Domain III substitution in Bacillus thuringiensis delta-endotoxin CryIA(b) results in superior toxicity for Spodoptera exigua and altered membrane protein recognition
Domain III substitution in Bacillus thuringiensis delta-endotoxin CryIA(b) results in superior toxicity for Spodoptera exigua and altered membrane protein recognition
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DOI：
10.1128/aem.62.5.1537-1543.1996
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发表时间：
1996-05-01
影响因子：
4.4
通讯作者：
Bosch, D
中科院分区：
文献类型：
--
作者：
deMaagd, RA;Kwa, MSG;Bosch, D
为了验证我们的假设，即苏云金芽孢杆菌三角洲内毒素（Cry）蛋白结构域III的替代可能提高对害虫（如夜蛾）的毒性，我们采用体内重组的方法产生了一些cryIA(b)-cryIC杂交基因。随后利用快速筛选试验筛选编码可溶性原毒素的杂交基因。120个重组基因的筛选得到了两个不同的杂交基因，编码CryIA结构域I和II (b)和CryIC结构域III的可溶性蛋白。这两种杂交原毒素在体外被胰蛋白酶激活后都产生了蛋白酶抗性毒素。生物实验表明，其中一种CryIA(b)-CryIC杂交蛋白（H04）与亲本CryIA(b)蛋白相比具有高毒性，且毒性明显高于CryIC。在与生物素标记的毒素和完整的山梨毛囊膜泡的半定量结合研究中，这种结构域III取代似乎不影响结合位点的特异性。然而，在制备溶解和印迹的刷状边界膜泡蛋白时，CryIA(b)与200-kDa蛋白的结合被III结构域取代完全取消，一个含有CryIC结构域I和II和CryIA结构域III (b)的互易杂合体确实与200-kDa蛋白结合，证实了CryIA(b)的结构域III对该反应至关重要。这些结果表明，CryIC蛋白的结构域III在毒力水平中起着重要作用，结构域III的替代可能是增加害虫可利用活性毒素库的有力工具，并且结构域III参与了与肠上皮膜蛋白的结合。
To test our hypothesis that substitution of domain III of Bacillus thuringiensis delta-endotoxin (Cry) proteins might improve toxicity to pest insects, e.g., Spodoptera exigua, in vivo recombination was used to produce a number of cryIA(b)-cryIC hybrid genes. A rapid screening assay was subsequently exploited to select hybrid genes encoding soluble protoxins. Screening of 120 recombinants yielded two different hybrid genes encoding soluble proteins with domains I and II of CryIA(b) and domain III of CryIC. These proteins differed by only one amino acid residue, Both hybrid protoxins gave a protease-resistant toxin upon in vitro activation by trypsin. Bioassays showed that one of these CryIA(b)-CryIC hybrid proteins (H04) was highly toxic to S. exigua compared with the parental CryIA(b) protein and significantly more toxic than CryIC, In semiquantitative binding studies with biotin-labelled toxins and intact brush border membrane vesicles of S. exigua, this domain III substitution appeared not to affect binding-site specificity. However, binding to a 200-kDa protein by CryIA(b) in preparations of solubilized and blotted brush border membrane vesicle proteins was completely abolished by the domain III substitution, A reciprocal hybrid containing domains I and II of CryIC and domain III of CryIA(b) did bind to the 200-kDa protein, confirming that domain III of CryIA(b) was essential for this reaction. These results show that domain III of CryIC protein plays an important role in the level of toxicity to S. exigua, that substitution of domain III may be a powerful tool to increase the repertoire of available active toxins for pest insects, and that domain III is involved in binding to gut epithelium membrane proteins of S. exigua.