Construction Of CDNA Library From Helicoverpa Armigera (Hübner) Larval Midgut And Cloning, Functional Analysis Of Its V-ATPase Subunit B

The midgut of insect is the main action position to Bacillus thuringiensis (Bt). Binding of the Bt toxins to specific receptors in the larval midgut is required for Bt toxicity, and the alterations of toxin-binding receptors are reported as main mechanisms of Bt resistance. As the growing area of Bt transgenic cotton has been increasing rapidly, the resistance problem of cotton bollworm Helicoverpa armigera (Hübner) to Bt has become the main restriction for long-term effective use of Bt cotton. So it is essential to study the molecular mechanism of Bt resistance. A more thorough understanding of Bt resistance mechanisms could provide necessary theoretical basis for devising resistance management strategies for H. armigera and prolong the service life of Bt cotton. Hence, we constructed a larval midgut cDNA library of H. armigera and searched many vacuolar ATPase subunit genes though library screening. Vacuolar ATPase subunit B has been reported is the probable binding protein of Bt toxin Cry1Ac. Thus we cloned the Vacuolar ATPase subunit B gene in H. armigera midgut and analysed its preliminary functions. The main results were as follows:1. Using Switching Mechanism at 5′end of the RNA Transcript (SMART) technology to construct the DNA library of larvae midgut in H. armigera. The quality evaluation showed the library had a complexity of 2×106 pfu/mL, and the recombination rate was 100%. The average length of inserted cDNA fragments was over 1000 bp and 50% were full-length form. About 1098 expressed sequence tags (ESTs) were generated successfully after sequencing. These suggested that one high quality cDNA library had been constructed.2. The full-length cDNAs-encoding V-ATPase subunit B was cloned by degenerative PCR combined with RACE techniques from the larval midgut of H. armigera. The length of cDNA sequence was 2091 bp, ORF was 1485bp which encoded a protein of 494 amino acid residues. The assession number was GU370066 in GenBank. The predicted molecular weight and isoelectric point were 55kD and 5.26. Though analyzed by software, V-ATPase subunit B had no signal peptide and no potential anchor site on C-terminal. This sequence had the highest homology with Heliothis virescens, the homology was about 99.8%. Compared with V-ATPase subunit B genes from human and mouse, the homologies were also high, with 85.2% and 81.3% respectively. The result indicated that V-ATPase subunit B was a conserved gene in many species.3. The V-ATPase subunit B was successfully expressed in prokaryotic cell. The results of Ligand blotting demonstrated that the expressed protein V-ATPase subunit B could bind with Bacillus thuringiensis Cry1Ac, Cry2Ab, Cry1C toxin respectively, but could not bind with Cry1B. This suggested that V-ATPase subunit B in the midgut of H. armigera is the binding protein of some Bt toxin. Bioassay results indicated that there were no significant difference in larval weight between larvae which fed on only containing Cry1Ac toxin and those fed on containing the mix of V-ATPase subunit B expressed protein and Cry1Ac toxin. Therefore, though the V-ATPase subunit B was a binding protein of Cry1Ac toxin, but it had no influence to the toxicity of Cry1Ac.4. The result of RT-qPCR indicated that the expression level of V-ATPase subunit B was different in every H. armigera development stages. The expression level in adult was the highest, and then was in larvae, in pupal stage the expression level was the lowest. The expression amounts of V-ATPase subunit B in different parts of the larval gut were also different. The expression amounts in foregut and midgut were obvious higher than that in hindgut, but the differenc between foregut and midgut was not significant. The expression amount in different strains were different, the expression level in susceptible strain was obvious higher than Cry1Ac-resistant strain. But whether there have relationship betwteen the resistance to Cry1Ac of H. armigera and the expression level of V-ATPase subunit B still need further verified.