棉铃虫中肠氨肽酶APN4与Cry1Ac、Cry2Aa结合能力的比较
Comparison of the binding affinity of the APN4 receptor in Helicoverpa armigera (Hübner) to the Cry1Ac and Cry2Aa insecticidal proteins
袁向东1** 葛朝虹2** 肖玉涛1 梁革梅1***
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DOI：10.7679/j.issn.2095-1353.2015.147
作者单位：1. 中国农业科学院植物保护研究所, 植物病虫害生物学国家重点实验室, 北京 100193；2. 西北农林科技大学植物保护学院, 杨凌 712100
中文关键词：棉铃虫，Bacillus thuringiensis，氨肽酶N（aminopeptidase N），结合动力学
英文关键词：Helicoverpa armigera, Bacillus thuringiensis, aminopeptidase N, binding dynamics
中文摘要：
【目的】 Bt杀虫蛋白（Bacillus thuringiensis）具有高度的靶标特异性，已经被广泛用于农业害虫防治。Bt杀虫蛋白要发挥杀虫活性，必须首先与其受体蛋白结合，氨肽酶N（Aminopeptidase N）是一类重要的Bt受体蛋白。因此，分析该受体与Bt杀虫蛋白的结合能力，可为进一步明确不同Bt的分子作用机制、Bt的抗性治理以及新Bt的开发应用等提供借鉴。【方法】 本文利用Ligand blot和Elisa方法比较了棉铃虫Helicoverpa armigera中肠APN4（Aminopeptidase N4，APN4）与Cry1Ac、Cry2Aa的结合能力。【结果】 原核表达的APN4片段与活化的Cry1Ac、Cry2Aa都可以结合，解离常数（Kd）分别是48.59 nmol/L和21.73 nmol/L。【结论】 APN4片段与Cry1Ac、Cry2Aa的结合能力在数量级上不存在显著性差异。
英文摘要： [Objectives] Bt (Bacillus thuringiensis) insecticidal proteins have been widely adopted to control agricultural pests because of their high target specificity. The binding of a Bt insecticidal protein to its specific receptor in the insect midgut plays a key role in the insecticidal action process. Aminopeptidase N (APN) is one of the major Bt protein receptors. To better characterize the molecular mechanism underlying the insecticidal activity of different Bt insecitcidal proteins, and lay the foundation for Bt resistance management and novel Bt insecticidal protein development, we analyzed the binding affinity of aminopeptidase N4 (APN4) to the Cry1Ac and Cry2Aa insecitcidal proteins in Helicoverpa armigera. [Methods] The binding affinity of aminopeptidase N4 (APN4) was assessed using ligand blot analysis and an ELISA binding assay, respectively. [Results] The results show that recombinant APN4 could bind to both Cry1Ac and Cry2A; their respective dissociation constants were 46.7 nmol/L and 26.5 nmol/L. [Conclusion] The results suggest that there was no significant difference in the binding affinity of APN4 to Cry1Ac and Cry2Aa in H. armigera.