| The crystalline glycoprotein toxins based on Bacillus thuringiensis and the cry 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 Cry proteins in integrated pest management. Here, the author has selected the resistant cultured insect cells to Bt Cry toxin instead of resistant insects, and studied the characterization of resistance and its possible mechanisms. The main results of this paper are detailed as follows:1. The developmental speed of resistance to in vitro-activated CrylAc inTnH5 cells was S-like curve. The resistance ratio reached 1, 280 fold after the selection at generation 56. This resistance ratio declined after the selection ceased. The higher the resistance ratio was, the more stable the resistance was. If the reselection was done, the resistance went up rapidly for the reduced resistant cells. The Cry1Ac-selected cells had high cross resistance to in vitro-activated multi-Cry toxins from B. thuringiensis aizawai GC-91, B. thuringiensis sub. aizawai and B. thuringiensis sub. kurstaki, respectively. The cells had 19.7-fold resistance to in vitro-activated CrylC, but had no cross-resistance to chemical pesticides.2. The binding of in vitro-activated CrylAc to the resistant and susceptible cells or their extracted protein was assessed using ELISA, ligand western-blot or labeling with FITC. The results of ELISA showed that the quantity of in vitro-activated CrylAc toxin binded to resistant cells reduced slightly; ligand Western blot revealed that there were five bands recognized by in vitro-activated CrylAc toxin in both the membrane protein of the susceptible cells and that of resistant cells, which were 207kD, 158kD, 119kD, 72kD and 39kD, respectively, and 119 kD positive bands from the resistant cell sample was weaker than that from susceptible cell sample. Observation under fluorescensin microscopy showed that the susceptible cells labeled by FITC-Cry1Ac toxin were brighter than the labeled resistant cells.3. The effects of chemicals on the action of in vitro-activated CrylAc toxin on the Trichoplusiani cell line TnH5 were studied in order to elucidate the mode of action of Bacillus thuringiensis insecticidal crystal protein on cultured insect cells. The susceptibility of the cells to in vitro-activated CrylAc toxin went up after it was preinocubated by tunicamycin, cycloheximide, monensin, or trypsin. However, N-acetylgalactosamine and galactose could not inhibit the toxicity of in vitro-activated CrylAc toxin to the cells. The tolerance of the hypotonic solution for the resistant cells were stronger than that for the susceptible cells.4. Two different APN gene fragments from TnH5 cells were amplified using degenerate primers by RT-PCR (GenBank-Accession: CD809324; GenBank-Accession: CD809326), respectively. Deduced protein sequences were highly similar to several receptor APNs in insects with sequence identities of 45-47% for the one and 61-63% for the other. The relative activity of APN for the resistant cells was weaker (0.84 fold) than that of susceptible cells. The relative activity of alkaline phosphatase has no difference by statistics between the susceptible and resistant cells.5. Differential display of mRNA between the resistant cells selected by in vitro-activated CrylAc and the non-selected susceptible cells was analyzed with DDRT-PCR technique, and 5 bands were proved to be specially expressed sequence-tags (EST) by RNA rev-Northern hybridization using DIG labeled second strand cDNA probes. Sequence analysis of ESTs was performed, two of the three special bands from the susceptible cells had no homology with the known ESTs (G...
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