苏云金芽胞杆菌Sigma H对芽胞形成的影响

Effect of Sigma H on sporulation in Bacillus thuringiensis

摘要:

[目的]检测苏云金芽胞杆菌HD73中的转录调控因子Sigma H（σ^H）对spo0A基因转录的调控作用；异源表达纯化Sigma H蛋白，验证其对spo0A基因启动子的直接结合；检测sigH基因的缺失对苏云金芽胞杆菌HD73芽胞形成和晶体蛋白产生的影响。[方法]通过测定spo0A基因启动子指导的β-半乳糖苷酶活性评价spo0A基因在苏云金芽胞杆菌HD73野生型和sigH缺失突变体中的转录水平；通过PCR扩增苏云金芽胞杆菌HD73的sigH基因并插入到表达载体pET21b上，将质粒转入到表达菌株BL21（DE3）中，得到重组菌株BL21（pETsigH）；利用镍柱亲和纯化和阴离子交换纯化得到纯化的Sigma H蛋白；通过凝胶迁移实验（electrophoretic mobility shift assay，EMSA）验证Sigma H蛋白与spo0A基因启动子的直接结合；通过显微镜观察、活芽胞计数的方法对突变株HDΔsigH进行表型特征分析。[结果]sigH缺失后，spo0A基因转录活性降低；在大肠杆菌中正确表达并纯化出大小约为28 kDa的Sigma H-His蛋白；EMSA结果表明纯化后的Sigma H-His蛋白可与spo0A基因启动子结合；镜检和活芽胞计数结果表明突变株HDΔsigH无法产生芽胞和蛋白晶体。[结论]Sigma H蛋白通过与spo0A基因启动子结合直接调控spo0A基因的表达且sigH基因的缺失阻断了苏云金芽胞杆菌中芽胞和晶体蛋白的产生。

关键词:苏云金芽胞杆菌;Sigma H;表达纯化;spo0A基因;芽胞形成

Abstract:

[Objective] To test the regulation of spo0A transcription by the regulatory protein Sigma H (σ^H) from Bacillus thuringiensis (Bt) HD73;Expressing and purifying Sigma H in Escherichia coli to verify its direct binding to the promoter of spo0A;To detect the effect of deletion of sigH on production of spores and crystal proteins in B.thuringiensis HD73.[Methods] The transcription level of spo0A was determined by measuring the β-galactosidase activities directed by the promoter of spo0A in Bt HD73 and sigH deletion mutant.The open reading frame of the sigH was amplified by PCR from Bt HD73 genome,and then ligated into the vector pET21b to generate pETsigH.The pETsigH was transformed into BL21(DE3) to obtain the recombinant strain BL21(pETsigH).The Sigma H protein was extracted and purified by Ni Sepharose 6 Fast Flow column purification and anion exchange purification.The electrophoretic mobility shift assay (EMSA) was carried out to verify the direct binding of Sigma H and the promoter of spo0A.The phenotypic characteristics of the sigH deletion mutant strain (HDΔsigH) were analyzed by microscopic observation and sporulation efficiency determination. [Results] The deletion of sigH decreased the transcription level of spo0A.The 28 kDa-Sigma H-His was expressed and purified from E.coli strain.EMSA results showed that the Sigma H-His could bind to the promoter of spo0A.Microscopic observation and sporulation efficiency determination demonstrated that the HDΔsigH failed to produce spores and crystal proteins.[Conclusion] The Sigma H-His protein directly regulates the expression of spo0A by binding to the promoter of spo0A.Deletion of the sigH blocks the spores and crystal proteins production in Bt strains.

Key words:Bacillus thuringiensis;sigma H protein;expression and purification;spo0A;sporulation