| The expression, exudation, residue in rhizosphere of Bt protein from Bt transgenic rice (KMD) and degradation of the Bt protein derived from KMD straws in soils were investigated in this paper. Degradation, adsorption and desorption of the Bt purified protein in soils were also studied. In addition, the probability of antibiotic marker genes (nptll and hpt) floating to soil bacteria was discussed, too. The results were as follows: l)The purification and extraction of Bt protein from KMD straws and soilA method of extracting and purifying CrylAb protein (Bt toxin) from crylAb transgenic rice was established. A solution of 0.1M Na2CO3-NaHCO3+5mM DTT effectively extracted most of Bt toxin presented in the tissue of crylAb transgenic rice. Bt crude protein was obtained after pretreatment with ultra-filtration, ammonium sulfate precipitation, desalinization by bagfilter and freeze-drying concentration. The dialysed crude protein was father separated on DEAE Sephadex A-50 columns and Sephadex G-150 columns. The purity and the bioactivity of the Bt toxin was determined by SDS-PAGE and larvicidal assay, respectively. The purity of the Bt protein obtained by this method was higher than 80%, and its insecticidal activity was retained after the toxin was purified.Efficiency of present methods (Palm and Envirologix Co.) on Bt protein extraction from soil was 4.6-35%. It was important to establish an effectively extracted method to study environmental behavior and fate of Bt protein in soil. A new method was improved in this study. Efficiency of Bt protein extracted from different soils by using the method was higher than that of Palm's and Envirologix Co.'s. Extraction recovery of Bt protein in soils amended with the purified protein and KMD straws were 46.18-81.73% and 47.72-82.25%, respectively. It suggested that Bt protein was easy to bound to the soil matrix based on the low efficiency( less than 50%). 2) Degradation of Bt purified protein and KMD straws in soilDegradation of Bt protein purified from KMD straws in soil was studied under lab condition. The results were as follows: degradation trends of Bt purified protein in 7 soils were in accord with first order kinetics equation(Y=Y0e-t); Half life of Bt protein in soils was among 15.2~97.6d; Degradation of purified Bt protein was rapid at the initial incubation time (30d), but slow at 150d incubation; The degradation of purified Bt protein in S7 (Intertidal sandy soil) was slowest with half-life of 97.6d. Despite incubation for 345d, Bt protein in the spiked soil amended with 1.25g/g could be still detected; The degradation of purified Bt protein in S5 (Coastal saline soil) and S6 (Aquic light saline sandy soil) were faster. Their half-lifes were 19.6d and 15.2d, respectively. The time of purified Bt protein residue in the soils was all more than 150d.Degradation trends of Bt protein in 5 soils amended with KMD straws (4%,W:W) were also in accord with equation(Y=Y0e-t). Their half-lifes were among 10.7-32.Id. The degradation of the Bt protein from the straws in S1 (paddy field on Quaternary red soil) and S2 (paddy field on red sandstone soil) was slow, with the half-lifes 32.Id and 31.1d, respectively. The results also showed that the Bt protein could be detected (detectable limit, 0.5ng/g.air-dried soil) after 146d and 138d incubation. Half life of Bt protein in S3 (Fluvio-marine yellow loamy soil) and S4 (powdery-muddy paddy soil) were 15.2d and 20.Id, respectively. However, the degradation of the Bt protein in S5 was fastest with half-life 10.7d, and the Bt protein couldn't be found in the soil after 60d incubation. After 150d, no Bt protein was detected in the amended soils.3) Adsorption and desorption of Bt purified protein in soilsAdsorption rate of purified Bt protein in different soils was decreasing with its concentration increasing (125~780ng/mL). Adsorption rate (125 and 780ng/mL) in S3, S4 and S5 were 9.12% and 31.67%, 24.85% and 40.81%, 12.47%and 30.75%, respectively. Desorption rate in the soils dropped with content of soil-absor... |
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