| Based on elucidating the action mechanism of Bacillus thuringiensis crystal toxin to Cultured insect cells, we choose single insecticidal crystal protein(ICPs) CrvIA(c) toxin to select continuously Tn-5B1 cells, and find out on the receptors level the reason caused resistance. All results as follows: 1.C~oning and expression of CryIA(c) toxin in Eco/i BL21. According to common CaC12 transformation,pUC 19 plasmid containing CryIA(c) toxin encoding gene and AmpT gene was cloned into E.coli BL2I.All E.coli strain containing CrylA(c) gene were gro~ in LB liquid medium in the presence of ampicillin, and inclusion bodies were purified by discontinuous surcrose density gradients.The E.coli cultures were examined in thin section with the electron microscope, crystal formed in E.coli has the same lattice as native crystal protein from B.thuringiensis. and crystal usually were bipyramid~ cube structures; Bioassay using crystal from E.coli against cultured insect cells showed compatible toxicity as those from B.thuringiensis; A little aliquots of trypsinized crystal were subjected to SDS-PAGE analysis. a 66-Kda protein band was observed. 2. SelectionofresistantBll桾n?BlceIIstoCryIA(c)toxinandevaluation of resistance. Selection of resistance in Tn-5B1 cells to single CiyLA(c) toxin in E.coli was attempted by laboratory MTT In T4tro standard assay.Very high resistance to CryIA(c) toxin could be achieved after 20 generation of selection. Compared to mixed ICPs selection, resistance development of Tn-5B 1 cells to single crylA(c) toxin was faster. Significant levels of resistance(873 .2 fold) to CryIA(c) inclusion bodies expression in E.coli were observed after 10 generations. Sequent selection of the CrylA(c)-resistance population with trypsinized CryIA(c) toxin mixed with E.coli protein resulted, after 20 generation of CryIA(c) selection, that took 5-6 h to exhibit 80%-90% motality at the iii :7 highest toxin concentration tested(6.Omg/ml), whereas the 50% lethal concentration was for 55.1 ii g抦l susceptible Tn-5B1 cells. Resistance to the CiyIA(c) toxin was still observed 20 generations after CryIA(c) selection was removed, and the higher the resistance, the more stable the resistance, which was similar to the results In flvo. 3. Studies on receptors of CrylA(c) toxin in R桾n?B1 cells and S桾n?B1 cel Is. I Membrane preparations from resistance Tn-SB 1 cells and sensitive Tn-SB 1 cells were isolated essentially by homogenation in MET buffer and centrifugation at 30,000 g for 30 mm. The pellet was analysized by SDS-PAGE, a clear difference in protein bands between R-Tn-5B1 cells and S-Tn-SB 1 was observed. A 37KDa protein band from R- Tn-SB 1 cells was not observed, whereas it is obvious in S-Tn-SB 1 cells. Ligand blot analysis of SDS-PAGE size-seperated proteins showed CiyJA(c) toxin specifically binds to two protein in S-Tn-SB I cells, MW34KDa,37KDa,respectively. CryIA(c) toxin only specifically bind to 34KDa protein in R-Tn-5B1 cells, and we suppose that the disappearance of the 37-KDa protein leads to the resistance of Tn-5Bl cells to CxyIA(c) toxin. At the same time, it also testified that even if the same crystal toxin has different receptors m different insects and cells, ev... |
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