Downregulation of APN1 and mutation in Bt Cry1Ac-resistant are genetically independent.

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.