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  

Identification of Cry4Ba and Cry11Aa Interacting Proteins in Aedes aegypti Midgut by GST-pull Down and Co-immunoprecipitation Combined with Mass Spectrometry

作　　者：王焌翔 杨小祯 何欢 张灵玲[1] 关雄[1] 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)

机构地区：[1]福建农林大学植物保护学院/生物农药与化学生物学教育部重点实验室/海峡两岸特色作物安全生产省部共建协同创新中心,福州350002

出　　处：《农业生物技术学报》2022年第9期1797-1809,共13页Journal of Agricultural Biotechnology

基　　金：科技部两岸联合资助研发项目(2017YFE0121700);福建省自然科学基金(2020J01550,2020I0031);福建省卫生联合攻关计划(2019-WJ-29);生物农药与化学生物学教育部重点实验室开放课题(Keylab2018-04);福建农林大学科技创新专项基金(KFA20124A)。

摘　　要：晶体蛋白(crystal protein,Cry)毒素是苏云金芽胞杆菌(Bacillus thuringiensis,Bt)分泌的主要杀虫蛋白,其中Cry4Ba和Cry11Aa对埃及伊蚊(Aedes aegypti)幼虫具有特异的毒杀活性。Cry毒素作用于昆虫肠道上皮细胞需要细胞膜受体的介导,而昆虫肠道的免疫应答因子通过识别Cry毒素,进而驱动免疫系统抵御毒素的侵染。为了系统探究Cry4Ba和Cry11Aa毒素与埃及伊蚊的分子互作机制,本研究通过制备GST-Cry4Ba和GST-Cry11Aa融合蛋白,提取Cry4Ba和Cry11Aa晶体蛋白,利用GST-pull down和免疫共沉淀技术挖掘埃及伊蚊中肠刷状缘膜泡囊蛋白中与Cry4Ba和Cry11Aa互作的结合蛋白。通过液相色谱串联质谱鉴定和数据库比对,发现133个与Cry4Ba互作蛋白和237个与Cry11Aa互作蛋白。通过生物信息学分析,从中筛选到16个膜结合或跨膜蛋白,包括5个氨肽酶N、1个碱性磷酸酶、1个钙黏蛋白、2个α-淀粉酶、1个B型清道夫受体、2个电压依赖性阴离子通道以及4个肌动蛋白。此外,筛选到7个与免疫或维持细胞稳态相关蛋白,包括1个肽聚糖识别蛋白、1个载脂蛋白、1个血粘蛋白、1个丝氨酸蛋白酶抑制剂以及3个热激蛋白。本研究为后续埃及伊蚊Bt受体的功能验证以及免疫应答机制的研究提供必要的靶标参考。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 GSTCry4Ba 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 5aminopeptidases N,1 alkaline phosphatase,1 cadherin-like,2α-amylases,1 class B scavenger receptor,2voltage-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.

关 键 词：苏云金芽胞杆菌 埃及伊蚊 GST-pull down 免疫共沉淀 Cry互作蛋白 

分 类 号：S476[农业科学—农业昆虫与害虫防治] Q965.9[农业科学—植物保护]