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Sıra DOSYA ADI Format Bağlantı
01. Toxin Different Study Toxin ppt Sunumu İndir

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Page 1Investigation of Bacillus thuringiensis Cry1Aa toxicity to Ephestia kuehniella (Lepidoptera: Pyralidae)Souad Rouis, Maissa Chakroun, Nouha Abdelmalek and Slim TounsiCENTRE DE BIOTECHNOLOGIE DE SFAX LABORATOIRE DE PROTECTION ET AMÉLIORATION DES PLANTES ********************EQUIPE DES BIOPESTICIDES
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Page 2Bacillus thuringiensisspore-forming bacteria Each crystal is formed by one or more -endotoxins (cry proteins) that define the nature of the spectrum insecticidal activityProducts crystals during the sporulation phasecristalspore
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Page 3δ-endotoxins Structure• 3 domains:Domain I: 7 α helicesDomain II: 3 β antiparallel feuilletsDomain III: 2 β antiparallel feuillets
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Page 4-endotoxins Domains specificity Model UmbrellaIt probably interacts with membrane receptors of the intestinal cellsRole is not clear???It is supposed to penetrate to the BBM?
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Page 5SolubilisationActivationActive ToxineBindingMidgut receptorsIngestionOligomerization et perforation Insect deathMode of action of -endotoxins
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Page 6Activation by proteolysis : Basal Membrane : Latéral Membrane: Apical MembraneToxin-receptor interaction The toxin creates pores in the membrane of apical microvilliExchange between the intra and extra cellular environment Changes of cell morphology Death of the insect Mode of action of -endotoxins
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Page 7Cry1Aa protein • Molecular Mass : 130 kDa.• Cry1Aa of BNS3 : Inactive against The flour moth Ephestia kuehniella (Lepidoptera) Cry1Ab Cry1Ac Cry1Ba Cry1Ca Cry1Da Cry3A Cry4A Cry4B Cry4CCry1Aa 90 82 55 67 71 25 27 27 22
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Page 8Previous work conducted in our laboratory has shown that, unlike the δ-endotoxin Cry1Ac, Cry1Aa shows no toxicity towards E. kuehniella even at high concentrations (Tounsi et al., 2005) However, it has been reported in the literature that Cry1Aa is seven times more toxic than Cry1Ac on Bombyx Mori (Wolfersberger, M 1996)BNS3 strain expressing toxins (Cry1Aa, Cry1Ac and Cry2), was isolated and characterized in our laboratory (Jaoua et al., 2000)
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Page 9Impact of the difference of Cry1Aa and Cry1Ac structure on their susceptibility to activation and / or inactivation by proteolysis(a) protoxins Cry1Ac and Cry1Aa digested by proteases of the gut juice of E.kuehniella T: protoxins of Cry1Ac and Cry1Aa(b) protoxins of Cry1Ac and Cry1Aa digested by the chymotrypsinVarious potential sites for digestion by chymotrypsinCry1Ac is fully activated by the proteases of the gut juice of E. kuehniella
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Page 10Comparative study by zymogramme of proteases of the gut juice of E. kuehniella involved in the activation of both toxinsThe protease activity of the juice of E. kuehniella (L3) against Cry1Ac involves two additional proteasesThis difference in protease activity could contribute to a relative resistance of E. kuehniella towards Cry1Aa
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Page 11Cry 1Aa Cry 1Ac5 544 332 21131 kDa14,4 kDa66,2 kDa45 kDa20 kDa6,5 kDaC+ C+Hybridization with anti-Cry1Aa 1 5: decreasing concentrations of Pronase Comparative study of the susceptibility of the two toxins Cry1Aa and Cry1Ac to digestion by PronaseThe Pronase is a mixture of different types of proteases, serine proteases, metalloproteases, carboxypeptidases, aminopeptidases, chymotrypsin, trypsin « Exfashion » Activity : prediction of differences in conformation Cry1Ac is more resistant to Pronase, suggesting a difference in conformation
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Page 12Marqueur de taille66,2 kDa 45 kDa 31 kDa 20 kDa 14,4 kDa 6,5 kDa15 4 3 2 15 4 3 2 C+C+Cry1Ac Cry1AaWestern BlotCry1Ac 4-5 peptide is more resistant to pronase than Cry1Aa one 1- additional cleavage site in Cry1Aa ?2- conformational change to protect a cleavage site in Cry1AcComparative study of the susceptibility of Cry1Aa and Cry1Ac a4-5 domains to digestion by Pronase
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Page 13Cry 1AaCry1Ac
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Page 15Lumen (Lu); membrane Apicale (Am); membrane Basale (Bm); grossissement 100x control Cry 1Aa Cry1AcHistopathological effects of the interaction between Cry1Aa and Cry1Ac with receptors on the intesti al cells of E. kuehniella Hypertrophy of Epithelial cellsComplete disintegration of the epithelial cells
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Page 16Lumen (Lu); membrane Apicale (Am); membrane Basale (Bm);Magnification 100x controle Cry1AcCry1AaImmunohistochemical localization of the toxins Cry1Aa and Cry1Ac in the intestine of E. kuehniellaClear localization of the toxin Cry1Ac in the basal membrane of epithelial cells. Slight localization of the toxin Cry1Aa in the apical membrane of epithelial cells
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Page 17Comparative study « in vitro » of the interaction between the toxins Cry1Aa, Cry1Ac and the receptors of E. kuehniella Cry1Ac binds to a number of receptors more important than Cry1Aa The intensity of the bands observed in the case of Cry1Ac suggests the presence of a large number of receptors and / or a greater affinity towards the same receptors that Cry1Aa BSA BBMV100kDa10080kDa BBMV BBMV Cry1Ac Cry1Aa
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Page 19Cry1Aa CL50 (g/g of semolina)Stage L1 429,048±123,28Stage L5 1420.483 ±594.794Positive control L50 (g/g of se olina)tage L1 76,1 28,341tage L5 9 ,1 8±27,308For Cry1Aa, the difference is clear between L1 and L5 larvea stagesComparative toxicity assay of recombinant strain harboring cry1Aa gene againt Ephestia kuehniella larvea
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Page 20 Stage L5 ON 5h 3h 2h 1h ND M ND 1h 2h 3h 5h ON Stage L1 ON 5h 3h 2h 1h ND M ND 1h 2h 3h 5h ON BLB1 BBP4 Positive control Cry1Aa60 kDaThe battery of proteases, that the insect has, is apparently different from a larval stage to another. Positive control Cry1AaComparative study of the susceptibility of recombinant Cry1Aa to Ephestia kuehniella midgut juce
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Page 2145 kDa97 kDa66 kDaA clear difference between proteases batteries of 1st and 5th instar E.kuehniella larvea gut juice Comparative study of proteases of 1st and 5th instar E.kuehniella larvea gut juice by zymogramme Positive control Cry1Aa casein
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Page 22Receptor Investigation of 1st and 5th instar E. kuehniella larvea Different receptor profiles between L1 and L5 stages suggesting differences in toxin receptor interactionM L5 L1 L5 L1 Anti APN1 Anti APN2 SDS PAGEWestern Blot
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Page 23L1Negative ControlComparative histopathological effects of Cry1Aa on 1st and 5th instar E. kuehniella larvea L5Hypertrophy of Epithelial cellsApparent disintegration of the epithelial cells
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