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Sabino Pacheco Isabel G��mez Ivan Arenas Gloria Saab-Rincon Claudia Rodr��guez-Almaz��n Sarjeet S. Gill Alejandra Bravo Mario Sober��n 《The Journal of biological chemistry》2009,284(47):32750-32757
Bacillus thuringiensis Cry toxins are used worldwide as insecticides in agriculture, in forestry, and in the control of disease transmission vectors. In the lepidopteran Manduca sexta, cadherin (Bt-R1) and aminopeptidase-N (APN) function as Cry1A toxin receptors. The interaction with Bt-R1 promotes cleavage of the amino-terminal end, including helix α-1 and formation of prepore oligomer that binds to APN, leading to membrane insertion and pore formation. Loops of domain II of Cry1Ab toxin are involved in receptor interaction. Here we show that Cry1Ab mutants located in domain II loop 3 are affected in binding to both receptors and toxicity against Manduca sexta larvae. Interaction with both receptors depends on the oligomeric state of the toxin. Monomers of loop 3 mutants were affected in binding to APN and to a cadherin fragment corresponding to cadherin repeat 12 but not with a fragment comprising cadherin repeats 7–12. In contrast, the oligomers of loop 3 mutants were affected in binding to both Bt-R1 fragments but not to APN. Toxicity assays showed that either monomeric or oligomeric structures of Cry1Ab loop 3 mutations were severely affected in insecticidal activity. These data suggest that loop 3 is differentially involved in the binding with both receptor molecules, depending on the oligomeric state of the toxin and also that possibly a “ping pong” binding mechanism with both receptors is involved in toxin action. 相似文献
3.
Peng Xu Mayira Islam Yutao Xiao Fei He Yi Li Jianxin Peng Huazhu Hong Chenxi Liu Kaiyu Liu 《Cytotechnology》2016,68(3):481-496
Bacillus thuringiensis (Bt) toxin receptors play important roles in the killing of pests, and investigation on characterization of the receptors is essential for utilization of Bt and management of insect resistance. Here, recombinant and mosaic receptors of Bt Cry1Ac toxin from Helicoverpa armigera were expressed in Spodoptera litura Sl-HP cells and their influences on cytotoxicity of activated Cry1Ac toxin were investigated. When H. armigera aminopeptidase N1 (APN1), alkaline phosphatase 2 (ALP2) and cadherin fused with or without GFP tag were, respectively, expressed in Sl-HP cells, live cell-immunofluorescence staining detection revealed that the quantity of the toxin binding to cadherin or cadherin-GFP was much more than that binding to ALP2 and APN1 or their fusion proteins with GFP, and only the cadherin- or cadherin-GFP-expressing cells showed aberrant cell morphology after the treatment of the toxin at low concentrations. ALP2 and APN1 fused with or without GFP tag did not significantly enhance the cadherin-mediated cytotoxicity of the toxin. The mosaic ALP-TBR-GFP-GPI was located on cell membrane, but did not bind to the toxin. The mosaic truncated cadherin-GFP-GPI was not located on cell membrane even if the signal peptide was sustained. The concentrations of the toxin resulting in swelling of 50 % cells for noncadherin-expressing Sl-HP cells and cadherin-expressing Hi5 cells were 5.08 and 9.50 µg/ml within 1 h, respectively. Taken together, our data have indicated that the binding affinity of ALP2 and APN1 to activated Cry1Ac toxin is much weaker than that of cadherin and both ALP2 and APN1 do not enhance the cytotoxicity of the toxin even though cadherin is co-expressed, and the mosaic receptor of ALP2 inserted with cadherin toxin binding domain does not mediate cytotoxicity of the toxin. In addition, the noncadherin-expressing Sl-HP cells are more susceptible to activated Cry1Ac than the cadherin-expressing Hi5 cells. 相似文献
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The Mode of Action of the Bacillus thuringiensis Vegetative Insecticidal Protein Vip3A Differs from That of Cry1Ab δ-Endotoxin
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Mi Kyong Lee Frederick S. Walters Hope Hart Narendra Palekar Jeng-Shong Chen 《Applied microbiology》2003,69(8):4648-4657
