Brush border membrane vesicles （BBMV） isolated from insect midguts have been widely used to study CrylA binding proteins. Sample preparation is important in two- dimensional electrophoresis （2-DE）, so to determine...Brush border membrane vesicles （BBMV） isolated from insect midguts have been widely used to study CrylA binding proteins. Sample preparation is important in two- dimensional electrophoresis （2-DE）, so to determine a suitable BBMV preparation method in Helicoverpa armigera for 2-DE, we compared three published BBMV preparation methods mostly used in sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS- PAGE）. All methods yielded similar types and numbers of binding proteins, but in different quantities. The Abdul-Rauf and Ellar protocol was the best of the three, but had limitations. Sufficient protein quantity is important for research involving limited numbers of insects, such as studies of insect resistance to Bacillus thuringiensis in the field. Consequently, we integrated the three BBMV isolation methods into a single protocol that yielded high quantities of BBMV proteins from H. armigera larval midguts, which proved suitable for 2- DE analysis.展开更多
The insect midgut epithelium is composed of columnar, goblet, and regenerative cells. Columnar epithelial cells are the most abundant and have membrane protrusions that form the brush border membrane (BBM) on their ap...The insect midgut epithelium is composed of columnar, goblet, and regenerative cells. Columnar epithelial cells are the most abundant and have membrane protrusions that form the brush border membrane (BBM) on their apical side. These increase surface area available for the transport of nutrients, but also provide opportunities for interaction with xenobiotics such as pathogens, toxins and host plant allelochemicals. Recent improvements in proteomic and bioinfbrmatics tools provided an opportunity to determine the proteome of the T. ni BBM in unprecedented detail. This study reports the identification of proteins from BBM vesicles (BBMVs) using single dimension polyacrylamide gel elec? trophoresis coupled with multi-dimensional protein identification technology. More than 3000 proteins were associated with the BBMV of which 697 were predicted to possess either a signal peptide, at least one transmembrane domain or a GPI-anchor signal. Of these, bioinfbrmatics analysis and manual curation predicted that 185 may be associated with the BBMV or epithelial cell plasma membrane. These are discussed with respect to their predicted functions, namely digestion, nutrient uptake, cell signaling, development, cell-cell interactions, and other functions. We believe this to be the most detailed proteomic analysis of the lepidopteran midgut epithelium membrane to date, which will provide information to better understand the biochemical, physiological and pathological processes taking place in the larval midgut.展开更多
The relatively low susceptibility ofHelicoverpa armigera to CrylAc, its history of resistance to chemical insecticides and the seasonal decline in expression of CrylAc in transgenic cotton necessitated the development...The relatively low susceptibility ofHelicoverpa armigera to CrylAc, its history of resistance to chemical insecticides and the seasonal decline in expression of CrylAc in transgenic cotton necessitated the development of cotton expressing two insecticidal proteins to provide sustainable control of this multinational pest. To manage the resistance issue, it was essential that the second insecticidal protein have a significantly different mode of action to CrylAc. A common feature of resistance to CrylA proteins in several species as well as H. armigera has been a change in the binding site. A study of binding sites for some Cry proteins in the brush border membrane vesicles （BBMV） ofH. armigera and Helicoverpa punctigera was undertaken. The binding affinity for CrylAc was higher than for CrylAb, matching their relative toxicities, and CrylAc and CrylAb were found to share at least one binding site in both I-1. armigera and I-1. punctigera. However Cry2Aa did not compete with CrylAc for binding and so could be used in transgenic cotton in combination with CrylAc to control H. armigera and manage resistance. Variation in the susceptibilities of three different H. armigera strains to CrylAc correlated with the parameter Bmax/Kcom.展开更多
Hemipteran group of sap sucking insect pests cause worldwide crop destruction. The role of mannose specific monocot lectins have recently been worked out in hemipteran pest management. The present article demonstrates...Hemipteran group of sap sucking insect pests cause worldwide crop destruction. The role of mannose specific monocot lectins have recently been worked out in hemipteran pest management. The present article demonstrates the insecticidal efficacy of a new mannose specific agglutinin, isolated from tubers of Amorphophallus paeonifolius (AMTL) against a wide range of hemipteran insects. The 25 kDa dimeric protein was found to inhibit the survivability of hemipteran insects namely, Lipaphis erysimi, Aphis gossypii and Dysdercus cingulatus quite efficiently, as analysed by synthetic diet based bioassay experiments. Surface Plasmon Resonance study detected binding of insecticidal AMTL to insect gut brush border membrane vesicle (BBMV) protein, an absolute prerequisite for conferring toxicity against target insects. Further ligand blot analysis spotted a ~74 kDa glycoprotein as putative receptor of AMTL from the total BBMV protein fraction of Lipaphis erysimi. Phylogenetic analysis showed a significant relatedness of AMTL to the previously established monocot lectin Galanthus nivalis agglutinin (GNA) in terms of their conserved mannose binding domains, agglutinating ability of rabbit erythrocytes and insecticidal efficacies. These information project AMTL as a promising candidate in preventing crop loss caused due to hemipteran insect attack.展开更多
