Identification of Cry4Ba and Cry11Aa Interacting Proteins in Aedes aegypti Midgut by GST-pull Down and Co-immunoprecipitation Combined with Mass Spectrometry
WANG Jun-Xiang, YANG Xiao-Zhen, HE Huan, ZHANG Ling-Ling, GUAN Xiong*
College of Plant Protection, Key Laboratory of Biopesticide and Chemical Biology, Ministry of Education, Ministerial and Provincial Joint Innovation Centre for Safety Production of Cross-Strait Crops, Fujian Agricultural and Forestry University, Fuzhou 350002, China
Abstract：Cry toxin is the main insecticidal protein produced by Bacillus thuringiensis (Bt) strain, Cry4Ba and Cry11Aa of which are highly toxic to the larvae of Aedes aegypti. Cry toxin infects the insect midgut epithelial cells by binding to the specific membrane receptors. The midgut immune factors can recognize and interact with Cry toxin, and further drive the immune system to resist Bt infection. Therefore, in order to further explore the molecular interaction mechanism of Cry4Ba and Cry11Aa toxins to Aedes aegypti, the GST-Cry4Ba and GST-Cry11Aa fusion proteins were prepared for the GST-pull down assay, and the Cry4Ba and Cry11Aa protoxins were prepared for the Co-immunoprecipitation assay. The 133 Cry4Ba-interacting proteins and 237 Cry11Aa-interacting proteins were identified from the midgut brush border membrane vesicles (BBMVs) by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and database searching. Bioinformatics analysis revealed that at least 16 membrane-binding or transmembrane proteins, including 5 aminopeptidases N, 1 alkaline phosphatase, 1 cadherin-like, 2 α-amylases, 1 class B scavenger receptor, 2 voltage-dependent anion-selective channel proteins and 4 actins. In addition, 7 Cry-binding proteins related to immune or maintenance of cell homeostasis were screened, including 1 peptidoglycan recognition protein (short), 1 apolipophorin, 1 hemomucin, 1 serpin and 3 heat shock proteins. These results provided the necessary reference for functional verification of Bt receptors and the immune response mechanism against Bt in Aedes aegypti.
王焌翔, 杨小祯, 何欢, 张灵玲, 关雄. GST-pull Down和免疫共沉淀联合质谱鉴定埃及伊蚊中肠的Cry4Ba和Cry11Aa互作蛋白[J]. 农业生物技术学报, 2022, 30(9): 1797-1809.
WANG Jun-Xiang, YANG Xiao-Zhen, HE Huan, ZHANG Ling-Ling, GUAN Xiong. Identification of Cry4Ba and Cry11Aa Interacting Proteins in Aedes aegypti Midgut by GST-pull Down and Co-immunoprecipitation Combined with Mass Spectrometry. 农业生物技术学报, 2022, 30(9): 1797-1809.
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