| Transgenic Bt cotton is very effective against cotton bollworm, Helicoverpa armigera and the Bt cotton planting area in China has expanded to two thirds of the total cotton area. However, the development of resistance to Bt toxin Cry 1 Ac in the cotton bollworm is considered the direct threat to the long-term effectiveness of Bt cotton. Investigation of mechanisms of Bt resistance in the cotton bollworm will be necessary for developing reliable resistance detection method at molecular level, which will help to monitor resistance development and design effective resistance management tactics.1. Laboratory selection for CrylAc resistance in Helicoverpa armigeraA resistant strain (GYBT) of Helicoverpa armigera was selected with activated CrylAc for 28 generations in laboratory and its resistant level increases to >800-fold compared with the original GY strain. That indicated the cotton bollworm has the potential to evolve resistance under the pressure of Bt toxin CrylAc. Geographic variation (about 10 times at LC50) in susceptibility to activated CrylAc was found in ten field populations collected across north China cotton area in 2002.2. Midgut protease activity, zymogram analysis and degradation of CrylAcThe midgut proteases from the susceptible (GY) and resistant (GYBT) strains of H. armigera were characterized and compared by use of chromomeric substrates for trypsin (BApNA), chymotrypsin (SAAPFpNA) and general proteases (azocasein). Aminopeptidase activity with substrate L-Leucine p-nitroanilide (L-pNA) was alsodetermined. The hydrolysis efficiency, determined by Vmax, and Michaelis constant (Km) shows no significant difference between GY and GYBT for substrates BApNA and L-pNA, and also the inhibition effect of TLCK to trypsin have no significant difference between GY and GYBT strains, while the Km and Vmax of chymotrypsin from GYBT strain and its inhibition by TPCK were significantly different from that of GY strain. The profile of midgut proteases by native PAGE and SDS-PAGE was different between GYBT and GY strains. SDS-PAGE analysis of degradation of activated Cry 1 Ac in vitro by midgut preparation revealed no difference between GYBT and GY strains, which suggested general proteases in GYBT strain may be changed to adapt to Cry 1 Ac-treated diet and not involved in resistance to CrylAc.3. Cloning of cadherin-like receptor gene in Helicoverpa armigea and linkage analysis of resistance to CrylAcThe full length cDNA of cadherin-like receptor in GYBT and GY strains of H. armigera were cloned with PCR and 3'RACE methods. The deduced amino acid of cDNA from GY strain was 1730 and showed 95.9% similarity with H.zea, 83% with H.Virescens, 61.5% with B.mori, 61.9% with L.diapar, 59.4% with M.Sexta, 58.3% with O.nubilalis, 54%with P.gossypiella and 53.7% with C.supperssli. The cadherin-like receptor gene in the GYBT strain were truncated for a prematured stop codon TAA at the position 1258, and the deduced amino acid was only 428 aa and the deduced binding region of CrylAc was lost. Linkage analysis in the backcross confirmed that the truncated cadherin-like receptor gene in GYBT strain was tightly linked to CrylAc resistance.Diagnostic methods at both cDNA and genomic DNA levels were developed to genotype Cadherin alleles in H. armigera.4. Binding analysis of CrylAc with BBMV of Helicoverpa armigeraLigand-receptor binding analysis with 125I labelled CrylAc to BBMV of Helicoverpa amigera indicated that the binding affinity (Kd) of BBMV of the resistant GYBT strain to CrylAc was lower than that of the susceptible GY strain and binding site concentration (Bmax) was similar in both strains. Hill factors wereintroduced to model the binding curves, indicating a positive interaction between toxin and receptor binding.Biotinylated Cry 1 Ac toxin was used for the western blotting of BBMV. Four major binding proteins (with molecular weight of 210Kd, 190Kd, 160Kd and 120Kd) and a 85Kd endogenous biotin-protein were found in the midgut of the susceptible GY strain. However, 210Kd and 190Kd binding proteins were absent in the midg...
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