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Research of Biochemical Characteristics of Lipid Raft in Larval Midgut of Helicoverpa Armigera H(?)bner and the Immunodetection of Cadherin

Author: WeiHongWei
Tutor: SuJianYa
School: Nanjing Agricultural College
Course: Pesticides
Keywords: Helicoverpa armigera BBMV lipid raft enzyme activity ELISA Dot-blot Western blotting
CLC: S435.622.3
Type: Master's thesis
Year: 2009
Downloads: 2
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Abstract

Lipid raft is a type of microdomain which enriches in cholesterol, sphingolipids, and glycosylphosphatidylinositol-anchored proteins. It is considered as the platform on the surface of cell membrane that pore-forming toxins aggregating and oligomers forming. Lipid rafts are characterizaed by the insolubility in detergents at 4℃.We used non-ionic detergent (Triton X-100) to treat with BBMV of Lepidoptera larval, the non-raft membrane would be solubilized. Then we utilized Optiprep gradient centrifugation, so that preparations of lipid rafts could be obtained. Glycosphingolipid (GM1) was used as the marker of lipid raft. GM1 dot revealed by probing with cholera toxin subunit B coupled to horse radish peroxidase (CTB-HRP) and followed by chemiluminiscent HRP substrate. Our results suggest that lipid rafts in brush border membrane vesicle from four kinds of Lepidoptera larval were successfully extracted.Enzymes from midgut juice, BBMV and lipid raft of BBMV were compared between Bacillus thuringiensis-susceptible and -resistant Helicoverpa armigera. Serine proteins, as trypsin and chymotrypsin, performed more activities in midgut juice than BBMV and more activities in BBMV than lipid raft, while aminopeptidase acitvies was opposite. To compare Bt susceptible & resistant strains, we found that most of the enzymes had higher activities in susceptible part, except alkaline phosphatase in midgut juice and total proteinase in lipid raft.Insect molecular systematics was complemented and enhanced the value of morphological and ecological data, but we found there was nearly no method referring to the enzyme detection field. In our study, we figured out that four kinds of lepidoptera insect larvae performed the same tendency of enzyme difference in four important enzymes. In hence, an udacious hypothesis that the enzyme analysis of BBMV and lipid raft in the insect larval was a new approach to study the relationships between insect species. Here we mention about the preparation of lipid raft referring to four kinds of lepidopteran larval-Helicoverpa armigera, Prodenia litura, Pieris Brassicae, Chilo suppressalis, and the analysis of the trypsin, chymotrypsin, aminopeptidase, alkaline phosphatase activities in therush border membrane vesicles (BBMVs) and lipid rafts of them. In the study, distributions of the enzymes and receptors in midgut played an important role on demonstrating the resistant mechanism of Bt toxin to insects. As an aspect to discuss the difference of enzymes and receptors on BBMV and lipid raft between four kinds of lepidopteran larval, we suggested a new way in detecting the evolution of insects.In the research, we investigated three methods as dot-blot, ELISA and Western blotting to detect the cadherin in susceptible & resistant Helicoverpa armigera. When Dot-blot and ELISA was adopt to analysis the existence of cadherin, the qualitation and quantitation of data could be got separately. In dot-blot detection, we found that the susceptible strain showed darker color than the resistant one, which was in accordance with the data got in ELISA detection, the susceptible strain existing a higher value of OD than the resistant one. ELISA had the characteristics of a large analyze capacity, and high degree of normalization, while dot-blot was easy to operate and possessed high sensibility. But to be compared with western blotting, there were still some shortages, such as not capable to determine the molecular weight of the antigens and antibodies. In order to know the information about the antigen further, we adopt the method of western blotting, a kind of technical commonly used in the identification of antigen and also commonly used in separation technique and detection method, to analyze and evaluate the advantages and disadvantages with other two methods in terms of sensitivity and specificity taking results of dot-blot as a standard. In the consequence, the susceptible & resistant strains performed the bands at the same mark level-130-kDa, which demonstrated that the characteristics of the antigens might differs little, the molecular construction at least indeed. One of the possiblity might be that the susceptible strain mixed in the course of extraction of BBMV of resistant strain.

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