicated that expression of crylAc gene in HD73 （△nprR） was stronger than that in HD-73 during transition phase and early stationary phase. However, Cry expressed product between HD-73 and HD73 （△nprR） in LB medium has no significant difference when crystal and spore were released. [ Conclusion] The deletion of nprR increased expression and transcription activity of crylAc during transition and early stationary phase in rich media.