Department of Entomology, Cornell University, New York State Agricultural Experiment Station, Geneva, New York, United States of America.
PLoS One. 2012;7(5):e35991. doi: 10.1371/journal.pone.0035991. Epub 2012 May 14.
Alteration of binding sites for Bacillus thuringiensis (Bt) toxins in insect midgut is the major mechanism of high-level resistance to Bt toxins in insects. The midgut cadherin is known to be a major binding protein for Bt Cry1A toxins and linkage of Bt-resistance to cadherin gene mutations has been identified in lepidopterans. The resistance to Bt toxin Cry1Ac evolved in greenhouse populations of Trichoplusia ni has been identified to be associated with the down-regulation of an aminopeptidase N (APN1) gene by a trans-regulatory mechanism and the resistance gene has been mapped to the locus of an ABC transporter (ABCC2) gene. However, whether cadherin is also involved with Cry1Ac-resistance in T. ni requires to be understood. Here we report that the Cry1Ac-resistance in T. ni is independent of alteration of the cadherin. The T. ni cadherin cDNA was cloned and the cadherin sequence showed characteristic features known to cadherins from Lepidoptera. Various T. ni cadherin gene alleles were identified and genetic linkage analysis of the cadherin alleles with Cry1Ac-resistance showed no association of the cadherin gene with the Cry1Ac-resistance in T. ni. Analysis of cadherin transcripts showed no quantitative difference between the susceptible and Cry1Ac-resistant T. ni larvae. Quantitative proteomic analysis of midgut BBMV proteins by iTRAQ-2D-LC-MS/MS determined that there was no quantitative difference in cadherin content between the susceptible and the resistant larvae and the cadherin only accounted for 0.0014% (mol%) of the midgut BBMV proteins, which is 1/300 of APN1 in molar ratio. The cadherin from both the susceptible and resistant larvae showed as a 200-kDa Cry1Ac-binding protein by toxin overlay binding analysis, and nano-LC-MS/MS analysis of the 200-kDa cadherin determined that there is no quantitative difference between the susceptible and resistant larvae. Results from this study indicate that the Cry1Ac-resistance in T. ni is independent of cadherin alteration.
昆虫中肠对苏云金芽孢杆菌（Bt）毒素结合位点的改变是昆虫对 Bt 毒素产生高水平抗性的主要机制。中肠钙粘蛋白已知是 Bt Cry1A 毒素的主要结合蛋白，并且在鳞翅目昆虫中已经确定了 Bt 抗性与钙粘蛋白基因突变的连锁关系。在烟粉虱温室种群中进化出的对 Bt 毒素 Cry1Ac 的抗性已被确定与一种跨调节机制下调氨肽酶 N（APN1）基因有关，并且抗性基因已被定位到 ABC 转运体（ABCC2）基因的位置。然而，钙粘蛋白是否也与 T.ni 中的 Cry1Ac 抗性有关仍需要进一步研究。在这里，我们报告称，T.ni 中的 Cry1Ac 抗性与钙粘蛋白的改变无关。克隆了 T.ni 的 Cry1Ac 抗性的钙粘蛋白 cDNA，并且钙粘蛋白序列表现出已知来自鳞翅目昆虫的钙粘蛋白的特征。鉴定了各种 T.ni 钙粘蛋白基因等位基因，并对钙粘蛋白等位基因与 Cry1Ac 抗性的遗传连锁分析表明，钙粘蛋白基因与 T.ni 中的 Cry1Ac 抗性没有关联。对敏感和 Cry1Ac 抗性 T.ni 幼虫的钙粘蛋白转录物的分析表明，两者之间没有定量差异。通过 iTRAQ-2D-LC-MS/MS 对中肠 BBMV 蛋白进行定量蛋白质组学分析，确定敏感和抗性幼虫之间的钙粘蛋白含量没有差异，并且钙粘蛋白仅占中肠 BBMV 蛋白的 0.0014%（摩尔%），而在摩尔比中仅占 APN1 的 1/300。毒素覆盖结合分析显示，来自敏感和抗性幼虫的钙粘蛋白均为 200kDa Cry1Ac 结合蛋白，并且对 200kDa 钙粘蛋白的 nano-LC-MS/MS 分析表明，敏感和抗性幼虫之间没有定量差异。本研究的结果表明，T.ni 中的 Cry1Ac 抗性与钙粘蛋白改变无关。
