The Mechanism Of Bt Cry1Ac Toxin To Helicoverpa Armigera

Bacillus thuringiensis(Bt) is a widespread gram-positive bacterium that can produce insecticidal crystal proteins(ICPs).When the spore is formed,Bt is used for pest prevention because of its toxicity to Lepidoptera,Diptera and Coleoptera insect.The Helicoverpa armigera is a worldwide agricultural pest,with the extensive usage of Bt and transgenic cotton,the resistance of Helicoverpa armigera is becoming the bottleneck of Bt pesticide development.The better understand of mechanism of Bt makes important sense for reasonable usage of Bt and delaying insect resistance.It was reported the method of extracting Bt HTX-42 Cry1Ac toxin, the liquid double-phase method and the isoelectric point method were done,then we purified the protein liquid with the gel filtration chromatography and collected the absorb peak,the result of SDS-PAGE showed that high purity and high concentration of Bt Cry1Ac toxin was obtained in the end.The Helicoverpa armigera midgut BBMV was successfully achieved by the differential centrifugation,then we used Bradford method to test the protein content of tissue debris,centrifugation supernatant and centrifugation precipitation,the result showed that almost all the protein existed in the midgut BBMV centrifugation precipitation.The homologous competition binding analysis was maken by using biotin-labled Cry1Ac toxin,the result showed that there was obviously a single 60kDa protein band,when the content of unlabeled Cry1Ac toxin increased,the protein band became weaker,and the combination between BBMV and biotin-labled Cry1Ac toxin decreased.The combination between BBMV and labled toxin decreased by 80%when the unlabeled toxin increased by 100 times.The result indicated that the Cry1Ac toxin and the Helicoverpa armigera midgut BBMV could finish the homologous competition binding analysis,both of them had physiological activity and were able to use in the coming experiment.SDS-PAGE and co-immumoprecipitation results showed that both the midgut total BBMV protein and the midgut receptor protein Aminopeptidase N(APN)are influenced greatly when the Helicoverpa armigera fed by Cry1Ac toxin.Compared with normal insect,some of the small molecular Cry1Ac toxin treated insect midgut protein was missed,and the content of protein band was different obviously.We successfully achieved a 120kDa protein which was idendified as APN by mass spectrometry,and the content of APN incerased obviously.The Western blot research of Cry1Ac toxin oligomer indicated that the toxin could form dimmer and tetramer in the Helicoverpa armigera BBMV,and the toxin oligomer dissociated to toxin monomer when treated by 100℃high temperature.We studied the pathological effect of Bacillus thuringiensis Cry1Ac toxin to Helicoverpa armigera,compared to normal insect,the appearance and development of Cry1Ac toxin treated Helicoverpa armigera was changed obviously.Using transmission electron microscope,it was found that Cry1Ac toxin mainly influenced Helicoverpa armigera midgut microvilli,maken the microvilli shorter and thicker,also there were some vacuolus on the apical membrane of microvilli while some microvilli fell off.