The Vip3A protein, secreted by Bacillus spp. during the vegetative stage of growth, represents a new family of insecticidal proteins. In our investigation of the mode of action of Vip3A, the 88-kDa Vip3A full-length toxin (Vip3A-F) was proteolytically activated to an approximately 62-kDa core toxin either by trypsin (Vip3A-T) or lepidopteran gut juice extracts (Vip3A-G). Biotinylated Vip3A-G demonstrated competitive binding to lepidopteran midgut brush border membrane vesicles (BBMV). Furthermore, in ligand blotting experiments with BBMV from the tobacco hornworm, Manduca sexta (Linnaeus), activated Cry1Ab bound to 120-kDa aminopeptidase N (APN)-like and 250-kDa cadherin-like molecules, whereas Vip3A-G bound to 80-kDa and 100-kDa molecules which are distinct from the known Cry1Ab receptors. In addition, separate blotting experiments with Vip3A-G did not show binding to isolated Cry1A receptors, such as M. sexta APN protein, or a cadherin Cry1Ab ecto-binding domain. In voltage clamping assays with dissected midgut from the susceptible insect, M. sexta, Vip3A-G clearly formed pores, whereas Vip3A-F was incapable of pore formation. In the same assay, Vip3A-G was incapable of forming pores with larvae of the nonsusceptible insect, monarch butterfly, Danaus plexippus (Linnaeus). In planar lipid bilayers, both Vip3A-G and Vip3A-T formed stable ion channels in the absence of any receptors, supporting pore formation as an inherent property of Vip3A. Both Cry1Ab and Vip3A channels were voltage independent and highly cation selective; however, they differed considerably in their principal conductance state and cation specificity. The mode of action of Vip3A supports its use as a novel insecticidal agent. 相似文献
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Xiaozhao Song Wendy Kain Douglas Cassidy Ping Wang 《Applied and environmental microbiology》2015,81(15):5184-5195
The resistance to the Bacillus thuringiensis (Bt) toxin Cry2Ab in a greenhouse-originated Trichoplusia ni strain resistant to both Bt toxins Cry1Ac and Cry2Ab was characterized. Biological assays determined that the Cry2Ab resistance in the T. ni strain was a monogenic recessive trait independent of Cry1Ac resistance, and there existed no significant cross-resistance between Cry1Ac and Cry2Ab in T. ni. From the dual-toxin-resistant T. ni strain, a strain resistant to Cry2Ab only was isolated, and the Cry2Ab resistance trait was introgressed into a susceptible laboratory strain to facilitate comparative analysis of the Cry2Ab resistance with the susceptible T. ni strain. Results from biochemical analysis showed no significant difference between the Cry2Ab-resistant and -susceptible T. ni larvae in midgut proteases, including caseinolytic proteolytic activity and zymogram profile and serine protease activities, in midgut aminopeptidase and alkaline phosphatase activity, and in midgut esterases and hemolymph plasma melanization activity. For analysis of genetic linkage of Cry2Ab resistance with potential Cry toxin receptor genes, molecular markers for the midgut cadherin, alkaline phosphatase (ALP), and aminopeptidase N (APN) genes were identified between the original greenhouse-derived dual-toxin-resistant and the susceptible laboratory T. ni strains. Genetic linkage analysis showed that the Cry2Ab resistance in T. ni was not genetically associated with the midgut genes coding for the cadherin, ALP, and 6 APNs (APN1 to APN6) nor associated with the ABC transporter gene ABCC2. Therefore, the Cry2Ab resistance in T. ni is conferred by a novel but unknown genetic mechanism. 相似文献
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《Peptides》2017
Bacillus thuringiensis Cry toxins are currently used for pest control in transgenic crops but evolution of resistance by the insect pests threatens the use of this technology. The Cry1AbMod toxin was engineered to lack the alpha helix-1 of the parental Cry1Ab toxin and was shown to counter resistance to Cry1Ab and Cry1Ac toxins in different insect species including the fall armyworm Spodoptera frugiperda. In addition, Cry1AbMod showed enhanced toxicity to Cry1Ab-susceptible S. frugiperda populations. To gain insights into the mechanisms of this Cry1AbMod-enhanced toxicity, we isolated the Cry1AbMod toxin binding proteins from S. frugiperda brush border membrane vesicles (BBMV), which were identified by pull-down assay and liquid chromatography-tandem mass spectrometry (LC–MS/MS). The LC–MS/MS results indicated that Cry1AbMod toxin could bind to four classes of aminopeptidase (N1, N3, N4 y N5) and actin, with the highest amino acid sequence coverage acquired for APN 1 and APN4. In addition to these proteins, we found other proteins not previously described as Cry toxin binding proteins. This is the first report that suggests the interaction between Cry1AbMod and APN in S. frugiperda. 相似文献
