棉铃虫对Bt毒素Cry1Ac和Cry2Ab交互抗性的遗传多样性及机理

Pyramided Bt crops produce two or more distinct Bt toxins that kill the same insect pest. The pyramiding strategy for Bt resistance management is expected to delay resistance evolution on the basis of “redundant killing”, which means the target insects will be killed twice. Target pests are possible to adapt to the pyramided Bt crops through evolution of cross-resistance to two or more Bt toxins, so it is very crucial to understand which mechanisms and pathways of cross-resistance the target pest will evolve to cope with multiple Bt toxins. To address this key scientific question, molecular mechanisms of cross-resistance to Bt Cry toxins Cry1Ac and Cry2Ab will be investigated in several laboratory-selected and field-derived resistant strains of Helicoverpa armigera. Multiple approaches, including positional cloning, association analysis of transcriptomic and proteomic data and interaction analysis between Bt toxins and midgut receptors, will be employed to elucidate genetic diversity and the underlying mechanisms of cross-resistance to Cry1Ac and Cry2Ab in H. armigera. Our results are not only expected to improve present models for mode of action of and resistance mechanisms to Bt Cry toxins, but also to provide the theoretical basis for both optimizing Bt gene pyramiding and developing new tactics for Bt resistance management in target insect pests in future.

在同一种作物中转入针对同一种害虫的两个或多个不同类型的Bt基因，对靶标害虫实现“饱和杀死”，从而可以延缓抗性发展，该抗性治理策略称为“基因聚合策略”。靶标害虫可以进化出交互抗性适应此策略，因此，弄清靶标害虫通过何种机制形成稳定遗传的交互抗性来适应多基因Bt作物是成功实施基因聚合策略的关键。为了探究这一重要科学问题，本项目将在建立室内筛选的以及田间分离的多个棉铃虫对Cry1Ac和Cry2Ab交互抗性品系的基础上，以抗性相关基因的高通量筛选为切入点，通过基因定位、转录组和蛋白组数据关联分析和毒素受体互作分析等技术手段揭示Bt毒素的毒理学机制和交互抗性机理，阐明棉铃虫对不同Bt毒素交互抗性的分子遗传机制。项目成果不仅可以提出Bt作用机制及交互抗性机理的新模式，同时还为Bt基因聚合抗虫作物选育中Bt基因的科学搭配及靶标害虫抗性治理新策略的研发提供重要理论依据。

在同一种作物中转入针对同一种害虫的两个或多个不同类型的Bt基因，对靶标害虫实现“饱和杀死”，从而可以延缓抗性发展，该抗性治理策略称为“基因聚合策略”。靶标害虫可以进化出交互抗性适应此策略。为了探究这一重要科学问题，本项目在建立室内筛选以及田间分离的多个棉铃虫对Cry1Ac和Cry2Ab交互抗性品系的基础上，通过基因定位、CRISPR/Cas9等技术手段揭示了Bt毒素作用机制和交互抗性机理。.通过田间分离与室内筛选相结合，共培育了3个对Cry1Ac和Cry2Ab具有不同程度交互抗性品系（AN-R, AY2-R及SCD-DR）。AY2-R品系的Bt抗性基因位于10号染色体10.80 Mb区域，AN-R和SCD-DR品系对Cry2Ab/Cry1Ac抗性主要与9号染色体7.81Mb区域连锁。该结果揭示了棉铃虫对Cry1Ac和Cry2Ab交互抗性的遗传多样性。.对AY2-R品系Chr10上的Bt抗性基因进行精细定位和图位克隆，发现HaTSPAN1基因点突变（T92C，导致L31S氨基酸突变）与AY2-R品系对Cry1Ac和Cry2Ab抗性紧密连锁。利用棉铃虫基因组编辑技术，证实了HaTSPAN1基因L31S点突变与AY2-R品系Cry1Ac抗性的因果关系。通过扩增子检测法对T92C突变检测结果表明，2016年黄河流域和长江流域棉区棉铃虫田间种群T92C突变的平均频率高达10%，较2006年的0.1%上升了100倍。模型模拟结果表明T92C突变频率变化的预测值与实际观测值相吻合，表明庇护所策略无法有效延缓显性抗性演化。.对AN-R和SCD-DR品系Chr09上的Cry2Ab/Cry1Ac抗性基因进行精细定位和图位克隆，发现编码胰凝乳蛋白酶类似物的基因HaOG200431上的两个点突变（E20G和d271I）与抗性连锁。通过CRISPR技术敲除该基因，抗性品系对2种Bt毒素的敏感性得到显著恢复，表明该基因突变与抗性相关。.上述3个抗性品系对Cry1Ac和Cry2Ab均为偏显性遗传。与敏感品系相比，抗性品系幼虫中肠蛋白酶、中肠BBMV与两种Bt毒素的互作未表现出显著差异。中肠组织病理研究表明，中肠细胞增殖是AY2-R品系L31S点突变导致Cry1Ac抗性的重要机理。