苏云金杆菌辅助蛋白P19对杀虫晶体蛋白Cry11Aa表达的影响

苏云金杆菌以色列亚种的p19基因、cry11Aa基因和p20基因位于同一操纵子上,据推测辅助蛋白P19可能与Cry11Aa蛋白的晶体化相关。本研究利用穿梭载体pHT3101构建了两个重组质粒pHcy1和pHcy3,两质粒均携带cry11Aa基因,但后者完全缺失了cry11Aa基因上游的p19基因。将重组质粒电激转化至苏云金杆菌无晶体突变株4Q7中进行蛋白表达,SDS-PAGE结果表明在4Q7(pHcy1)和4Q7(pHcy3)中均能检测到正常表达的Cry11Aa蛋白,但单位体积培养液的Cry11Aa蛋白在辅助蛋白P19存在时的表达量明显高于其单独表达的表达量;透射电镜观察显示两菌株中的Cry11Aa蛋白形成了大小相近、形状相似的双梯形晶体;另外,生物测定结果表明重组菌株4Q7(pHcy1)和4Q7(pHcy3)对三龄致倦库蚊的杀虫活性没有显著性差异。该现象说明辅助蛋白P19的缺失对Cry11Aa蛋白的晶体形成和杀蚊活性没有影响,但P19作为分子伴侣在一定程度上帮助提高了Cry11Aa蛋白的表达水平。

Influence of accessory protein P19 from Bacillus thuringiensis on insecticidal crystal protein Cry11Aa

p19 gene,cry11Aa gene and p20 gene from Bacillus thuringienesis subsp.israelensis are organized as a single operon.It is reported that P20 polypeptide is not required for high-level expression of Cry11Aa and crystal formation in B.thuringiensis.It is deduced that P19 might relate to Cry11Aa crystallization.In this study,two recombinant plasmids pHcy1 and pHcy3 containing cry11Aa gene were constructed,the latter absent from p19 gene encoding a possible accessory protein between cry11Aa promoter and cry11Aa gene.The recombinant plasmids were introduced into an acrystalliferous mutant 4Q7 of B.thuringiensis subsp.israelensis.SDS-PAGE showed that Cry11Aa protein per unit of culture medium had a higher expression level in 4Q7(pHcy1) with p19 and cry11Aa genes than in 4Q7(pHcy3) with only cry11Aa gene.Both two B.thuringiensis strains formed Cry11Aa crystals in a similar size and shape during sporulation.Toxicity bioassay showed 4Q7(pHcy1) and 4Q7(pHcy3) exhibited a comparable mosquito-larvicidal activity against 3~(rd)-instar Culex quinquefasciatus.It indicated that accessory protein P19 did not have an effect on cry11Aa crystallization and high mosquitocidal toxicity.However,it could enhance Cry11Aa expression amount to a certain extent.