Department of Entomology, Cornell University, Geneva, New York, USA.
Department of Genetics, Instituto Universitario de Biotecnología y Biomedicina (BIOTECMED), Universitat de València, Burjassot, Spain.
Appl Environ Microbiol. 2024 Oct 23;90(10):e0074224. doi: 10.1128/aem.00742-24. Epub 2024 Sep 18.
The resistance to the insecticidal protein Cry1Ac from the bacterium (Bt) in the cabbage looper, , has previously been identified to be associated with a frameshift mutation in the ABC transporter ABCC2 gene and with altered expression of the aminopeptidase N (APN) genes and , shown as missing of the 110-kDa APN1 (phenotype APN1¯) in larval midgut brush border membrane vesicles (BBMV). In this study, genetic linkage analysis identified that the APN1¯ phenotype and the mutation in Cry1Ac-resistant segregated independently, although they were always associated under Cry1Ac selection. The mutation and APN1¯ phenotype were separated into two strains respectively. Bioassays of the strains with Cry1Ac determined that the with the APN1¯ phenotype showed a low level resistance to Cry1Ac (3.5-fold), and the associated resistance is incompletely dominant in the background of the mutation. Whereas the mutation-associated resistance to Cry1Ac is at a moderate level, and the resistance is incompletely recessive in the genetic background of downregulated APN1. Analysis of Cry1Ac binding to larval midgut BBMV indicated that the midgut in larvae with the APN1¯ phenotype had reduced binding affinity for Cry1Ac, but the number of binding sites remained unchanged, and the midgut in larvae with the mutation had both reduced binding affinity and reduced number of binding sites for Cry1Ac. The reduced Cry1Ac binding to BBMV from larvae with the mutation or APN1¯ phenotype correlated with the lower levels of resistance.IMPORTANCEThe soil bacterium (Bt) is an important insect pathogen used as a bioinsecticide for pest control. Bt genes coding for insecticidal proteins are the primary transgenes engineered into transgenic crops (Bt crops) to confer insect resistance. However, the evolution of resistance to Bt proteins in insect populations in response to exposure to Bt threatens the sustainable application of Bt biotechnology. Cry1Ac is a major insecticidal toxin utilized for insect control. Genetic mechanisms of insect resistance to Cry1Ac are complex and require to be better understood. The resistance to Cry1Ac in is associated with a mutation in the gene and also associated with the APN expression phenotype APN1¯. This study identified the genetic independence of the APN1¯ phenotype from the mutation and isolated and analyzed the mutation-associated and APN1¯ phenotype-associated resistance traits in to provide new insights into the genetic mechanisms of Cry1Ac resistance in insects.
先前已鉴定出，在甘蓝夜蛾中，对来自细菌 (Bt) 的杀虫蛋白 Cry1Ac 的抗性与 ABC 转运蛋白 ABCC2 基因的移码突变以及氨肽酶 N (APN) 基因 和 的表达改变有关，表现为幼虫中肠刷状缘膜囊泡 (BBMV) 中缺失 110 kDa APN1（APN1¯表型）。在这项研究中，遗传连锁分析表明，APN1¯表型和 Cry1Ac 抗性中的 突变是独立分离的，尽管它们在 Cry1Ac 选择下总是相关的。 突变和 APN1¯表型分别分离成两个 菌株。Cry1Ac 对 菌株的生物测定表明，具有 APN1¯表型的 对 Cry1Ac 表现出低水平抗性（3.5 倍），并且在 突变背景下，抗性不完全显性。而 Cry1Ac 抗性的 突变相关水平中等，并且在 APN1 下调的遗传背景下，抗性不完全隐性。Cry1Ac 与幼虫中肠 BBMV 结合的分析表明，APN1¯表型幼虫的中肠对 Cry1Ac 的结合亲和力降低，但结合位点数量保持不变，而 突变幼虫的中肠对 Cry1Ac 的结合亲和力和结合位点数量均降低。突变或 APN1¯表型幼虫的 BBMV 中 Cry1Ac 结合减少与抗性水平降低相关。
重要性土壤细菌 (Bt) 是一种重要的昆虫病原体，用作害虫防治的生物杀虫剂。编码杀虫蛋白的 Bt 基因是工程转入转基因作物（Bt 作物）中的主要转基因，以赋予昆虫抗性。然而，昆虫种群对暴露于 Bt 中 Bt 蛋白的抗性进化对 Bt 生物技术的可持续应用构成了威胁。Cry1Ac 是一种用于控制昆虫的主要杀虫毒素。昆虫对 Cry1Ac 的抗性遗传机制很复杂，需要更好地理解。在 中，对 Cry1Ac 的抗性与 基因中的突变有关，也与 APN 表达表型 APN1¯有关。本研究鉴定了 APN1¯表型与 突变的遗传独立性，并分离和分析了 中与 突变相关和与 APN1¯表型相关的抗性特征，为昆虫对 Cry1Ac 抗性的遗传机制提供了新的见解。
