Department of Entomology, Cornell University, Geneva, NY, 14456, USA.
Boyce Thompson Institute, Ithaca, NY, 14853, USA.
Insect Biochem Mol Biol. 2022 Jan;140:103678. doi: 10.1016/j.ibmb.2021.103678. Epub 2021 Nov 13.
The three-domain Cry toxin Cry1Ac from Bacillus thuringiensis （Bt） is an important insecticidal toxin in Bt sprays and has been used in transgenic Bt-crops to confer insect resistance. The cabbage looper, Trichoplusia ni, has developed resistance to Bt sprays in commercial greenhouses, and the resistance to Cry1Ac has been previously identified to be associated with altered expression of the APN1 and APN6 genes and be genetically linked to a locus on chromosome 15. In this study, the Cry1Ac resistance locus in T. ni was further finely mapped, and the specific Cry1Ac resistance-conferring mutation in the resistance locus was identified to be a 4 bp frameshift insertion in the ABCC2 gene by whole genome resequencing, midgut transcriptome analysis, candidate gene cDNA sequencing and mutation site genomic DNA sequencing. By CRISPR/Cas9 mutagenesis, a series of ABCC2 and ABCC3 mutant T. ni strains were generated, and the role of ABCC2 in the toxicity of Cry1Ac in T. ni was confirmed. The results from this study also showed that knockout of ABCC2 in T. ni conferred resistance to Cry1Ac at a level lower than that in the greenhouse-derived resistant T. ni strain and that the Cry1Ac resistance-associated alteration of APN1 and APN6 expression was independent of ABCC2 gene mutations, indicating that the altered expression of APN1 and APN6 was controlled by another gene mutation in Cry1Ac resistant T. ni. Furthermore, T. ni larval bioassays showed that the level of Cry1Ac resistance in F families from reciprocal crosses of the Cry1Ac resistant strain with an ABCC2 knockout CRISPR strain was significantly higher than that in ABCC2 knockout strain, indicating the presence of additional Cry1Ac resistance-conferring mutation(s) in the Cry1Ac resistant strain. Therefore, the resistance to Cry1Ac in T. ni is conferred by a mutation in ABCC2 and an additional mutation (or mutations) which leads to altered expression of APN1 and APN6. The additional Cry1Ac resistance mutation or mutations remain to be identified.
苏云金芽孢杆菌（Bt）的三域 Cry1Ac 毒素是 Bt 喷雾中的一种重要杀虫毒素，已被用于转基因 Bt 作物中以赋予昆虫抗性。白菜夜蛾（Trichoplusia ni）在商业温室中对 Bt 喷雾产生了抗性，并且先前已经确定 Cry1Ac 抗性与 APN1 和 APN6 基因表达的改变有关，并且与染色体 15 上的一个基因座遗传连锁。在这项研究中，进一步对 T. ni 中的 Cry1Ac 抗性基因座进行了精细定位，通过全基因组重测序、中肠转录组分析、候选基因 cDNA 测序和突变位点基因组 DNA 测序，确定了抗性基因座中特定的 Cry1Ac 抗性赋予突变是 ABCC2 基因中的 4bp 移框插入。通过 CRISPR/Cas9 诱变，生成了一系列 ABCC2 和 ABCC3 突变的 T. ni 菌株，并证实了 ABCC2 在 T. ni 中 Cry1Ac 毒性中的作用。这项研究的结果还表明，T. ni 中 ABCC2 的敲除赋予了对 Cry1Ac 的抗性，其水平低于从温室获得的抗性 T. ni 菌株，并且与 ABCC2 基因突变无关的 APN1 和 APN6 表达的 Cry1Ac 抗性改变表明，APN1 和 APN6 的表达改变由 Cry1Ac 抗性 T. ni 中的另一个基因突变控制。此外，T. ni 幼虫生物测定表明，来自 Cry1Ac 抗性菌株与 ABCC2 敲除 CRISPR 菌株的正反交 F 代的 Cry1Ac 抗性水平明显高于 ABCC2 敲除菌株，表明 Cry1Ac 抗性菌株中存在额外的 Cry1Ac 抗性赋予突变（或突变）。因此，T. ni 对 Cry1Ac 的抗性是由 ABCC2 中的突变和导致 APN1 和 APN6 表达改变的额外突变（或突变）赋予的。额外的 Cry1Ac 抗性突变或突变仍有待确定。
