Krishnamoorthy M, Jurat-Fuentes J L, McNall R J, Andacht T, Adang Michael J
Departments of Entomology, University of Georgia, Athens, GA 30602-2603, USA.
Insect Biochem Mol Biol. 2007 Mar;37(3):189-201. doi: 10.1016/j.ibmb.2006.10.004. Epub 2006 Oct 28.
Proteins such as aminopeptidases and alkaline phosphatases, both glycosyl-phosphatidyl-inositol (GPI) anchored proteins, were previously identified as Cry1Ac binding proteins in the Heliothis virescens midgut. To identify additional toxin binding proteins, brush border membrane vesicles from H. virescens larvae were treated with phosphatidyl inositol phospholipase C, and released proteins were resolved by two-dimensional electrophoresis. Protein spots selected by their ability to bind Cry1Ac were identified by MALDI-TOF mass spectrometry coupled to peptide mass fingerprinting (PMF) and database searching. As in previous studies, H. virescens alkaline phosphatase was identified as a Cry1Ac binding protein. V-ATP synthase subunit A and actin were identified as novel Cry1Ac binding proteins in H. virescens. Additional toxin-binding proteins were predicted based on MS/MS fragmentation and de novo sequencing, providing amino acid sequences that were used in database searches to identify a phosphatase and a putative protein of the cadherin superfamily as additional Cry1Ac binding proteins.
诸如氨肽酶和碱性磷酸酶等蛋白质，二者均为糖基磷脂酰肌醇（GPI）锚定蛋白，先前在烟芽夜蛾中肠中被鉴定为Cry1Ac结合蛋白。为了鉴定其他毒素结合蛋白，用磷脂酰肌醇磷脂酶C处理烟芽夜蛾幼虫的刷状缘膜囊泡，释放出的蛋白质通过二维电泳进行分离。通过与肽质量指纹图谱（PMF）联用的基质辅助激光解吸电离飞行时间质谱（MALDI-TOF）和数据库搜索，鉴定出具有结合Cry1Ac能力的蛋白质斑点。与先前的研究一样，烟芽夜蛾碱性磷酸酶被鉴定为Cry1Ac结合蛋白。V-ATP合酶亚基A和肌动蛋白被鉴定为烟芽夜蛾中新的Cry1Ac结合蛋白。基于串联质谱（MS/MS）片段分析和从头测序预测了其他毒素结合蛋白，所提供的氨基酸序列用于数据库搜索，以鉴定一种磷酸酶和一种钙黏蛋白超家族的推定蛋白作为其他Cry1Ac结合蛋白。
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