Analysis of a novel mode of action of the dipteran-specific insecticidal protein

对Cry11A 70 kDa原毒素的加工过程进行了分析。Cry11A是苏云金芽孢杆菌产生的一种双翅目昆虫特异性杀虫蛋白。胰酶消化70 kDa的原毒素后，产生36 kDa的片段和32 kDa的片段。根据这两个片段的N端氨基酸序列，推测这两个片段是由70 kDa原毒素分子内裂解而来的。构建了编码融合蛋白GST11A-36k和GST11A-32k的基因，其中GST片段分别连接在36 kDa和32 kDa片段的N端。构建了另一种融合蛋白32K-His，将组氨酸六聚体连接到32 kDa片段的C末端。GST11A-36K和GST111A-32K对蚊虫幼虫均无杀虫作用。这两个片段被证明结合在一起形成了异二聚体。由于由36 kDa和32 kDa片段组成的复合体对蚊子幼虫具有显著的杀虫活性，因此推测该复合体是Cry11A的活性形式之一。此外，我们还在蚊子幼虫的刷状缘膜泡中发现了Cry11A特异性结合蛋白。也有人认为靶细胞膜上的Cry 11A特异性结合部位与Cry4A特异性结合部位是分开的。由此可见，Cry11A的杀虫机理不同于苏云金芽孢杆菌具有代表性的双翅目杀虫蛋白Cry4A。

The course of processing of the 70-kDa protoxin of Cry11A was analysed. Cry11A is one of the dipteran-specific insecticidal protein produced by Bacillus thuringiensis. Upon tryptic digestion of the 70-kDa protoxin, a 36-kDa fragment and a 32-kDa fragment were produced. Based on the N-terminal amino acid sequences of the fragments, it was strongly suggested that the two fragments were produced by intramolecular cleavage of the 70-kDa protoxin. By engineering the cry11A gene, the genes encoding fusion proteins GST11A-36k and GST11A-32k were constructed, in which the GST tract was attached onto the N-termini of the 36-kDa and the 32-kDa fragments, respectively. Another fusion protein 32k-His was constructed, in which a histidine hexamer was attached onto the C-terminus of the 32-kDa fragment. Neither GST11A-36k nor GST11lA-32k was insecticidal toward the mosquito larvae. The two fragments proved to associate to form a heterodimer. Because the complex consisting of the 36-kDa and the 32-kDa fragments exhibited the significant insecticidal activity toward the mosquito larvae, it was suggested that the complex was one of the active forms of Cry11A. Moreover, we have found Cry11A-specific binding proteins in the brush border membrane vesicle from the mosquito larvae. It was also suggested that the Cry 11A-specific binding site on the target cell membrane is separate from the Cry4A-specific one. Thus, it was strongly suggested that the insecticidal mechanism of Cry11A is novel one different from that of Cry4A, a representative dipteran-specific insecticidal protein of B.thuringiensis.10.