Jurat-Fuentes Juan L, Adang Michael J
Department of Entomology and Plant Pathology, University of Tennessee, Knoxville, TN 37996-4560, USA.
J Invertebr Pathol. 2007 Jul;95(3):187-91. doi: 10.1016/j.jip.2007.01.008. Epub 2007 Mar 25.
Binding of the Bacillus thuringiensis Cry1Ac toxin to specific receptors in the midgut brush border membrane is required for toxicity. Alteration of these receptors is the most reported mechanism of resistance. We used a proteomic approach to identify Cry1Ac binding proteins from intestinal brush border membrane (BBM) prepared from Heliothis virescens larvae. Cry1Ac binding BBM proteins were detected in 2D blots and identified using peptide mass fingerprinting (PMF) or de novo sequencing. Among other proteins, the membrane bound alkaline phosphatase (HvALP), and a novel phosphatase, were identified as Cry1Ac binding proteins. Reduction of HvALP expression levels correlated directly with resistance to Cry1Ac in the YHD2-B strain of H. virescens. To study additional proteomic alterations in resistant H. virescens larvae, we used two-dimensional differential in-gel electrophoresis (2D-DIGE) to compare three independent resistant strains with a susceptible strain. Our results validate the use of proteomic approaches to identify toxin binding proteins and proteome alterations in resistant insects.
苏云金芽孢杆菌Cry1Ac毒素与中肠刷状缘膜上的特定受体结合是产生毒性所必需的。这些受体的改变是最常报道的抗性机制。我们采用蛋白质组学方法从绿铃夜蛾幼虫制备的肠道刷状缘膜（BBM）中鉴定Cry1Ac结合蛋白。在二维印迹中检测到Cry1Ac结合的BBM蛋白，并使用肽质量指纹图谱（PMF）或从头测序进行鉴定。在其他蛋白质中，膜结合碱性磷酸酶（HvALP）和一种新型磷酸酶被鉴定为Cry1Ac结合蛋白。在绿铃夜蛾的YHD2-B品系中，HvALP表达水平的降低与对Cry1Ac的抗性直接相关。为了研究抗性绿铃夜蛾幼虫的其他蛋白质组学变化，我们使用二维差异凝胶电泳（2D-DIGE）来比较三个独立的抗性品系和一个敏感品系。我们的结果验证了使用蛋白质组学方法来鉴定抗性昆虫中的毒素结合蛋白和蛋白质组变化。
