Proteomic Analysis Of Larval Midgut And Identification Of Cry1Ab Binding Proteins In Chilo Suppressalis (Walker)

The striped stem borer, Chilo suppressalis (Walker) is one of the most serious pests threatening rice production in China. The striped stem borer has developed resistance to chemical insecticides with extensive use of insecticides. Transgenic Bt rice offers a new effective and safe weapon to control rice stem borers including C. suppressalis. Although Bt rice has not been released for commercial planting in China, it is important and necessary to study potential resistance risk and resistance mechanisms of the target pests. Insect midgut is the major digestive organs and the important target of unfavorable xenobiotics (such as toxins, pesticides, and pathogenic microorganisms), and alteration of Bt toxin receptors in insect midguts is recognized as a key mechanism of resistance. In order to understand basic knowledge of midgut proteins, we combined two-dimensional electrophoresis and mass spectrometry to separate and identify proteome of midgut of the striped stem borer larvae. Further, we separated and identified CrylAb binding proteins in BBMV of the midgut by using ligand blotting. The results from present study would provide methodological and technical preparation for investigating mode of action and resistance mechanisms of CrylAb in C. suppressalis in future.1. Proteomic analysis of midgut of C. suppressalis larvaeMidgut proteins were prepared from the5th instar larvae. The midgut protein preparation was separated by using two-dimensional electrophoresis, and about300protein spots were seperated. These protein spots were distributed mainly in pI5-8and the area of MW from10-100kDa. Most of the spots were located in acidic area.One hundred and three protein spots were digested by trypsin and analysised by MALDI-TOF/TOF. We identified71proteins from these spots. According to the predicted physiological functions of these proteins, they were divided into six types:protein metabolism, carbohydrate metabolism, energy metabolism, nutrition storge, muscle related proteins, and other proteins. 2. Identification of CrylAb binding proteins in BBMVs of C. suppressalis larvaeMidgut BBMV proteins were prepared from the midguts of C. suppressalis. Two-dimensional electrophoresis and Ligand blotting were used to separate and detect the CrylAb-binding proteins, and MALDI-TOF mass spectrometry was used to identify these proteins. When the midgut BBMVs were blotted with streptavidin, no binding protein was detected. It suggests there is no endogeneous biotin present in C. suppressalis midgut BBMVs. Twenty five CrylAb binding protein spots were detected by two-dimensional electrophoresis and ligand blotting with streptavidin. These proteins distributed in the area between40-100kDa.By matching two-dimensional electrophoresis and ligand blot spots, we selected17points for mass spectrometry identification. Eight proteins were successfully identified. These8Cry1Ab binding proteins included actin, V-ATPase subunit A, V-ATPase subunit B, CALNUC, NADH-ubiquinone oxidoreductase75kDa subunit, ETF-ubiquinone oxidoreductase, Enolase, and an uncharacterized protein.