Effects Of Rnai-Mediated Gene Silencing Of Aminopeptidase N (Haapnl) And Cadherin (Ha_BtR) On Cry1Ac Toxicity Against Helicoverpa Armigera

The cotton bollworm, Helicoverpa armigera, is one of the most serious pests on cotton. Widespread cultivation of transgenic Bt cotton results in effective control of this insect and reduced usage of chemical insecticides. Large-scale planting of transgenic Bt cotton creates intensive selection pressure on cotton bollworm in the field, and the development of resistance to Bt toxin by this pest should be considered as a major threat to Bt cotton. The Aminopeptidase N (Haapn1) and cadherin (Ha_BtR) are key receptors of Bacillus thuringiensis Cry1A toxin, and mutations in either Haapnl or Ha_BtR could result in resistance to Cry1Ac. In order to study and thus better undertand the interaction between these two receptors in mode of action of Bt toxin CrylAc, effects of RNAi-mediated gene silencing of Haapnl and Ha_BtR on CrylAc toxicity were investigated by injecting dsRNA of these two genes into the 4th instar larvae of H. armigera.1. Determination of injecting doses of Haapnl and Ha_BtR dsRNAThe cDNA fragments of both Haapn1 and Ha_BtR were PCR amplified and the dsRNA of them used for RNAi was prepared with the cDNA fragments as templates. A series of doses of Haapnl and Ha_BtR dsRNA (1,3,6,12μg) were injected respectively into 4th instar larvae, and larvae injected with elution solution (ES) were used as control. Real time PCR results at 48h after injection of 1μg dsRNA showed that the mRNA expression levels of Haapn1 and Ha_BtR were reduction to 49% and 70% respectively compared with the control. Silencing efficacy at 1 dsRNA was better than at other doses of dsRNA, so the dose of 1μg dsRNA was used in the present study.2. Effects of individual silencing of Haapn1 and Ha_BtR on Cry1Ac toxicityToxicity of Cry1Ac activated toxin to the fourth instar larvae injected with Haapn1 dsRNA was significantly lower (student's t test, P≤0.05) than that to the larvae injected with ES, but toxicity of Cry1Ac prtoxin was not signicantly different between the larvae injected with ds RNA and with ES. However. RNAi-mediated gene silencing by injecting Ha_BtR dsRNA had no effect on toxicity of both activated and protoxin Cry 1 Ac.The F] progeny between the SCD strain and SCD-rl strain (homozygous for Ha_BtR deletion mutation) was silenced by either Haapnl or Ha_BtR dsRNA, in order to investigate their effects on Cry1Ac toxicity under a low level of Ha_BtR mRNA expression. The mortality of Cry1Ac activated toxin against the F1 larvae injected with Haapnl dsRNA was significantly lower (P<0.01) than that of the F1 larvae injected with ES, and there was no effects on the toxicity of Cry1Ac protoxin to the F1 larvae injected with Haapnl dsRNA. Susceptibility to Cry1Ac activated toxin of the F1 larvae injected with Ha_BtR dsRNA was lower than the control at the fourth and fifth days, and the susceptibility to Cry1Ac protoxin was lower than the control at the third and fourth days.3. Effects of simultaneous silencing of Haapnl and Ha_BtR on CrylAc toxityIn the SCD strain, the mRNA expression levels of Haapnl and Ha_BtR in the larvae injected with a mixture of Haapnl dsRNA and Ha_BtR dsRNA reduced to 54% and 63% compared respectively to that of the control larvae injected with ES only. Toxicity of both activated and protoxin CrylAc was significantly reduced against the 4th instar larvae injected with the dsRNA mixture.In the F1 progeny, the mRNA expression levels of Haapnl and Ha_BtR in the larvae injected with a mixture of Haapnl dsRNA and Ha_BtR dsRNA reduced to 66% and 36% compared respectively to that of the control larvae injected with ES only. Toxicity of both activated and protoxin CrylAc was also significantly reduced against the 4th instar larvae injected with the dsRNA mixture.In conclusion, knockdown of Haapnl by RNAi reduced CrylAc toxicity significantly, but limited effects on CrylAc toxicicity was observed from knockdown of Ha_BtR by RNAi. Compared with individual silencing of Haapnl and Ha_BtR, simultaneous silencing of both genes produced more profound effects on CrylAc toxicity. These results further confirm that both Haapnl and Ha_BtR are functional receptors of CrylAc in H. armigera, and both of them are involved in intoxication of CrylAc. Our results also suggest that mutations occurring in either Haapnl or Ha_BtR may result in resistance to CrylAc in H. armigera.