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Bacillus thuringiensis Cry1A toxins, in contrast to other pore-forming toxins, bind two putative receptor molecules, aminopeptidase N (APN) and cadherin-like proteins. Here we show that Cry1Ab toxin binding to these two receptors depends on the toxins' oligomeric structure. Toxin monomeric structure binds to Bt-R1, a cadherin-like protein, that induces proteolytic processing and oligomerization of the toxin (Gómez, I., Sánchez, J., Miranda, R., Bravo A., Soberón, M., FEBS Lett. (2002) 513, 242-246), while the oligomeric structure binds APN, which drives the toxin into the detergent-resistant membrane (DRM) microdomains causing pore formation. Cleavage of APN by phospholipase C prevented the location of Cry1Ab oligomer and Bt-R1 in the DRM microdomains and also attenuates toxin insertion into membranes despite the presence of Bt-R1. Immunoprecipitation experiments demonstrated that initial Cry1Ab toxin binding to Bt-R1 is followed by binding to APN. Also, immunoprecipitation of Cry1Ab toxin-binding proteins using pure oligomeric or monomeric structures showed that APN was more efficiently detected in samples immunoprecipitated with the oligomeric structure, while Bt-R1 was preferentially detected in samples immunoprecipitated with the monomeric Cry1Ab. These data agrees with the 200-fold higher apparent affinity of the oligomer than that of the monomer to an APN enriched protein extract. Our data suggest that the two receptors interact sequentially with different structural species of the toxin leading to its efficient membrane insertion. 相似文献
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Employing phage display to study the mode of action of Bacillus thuringiensis Cry toxins 总被引:1,自引:0,他引:1
Phage display is an in vitro method for selecting polypeptides with desired properties from a large collection of variants. The insecticidal Cry toxins produced by Bacillus thuringiensis are highly specific to different insects. Various proteins such as cadherin, aminopeptidase-N (APN) and alkaline phosphatase (ALP) have been characterized as potential Cry-receptors. We used phage display to characterize the Cry toxin-receptor interaction(s). By employing phage-libraries that display single-chain antibodies (scFv) from humans or from immunized rabbits with Cry1Ab toxin or random 12-residues peptides, we have identified the epitopes that mediate binding of lepidopteran Cry1Ab toxin with cadherin and APN receptors from Manduca sexta and the interaction of dipteran Cry11Aa toxin with the ALP receptor from Aedes aegypti. Finally we displayed in phages the Cry1Ac toxin and discuss the potential for selecting Cry variants with improved toxicity or different specificity. 相似文献
9.
Binding Analyses of Bacillus thuringiensis Cry δ-Endotoxins Using Brush Border Membrane Vesicles of Ostrinia nubilalis
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Gang Hua Luke Masson Juan Luis Jurat-Fuentes George Schwab Michael J. Adang 《Applied microbiology》2001,67(2):872-879
Transgenic corn expressing the Bacillus thuringiensis Cry1Ab gene is highly insecticidal to Ostrinia nubilalis (European corn borer) larvae. We ascertained whether Cry1F, Cry9C, or Cry9E recognizes the Cry1Ab binding site on the O. nubilalis brush border by three approaches. An optical biosensor technology based on surface plasmon resonance measured binding of brush border membrane vesicles (BBMV) injected over a surface of immobilized Cry toxin. Preincubation with Cry1Ab reduced BBMV binding to immobilized Cry1Ab, whereas preincubation with Cry1F, Cry9C, or Cry9E did not inhibit BBMV binding. BBMV binding to a Cry1F-coated surface was reduced when vesicles were preincubated in Cry1F or Cry1Ab but not Cry9C or Cry9E. A radioligand approach measured 125I-Cry1Ab toxin binding to BBMV in the presence of homologous (Cry1Ab) and heterologous (Cry1Ac, Cry1F, Cry9C, or Cry9E) toxins. Unlabeled Cry1Ac effectively competed for 125I-Cry1Ab binding in a manner comparable to Cry1Ab itself. Unlabeled Cry9C and Cry9E toxins did not inhibit 125I-Cry1Ab binding to BBMV. Cry1F inhibited 125I-Cry1Ab binding at concentrations greater than 500 nM. Cry1F had low-level affinity for the Cry1Ab binding site. Ligand blot analysis identified Cry1Ab, Cry1Ac, and Cry1F binding proteins in BBMV. The major Cry1Ab signals on ligand blots were at 145 kDa and 154 kDa, but a strong signal was present at 220 kDa and a weak signal was present at 167 kDa. Cry1Ac and Cry1F binding proteins were detected at 220 and 154 kDa. Anti-Manduca sexta aminopeptidase serum recognized proteins of 145, 154, and 167 kDa, and anti-cadherin serum recognized the 220 kDa protein. We speculate that isoforms of aminopeptidase and cadherin in the brush border membrane serve as Cry1Ab, Cry1Ac, and Cry1F binding proteins. 相似文献
10.
