Molecular Mechanism And Detection Technology Of Resistance To Transgenic Bt Cotton In Helicoverpa Armigera H(?)bner

cDNAs of Bt toxin receptor proteins from larvae midgut of Helicoverpa armigera Htibner susceptible and resistant to Bt transgenic cotton were cloned and sequenced. Four members in aminopeptidase N (APN) family were identified. APN1 (AY358034), APN2(AY346383), APN3(AY279537) and APN4(AY279534) have 3220, 3209, 3053, and 2862bp nucleotides with 3042, 3096, 3045, and 2853bp open reading frame that encode proteins of 1014, 1032, 1014 and 951 amino acids, respectively.The putative amino acid sequences of the four cDNAs shared the consensus zinc binding/gluzincin motif HEXXHX18E and GAMEN sequence common to gluzincin aminopeptidases. And hydrophobic signal sequence with 17-20 amino acids in N-terminal region were present in all the four APNs, and GPI anchor site in C-terminal region were found in APN2, APN3 and APN4 except in APN1.The deduced amino acid sequences by the four cDNAs were aligned with 31 aminopeptidase Ns from other insects, human, rats and E. coli with ClustalX and the phylogenetic tree was constructed. The Lepidopteran APNs were grouped into five classes on the phylogenetic tree derived from a ClustalX alignment. The four APNs reported here were distributed in four classes respectively, and only two members from H. armigera (AY346383) and Plutella xylostella (AJ222699) belonged to class 2, which predict there would one APN not yet cloned in cotton bollworm. The other classes had 5-9 APNs.Comparing the amino acid sequences of APNs from cotton bollworm susceptible and resistant to Bt transgenic cotton revealed some amine acid mutations. APN1 from resistant strain had two mutations compared with that from susceptible, but these two mutations were not located in the region for Bt toxin binding, It can be believed that mutations on APN1 was not related with the resistance to Bt transgenic cotton. APN2 had 15 substitutions of amino acid in resistant strain, among which Gln137 Glu and Val173 Thr were located in the region responsible for CrylA toxin binding. APN3 had 9amino acid mutations among which only Thr189 Asn was in CrylA toxin binding region. APN4 had seven amino acid changes, among which His162 Tyr and Pro185 Ser were located in the region that bind Cryl A toxin. The possibility of relation between mutations in the toxin binding region of APN2, APN3 and APN4 and resistance to bt transgenic cotton couldn't be excluded, and need further experimental verfication.The cDNA (AY351904) of E-cadherin from larvae midgut of cotton bollworm had 5541 bp nucleotides with an open read frame of 5190 bp that encode 1730 amino acids. The deduced amino acid sequence was a typical transmembrane protein containing a putative singal sequence with 22 amino acids, extracellular region composed of 12 cadherin repeats (EC1-EC12), transmembrane region with 22 amino acids and cytoplasmic domain with 126 amino acids. The E-cadherin protein of cotton bollworm was 50-80% identical to the E-cadherin from other insects, and 22% identical to that from human. The genomic DNA sequence of E-caderin from cotton bollworm was also cloned; the genomic DNA sequence (AY714876) encoding E-cadherin mRNA was composed of more than 33 exons. The longest exon was 308 bp, and the shortest only 18 bp. The exon of each domain or cadherin repeats was disrupted by 1-2 introns. The longest intron was 1731 bp. And there were great variations in the length of intron in the region of EC8-EC11 in population of cotton bollworm.Comparing the genomic DNA sequences of E-cadherin from susceptible and resistant cotton bollworn revealed a long deletion mutation in YCR resistant strain. The resistant cotton bollworm lost almost 10kb (9959bp) from nucleotides 4911 to 14870 which result in an E-cadherin mRNA of 1581 bp much shorter than that from susceptible. The truncated transcript of E-cadherin which lost a long region from nucleotide 1484 to the 3' end of E-cadherin mRNA from susceptible larvae encode a small protein composed of only 428 amino acids shorter than that from susceptible larvae. The deduced amino acid sequence of resistant E-cadherin mRNA lost cytoplasma...