Jiangsu Key Laboratory of Sericultural Biology and Biotechnology, School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang, Jiangsu, China.
Key Laboratory of Silkworm and Mulberry Genetic Improvement, Ministry of Agriculture and Rural Affairs, Sericultural Research Institute, Chinese Academy of Agricultural Science, Zhenjiang, Jiangsu, China.
Arch Insect Biochem Physiol. 2022 Jun;110(2):e21886. doi: 10.1002/arch.21886. Epub 2022 Mar 20.
Bt toxins are parasporal crystals produced by Bacillus thuringiensis (Bt). They have specific killing activity against various insects and have been widely used to control agricultural pests. However, their widespread use has developed the resistance of many target insects. To maintain the sustainable use of Bt products, the resistance mechanism of insects to Bt toxins must be fully clarified. In this study, Bt-resistant and Bt-susceptible silkworm strains were used to construct genetic populations, and the genetic pattern of silkworm resistance to Cry1Ac toxin was determined. Sequence-tagged site molecular marker technology was used to finely map the resistance gene and to draw a molecular genetic linkage map, and the two closest markers were T1590 and T1581, indicating the resistance gene located in the 155 kb genetic region. After analyzing the sequence of the predicted gene in the genetic region, an ATP binding cassette transporter (ABCC2) was identified as the candidate gene. Molecular modeling and protein-protein docking result showed that a tyrosine insertion in the mutant ABCC2 might be responsible for the interaction between Cry1Ac and ABCC2. Moreover, CRISPR/Cas9-mediated genome editing technology was used to knockout ABCC2 gene. The homozygous mutant ABCC2 silkworm was resistant to Cry1Ac toxin, which indicated ABCC2 is the key gene that controls silkworm resistance to Cry1Ac toxin. The results have laid the foundation for elucidating the molecular resistance mechanism of silkworms to Cry1Ac toxin and could provide a theoretical basis for the biological control of lepidopteran pests.
苏云金芽孢杆菌（Bt）产生的伴孢晶体毒素（Bt 毒素）对多种昆虫具有特异性杀虫活性，已被广泛用于防治农业害虫。然而，其广泛使用导致了许多靶标昆虫产生了抗性。为了维持 Bt 产品的可持续使用，必须充分阐明昆虫对 Bt 毒素的抗性机制。本研究利用对 Bt 毒素产生抗性和敏感的家蚕品系构建遗传群体，明确了家蚕对 Cry1Ac 毒素的抗性遗传模式。采用序列标签位点（STS）分子标记技术精细定位抗性基因，并绘制了分子遗传连锁图谱，两个最接近的标记为 T1590 和 T1581，表明抗性基因位于 155kb 的遗传区域内。分析该遗传区域内预测基因的序列后，鉴定出一个三磷酸腺苷结合盒转运蛋白（ABCC2）为候选基因。分子建模和蛋白-蛋白对接结果显示，突变 ABCC2 中的一个酪氨酸插入可能是 Cry1Ac 与 ABCC2 相互作用的原因。此外，还利用 CRISPR/Cas9 介导的基因组编辑技术敲除了 ABCC2 基因。ABCC2 基因纯合突变的家蚕对 Cry1Ac 毒素表现出抗性，表明 ABCC2 是控制家蚕对 Cry1Ac 毒素抗性的关键基因。该研究结果为阐明家蚕对 Cry1Ac 毒素的分子抗性机制奠定了基础，可为鳞翅目害虫的生物防治提供理论依据。
