Bacillus thuringiensis produces insecticidal Cry toxins used in the control of multiple insect pests. Evolution of insect resistance to Bt toxins endangers the use of Cry toxins for pest control. Analysis of the Cry1Ah-binding proteins from brush border membrane vesicles (BBMV) of Ostrinia furnacalis, Asian corn borer (ACB) from the Cry1Ah-resistant (ACB-AhR) and susceptible (ACB-BtS) strains was performed by an improved pull down assay that includes coupling Cry1Ah to NHS-activated Sepharose combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Our data show that Cry1Ah bound to alkaline phosphatase (ALP), cadherin-like (CAD), actin, aminopeptidase-N (APN), prophenoloxidase (proPO), serine proteinase inhibitor (SPI), immulectin, and V-ATPase and to other proteins that were not previously characterized as Cry-binding proteins in ACB-BtS strain. Analysis of Cry1Ah-pulled down proteins of the BBMV from ACB-AhR revealed that Cry1Ah toxin did not bind to ALP in ACB-AhR strain, suggesting that this protein may correlate with the resistant phenotype of this strain. Additionally, we analyzed the expression of representative genes coding for Cry1Ah-binding proteins such as ALP, APN, CAD, proPO, SPI, and immulectin by qRT-PCR. ACB-AhR showed increased expression levels of proPO (7.5 fold), ALP (6.2 fold) and APN (1.4 fold) in comparison to ACB-BtS strain. In contrast, the cad gene showed slight decreased expression in ACB-AhR strain (0.7 fold) compared with ACB-BtS strain. Our data suggest that differences in the susceptibility to Cry1Ah toxin in the ACB-AhR strain may be associated with reduced ALP binding sites and with an increased immune response. This study also brings evidence of a possible binding interaction of Cry1Ah toxin to immune related proteins like proPO.

中文翻译：

---

通过下拉和基因表达分析鉴定 Cry1Ah 抗性和易感 Ostrinia furnacalis 菌株的 Cry1Ah 结合蛋白

苏云金芽孢杆菌产生用于控制多种害虫的杀虫 Cry 毒素。昆虫对 Bt 毒素抗性的进化危及 Cry 毒素用于害虫控制的用途。通过改进的下拉法对来自 Ostrinia furnacalis、来自 Cry1Ah 抗性 (ACB-AhR) 和敏感 (ACB-BtS) 菌株的亚洲玉米螟 (ACB) 的刷状缘膜囊泡 (BBMV) 的 Cry1Ah 结合蛋白进行分析包括将 Cry1Ah 与 NHS 活化的 Sepharose 结合，并结合液相色谱-串联质谱 (LC-MS/MS)。我们的数据显示 Cry1Ah 与碱性磷酸酶 (ALP)、钙粘蛋白样 (CAD)、肌动蛋白、氨肽酶-N (APN)、酚氧化酶原 (proPO)、丝氨酸蛋白酶抑制剂 (SPI)、免疫凝集素、和 V-ATPase 以及之前在 ACB-BtS 菌株中未表征为 Cry 结合蛋白的其他蛋白质。对来自 ACB-AhR 的 BBMV 的 Cry1Ah 下拉蛋白的分析表明，Cry1Ah 毒素不与 ACB-AhR 菌株中的 ALP 结合，表明该蛋白可能与该菌株的抗性表型相关。此外，我们通过 qRT-PCR 分析了编码 Cry1Ah 结合蛋白（如 ALP、APN、CAD、proPO、SPI 和 immulectin）的代表性基因的表达。与 ACB-BtS 菌株相比，ACB-AhR 显示出 proPO（7.5 倍）、ALP（6.2 倍）和 APN（1.4 倍）的表达水平增加。相比之下，与 ACB-BtS 菌株相比，cad 基因在 ACB-AhR 菌株中的表达略有下降（0.7 倍）。我们的数据表明，ACB-AhR 菌株对 Cry1Ah 毒素的敏感性差异可能与 ALP 结合位点减少和免疫反应增加有关。这项研究还提供了 Cry1Ah 毒素可能与 proPO 等免疫相关蛋白结合相互作用的证据。