Brown planthopper, the sap sucking hemipteran pest, is one of the major contributors to the yield loss of rice through the world. To combat the situation researchers are interested identifying genes from plant origin ...Brown planthopper, the sap sucking hemipteran pest, is one of the major contributors to the yield loss of rice through the world. To combat the situation researchers are interested identifying genes from plant origin having potentiality to develop hemipteran pest resistance. Interestingly, it was observed that rice plants expressing ASAL, a monocot mannose binding lectin, showed significant resistance to brown planthopper and green leafhopper. Additionally, antibiotic resistant marker gene free ASAL expressing rice lines were developed to overcome the biosafety issues. However, the basis behind the resistance against planthoppers is still not clearly understood. Ligand blot assay was performed with total BBMV protein from BPH and a ~56 kDa receptor protein was detected. LC MS/MS analysis revealed that the receptor protein is NADH quinone oxidoreductase (NQO), a key player in electron transport chain, insect defense response and male/female gametogenesis. Presumably interaction of ASAL with NQO may lead to toxicity and loss of fecundity among BPH feeding on ASAL expressing transgenic rice plants. These findings provide a stable scientific basis for considering these transgenic ASAL expressing rice plants as significant product for combating BPH attack associated yield loss of rice.展开更多
Prepore formation is hypothesized to be an obligate step in the insertion of Cry1Ab toxin into insect brush border membrane vesicles. We examined the architecture of the putative prepore when isolated using the publis...Prepore formation is hypothesized to be an obligate step in the insertion of Cry1Ab toxin into insect brush border membrane vesicles. We examined the architecture of the putative prepore when isolated using the published protocols [1] [2]. Our results demonstrate that the putative prepore form of Cry1Ab is a combination of receptor proteins attached to the toxin, when purified. The results also suggest that this prepore form as prepared by the methods published is different from other membrane-extracted oligomeric forms of Cry toxins and prepore of other toxins in general. While most other known prepores are composed of multimers of a single protein, the Cry1Ab prepore, as generated, is a protein-receptor complex oligomer and monomers of Cry toxins.展开更多
Alkaline phosphatases （ALPs） attached to the midgut membrane with glycosyl phosphotidyl inositol （GPI） have been proposed as the putative CrylAc toxin receptor in Helicoverpa armigera. Activated toxins bind to ALP...Alkaline phosphatases （ALPs） attached to the midgut membrane with glycosyl phosphotidyl inositol （GPI） have been proposed as the putative CrylAc toxin receptor in Helicoverpa armigera. Activated toxins bind to ALP receptors on the brush border membrane vesicle （BBMV） of the midgut epithelium, which activates intracellular oncotic pathways and leads to cell death. However, with the long-term use of Cry toxin, insects can develop a strong resistance to insecticidal delta-endotoxins. Although the molecular mechanism of insect resistance has not been fully understood, insects develop resistance to biopesticides due to changes of toxins binding to midgut receptors. So, it is a good idea to investigate the molecular mechanism of insect resistance by analyzing ALP receptor from Helicoverpa armigera （Ha-ALP）. Based on crystal structure of shrimp alkaline phosphatase, the three-dimensional structure of the CrylAc toxin-binding Ha-ALP receptor was obtained by homology modeling and the model was further evaluated using PROSA energy and ERRAT. The important role of binding of toxin to GalNAc on Ha-ALP was discussed in the aspect of CrylAc toxicity. Specific recognition sites of the binding of oligosaccharides to Ha-ALP were predicted. Post-translational modification of ALP provides insights into the functional properties of ALP and leads to profound understanding of receptor and toxin interactions.展开更多
基金This paper was contributed to the International Symposium on Insect Midgut Biology, April 7-11, 2008, Guangzhou, China.Acknowledgments This research was supported by the National Natural Science Foundation of China （30771424） and National Basic Research Program of China （973-2007CB 109204）. We thank Dr. Jun Zhao （University of West Virginia, USA） for reviewing this manuscript.
