College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China.
Toxins (Basel). 2020 Apr 11;12(4):246. doi: 10.3390/toxins12040246.
The adoption of transgenic crops expressing (Bt) insecticidal crystalline (Cry) proteins has reduced insecticide application, increased yields, and contributed to food safety worldwide. However, the efficacy of transgenic Bt crops is put at risk by the adaptive resistance evolution of target pests. Previous studies indicate that resistance to Cry1A and Cry1F toxins was genetically linked with mutations of ATP-binding cassette (ABC) transporter subfamily C gene in at least seven lepidopteran insects. Several strains selected in the laboratory of the Asian corn borer, , a destructive pest of corn in Asian Western Pacific countries, developed high levels of resistance to Cry1A and Cry1F toxins. The causality between the () gene and resistance to Cry1A and Cry1F toxins remains unknown. Here, we successfully generated a homozygous strain (OfC2-KO) of with an 8-bp deletion mutation of by the CRISPR/Cas9 approach. The 8-bp deletion mutation results in a frame shift in the open reading frame of transcripts, which produced a predicted protein truncated in the TM4-TM5 loop region. The knockout strain OfC2-KO showed much more than a 300-fold resistance to Cry1Fa, and low levels of resistance to Cry1Ab and Cry1Ac (<10-fold), but no significant effects on the toxicities of Cry1Aa and two chemical insecticides (abamectin and chlorantraniliprole), compared to the background NJ-S strain. Furthermore, we found that the Cry1Fa resistance was autosomal, recessive, and significantly linked with the 8-bp deletion mutation of in the OfC2-KO strain. In conclusion, functional investigation demonstrates the causality of the truncating mutation with high-level resistance to the Cry1Fa toxin in . Our results suggest that the protein might be a functional receptor for Cry1Fa and reinforces the association of this gene to the mode of action of the Cry1Fa toxin.
转 Bt 杀虫晶体蛋白基因作物的采用减少了杀虫剂的使用，提高了产量，有助于全球食品安全。然而，目标害虫的适应性抗药性进化使转基因 Bt 作物的功效面临风险。先前的研究表明，至少有七种鳞翅目昆虫的 ABC 转运蛋白亚家族 C 基因的突变与对 Cry1A 和 Cry1F 毒素的抗性遗传相关。在亚洲玉米螟的实验室中选择的几个品系，亚洲西太平洋国家玉米的破坏性害虫，对 Cry1A 和 Cry1F 毒素产生了高水平的抗性。基因 ()与对 Cry1A 和 Cry1F 毒素的抗性之间的因果关系尚不清楚。在这里，我们成功地通过 CRISPR/Cas9 方法生成了一个具有 8-bp 缺失突变的品系（OfC2-KO）。8-bp 缺失突变导致转录本的开放阅读框发生移码，从而产生 TM4-TM5 环区截断的预测蛋白。敲除株 OfC2-KO 对 Cry1Fa 的抗性超过 300 倍，对 Cry1Ab 和 Cry1Ac 的抗性较低（<10 倍），但对 Cry1Aa 和两种化学杀虫剂（阿维菌素和氯虫苯甲酰胺）的毒性没有显著影响与背景 NJ-S 株相比。此外，我们发现 Cry1Fa 抗性是常染色体隐性的，与 OfC2-KO 株中 8-bp 缺失突变显著相关。总之，功能研究表明基因 ()截断突变与对 Cry1Fa 毒素的高水平抗性之间存在因果关系。我们的结果表明，该蛋白可能是 Cry1Fa 毒素的功能受体，并加强了该基因与 Cry1Fa 毒素作用模式的关联。
