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

双翅目特异性杀虫蛋白的新作用模式分析

基本信息

批准号：
13836008
负责人：
SAKAI Hiroshi
金额：
$ 2.3万
依托单位：
OKAYAMA UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13836008/
关键词：
Bacillus thruingiensis Cry11A specific insecticidal protein Cry4A dipteran insect mosquito

项目摘要

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.

分析了Cry11A原蛋白70kda的加工过程。Cry11A是苏云金芽孢杆菌产生的双翅虫特异性杀虫蛋白之一。70 kda原蛋白经胰蛋白酶消化后，分别产生36 kda片段和32 kda片段。根据片段的n端氨基酸序列，强烈提示这两个片段是由70 kda原蛋白的分子内裂解产生的。通过对cry11A基因进行工程改造，构建了编码融合蛋白GST11A-36k和GST11A-32k的基因，其中GST区分别附着在36-kDa和32-kDa片段的n端。构建了另一个融合蛋白32k-His，其中一个组氨酸六聚体连接在32kda片段的c端。GST11A-36k和GST11lA-32k对蚊幼虫均无杀虫作用。这两个片段被证明结合形成异二聚体。36-kDa和32-kDa片段组成的复合物对蚊子幼虫具有明显的杀虫活性，表明该复合物是Cry11A的活性形式之一。此外，我们还在蚊子幼虫的刷缘膜泡中发现了cry11a特异性结合蛋白。同时也提示靶细胞膜上的cry11a特异性结合位点与cry4a特异性结合位点是分离的。因此，强烈提示Cry11A的杀虫机制不同于苏云金芽孢杆菌双翅虫特异性杀虫蛋白Cry4A的杀虫机制。