Shi Yongxia, Zeng Shaoling, Yuan Meijin, Sun Fan, Pang Yi
State Key Laboratory for Biocontrol, Zhongshan (Sun Yat-sen) University, Guangzhou 510275, China.
Wei Sheng Wu Xue Bao. 2008 May;48(5):672-6.
To analyze the coordination function of cry2A sporulation-dependent promoter and the enhanced expression of molecular chaperone ORF1-ORF2 to crystal protein Cry11Aa.
We constructed three recombinant plasmids pHcy1, pHcy2 and pHcy4 containing cry11Aa gene. pHcy1 carried cry11Aa own promoter and p19 gene, and pHcy2 carried cry2A sporulation-dependent promoter and orf1-orf2 gene. pHcy4 inserted cry2A promoter and orf1-orf2 gene upstream pHcy1 plasmid. The recombinant plasmids were introduced into an acrystalliferous mutant 4Q7 of Bacillus thuringiensis sub sp. israelensis. We performed SDS-PAGE to analyze Cry11Aa protein expression in the recombinant Bt strains and carried out the mosquitocidal bioassay.
SDS-PAGE showed that Cry11Aa protein was detected in 4Q7(pHcy1) and 4Q7(pHcy4), but not in 4Q7(pHcy2). The cry11Aa gene could not be regulated under cry2A promoter. Cry11Aa protein had a 1.25 fold expression amount in the equal volume culture of 4Q7(pHcy4) to that of 4Q7(pHcy1), which indicated that molecular chaperone ORF1-ORF2 could enhance Cry11Aa expression amount to a certain extent. Both 4Q7(pHcy1) and 4Q7(pHcy4) formed Cry11Aa crystals in a similar size and shape during sporulation under the transmission electron microscope. Their LC50s against 3rd-instar Culex quinquefasciatus were 59.33 ng/mL and 66.21 ng/mL respectively.
Whether crystal protein from B. thuringiensis could successfully express might relate to the type of the used promoter and their coordination. Molecular chaperone ORF1-ORF2 could enhance Cry11Aa expression amount to a certain extent with an unknown mechanism, but did not have an effect on high mosquitocidal toxicity of Cry11Aa protein. This research might play an important role to search the best collocation between ICP promoter or chaperone gene and ICP gene and to construct high-toxic Bacillus thuringiensis engineering strain by chaperone gene.
分析cry2A芽孢形成依赖型启动子的协同功能以及分子伴侣ORF1 - ORF2对晶体蛋白Cry11Aa的增强表达作用。
构建了三个含cry11Aa基因的重组质粒pHcy1、pHcy2和pHcy4。pHcy1携带cry11Aa自身启动子和p19基因，pHcy2携带cry2A芽孢形成依赖型启动子和orf1 - orf2基因。pHcy4在pHcy1质粒上游插入cry2A启动子和orf1 - orf2基因。将重组质粒导入苏云金芽孢杆菌以色列亚种无晶体突变体4Q7中。进行SDS - PAGE分析重组Bt菌株中Cry11Aa蛋白的表达，并进行杀蚊生物测定。
SDS - PAGE显示在4Q7(pHcy1)和4Q7(pHcy4)中检测到Cry11Aa蛋白，而在4Q7(pHcy2)中未检测到。cry11Aa基因在cry2A启动子调控下无法表达。在等体积培养条件下，4Q7(pHcy4)中Cry11Aa蛋白的表达量是4Q7(pHcy1)的1.25倍，这表明分子伴侣ORF1 - ORF2能在一定程度上增强Cry11Aa的表达量。在透射电子显微镜下，4Q7(pHcy1)和4Q7(pHcy4)在芽孢形成过程中形成的Cry11Aa晶体大小和形状相似。它们对致倦库蚊三龄幼虫的LC50分别为59.33 ng/mL和66.21 ng/mL。
苏云金芽孢杆菌晶体蛋白能否成功表达可能与所使用启动子的类型及其协同作用有关。分子伴侣ORF1 - ORF2能在一定程度上增强Cry11Aa的表达量，其机制尚不清楚，但对Cry11Aa蛋白的高杀蚊毒性没有影响。本研究对于寻找ICP启动子或伴侣基因与ICP基因之间的最佳搭配以及通过伴侣基因构建高毒力苏云金芽孢杆菌工程菌株可能具有重要作用。
