RNAi介导的棉铃虫氨肽酶N基因Haapn1和钙粘蛋白基因Ha_BtR沉默对Cry1Ac毒力的影响

氨肽酶N（aminopeptidase N, APN）和钙粘蛋白（cadherin）是存在于鳞翅目昆虫中肠刷状缘膜囊（brush border membrane vesicles, BBMV）上Bt毒素Cry1A的受体。本实验将棉铃虫Helicoverpa armigera氨肽酶N1基因Haapn1和钙粘蛋白基因Ha_BtR双链RNA（dsRNA）注入棉铃虫4龄幼虫体内, 以研究这两种受体基因沉默后对Cry1Ac毒力的影响。结果表明: 注射dsRNA（1 μg/头）进行基因沉默后, Haapn1 mRNA表达量比注射缓冲液（elution solution, ES）的对照下降了30%~49%, Ha_BtR mRNA表达量下降了30%~37%。注射Haapn1 dsRNA的幼虫在40和70 μg/cm² Cry1Ac活化毒素下的死亡率显著低于注射ES的幼虫, 而在 100 和 170 μg/cm² Cry1Ac原毒素处理下两者死亡率无显著差异; Cry1Ac活化毒素以及原毒素对注射Ha_BtR dsRNA幼虫与注射ES幼虫的毒力均无显著差异。当同时注射Haapn1及Ha_BtR dsRNA后, 干扰后的幼虫对Cry1Ac活化毒素和原毒素的敏感性均显著下降。本研究进一步证明了棉铃虫Haapn1和Ha_BtR均是Bt毒素Cry1Ac的功能受体, 这两种受体蛋白共同参与Cry1Ac的毒杀作用过程。该结果也提示, Haapn1或Ha_BtR基因产生突变都可能导致棉铃虫对Cry1Ac产生抗性。

Effects of RNAi-mediated silencing of an aminopeptidase N gene Haapn1 and a cadherin gene Ha_BtR on Cry1Ac toxicity against Helicoverpa armigera (Lepidoptera: Noctuidae)

Aminopeptidase N (APN) and cadherin are key receptors of Bacillus thuringiensis Cry1A toxins in brush border membrane vesicles (BBMVs) of lepidopteran insects. Effects of RNAi-mediated silencing of an APN gene Haapn1 and a cadherin gene Ha_BtR on Cry1Ac toxicity were investigated by injecting dsRNAs of these two genes into the 4th instar larvae of Helicoverpa armigera in this experiment. Reduction of mRNA expression of Haapn1 (30%-49%) and Ha_BtR (30%-37%) was observed in the larvae injected respectively with Haapn1 dsRNA and Ha_BtR dsRNA (1 μg/larva) compared with the control larvae injected with elution solution (ES) only. Mortality of larvae injected with Haapn1 dsRNA was significantly lower than that of the control larvae injected with ES in treatments of 40 and 70 μg/cm² of activated Cry1Ac, but there was no difference in mortality of larvae injected with either Haapn1 dsRNA or ES in treatments of 100 and 170 μg/cm² of Cry1Ac protoxin. RNAi-mediated gene silencing by injecting Ha_BtR dsRNA had no effect on toxicity of both activated Cry1Ac and Cry1Ac protoxin. However, toxicity of both activated Cry1Ac and Cry1Ac protoxin against the 4th instar larvae injected with a mixture of Haapn1 dsRNA and Ha_BtR dsRNA was significantly reduced. These results further confirm that both Haapn1 and Ha_BtR are functional receptors of Cry1Ac in H. armigera, and both of them are involved in intoxication of Cry1Ac. Our results also suggest that mutations occurring in either Haapn1 or Ha_BtR may result in resistance to Cry1Ac in H. armigera.