USDA ARS, U.S. Arid Land Agricultural Research Center, 21881 N. Cardon Lane, Maricopa, AZ, 85138, USA.
Department of Entomology, University of Arizona, Tucson, AZ, 85721, USA.
Sci Rep. 2022 Oct 6;12(1):16706. doi: 10.1038/s41598-022-21061-2.
Evolution of pest resistance reduces the benefits of widely cultivated genetically engineered crops that produce insecticidal proteins derived from Bacillus thuringiensis (Bt). Better understanding of the genetic basis of pest resistance to Bt crops is needed to monitor, manage, and counter resistance. Previous work shows that in several lepidopterans, resistance to Bt toxin Cry2Ab is associated with mutations in the gene encoding the ATP-binding cassette protein ABCA2. The results here show that mutations introduced by CRISPR/Cas9 gene editing in the Helicoverpa zea (corn earworm or bollworm) gene encoding ABCA2 (HzABCA2) can cause resistance to Cry2Ab. Disruptive mutations in HzABCA2 facilitated the creation of two Cry2Ab-resistant strains. A multiple concentration bioassay with one of these strains revealed it had > 200-fold resistance to Cry2Ab relative to its parental susceptible strain. All Cry2Ab-resistant individuals tested had disruptive mutations in HzABCA2. We identified five disruptive mutations in HzABCA2 gDNA. The most common mutation was a 4-bp deletion in the expected Cas9 guide RNA target site. The results here indicate that HzABCA2 is a leading candidate for monitoring Cry2Ab resistance in field populations of H. zea.
昆虫对苏云金芽孢杆菌（Bt）杀虫蛋白产生的抗药性的进化降低了广泛种植的基因工程作物的效益。为了监测、管理和对抗抗药性，需要更好地了解昆虫对 Bt 作物产生抗药性的遗传基础。以前的工作表明，在几种鳞翅目昆虫中，对 Bt 毒素 Cry2Ab 的抗性与编码 ABCA2 蛋白的基因中的突变有关。这里的结果表明，CRISPR/Cas9 基因编辑在编码 ABCA2（HzABCA2）的 Helicoverpa zea（玉米穗虫或棉铃虫）基因中引入的突变可导致对 Cry2Ab 的抗性。HzABCA2 中的破坏突变有助于创建两个 Cry2Ab 抗性株系。对其中一个株系进行的多浓度生物测定显示，与亲本敏感株系相比，其对 Cry2Ab 的抗性超过 200 倍。所有测试的 Cry2Ab 抗性个体在 HzABCA2 中均存在破坏突变。我们在 HzABCA2 的 gDNA 中鉴定出五个破坏突变。最常见的突变是在预期的 Cas9 向导 RNA 靶位点处发生 4 个碱基对的缺失。这里的结果表明，HzABCA2 是监测 H. zea 田间种群中 Cry2Ab 抗性的一个主要候选基因。
