Mutation of an aminopeptidase N gene is associated with Helicoverpa armigera resistance to Bacillus thuringiensis Cry1Ac toxin.
A Cry1Ac-resistant strain (Bt-R) of Helicoverpa armigera, with 2971-fold resistance, was derived by selection with Cry1Ac toxin for 75 generations. We used cDNA-amplified fragment length polymorphism analysis to identify those genes differentially expressed in the Cry1Ac-resistant and -susceptible strains, which revealed 212 differentially expressed transcripts among 2000 screened cDNAs. Among these transcript-derived fragments (TDFs), 37 showed some homology to known sequences, including Aminopeptidase N (APN), which is expressed in the midgut epithelium and has been implicated as a Cry1A subfamily receptor in several moths, including H. armigera. We confirmed the TDF by RT-PCR and identified a deletion mutation of apn1 in the Bt-R strain. We expressed the TDF in bacteria. The partial HaAPN1-96S wild-type protein, bound to Cry1Ac on ligand blots, whereas HaAPN1-BtR did not. This suggested that HaAPN1 is a receptor for Bt Cry1Ac and that its deletion mutation is associated with Cry1Ac resistance in H. armigera. The absence of one binding site is responsible for its resistance to Cry1Ac. We developed an allele-specific PCR to monitor whether the apn1 gene in an H. armigera field population produced a similar mutation. No deleted mutants were found in 2250 individuals collected from the field in 2006-2007.
State Key Laboratory for the Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China.
Shaoping
S
Zhang
Hongmei
H
Cheng
Yulin
Y
Gao
Guirong
G
Wang
Gemei
G
Liang
Kongming
K
Wu
D000595:Amino Acid Sequence; D000818:Animals; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000494:pharmacology*; D001665:Binding Sites; D018826:CD13 Antigens / Q000235:genetics* / Q000378:metabolism; D004731:Endotoxins / Q000494:pharmacology*; D015870:Gene Expression; D006460:Hemolysin Proteins / Q000494:pharmacology*; D019476:Insect Proteins / Q000235:genetics* / Q000378:metabolism; D007305:Insecticide Resistance; D008969:Molecular Sequence Data; D009036:Moths / Q000187:drug effects* / Q000201:enzymology* / Q000235:genetics / Q000502:physiology; D011485:Protein Binding; D016415:Sequence Alignment; D017384:Sequence Deletion
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D004731:Endotoxins; D006460:Hemolysin Proteins; D019476:Insect Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D018826:CD13 Antigens