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首页> 外文期刊>Applied and Environmental Microbiology >Binding Analyses of Bacillus thuringiensis Cry δ-Endotoxins Using Brush Border Membrane Vesicles of Ostrinia nubilalis
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Binding Analyses of Bacillus thuringiensis Cry δ-Endotoxins Using Brush Border Membrane Vesicles of Ostrinia nubilalis

机译：苏云金芽孢杆菌Cryδ-内毒素的结合分析利用猪牙Ost的刷状缘膜囊泡

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摘要

Transgenic corn expressing the Bacillus thuringiensisCry1Ab gene is highly insecticidal to Ostrinia nubilalis(European corn borer) larvae. We ascertained whether Cry1F, Cry9C, or Cry9E recognizes the Cry1Ab binding site on the O. nubilalis brush border by three approaches. An optical biosensor technology based on surface plasmon resonance measured binding of brush border membrane vesicles (BBMV) injected over a surface of immobilized Cry toxin. Preincubation with Cry1Ab reduced BBMV binding to immobilized Cry1Ab, whereas preincubation with Cry1F, Cry9C, or Cry9E did not inhibit BBMV binding. BBMV binding to a Cry1F-coated surface was reduced when vesicles were preincubated in Cry1F or Cry1Ab but not Cry9C or Cry9E. A radioligand approach measured 125I-Cry1Ab toxin binding to BBMV in the presence of homologous (Cry1Ab) and heterologous (Cry1Ac, Cry1F, Cry9C, or Cry9E) toxins. Unlabeled Cry1Ac effectively competed for 125I-Cry1Ab binding in a manner comparable to Cry1Ab itself. Unlabeled Cry9C and Cry9E toxins did not inhibit 125I-Cry1Ab binding to BBMV. Cry1F inhibited125I-Cry1Ab binding at concentrations greater than 500 nM. Cry1F had low-level affinity for the Cry1Ab binding site. Ligand blot analysis identified Cry1Ab, Cry1Ac, and Cry1F binding proteins in BBMV. The major Cry1Ab signals on ligand blots were at 145 kDa and 154 kDa, but a strong signal was present at 220 kDa and a weak signal was present at 167 kDa. Cry1Ac and Cry1F binding proteins were detected at 220 and 154 kDa. Anti-Manduca sexta aminopeptidase serum recognized proteins of 145, 154, and 167 kDa, and anti-cadherin serum recognized the 220 kDa protein. We speculate that isoforms of aminopeptidase and cadherin in the brush border membrane serve as Cry1Ab, Cry1Ac, and Cry1F binding proteins.
机译：表达苏云金芽孢杆菌Cry1Ab基因的转基因玉米对欧洲玉米bore幼虫具有很高的杀虫性。我们通过三种方法确定了Cry1F，Cry9C或Cry9E是否识别O.nubilalis刷缘上的Cry1Ab结合位点。一种基于表面等离振子共振的光学生物传感器技术，测量了固定化Cry毒素表面上注入的刷状缘膜囊泡（BBMV）的结合。与Cry1Ab预温育可降低BBMV与固定化Cry1Ab的结合，而与Cry1F，Cry9C或Cry9E预温育不会抑制BBMV的结合。当将囊泡在Cry1F或Cry1Ab中而不是Cry9C或Cry9E中预孵育时，BBMV与Cry1F包被的表面的结合减少。放射性配体方法在存在同源（Cry1Ab）和异源（Cry1Ac，Cry1F，Cry9C或Cry9E）毒素的情况下，测量了125I-Cry1Ab毒素与BBMV的结合。未标记的Cry1Ac以与Cry1Ab本身相当的方式有效竞争125I-Cry1Ab结合。未标记的Cry9C和Cry9E毒素不抑制125I-Cry1Ab与BBMV的结合。 Cry1F在浓度大于500 nM时抑制了125I-Cry1Ab的结合。 Cry1F对Cry1Ab结合位点具有低水平的亲和力。配体印迹分析确定了BBMV中的Cry1Ab，Cry1Ac和Cry1F结合蛋白。配体印迹上的主要Cry1Ab信号为145 kDa和154 kDa，但在220 kDa处存在强信号，在167 kDa处存在弱信号。在220和154 kDa处检测到Cry1Ac和Cry1F结合蛋白。抗Manduca sexta氨肽酶血清可识别145、154和167 kDa的蛋白质，而抗钙粘蛋白血清可识别220 kDa的蛋白质。我们推测在刷状缘膜中的氨基肽酶和钙黏着蛋白的同工型充当Cry1Ab，Cry1Ac和Cry1F结合蛋白。

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