Department of Entomology, Cornell University, New York State Agricultural Experiment Station, Geneva, NY, 14456, USA.
Boyce Thompson Institute, Ithaca, NY, 14853, USA.
Insect Biochem Mol Biol. 2019 Sep;112:103209. doi: 10.1016/j.ibmb.2019.103209. Epub 2019 Aug 15.
Insecticidal proteins from Bacillus thuringiensis (Bt) are the primary recombinant proteins expressed in transgenic crops (Bt-crops) to confer insect resistance. Development of resistance to Bt toxins in insect populations threatens the sustainable application of Bt-crops in agriculture. The Bt toxin Cry2Ab is a major insecticidal protein used in current Bt-crops, and resistance to Cry2Ab has been selected in several insects, including the cabbage looper, Trichoplusia ni. In this study, the Cry2Ab resistance gene in T. ni was mapped to Chromosome 17 by genetic linkage analyses using a whole genome resequencing approach, and was then finely mapped using RNA-seq-based bulked segregant analysis (BSA) and amplicon sequencing (AmpSeq)-based fine linkage mapping to a locus containing two genes, ABCA1 and ABCA2. Mutations in ABCA1 and ABCA2 in Cry2Ab resistant T. ni were identified by both genomic DNA and cDNA sequencing. Analysis of the expression of ABCA1 and ABCA2 in T. ni larvae indicated that ABCA2 is abundantly expressed in the larval midgut, but ABCA1 is not a midgut-expressed gene. The mutation in ABCA2 in Cry2Ab resistant T. ni was identified to be an insertion of a transposon Tntransib in ABCA2. For confirmation of ABCA2 as the Cry2Ab-resistance gene, T. ni mutants with frameshift mutations in ABCA1 and ABCA2 were generated by CRISPR/Cas9 mutagenesis. Bioassays of the T. ni mutants with Cry2Ab verified that the mutations of ABCA1 did not change larval susceptibility to Cry2Ab, but the ABCA2 mutants were highly resistant to Cry2Ab. Genetic complementation test of the ABCA2 allele in Cry2Ab resistant T. ni with an ABCA2 mutant generated by CRISPR/Cas9 confirmed that the ABCA2 mutation in the Cry2Ab resistant strain confers the resistance. The results from this study confirmed that ABCA2 is essential for the toxicity of Cry2Ab in T. ni and mutation of ABCA2 confers the resistance to Cry2Ab in the resistant T. ni strain derived from a Bt resistant greenhouse population.
苏云金芽孢杆菌（Bt）的杀虫蛋白是转基因作物（Bt 作物）中表达的主要重组蛋白，用于赋予昆虫抗性。昆虫种群对 Bt 毒素产生抗性，威胁到 Bt 作物在农业中的可持续应用。Cry2Ab 毒素是当前 Bt 作物中使用的主要杀虫蛋白，包括斜纹夜蛾（Trichoplusia ni）在内的几种昆虫已对 Cry2Ab 产生抗性。在这项研究中，通过全基因组重测序方法进行遗传连锁分析，将 T. ni 中的 Cry2Ab 抗性基因定位到染色体 17 上，然后使用 RNA-seq 为基础的 bulked segregant analysis (BSA) 和基于扩增子测序（AmpSeq）的精细连锁作图将其精细定位到包含两个基因 ABCA1 和 ABCA2 的基因座上。通过基因组 DNA 和 cDNA 测序鉴定 Cry2Ab 抗性 T. ni 中的 ABCA1 和 ABCA2 突变。分析 T. ni 幼虫中 ABCA1 和 ABCA2 的表达表明，ABCA2 在幼虫中肠中大量表达，但 ABCA1 不是中肠表达基因。Cry2Ab 抗性 T. ni 中的 ABCA2 突变被鉴定为 ABCA2 中的转座子 Tntransib 插入。为了确认 ABCA2 是 Cry2Ab 抗性基因，通过 CRISPR/Cas9 诱变产生了 ABCA1 和 ABCA2 移码突变的 T. ni 突变体。Cry2Ab 对 T. ni 突变体的生物测定证实，ABCA1 的突变不会改变幼虫对 Cry2Ab 的敏感性，但 ABCA2 突变体对 Cry2Ab 具有高度抗性。通过 CRISPR/Cas9 产生的 ABCA2 突变体对 Cry2Ab 抗性 T. ni 中的 ABCA2 等位基因进行遗传互补测试，证实了 Cry2Ab 抗性株系中的 ABCA2 突变赋予了对 Cry2Ab 的抗性。本研究结果证实，ABCA2 是 Cry2Ab 在 T. ni 中毒性所必需的，ABCA2 的突变赋予了源自 Bt 抗性温室种群的抗性 T. ni 菌株对 Cry2Ab 的抗性。
