Tanaka Shiho, Miyamoto Kazuhisa, Noda Hiroaki, Endo Haruka, Kikuta Shingo, Sato Ryoichi
Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Koganei, Tokyo, Japan.
National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, Japan.
Peptides. 2016 Apr;78:99-108. doi: 10.1016/j.peptides.2016.01.006. Epub 2016 Feb 27.
In a previous report, seven Cry1Ab-resistant strains were identified in the silkworm, Bombyx mori; these strains were shown to have a tyrosine insertion at position 234 in extracellular loop 2 of the ABC transporter C2 (BmABCC2). This insertion was confirmed to destroy the receptor function of BmABCC2 and confer the strains resistance against Cry1Ab and Cry1Ac. However, these strains were susceptible to Cry1Aa. In this report, we examined the mechanisms of the loss of receptor function of the transporter by expressing mutations in Sf9 cells. After replacement of one or two of the five amino acid residues in loop 2 of the susceptible BmABCC2 gene [BmABCC2_S] with alanine, cells still showed susceptibility, retaining the receptor function. Five mutants with single amino acid insertions at position 234 in BmABCC2 were also generated, resulting in loop 2 having six amino acids, which corresponds to replacing the tyrosine insertion in the resistant BmABCC2 gene [BmABCC2_R(+(234)Y)] with another amino acid. All five mutants exhibited loss of function against Cry1Ab and Cry1Ac. These results suggest that the amino acid sequence in loop 2 is less important than the loop size (five vs. six amino acids) or loop structure for Cry1Ab and Cry1Ac activity. Several domain-swapped mutant toxins were then generated among Cry1Aa, Cry1Ab, and Cry1Ac, which are composed of three domains. Swapped mutants containing domain II of Cry1Ab or Cry1Ac did not kill Sf9 cells expressing BmABCC2_R(+(234)Y), suggesting that domain II of the Cry toxin is related to the interaction with the receptor function of BmABCC2. This also suggests that different reactions against Bt-toxins in some B. mori strains, that is, Cry1Ab resistance or Cry1Aa susceptibility, are attributable to structural differences in domain II of Cry1A toxins.
在之前的一份报告中，在家蚕（Bombyx mori）中鉴定出了7种对Cry1Ab具有抗性的品系；这些品系在ABC转运蛋白C2（BmABCC2）细胞外环2的第234位有一个酪氨酸插入。该插入被证实破坏了BmABCC2的受体功能，并赋予这些品系对Cry1Ab和Cry1Ac的抗性。然而，这些品系对Cry1Aa敏感。在本报告中，我们通过在Sf9细胞中表达突变来研究转运蛋白受体功能丧失的机制。在用丙氨酸替换敏感的BmABCC2基因[BmABCC2_S]环2中的五个氨基酸残基中的一个或两个后，细胞仍然表现出敏感性，保留了受体功能。还产生了五个在BmABCC2第234位有单个氨基酸插入的突变体，导致环2有六个氨基酸，这相当于用另一种氨基酸替换抗性BmABCC2基因[BmABCC2_R(+(234)Y)]中的酪氨酸插入。所有五个突变体对Cry1Ab和Cry1Ac均表现出功能丧失。这些结果表明，对于Cry1Ab和Cry1Ac的活性，环2中的氨基酸序列不如环大小（五个与六个氨基酸）或环结构重要。然后在由三个结构域组成的Cry1Aa、Cry1Ab和Cry1Ac之间产生了几种结构域交换突变毒素。含有Cry1Ab或Cry1Ac结构域II的交换突变体不能杀死表达BmABCC2_R(+(234)Y)的Sf9细胞，这表明Cry毒素的结构域II与BmABCC2的受体功能相互作用有关。这也表明，一些家蚕品系对Bt毒素的不同反应，即Cry1Ab抗性或Cry1Aa敏感性，归因于Cry1A毒素结构域II的结构差异。
