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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 bordermembrane 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(1μg/头)进行基因沉默后,Haapn1mRNA表达量比注射缓冲液(elution solution,ES)的对照下降了30%～49%,Ha_BtRmRNA表达量下降了30%～37%。注射Haapn1dsRNA的幼虫在40和70μg/cm2Cry1Ac活化毒素下的死亡率显著低于注射ES的幼虫,而在100和170μg/cm2Cry1Ac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_BtRdsRNA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_BtRdsRNA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。
Aminopeptidase N (APN) and cadherin are receptors for Bt toxin CrylA present in the brush border membrane vesicles (BBMV) of Lepidoptera. In this study, the Helicoverpa armigera N1 gene Haapn1 and the cadherin gene Ha_BtR double-stranded RNA (dsRNA) were injected into the fourth instar larvae of Helicoverpa armigera to study the effects of the two receptor genes on the virulence of Cry1Ac after silencing. The results showed that the expression of Haapn1 mRNA was reduced by 30% -49% and the expression of Ha_BtR mRNA was reduced by 30% -37% compared with elution solution (ES) after gene silencing with dsRNA (1μg / head). The mortality of larvae injected with Haapn 1 dsRNA at 40 and 70 μg / cm2Cry1Ac activating toxin was significantly lower than that of ES-injected larvae, while there was no significant difference in mortality between 100 and 170 μg / cm2Cry1Ac protoxin; Cry1Ac activating toxin and protoxin There was no significant difference in virulence between injected Ha_BtRdsRNA larvae and ES larvae. After simultaneous injection of Haapn1 and Ha_BtRdsRNA, the susceptibility of interfering larvae to Cry1Ac-activating toxin and protoxin was significantly reduced. This study further proves that both H.pylori Haapn1 and Ha_BtR are functional receptors of Bt toxin Cry1Ac, and both of these receptor proteins are involved in the toxic effect of Cry1Ac. This result also suggests that mutations in the Haapn1 or Ha_BtR genes may cause the cotton bollworm to develop resistance to Cry1Ac.