Effects of Midgut-Protein-Preparative and Ligand Binding Procedures on the Toxin Binding Characteristics of BT-R1, a Common High-Affinity Receptor in Manduca sexta for Cry1A Bacillus thuringiensis Toxins
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Timothy P. Keeton Brian R. Francis Walid S. A. Maaty Lee A. Bulla Jr. 《Applied microbiology》1998,64(6):2158-2165
The identity of the physiologically important Cry1A receptor protein(s) in the lepidopteran Manduca sexta has been a matter of dispute due to the multiple proteins which bind the Cry1Ac toxin. Cry1Aa, Cry1Ab, and Cry1Ac exhibit essentially identical toxicities toward M. sexta larvae and show a high degree of sequence and presumed structural identities. These similarities make it likely that there is a common mechanism of toxicity in these lepidopteran-specific toxins in terms of both mode of action and the receptor proteins through which these toxins exert their lepidopteran-specific toxicity. Investigators in our laboratory previously demonstrated that the cloned 210-kDa glycoprotein BT-R1 binds all three Cry1A toxins (T. P. Keeton and L. A. Bulla, Jr., Appl. Environ. Microbiol. 63:3419–3425, 1997). This protein remains a common binding protein even after being subjected to various midgut membrane preparation and processing protocols. The method used to isolate proteins from the M. sexta larval midgut in no significant way affects the results of ligand binding and vacuum blotting experiments, and we have been unable to detect specific, high-affinity binding of any Cry1A toxin to Cry1Ac binding proteins other than BT-R1. Alterations in blot substrate and blocking, hybridization, and washing buffers support these conclusions. Collectively, these results indicate that in M. sexta the cadherin-like BT-R1 protein is a common high-affinity receptor protein for the Cry1A family of toxins. 相似文献
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苏云金芽孢杆菌(Bacillus thuringiensis,Bt)产生的内毒素具有杀虫活性,Cry2Ab毒素作为Bt棉花的杀虫活性蛋白,其在靶标昆虫体内的结合受体及作用位点尚不清楚,本研究采用噬菌体展示(phage display)的方法,经4轮的"吸附—洗脱—扩繁"筛选,并对阳性克隆所携带的外源DNA片段进行序列测定后,得到2段能够与活化Cry2Ab毒素相互作用的多肽序列,通过酶联免疫结合试验(ELISA)进一步证明,这2段多肽序列与活化Cry2Ab毒素具有较高的亲和力和特异性,结果表明,利用该方法能够由噬菌体随机肽库中高效捕获亲和序列,筛选到与活化Cry2Ab毒素具有高亲和力的多肽,该序列可以模拟Cry2Ab毒素的受体表位,为进一步研究Cry2Ab毒素作用机制奠定了基础,并为今后田间抗性基因频率检测,以及毒素—受体作用机制研究工作提供更有力的技术支持。 相似文献
13.
