Functional Analysis Of The V-ATPase Subuint A Involved In The Cry1ac Toxicity And Resistance Revolution In Helicoverpa Armigera(Hübner)

The midgut of insect plays an important role in the toxicity of Bacillus thuringiensis(Bt) insecticide crystal protein. The binding of the Bt toxins to the specific receptors in the larval midgut brush border membrane vesicles(BBMV) is a key step for the mechanism of action of Cry toxin. As for, alteration of the toxin-binding receptor are reported to be the main mechanism of resistance to Cry toxin. Widely-planted Bt cotton has achived effective control for Helicoverpa armigera(cotton bollworm) hazards as well as the evolution of cotton bollworm resistance to it, and the latter will be a great threat to the long-term effective utilization of Bt cotton. So it is important to have a more thorough understanding for the mechanism of resistance to Cry toxin and make corresponding resistance management policy to prolong the service life of Bt cotton. In order to explore resistance mechanism of cotton bollworm, we have discovered there were many Vacuolar H+-ATPase proteins, which were significantly differently expressed between the sensitive and resistant strains, when use the Isobaric Tags for Relative and Absolute Quantification(iTRAQ) technique to compare the differentially expressed protein in the midgut BBMV between the sensitive and resistant strains of H. armigera. V-ATPase subunit A has been reported to be a binding protein of Bt toxin Cry1 Ac. In this research, we cloned the V-ATPase subunit A gene in H. armigera midgut and analysed its preliminary functions. The main method and results were as followed:1. The full-length sequence of the V-ATPase subunit A coding gene from the midgut in H. armigera was obtained using PCR(polymerase chain reaction) and RACE(rapid amplification of cDNA ends) technology. The length of cDNA sequence was 2578 bp(GenBank accession number KP090287), the ORF was 1863 bp which encoded a protein of 621 amino acid residues. The predicted molecular weight and isoelectric point were 68 KD and 5.13. There were no signal peptide on N-terminal and no potential anchor site on C-terminal among the amino acid sequence. The result of Protein affinity-disaffinity water-based prediction is hydrophilic. And it mainly localized in the cytoplasm, which is confronted to the structure of V-ATPase, using on-line subcellular localization analysis. This amino acid sequence had a very high homology compared with other species, the homology was more 90%, among which the homology with Manduca sexta was the highest, obtaining for 95.48%. The difference of amino acid sequence in different species main lied in the N-terminal. The results indicated that V-ATPase subunit A gene was highly conserved.2. The result of qRT-PCR indicted that V-ATPase subunit A was expressed differently in the growth development of H. armigera. It mainly expressed in the larval stage, among which the expression level in the 4th larvae was the highest, the second highest expressed was in the 2nd larvae, the expression level in egg, pupa and adult stage were low. In different tissues of larval digestive tract, the highest expression occured in the midgut, the second was the foregut, the expression in hindgut was significantly lower than that in the midgut and foregut. After the 4th larvae fed on artificial diet containing Cry1 Ac toxin, the expression of V-ATPase subunit A gene was suppressed dramatically.3. The full-length of V-ATPase subunit A was successfully expressed in the prokaryotic cell. The western blot results showed that the protein was in the BBMV with a relatively high expression. The results of ligand blot demonstrated that the prokaryotic expressed protein V-ATPase subunit A could not bind with Bt toxin-Cry1 Ac.4. When compares the nucleotide sequence and amino acid sequence between the sensitive strains and resistant strains, there were about 75 nucleotide but no amino acid change in these four strains of H. armigera. The mRNA level and protein level in the midgut between the sensitive strain LFs and resistant strain LF120.0 were expressed differently, which in the LF120.0 was significantly lower than that in the LFs.5. Silencing the expression of V-ATPase subunit A using the RNAi(RNA interference), then detected the effect of the RNAi-mediated silencing of the V-ATPase subunit A gene on Cry1 Ac toxicity against H. armigera. The sesults showed that the siRNA-A1 had a relatively high siliencing effect, with a 42%~55% mRNA expression reduction in different period after injecting siRNA-A1. The rate of weight inhibition of the sensitive larvae injected with siRNA-A1 increased, expect for the traeatment of the 3 day 60 μg/ml, the weight inhibition of other treatments were significantly higher than that of the control. After silencing the gene expression, the sensitive larval became more sensitive to Cry1 Ac.In conclusion, the V-ATPase subunit A expressed highly in the midgut. In the 4th sensitive larval, the V-ATPase subunit A gene expression decreased in the treatment of Cry1 Ac toxin, and it became more sensitive to Cry1 Ac when silencing the gene expression by RNAi. While cotton bollworm became resistant to Cry1 Ac, the mRNA and protein level both reduced. These results showed that V-ATPase subunit A played an important role in growth and energy metabolism, it may affect the protease synthesize and secretion, or participate in ATP hydrolysis and activation of the protein kinase A in the signaling pathway of Bt mechanism of action. The definite mechanism need further study.