Candas Mehmet, Loseva Olga, Oppert Brenda, Kosaraju Pradeepa, Bulla Lee A
Biological Targets, Inc, Tioga, Texas 76271, USA.
Mol Cell Proteomics. 2003 Jan;2(1):19-28. doi: 10.1074/mcp.m200069-mcp200.
Insect resistance to the Cry toxins of Bacillus thuringiensis (Bt) has been examined previously using a number of traditional biochemical and molecular techniques. In this study, we utilized a proteomic approach involving two-dimensional differential gel electrophoresis, mass spectrometry, and function-based activity profiling to examine changes in the gut proteins from the larvae of an Indianmeal moth (IMM, Plodia interpunctella) colony exhibiting resistance to Bt. We found a number of changes in the levels of certain specific midgut proteins that indicate increased glutathione utilization, elevation in oxidative metabolism, and differential maintenance of energy balance within the midgut epithelial cells of the Bt-resistant IMM larva. Additionally, the electrophoretic migration pattern of a low molecular mass acidic protein, which apparently is an ortholog of F(1)F(0)-ATPase, was considerably altered in the Bt-resistant insect indicating that variations in amino acid content or modifications of certain proteins also are important components of the resistance phenomenon in the IMM. Furthermore, there was a dramatic decrease in the level of chymotrypsin-like proteinase in the midgut of the Bt-resistant larva, signifying that reduction of chymotrypsin activity, and subsequently decreased activation of Cry toxin in the insect midgut, is an important factor in the resistant state of the IMM. The proteomic analysis of larval gut proteins utilized in this study provides a useful approach for consolidating protein changes and physiological events associated with insect resistance to Bt. Our results support the hypothesis that physiological adaptation of insects and resistance to Bt is multifaceted, including protein modification and changes in the synthesis of specific larval gut proteins. We believe that increased oxidative metabolism may be an adaptive response of insects that undergo survival challenge and that it could mediate detoxification as well as higher rates of generalized and localized mutations that enhance their resistance and provide survival advantage.
此前已使用多种传统生化和分子技术研究了昆虫对苏云金芽孢杆菌（Bt）Cry毒素的抗性。在本研究中，我们采用了一种蛋白质组学方法，包括二维差异凝胶电泳、质谱分析和基于功能的活性分析，以研究印度谷螟（IMM，Plodia interpunctella）对Bt产生抗性的群体幼虫肠道蛋白质的变化。我们发现某些特定中肠蛋白质水平发生了许多变化，这表明Bt抗性IMM幼虫中肠上皮细胞内谷胱甘肽利用增加、氧化代谢增强以及能量平衡的差异维持。此外，一种低分子量酸性蛋白质（显然是F(1)F(0)-ATP酶的直系同源物）的电泳迁移模式在Bt抗性昆虫中发生了显著改变，这表明氨基酸含量的变化或某些蛋白质的修饰也是IMM抗性现象的重要组成部分。此外，Bt抗性幼虫中肠中类胰凝乳蛋白酶样蛋白酶的水平显著降低，这表明胰凝乳蛋白酶活性的降低以及随后昆虫中肠中Cry毒素激活的减少是IMM抗性状态的一个重要因素。本研究中对幼虫肠道蛋白质进行的蛋白质组学分析为整合与昆虫对Bt抗性相关的蛋白质变化和生理事件提供了一种有用的方法。我们的结果支持这样的假设，即昆虫对Bt的生理适应和抗性是多方面的，包括蛋白质修饰和特定幼虫肠道蛋白质合成的变化。我们认为，氧化代谢增强可能是经历生存挑战的昆虫的一种适应性反应，它可以介导解毒以及更高频率的普遍和局部突变，从而增强它们的抗性并提供生存优势。