Bravo A Gómez I Conde J Muñoz-Garay C Sánchez J Miranda R Zhuang M Gill SS Soberón M 《Biochimica et biophysica acta》2004,1667(1):38-46
Bacillus thuringiensis Cry1A toxins, in contrast to other pore-forming toxins, bind two putative receptor molecules, aminopeptidase N (APN) and cadherin-like proteins. Here we show that Cry1Ab toxin binding to these two receptors depends on the toxins' oligomeric structure. Toxin monomeric structure binds to Bt-R1, a cadherin-like protein, that induces proteolytic processing and oligomerization of the toxin (Gomez, I., Sanchez, J., Miranda, R., Bravo A., Soberon, M., FEBS Lett. (2002) 513, 242-246), while the oligomeric structure binds APN, which drives the toxin into the detergent-resistant membrane (DRM) microdomains causing pore formation. Cleavage of APN by phospholipase C prevented the location of Cry1Ab oligomer and Bt-R1 in the DRM microdomains and also attenuates toxin insertion into membranes despite the presence of Bt-R1. Immunoprecipitation experiments demonstrated that initial Cry1Ab toxin binding to Bt-R1 is followed by binding to APN. Also, immunoprecipitation of Cry1Ab toxin-binding proteins using pure oligomeric or monomeric structures showed that APN was more efficiently detected in samples immunoprecipitated with the oligomeric structure, while Bt-R1 was preferentially detected in samples immunoprecipitated with the monomeric Cry1Ab. These data agrees with the 200-fold higher apparent affinity of the oligomer than that of the monomer to an APN enriched protein extract. Our data suggest that the two receptors interact sequentially with different structural species of the toxin leading to its efficient membrane insertion. 相似文献
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Insecticidal crystal (Cry) proteins from Bacillus thuringiensis (Bt) are highly active against Lepidoptera. However, field-evolved resistance to Bt toxins is on the rise. The 12-cadherin domain protein HevCaLP and the ABC transporter HevABCC2 are both genetically linked to Cry toxin resistance in Heliothis virescens. We investigated their interaction using stably expressing non-lytic clonal Sf9 cell lines expressing either protein or both together. Untransfected Sf9 cells are innately sensitive to Cry1Ca toxin, but not to Cry1A toxins; and quantitative PCR revealed negligible expression of genes involved in Cry1A toxicity such as cadherin, ABCC2, alkaline phosphatase (ALP) and aminopeptidase N (APN). Cry1Aa, Cry1Ab or Cry1Ac caused swelling of Sf9 cells expressing HevABCC2, and caused faster swelling, lysis and up to 86% mortality in cells expressing both proteins. No such effect was observed in control Sf9 cells or in cells expressing only HevCaLP. The results of a mixing experiment demonstrated that both proteins need to be expressed within the same cell for high cytotoxicity, and suggest a novel role for HevCaLP. Binding assays showed that the toxin-receptor interaction is specific. Our findings confirm that HevABCC2 is the central target in Cry1A toxin mode of action, and that HevCaLP plays a supporting role in increasing Cry1A toxicity. 相似文献
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Donghai Peng Xiaohui Xu Weixing Ye Ziniu Yu Ming Sun 《Applied microbiology and biotechnology》2010,85(4):1033-1040
The interaction between Bacillus thuringiensis insecticidal crystal protein Cry1A and cadherin receptors in lepidopteran insects induces toxin oligomerization, which is
essential for membrane insertion and mediates Cry1A toxicity. It has been reported that Manduca sexta cadherin fragment CR12-MPED and Anopheles gambiae cadherin fragment CR11-MPED enhance the insecticidal activity of Cry1Ab and Cry4Ba to certain lepidopteran and dipteran larvae
species, respectively. This study reports that a Helicoverpa armigera cadherin fragment (HaCad1) containing its toxin binding region, expressed in Escherichia coli, enhanced Cry1Ac activity against H. armigera larvae. A binding assay showed that HaCad1 was able to bind to Cry1Ac in vitro and that this event did not block toxin binding
to the brush border membrane microvilli prepared from H. armigera. When the residues 1423GVLSLNFQ1430 were deleted from the fragment, the subsequent mutation peptide lost its ability to bind Cry1Ac and the toxicity enhancement
was also significantly reduced. Oligomerization tests showed that HaCad1 facilitates the formation of a 250-kDa oligomer of
Cry1Ac-activated toxin in the midgut fluid environment. Oligomer formation was dependent upon the toxin binding to HaCad1,
which was also necessary for the HaCad1-mediated enhancement effect. Our discovery reveals a novel strategy to enhance insecticidal
activity or to overcome the resistance of insects to B. thuringiensis toxin-based biopesticides and transgenic crops. 相似文献
16.
