| Bacillus thuringensis is Gram positive bacillus, in spores formation process, which can produce spore crystals, the main ingredient of pesticideThe ICPs from Bacillus thuringiensis and the Cry1Ac gene products expressed in plants are especially toxic to many susceptible insects. This biopesticides are being widely used effectively for control of agriculturally and biomedically detrimental pests because the bio-insecticides are environmentally friendly. However, the pests might develop high-level resistance to Bt toxins rapidly, and little is known about the mechanism of resistance until today. Further understanding of the mechanism is fundamental in sustaining the use of Cry1Ac proteins in integrated pest management. Here, the author has selected the resistant cultured insect cells to Bt Cry1Ac toxin instead of resistant insects, and studied the characterization of resistance and its possible mechanisms.In order to elucidating the action mechanism of Bacillus thuringiensis crystal toxin to cultured insect cells, we choose single insecticidal crystal protein Cry1Ac toxin to select continuously Tn-5B1cells, and find out the mechanism of resistance based on proteomics technique. All results are as follows:1.Expression of Cry1Ac toxin in E.coli BL21.According to common CaCl2transformation,pHT304plasmid containing Cry1Ac toxin encoding gene and Ampr gene was cloned into E. coli BL21.All E. coli strain containing Cry1Ac gene were grown in LB liquid medium in the presence of ampencillin, 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, protein band was observed.2.Selection of R-Tn-5B1cells to Cry1Ac toxin and evaluation of resistance.Test results show that Tn-5B1cells were sensitive to Cry1Ac toxins. During the selection of Tn-5B1cells with Cry1Ac toxins continuously, every five generations we determinated the resistance of Tn-5B1cell to Cry1Ac toxins with MTT method. The resistance ratio reached128fold after the selection at generation122in Tn-5B1cells, and181.9fold after the selection at generation124. After selection at generation130,the resistance ratio reached to1298.6fold.3.two-dimensional electrophoresis and map analysisThis study is based on the work of toxicity mensuration,filtration and stability of cell resistance, studying differences in gene expression and identification of different proteins, using two-dimention electrophoresis to separate the total proteins of the sensitive and resistant cells, silver staining color, Image Master2D Platinum6.0software analysis, statistics of the gel on all the differences in protein spots. Referring to2D map of Trichoplusiani cells R-Tn-5B1cell, we found2000different protein dots,Differential Dots were mainly concentrated in alkaline and molecular weight between35.0to66.2KD. the expression increase of two to four times is24, the expression increase of more than four times is14; the decrease is15and18homologously through analyzing software. With wonderful repeatability and good significance, we can get7dots. Maybe these differential proteins were related to resistance development directly or indirectly.4. Comparison of activity of alkaline phosphatase, one of Cry1Ac receptors, between R-Tn-5B1and Tn-5B1cellsAlkaline phosphatase is one of Cry1Ac receptors. Based on the total protein extracted from two kinds of cells, individual cell alkaline phosphatase activity was measured, alkaline phosphatase activity of sensitive cells was higher than that of resistant cells. However, its activity is not high in both of cells. |
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