PDF(Pubmed)
Abstract：
The Aedes aegypti cadherin-like protein (Aae-Cad) and the membrane-bound alkaline phosphatase (Aae-mALP) are membrane proteins identified as putative receptors for the larvicidal Cry toxins produced by Bacillus thuringiensis subsp. israelensis bacteria. Cry toxins are the most used toxins in the control of different agricultural pest and mosquitos. Despite the relevance of Aae-Cad and Aae-mALP as possible toxin-receptors in mosquitoes, previous efforts to establish a clear functional connection among them and Cry toxins activity have been relatively limited. In this study, we used CRISPR-Cas9 to generate knockout (KO) mutations of Aae-Cad and Aae-mALP. The Aae-mALP KO was successfully generated, in contrast to the Aae-Cad KO which was obtained only in females. The female-linked genotype was due to the proximity of aae-cad gene to the sex-determining loci (M:m). Both A. aegypti KO mutant populations were viable and their insect-development was not affected, although a tendency on lower egg hatching rate was observed. Bioassays were performed to assess the effects of these KO mutations on the susceptibility of A. aegypti to Cry toxins, showing that the Aae-Cad female KO or Aae-mALP KO mutations did not significantly alter the susceptibility of A. aegypti larvae to the mosquitocidal Cry toxins, including Cry11Aa, Cry11Ba, Cry4Ba, and Cry4Aa. These findings suggest that besides the potential participation of Aae-Cad and Aae-mALP as Cry toxin receptors in A. aegypti, additional midgut membrane proteins are involved in the mode of action of these insecticidal toxins.
摘要：
埃及伊蚊钙粘蛋白样蛋白（Aae-Cad）和膜结合碱性磷酸酶（Aae-mALP）是被鉴定为苏云金芽孢杆菌亚种产生的幼虫Cry毒素的推定受体的膜蛋白。以色列细菌。哭泣毒素是控制不同农业害虫和蚊子的最常用毒素。尽管Aae-Cad和Aae-mALP可能是蚊子的毒素受体，以前在它们与Cry毒素活性之间建立明确的功能联系的努力相对有限。在这项研究中,我们使用CRISPR-Cas9产生Aae-Cad和Aae-mALP的敲除(KO)突变。成功生成了Aae-mALPKO,与仅在雌性中获得的Aae-CadKO相反。与女性相关的基因型是由于aae-cad基因与性别决定基因座（M：m）的接近性。两个埃及伊蚊KO突变体种群都是可行的，它们的昆虫发育不受影响，尽管观察到卵孵化率较低的趋势。进行生物测定以评估这些KO突变对埃及伊蚊对Cry毒素的敏感性的影响。显示Aae-Cad雌性KO或Aae-mALPKO突变并没有显着改变埃及伊蚊幼虫对杀蚊性Cry毒素的敏感性，包括Cry11Aa,Cry11Ba,Cry4Ba,Cry4Aa这些发现表明，除了Aae-Cad和Aae-mALP作为Cry毒素受体的潜在参与，这些杀虫毒素的作用方式涉及其他中肠膜蛋白。
