| Helicoverpa armigera (Hubner) is one of the important worldwide agricultural pests. Bacillus thuringiensis (Bt) insecticidal crystal proteins (ICP) have high specialty to target insects and no harm to human being and livestock, so its related genes are wildly applied to control insect pests. As the planting area of Bt cotton has been increasing rapidly and the transformed insecticidal gene in cotton are expressing continuously, H. armigera got high selected pressure during whole growing stage of cotton and the resistance risk is unavoidable. So the resistance of H. armigera to Bt can not be ignored. Research on mechanism of resistance in H. armigera to Bt will provide the theoretical guidance for establishing resistance management and have important meaning for suggesting rational planting Bt cotton.In this dissertation, the aminopeptidase N1 (APN1) in the susceptible strain of H. armigera to Bt (96-S) and the resistant strain (Bt-R) were cloned separately using PCR method. The APN1 gene sequences mutation, between 96-S and Bt-R strain were compared. The APN1 in both strain of H. armigera were successfully expressed in baculovirus-insect expression system. The results were as follows:The gene sequences were analyzed and were made sure they belong to APN gene family. The length of this APN1 was 2,982 bp, encoding 993 amino acid residues. The first 25 amino acid residues at C terminal form had hydrophobic trans-membrane helix.The mutations between 96-S and Bt-R strain of H. armigera were compared using DNAMAN program. Some nuclear changes were found: A in the 579~th site changed into T, 803C→T, 854G→A, 1250C→T, 1460T→C, 1486A→T, 1497C→T, 1647G→C, 2135T→C, 2231G→A, 2586T→C, 2597A→T, 2615A→G, 2703G→C, 2831G→T, 2859A→C, and three nuclears were lost in resistant strain, that were, the AC A (36-38) deficiencies. These changes induced some amino acid changes: 194Thr→Ser, 550Val→Ile, 861Ile→Thr, 902Ala→Pro, 954Thr→Pro, and 45Thr amino acid was lost. In the activation region contacting Bacillus thuringiensis toxin Cry 1 Ac, Thr in the 194~th site changed into Ser, which may account for the resistance of H. armigera to Cry 1 Ac. Thr is a potential phosphorylation site for post-translational modify, the mutation of 861 Ile →Thr may undermine the disulfide bride formation ability of its adjacent cysteines, break tertiary structure of receptor protein and result in resistance.The fragments encoding APN1 without signal peptides were cloned into the Bac-to-Bac baculovirus expression system with transfer vector pFastBacHTb under the polyhedron gene promoter. The recombinant transposing plasmid pFastBacHTb/APN1 was screened and then transformed into DHlOBac Escherichia coli. It was cultured in LB medium, which contained Te~+, Kan~+, Gen~+, X-gal and IPTG The resulting recombinant bacmid was transfected into cells of the insect Trichoplusia ni (Tn) and recombinant baculoviruse was obtained. The lysate of cells infected with recombinant baculoviruse was analyzed by SDS-PAGE and dot-blotting. The results showed that the recombinant baculoviruse was fully capable of expressing APN1. These results will provide the basic knowlodges to separate and purify receptor protein, analyze the relationship between receptor and Bt toxin, and to establish the ground for advanced research on resistance mechanism of H. armigera to Bt.
|
PDF Full Text Request