文摘Brush border membrane vesicles （BBMV） isolated from insect midguts have been widely used to study CrylA binding proteins. Sample preparation is important in two- dimensional electrophoresis （2-DE）, so to determine a suitable BBMV preparation method in Helicoverpa armigera for 2-DE, we compared three published BBMV preparation methods mostly used in sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS- PAGE）. All methods yielded similar types and numbers of binding proteins, but in different quantities. The Abdul-Rauf and Ellar protocol was the best of the three, but had limitations. Sufficient protein quantity is important for research involving limited numbers of insects, such as studies of insect resistance to Bacillus thuringiensis in the field. Consequently, we integrated the three BBMV isolation methods into a single protocol that yielded high quantities of BBMV proteins from H. armigera larval midguts, which proved suitable for 2- DE analysis.
文摘The insect midgut epithelium is composed of columnar, goblet, and regenerative cells. Columnar epithelial cells are the most abundant and have membrane protrusions that form the brush border membrane (BBM) on their apical side. These increase surface area available for the transport of nutrients, but also provide opportunities for interaction with xenobiotics such as pathogens, toxins and host plant allelochemicals. Recent improvements in proteomic and bioinfbrmatics tools provided an opportunity to determine the proteome of the T. ni BBM in unprecedented detail. This study reports the identification of proteins from BBM vesicles (BBMVs) using single dimension polyacrylamide gel elec? trophoresis coupled with multi-dimensional protein identification technology. More than 3000 proteins were associated with the BBMV of which 697 were predicted to possess either a signal peptide, at least one transmembrane domain or a GPI-anchor signal. Of these, bioinfbrmatics analysis and manual curation predicted that 185 may be associated with the BBMV or epithelial cell plasma membrane. These are discussed with respect to their predicted functions, namely digestion, nutrient uptake, cell signaling, development, cell-cell interactions, and other functions. We believe this to be the most detailed proteomic analysis of the lepidopteran midgut epithelium membrane to date, which will provide information to better understand the biochemical, physiological and pathological processes taking place in the larval midgut.
文摘The relatively low susceptibility ofHelicoverpa armigera to CrylAc, its history of resistance to chemical insecticides and the seasonal decline in expression of CrylAc in transgenic cotton necessitated the development of cotton expressing two insecticidal proteins to provide sustainable control of this multinational pest. To manage the resistance issue, it was essential that the second insecticidal protein have a significantly different mode of action to CrylAc. A common feature of resistance to CrylA proteins in several species as well as H. armigera has been a change in the binding site. A study of binding sites for some Cry proteins in the brush border membrane vesicles （BBMV） ofH. armigera and Helicoverpa punctigera was undertaken. The binding affinity for CrylAc was higher than for CrylAb, matching their relative toxicities, and CrylAc and CrylAb were found to share at least one binding site in both I-1. armigera and I-1. punctigera. However Cry2Aa did not compete with CrylAc for binding and so could be used in transgenic cotton in combination with CrylAc to control H. armigera and manage resistance. Variation in the susceptibilities of three different H. armigera strains to CrylAc correlated with the parameter Bmax/Kcom.
