长足大竹象信息素结合蛋白CbuqPBP2互作蛋白的筛选与验证

Screening and verification of proteins interacting with CbuqPBP2 in Cyrtotrachelus buqueti

【目的】筛选长足大竹象(Cyrtotrachelus buqueti)信息素结合蛋白CbuqPBP2的互作蛋白,为实现利用信息物质对长足大竹象种群持续控制提供参考。【方法】利用GST pull-down联合质谱技术,对长足大竹象信息素结合蛋白CbuqPBP2的互作蛋白进行筛选、鉴定和分析,并采用酵母双杂交试验验证了CbuqPBP2与信息素结合蛋白CbuqPBP1的特异性互作。【结果】GST pull-down共筛选出45个与长足大竹象信息素结合蛋白CbuqPBP2特异性结合的候选互作蛋白,包括CbuqPBP1、ND2、CYTB。这些互作蛋白主要参与细胞过程、定位、代谢过程、应激反应以及生物调控等多个生物学过程。使用酵母双杂交体系,构建了pGADT7-PBP1重组猎物质粒与pGBKT7-PBP2重组诱饵质粒,通过诱饵质粒毒性检测和自激活检测,表明重组诱饵质粒对Y2HGold酵母菌无毒性作用。共转化验证结果显示,诱饵质粒pGBKT7-PBP2共转化酵母菌株能够在TDO培养基上生长。【结论】CbuqPBP2和CbuqPBP1之间有相互作用,不同信息素结合蛋白间的互作对深入理解长足大竹象嗅觉感受机制提供了新的思路。

【Objective】The interaction proteins of Cyrtotrachelus buqueti pheromone binding protein CbuqPBP2 were screened to provide references for sustainable control of C.buqueti population using information substances.【Method】The interaction proteins of CbuqPBP2 were screened,identified and analyzed by GST pull-down mass spectrometry,and the specific interaction between CbuqPBP2 and CbuqPBP1 was verified by yeast two-hybrid assay.【Result】A total of 45 candidate interacting proteins specifically bounded to CbuqPBP2 were selected by GST pull-down,including CbuqPBP1,ND2 and CYTB.These interacting proteins were mainly involved in biological processes,such as cellular process,localization,metabolic process,stress response and biological regulation.Using the yeast two-hybrid system,the recombinant hunter plasmid pGADT7-PBP1 and the recombinant bait plasmid pGBKT7-PBP2 were constructed.The toxicity and self-activation detection of the bait plasmid showed that the recombinant bait plasmid had no toxicity to yeast Y2HGold,and the co-transformation verification results showed that the bait plasmid pGBKT7-PBP2 co-transformed yeast strains could grow on TDO medium.【Conclusion】There were interactions between CbuqPBP2 and CbuqPBP1,and the interactions between different pheromone binding proteins provided new ideas for further understanding the olfactory mechanism of C.buqueti.