| Bt transgenic cotton is the most comprehensive being used in our country, because of its wilder and higher resistance to insects in lepidop term and its boll number per plant was more. The Bt gene, which is transferred into cotton by gene engineering, can express efficiently in all tissues and organs of cotton, and synthetic Bt protein can kill lepidopteran. But if still using the routine law of planting and management, the output potential cann't be worked out normally, and there might have been a cut in output in different cotton ecosystem. In this paper, the author used the pesticide powder sold in the market to purify and crystallize the Bt protein with higher purity, then prepared the specific protein-antibody and enzyme-linked immunosorbent assay, and studied the resistance of transgenic cotton and non-transgenic cotton to insects in different time and spheres. In the research,the author wanted to find a quick and effective method to inspect the transgenic cotton, and provide thetheoritical foundation of field management of transgenic cotton. Results showed as follows:1. The author used the pesticide powder sold in the market as the material, separated and purified the Bt protein by resolving in Alkaline solution, chromatography and ethanol precipitation. The results showed: when pH=10.5, the degree of resolving in Alkaline solution was highest; when solid ammonium sulphate was added in the solution until 60% saturation, it can split off most amylase; Separating Bt protein (130KD) from other protein clutter by Sephadex G-50,and then gaining crystalline Bt protein by adding 1.2-1.5 times of 95% ethanol in the solution, freeze-drying and weighing, the harvest was 1.338g/Kg Bt protein powder.2. Biological experimentation was used by breeding bollworm in laboratory. The death rate of the bollworm with larva to second instar larva was 100% in 48h, and the death rate of the bollworm with third instar larva was 88%, and the death rate of the bollworm with fourth instar larva was 76%, and biological activity is 18000IU/mg.3.The big white rabbit from New Zealand, which was 2Kg,was used in experiment. Withdrew the protein from the rabbit blood serum by the saturated ammonium sulfate precipitation, then added the cold 95% ethyl alcohol precipitation after the A-50 chromatographic analyses, and the antibody was preparated by this method of immunology. The antibody' titer was 32(Agar-agar gelatin diffusion process),and it can be completely used as ELISA-reagent(The titer needed was not lower than 16).4. The ELISA-reagent preparation contrasted with Agdia from overseas, the results showed the linear scope of ELISA-reagent' protein density was 1750～28000 ng·ml-1 and the linear scope of Agdia' protein density was 2250～36000 ng·ml-1.It was obvious that the ELISA-reagent' examination sensitivity was higher than Agdia',but the ELISA-reagent' examination scope was narrower than Agdia'. The ELISA-reagent' precision was misses slightly and its stability was not very good, so it should be explored for further tests.5. Sampling from different apparatus of transgenic and non- transgenic cotton in different time, and examining by the ELISA-reagent preparation (branch stem and leaf of cotton' leaf was choosed). The results showed that at the same time in the same cotton plant, the content of Bt toxin protein was lower in the leaf of peak one and in the leaf of peak two was higher, in the leaf of peak three was highest, but in the leaf of peak five was lower than in the leaf of peak three and four, so it indicated the product which was expressed by Bt gene in the leaf blade was changed along with the maturity degree of young plants. In different periods of growth, Bt protein expression quantity also was increasing while the seedling was growing, and they all can reach the anti-insect's threshold. Especially in the flower bud time and ties the bell time the content of Bt protein was highest, but after the boll opening time, Bt protein expression quantity was reducing gradually and the content of Bt protein in the cotton leaf was lowest, even it already was lower than the threshold which was the anti-insect requested. The Bt protein expression tendency from cotton leaf in different periods of growth was: Initially flowering season> Bud stage > Colored bell time > Seedling stage > Boll opening time. In nine tissues, the resistant order to initially hatches larva was: Functional leaf > Tender leaf > Young flower bud > Young bell leaf> Young leaf > Flower petal > Stamen and pistil > Binder heaf > Hard mulberry leaf.6. The cotton leaves which were in the identical position but in different time(peak two, launches function leaf completely) ,was examed by the ELISA ,and the results showed that in the anti-insect cotton' growth initial period, the expression started actively, and with cotton plants' unceasing growth, the expression ability of the anti-insect cotton also gradually strengthened, and it would achieve the peak when bud stage and flowering season. But expression quantity of the anti-insect cotton started to drop fast after cotton wool time, so the Bt protein expression level in later period was lower than in the growth initial period by far. |
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