| A separation method for Bt toxic protein CrylAb from Bt transgenic rice has been developed and validated by twice employing Ion- Exchange High Performance Liquid Chromatography (IEHPLC). The conformations and insecticidal activities of the purified CrylAb protein in different buffers have been studied. After that, the highly purified protein was labeled by 125I- NaI as a radioactive probe. The Western hybridization experiment by the probe gave the evidence that the midgut of Chilo suppressalis exists CrylAb receptor protein or proteins. Further, we cloned a kind of CrylAb receptor gene, Bt-R3, getting its fully length cDNA sequence and expressing the extracellular part of the gene in baculovirus- insect system. Western blot experiment showed that the expressing product is able to combine efficiently with CrylAb, validating the Bt-R3 do is CrylAb receptor protein. Based on these research, Bt-R3 structural properties, the molecular evolution relationship of Bt-R3 and other Bt receptors reported and conservation of these receptor proteins have been analyzed by bioinformation methods. The putative physiology of Bt-R3 protein in intro insect body was suggested on these analyses. Main results of this paper includes following five parts1. The separation and purification of Bacillus Thuringiensis (Bt) toxic protein CrylAb from transgenic rice by IEHPLC.A new separation and enriched method for Bt toxic protein CrylAb from Bt transgenic rice by IEHPLC has been developed and validated. The purified product has highly insecticidal activity. Two IEHPLC processes were engaged. The first IEHPLC with a weak anion-exchange column was performed with a gradient elution as: 0 to 20 min from 100 to 65% A, 20 to 60 min from 65 to 45% A, 60 to 90 min from 45 to 45% A, the mobile phase components of 10 mM Trisbase, pH = 9.8 (solvent A), and 10 mM Trisbase solvent containing 0.18 M NaCl, pH = 9.8 (solvent B). The flow rate is 1 mL.min-1 and Uv/Vis detector set to 215 nm, giving retention time 52.16 min. pHwas sensitive to the retention time of CrylAb and resolution, optimal selective as 9.8. The second HPLC with a cation-exchange column was operated by isocratic elution, with detecting wavelength being 215 nm, the flow rate being 1mL-min-1 and pH of the elution solvent being 7.5, giving the CrylAb retention time 5.21 min. The HPLC operation was suggested at temperature range from 8 to 10 C in a cool room.2. Studies on the solution conformations and insecticidal activity of Bt toxic CrylAb.The conformations of the toxic protein CrylAb in its solution have been studied by fluorescence. The results showed that the toxic protein fluorescence intensity is improved with its pH value increase and reaches its maximum at pH equivalent to 11. Adding some organic solvents such as methanol, ethanol, isopropanol result in the emission wavelength of fluorescence of the toxic protein blue shift and its fluorescence intensity increase. However, the toxic protein fluorescence emission wavelength no long blue shift as these organic solvents concentration beyond certain values. Acetone can quench all fluorescence of the toxic protein CrylAb and acrylamide quench 86% of the total fluorescence of the toxic protein, but KI can't quench any fluorescence. It implicates that the most of tryptophan residuals of CrylAb are located on the toxic protein surface where are rich in negative charge. The insecticidal activity of CrylAb in four different buffer are similar.3. Identification of CrylAb receptor protein in midgut of Chilo suppressalis, cloning thereceptor gene and analyzing its nucleotide sequence property.The protein extract was separated by SDS- (7.5%) PAGE and was electrotransferred to nitrocellulose membranes. After renaturation and blocking, western blots was conducted by incubated with 125I- CrylAb, a radioactive probe. The experiment showed that this is a 195 kDa protein in midgut of Chilo suppressalis , which specifically binding with Cry1Ab. We designed a pair of degenerated primers based on Bt- R1 and Bt -R175 amino aci... |
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