Cloning, M Rna Expression And Function Analysis Of Polycalin Gene In Helicoverpa Armigera

The bindings between Bt(Bacillus thuringiensis) with receptor proteins in midgut of insect pests play vital roles in Bt insecticidal action process. The reduced or loss of binding ability in insect cause the resistance to Bt. Polycalin is a kind of binding protein with Cry1 Ac in midgut of insect has been reported before. The research on cloning, m RNA expression and function analysis of Polycalin gene in cotton bollworm Helicoverpa armigera were studied. The full-length sequence of the Polycalin coding gene was obtained using RT-PCR(reverse transcription-polymerase chain reaction) and RACE(rapid amplification of c DNA ends) technology. The expression levels of Polycalin in different development stages and different tissues of larval digestive tract were analyzed by real-time quantitative PCR. The expression changes of Polycalin in larval midgut after 4th intar larvae fed on artificial diet containing Bt insecticide protein Cry1 Ac were compared. Part of Polycalin gene was expressed in prokaryotic expression system, the antibody was prepared, the binding character of Polycalin protein with Cry1 Ac was detected using western blot and ligand blot. The differences of Polycalin between resistant and susceptible H. armigera were compared by comparision of gene sequence, m RNA expression and i TRAQ method. The results were summarized as follows:1. The results indicated that the full-length of the Polycalin from H. armigear coding gene was 2955 bp(Gen Bank accession number KP100652), the open reading frame was 2781 bp in length, encoding 926 amino acid residues. The predicted molecular weight was 101.68 KD and isoelectric point was 4.95. The putative protein sequence contained a N-terminal signal peptide with 20 amino acids, three N-linked and eight O-linked glycosylation sites, and a GPI anchor signal peptide with 2 amino acids at the C-terminus.2. Polycalin were expressed in all growth stages of H. armigera, and the expression in larvae were higher than that in other stages, especially in 1st-3rd instar larvae were the highest. The lowest expression levels occurred in egg, adult and pupae. After larvae fed on Cry1 Ac toxin, the expression of Polycalin gene was suppressed dramatically.3. Part of Polycalin gene was expressed in prokaryotic expression system, the antibody was prepared, the results from western blot and ligand blot showed that Polycalin protein could bind with Cry1 Ac. The nucleotide sequence had four mutations in the coding region while the amino acid was completely consistent between susceptible and resistant H. armigera strains. The expression of Polycalin in the resistant strain were higher than that in the susceptible strain except in Polycalin2 nd instar larvae and adult. Polycalin protein expression level was higher in the resistant strain using western blot and i TRAQ.The Polycalin was confirmed as a binding protein with Cry1 Ac in this study. It was suggested that the higher expression of Polycalin m RNA and protein maybe one of the major reason involved in H. armigera resistance to Cry1 Ac toxin. These results could provide basis for further clarifying the function of Polycalin and its role in action mode of Bt toxin and in resistance machnism.