《Peptides》2013
Bacillus thuringiensis Cry toxins recognizes their target cells in part by the binding to glycosyl–phosphatidyl–inositol (GPI) anchored proteins such as aminopeptidase-N (APN) or alkaline phosphatases (ALP). Treatment of Tenebrio molitor brush border membrane vesicles (BBMV) with phospholipase C that cleaves out GPI-anchored proteins from the membranes, showed that GPI-anchored proteins are involved in binding of Cry3Aa toxin to BBMV. A 68 kDa GPI-anchored ALP was shown to bind Cry3Aa by toxin overlay assays. The 68 kDa GPI-anchored ALP was preferentially expressed in early instar larvae in comparison to late instar larvae. Our work shows for the first time that GPI-anchored ALP is important for Cry3Aa binding to T. molitor BBMV suggesting that the mode of action of Cry toxins is conserved in different insect orders. 相似文献
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Gómez I Arenas I Benitez I Miranda-Ríos J Becerril B Grande R Almagro JC Bravo A Soberón M 《The Journal of biological chemistry》2006,281(45):34032-34039
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A cadherin-like protein has been identified as a putative receptor for Bacillus thuringiensis (Bt) Cry1Ac toxin in Helicoverpa armigera and plays a key role in Bt insecticidal action. In this study, we produced a fragment from this H. armigera Cry1Ac toxin-binding cadherin that included the predicted toxin-binding region. Binding of Cry1Ac toxin to this cadherin fragment facilitated the formation of a 250-kDa toxin oligomer. The cadherin fragment was evaluated for its effect on Cry1Ac toxin-binding and toxicity by ligand blotting, binding assays, and bioassays. The results of ligand blotting and binding assays revealed that the binding of Cry1Ac to H. armigera midgut epithelial cells was reduced under denaturing or native conditions in vitro. Bioassay results indicated that toxicities from Cry1Ac protoxin or activated toxin were reduced in vivo by the H. armigera cadherin fragment. The addition of the cadherin fragment had no effect on Cry2Ab toxicity. 相似文献
19.
Yolanda Bel Herbert A.A. Siqueira Blair D. Siegfried Juan Ferré Baltasar Escriche 《Insect biochemistry and molecular biology》2009,39(3):218-223
Transgenic corn expressing Cry1Ab (a Bacillus thuringiensis toxin) is highly effective in the control of Ostrinia nubilalis. For its toxic action, Cry1Ab has to bind to specific insect midgut proteins. To date, in three Lepidoptera species resistance to a Cry1A toxin has been conferred by mutations in cadherin, a protein of the Lepidoptera midgut membrane. The implication of cadherin in the resistance of an Ostrinia nubilalis colony (Europe-R) selected with Bacillus thuringiensis Cry1Ab protoxin was investigated. Several major mutations in the cadherin (cdh) gene were found, which introduced premature termination codons and/or large deletions (ranging from 1383 to 1701 bp). The contribution of these major mutations to the resistance was analyzed in resistant individuals that survived exposure to a high concentration of Cry1Ab protoxin. The results indicated that the presence of major mutations was drastically reduced in individuals that survived exposure. Previous inheritance experiments with the Europe-R strain indicated the involvement of more than one genetic locus and reduced amounts of the cadherin receptor. The results of the present work support a polygenic inheritance of resistance in the Europe-R strain, in which mutations in the cdh gene would contribute to resistance by means of an additive effect. 相似文献
20.
S. E. Franklin L. Young D. Watson A. Cigan T. Meyer L. A. Bulla Jr. 《Molecular genetics and genomics : MGG》1997,256(5):517-524
Various subspecies of the gram-positive bacterium Bacillus thuringiensis are known to produce a wide array of insecticidal crystal proteins (ICPs) upon sporulation. These ICPs act primarily on the brush border of midgut epithelial cells of susceptible larvae. Recently, a protein of 210?kDa, isolated from the midgut of Manduca sexta, has been demonstrated to bind the Cry1Ab toxin produced by B. thuringiensis subsp. berliner and is therefore postulated to be involved in mediating the toxicity of Cry1Ab. The cDNA encoding the 210?kDa protein, termed BT-R1 (Bacillus thuringiensis receptor-1), was recently cloned, and shows limited homology to the cadherin superfamily of proteins. Quite naturally, there is a great deal of interest in the characterization of BT-R 1 , the gene encoding the 210?kDa Cry1Ab binding protein. The studies presented here involve the use of various restriction fragments prepared from the cDNA encoding BT-R1 as probes of Southern blots bearing M. sexta genomic DNA cleaved with a variety of restriction endonucleases. These Southern blot data reveal that there are two discrete regions within the M. sexta genome which encode sequences homologous to BT-R1. On the basis of the signal intensities seen on Southern blots, it appears that only one of these genes encodes BT-R1, whereas the other is a closely related homologue. 相似文献