文摘Hemipteran group of sap sucking insect pests cause worldwide crop destruction. The role of mannose specific monocot lectins have recently been worked out in hemipteran pest management. The present article demonstrates the insecticidal efficacy of a new mannose specific agglutinin, isolated from tubers of Amorphophallus paeonifolius (AMTL) against a wide range of hemipteran insects. The 25 kDa dimeric protein was found to inhibit the survivability of hemipteran insects namely, Lipaphis erysimi, Aphis gossypii and Dysdercus cingulatus quite efficiently, as analysed by synthetic diet based bioassay experiments. Surface Plasmon Resonance study detected binding of insecticidal AMTL to insect gut brush border membrane vesicle (BBMV) protein, an absolute prerequisite for conferring toxicity against target insects. Further ligand blot analysis spotted a ~74 kDa glycoprotein as putative receptor of AMTL from the total BBMV protein fraction of Lipaphis erysimi. Phylogenetic analysis showed a significant relatedness of AMTL to the previously established monocot lectin Galanthus nivalis agglutinin (GNA) in terms of their conserved mannose binding domains, agglutinating ability of rabbit erythrocytes and insecticidal efficacies. These information project AMTL as a promising candidate in preventing crop loss caused due to hemipteran insect attack.
文摘Brown planthopper, the sap sucking hemipteran pest, is one of the major contributors to the yield loss of rice through the world. To combat the situation researchers are interested identifying genes from plant origin having potentiality to develop hemipteran pest resistance. Interestingly, it was observed that rice plants expressing ASAL, a monocot mannose binding lectin, showed significant resistance to brown planthopper and green leafhopper. Additionally, antibiotic resistant marker gene free ASAL expressing rice lines were developed to overcome the biosafety issues. However, the basis behind the resistance against planthoppers is still not clearly understood. Ligand blot assay was performed with total BBMV protein from BPH and a ~56 kDa receptor protein was detected. LC MS/MS analysis revealed that the receptor protein is NADH quinone oxidoreductase (NQO), a key player in electron transport chain, insect defense response and male/female gametogenesis. Presumably interaction of ASAL with NQO may lead to toxicity and loss of fecundity among BPH feeding on ASAL expressing transgenic rice plants. These findings provide a stable scientific basis for considering these transgenic ASAL expressing rice plants as significant product for combating BPH attack associated yield loss of rice.
文摘Prepore formation is hypothesized to be an obligate step in the insertion of Cry1Ab toxin into insect brush border membrane vesicles. We examined the architecture of the putative prepore when isolated using the published protocols [1] [2]. Our results demonstrate that the putative prepore form of Cry1Ab is a combination of receptor proteins attached to the toxin, when purified. The results also suggest that this prepore form as prepared by the methods published is different from other membrane-extracted oligomeric forms of Cry toxins and prepore of other toxins in general. While most other known prepores are composed of multimers of a single protein, the Cry1Ab prepore, as generated, is a protein-receptor complex oligomer and monomers of Cry toxins.
基金Project supported by the National High Technology Research and Development Program of China （Nos. 2006AA02Z 187, 2006AA 10A212）, the Na- tional Natural Science Foundation of China （Nos. 30670052, 30870064, 30970066） and Ph.D. Programs Foundation of Ministry of Education of China （No. 20060452006）.
文摘Alkaline phosphatases （ALPs） attached to the midgut membrane with glycosyl phosphotidyl inositol （GPI） have been proposed as the putative CrylAc toxin receptor in Helicoverpa armigera. Activated toxins bind to ALP receptors on the brush border membrane vesicle （BBMV） of the midgut epithelium, which activates intracellular oncotic pathways and leads to cell death. However, with the long-term use of Cry toxin, insects can develop a strong resistance to insecticidal delta-endotoxins. Although the molecular mechanism of insect resistance has not been fully understood, insects develop resistance to biopesticides due to changes of toxins binding to midgut receptors. So, it is a good idea to investigate the molecular mechanism of insect resistance by analyzing ALP receptor from Helicoverpa armigera （Ha-ALP）. Based on crystal structure of shrimp alkaline phosphatase, the three-dimensional structure of the CrylAc toxin-binding Ha-ALP receptor was obtained by homology modeling and the model was further evaluated using PROSA energy and ERRAT. The important role of binding of toxin to GalNAc on Ha-ALP was discussed in the aspect of CrylAc toxicity. Specific recognition sites of the binding of oligosaccharides to Ha-ALP were predicted. Post-translational modification of ALP provides insights into the functional properties of ALP and leads to profound understanding of receptor and toxin interactions.
