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【目的】Cry1A和Cry2A类Bt蛋白通过特异性地与昆虫中肠上的受体蛋白结合而发挥杀虫作用,现已广泛应用于转基因抗虫作物。本研究旨在进一步明确Cry2A类蛋白的作用机制和Cry1A受体蛋白在Cry2A发挥毒力中的作用。【方法】本研究首先提取了棉铃虫Helicoverpa armigera的BBMV,制备了钙粘蛋白(CAD)、氨肽酶N(APN)和碱性磷酸酯酶(ALP)3种受体蛋白的抗体和抗血清;然后,利用Western blot检测BBMV上这3种受体蛋白后,利用抗体封闭技术比较了敏感棉铃虫和Cry1Ac抗性棉铃虫(BtR)中3种受体蛋白的抗血清对Cry1Ac和Cry2Aa毒力的影响。【结果】对敏感品系棉铃虫,这3种已知的Cry1Ac受体蛋白抗血清显著地降低了Cry1Ac和Cry2Aa的毒力。其中APN抗血清对Cry1Ac毒力的影响最大,棉铃虫幼虫的死亡率降低了84.44%;ALP抗血清对Cry2Aa的毒力影响最大,棉铃虫幼虫死亡率比对照降低了71.04%。Cry1Ac对Cry1Ac抗性棉铃虫(BtR)的毒力显著降低,Cry2Aa的毒性也减弱。在Cry1Ac抗性棉铃虫(BtR)中,3种受体抗血清对Cry1Ac的影响比在敏感棉铃虫中的影响小,尤其是CAD和APN抗血清对Cry1Ac毒力的抑制率显著低于在敏感棉铃虫中的抑制作用;CAD和ALP抗血清对Cry2Aa毒力的影响与在敏感棉铃虫中的影响差异不显著,但APN抗血清可以显著降低Cry2Aa对Cry1Ac抗性棉铃虫(BtR)的毒力。【结论】棉铃虫CAD,APN和ALP不仅参与了Cry1Ac的杀虫过程,也对Cry2Aa毒力有一定的影响,而且这3种蛋白可能与棉铃虫对Cry1Ac和Cry2Aa产生抗性及交互抗性相关。
【Objective】 Cry1A and Cry2A Bt proteins play a key role in killing insects by specifically binding to the receptor proteins in the midgut of insects and are now widely used in transgenic insect-resistant crops. The purpose of this study is to further clarify the mechanism of action of Cry2A protein and the role of Cry1A receptor protein in Cry2A exerting virulence. 【Method】 In this study, the BBMV of Helicoverpa armigera was extracted and the antibodies against cadherin (CAD), aminopeptidase N (APN) and alkaline phosphatase (ALP) and antisera ; Then, using western blot to detect the three receptor proteins on BBMV, the antiserum of three receptor proteins in sensitive cotton bollworm and Cry1A-resistant cotton bollworm (BtR) was compared to Cry1Ac and Cry2Aa virulence Impact. [Results] The three known antiserums against Cry1Ac receptor protein significantly reduced the virulence of Cry1Ac and Cry2Aa against the susceptible strain of cotton bollworm. The APN antiserum had the greatest impact on the toxicity of Cry1Ac, the mortality of H. armigera larvae was reduced by 84.44%, and the ALP antiserum had the greatest impact on the toxicity of Cry2Aa. The mortality of H. armigera larvae was reduced by 71.04% compared with the control. Cry1Ac significantly reduced the virulence of Cry1Ac-resistant cotton bollworm (BtR) and also reduced the toxicity of Cry2Aa. In Cry1Ac resistant cotton bollworm (BtR), the effect of three kinds of receptor antiserum on Cry1Ac was less than that in the susceptible cotton bollworm. Especially, the inhibition rates of Cry1Ac virulence of CAD and APN antisera were significantly lower than those in the sensitive The effect of Cry1Ac-resistant Cry1Ac-resistant cotton bollworm (BtR) on Cry1Ac-resistant cotton bollworm (BtR) was significantly inhibited by APN antiserum . 【Conclusion】 The results showed that the cotton bollworm CAD, APN and ALP not only participated in the insecticidal process of Cry1Ac, but also had some effects on the virulence of Cry2Aa, and these three proteins may be related to the resistance and cross resistance of Cry1Ac and Cry2Aa to H. armigera .