ABC转运蛋白介导亚洲玉米螟调控Cry1Ab抗性的分子机制研究

转Bt基因玉米的长期大量种植使害虫产生抗药性。亚洲玉米螟是我国玉米上最严重的害虫，转Bt基因玉米为其危害提供了可行防控策略。前期研究证明94个ABC转运蛋白中有3个，即ABCC2、ABCC3和ABCG1(简称ABCs)参与玉米螟对Cry1Ab的抗性调控。研究目的为阐明ABCs介导玉米螟调控Cry1Ab的抗性机制。提出假设为ABCs在调节Cry1Ab抗性中具有独立的功能和协同增效作用。研究目标为①克隆感/抗玉米螟ABCs基因，分析抗性位点，通过Sf9细胞表达证明其独立的受体作用; ②原核表达感/抗ABCs，确定突变与毒素的结合力变化导致抗性；③通过CRISPR/Cas9构建ABCs纯合体，研究ABCs对Cry1Ab的体内功能性作用；④通过双ABCs突变体和竞争结合实验证明ABCs对Cry1Ab具有协同增效作用。本研究将为抗性治理确定新的分子靶点，促进Bt制剂和转Bt基因作物的长期可持续利用。

Long-term and large-scale cultivation of Bt transgenic maize have led the vast development of Bt resistance in the field. The Asian corn borer, Ostrinia furnacalis (Guenée), is the most devastating insect pest on maize in China, and Bt transgenic maize offers a viable control alternative to combat O. furnacalis infestation. In the proceeding phase of the research, the applicant demonstrated that 3 out of 94 ABC transporters, namely ABCC2, ABCC3, and ABCG1, were involved in Cry1Ab resistance in O. furnacalis. The overall goal of this proposed research is to characterize the mechanism underlying Cry1Ab resistance mediated by ABC transporters in O. furnacalis. We hypothesize that ABCC2, ABCC3, and ABCG1 have independent roles but function synergistically in regulating Cry1Ab resistance. To examine this overarching hypothesis, we propose to carry out the following objectives: ①By cloning the ABCC2, ABCC3, and ABCG1 genes from susceptible and resistant strain of O. furnacalis, we demonstrate their independent roles as Cry1Ab toxin receptors via in vitro cell expression system; identify the mutation sites leading to altered susceptibility to Cry1Ab toxin. ②Construct recombinant ABCs and perform binding assay with Cry1Ab to measure binding ability of resistant and susceptible ABCs to Cry1Ab to know if the mutation led to altered binding affinity, as a result leading to toxin resistance. ③Establish homozygous strains of ABCC2, ABCC3, and ABCG1 through CRISPR/Cas9 gene editing system to investigate their in vivo roles as Cry1Ab receptors. ④Demonstrate the synergistic effects of ABCs in mediating O. furnacalis against Cry1Ab toxin through combination by cross to construct double ABC mutant strains, and protein-protein analysis through competition binding assay. .A better understanding of the mechanisms governing the Bt resistance in O. furnacalis will allow us to identify novel molecular targets for resistance management strategies, and facilitate the long-term sustainable management of O. furnacalis using Bt biopesticides and Bt transgenic traits.