【作者】 姚艳玲；

【导师】 崔海瑞；

【作者基本信息】 浙江大学 ， 生物物理学， 2006， 硕士

【摘要】 以转cry1Ab基因高粱为材料,研究了cry1Ab表达产物的土壤环境行为与效应。本论文建立了从转cry1Ab基因高粱植株中提取Cry1Ab蛋白的适宜方法,分析了Cry1Ab蛋白在6中不同土壤中的吸附、解吸与降解,测定了转cry1Ab基因高粱不同生长发育期间根部分泌Cry1Ab蛋白,研究了秸秆还田释放Cry1Ab蛋白在土壤中的降解动态,并揭示了转cry1Ab基因高粱根部土壤中的生物学效应。主要结果如下: 采用碳酸钠-碳酸氢钠、甘氨酸-氢氧化钠、硼砂-氢氧化钠、硼酸-硼砂、碳酸氢钠-氢氧化钠五种提取缓冲液,比较了不同条件下Bt蛋白的提取效率。Bt蛋白的提取效率在前1h内表现较高,随后提取效率逐渐降低,当提取时间超过1h后提取效率基本保持稳定:而pH值对其影响表现各不相同。不同提取缓冲液的提取效率相差较大,其中甘氨酸-氢氧化钠提取缓冲液的提取效率最高,其次是碳酸钠-碳酸氢钠、碳酸氢钠-氢氧化钠、硼砂-氢氧化钠和硼酸-硼砂。考虑到提取效率和实验成本,碳酸钠-碳酸氢钠提取液是从转cry1Ab基因高粱中大量提取Cry1Ab蛋白的适宜缓冲液。 在6种不同土壤中添加已提取的Cry1Ab蛋白,测定了Cry1ab蛋白在土壤中的吸附与解吸,分析了Cry1Ab蛋白溶液浓度、土壤理化性质对Cry1Ab蛋白吸附与解吸的影响。不同类型的土壤对Cry1Ab的吸附与解吸存在明显的差异,吸附量表现为红泥土>灰化土>青紫泥>黄筋泥>小粉土>红砂土,解吸量表现为灰化土>青紫泥>红砂土>黄筋泥>红泥土>小粉土;Cry1Ab蛋白溶液的浓度与吸附量和解吸量呈显著正相关,相关系数分别为0.86和0.99:土壤有机质、pH值与土壤吸附Cry1Ab蛋白的能力呈显著正相关,相关系数分别为0.83和0.82:土壤全氮、有效磷的含量与土壤吸附Cry1Ab蛋白呈正相关,土壤全氮、有效磷和速效钾的含量与解吸均呈负相关。土壤吸附、解吸Cry1Ab蛋白是加入Cry1Ab蛋白溶液的浓度及不同土壤的理化性质共同作用的结果。 将粉碎后转cry1Ab基因高粱秸秆还田,同时把提纯的Cry1Ab蛋白添加到6种不同土壤中,从田间和室内实验分析了Cry1Ab蛋白在土壤中的残留与降解。转cry1Ab基因高粱秸秆还田后,其秸秆释放到田间土壤的Cry1Ab蛋白整个残更多还原

【Abstract】 Environmental behavior and effects of expression product of Cry1Ab gene in soil were studied by using transgenic sorghum with a cry1Ab gene as tested material. The suitable method for extracting Cry1Ab protein from transgenic sorghum.was established, adsorption, desorption and degradation of Cry1Ab protein in six types of soils were detected, the degradation trend of Cry1Ab protein released from crushed straw of transgenic sorghum in field was analyzed, content of Cry1Ab protein released from the root of transgenic sorghum in different periods were mensurated, and the effects of Cry1Ab protein on the soil enzyme activities and microorganism were studied also in this paper. Results can be summarized as the follows:Five kinds of extraction buffer, such as Glycin-NaOH, Na₂CO₃-NaHCO₃, NaHCO₃-Na0H, Na₂B₄O₇- NaOH and H₃BO₃- Na₂B₄O₇, were compared in efficiency for extracting Cry1Ab protein under different conditions. The efficiency of Cry1Ab extraction was higher within 1h, and after 1h it was slight lower and kept stable. The effect of pH on the efficiency of Cry1Ab extraction varied with the buffer. There were great differences in extraction efficiency among buffers, with the highest by using Glycin-NaOH, followed by Na₂CO₃-NaHCO₃ 、 NaHCO₃-NaOH、 Na₂B₄O₇-NaOH and H₃BO₃-Na₂B4O₇ in order. Based on both the extraction efficiency and test costs, Na₂CO₃-NaOH buffer can be considered as sutable one for extraction of Cry1Ab protein with large amounts from transgenic sorghum.Adsorption and desorption of Cry1Ab crystal protein extracted from transgenic sorghum were determined in six types of soils, and effects of concentration of Cry1Ab protein in the solution and physical and chemical property of 6 types of soils on adsorption/desorption of the protein were studied. The adsorption anddesorption of CrylAb protein in the 6 soils were obviously different. Paddy soil from red clay was the highest in CrylAb adsorption, and followed by typic podzolic soil, blue purple clay soil, paddy soil from quaternary red clay, powdery-muddy paddy soil and paddy soil derived from red sandstone in a decreasing order, while tipic podzolic soil was found the highest in CrylAb desorption and followed by blue purple clay soil, paddy soil derived from red sandstone, paddy soil from quaternary red clay, paddy* soil from red clay and powdery-muddy paddy soil. A significant positive correlation was found between the concentration of CrylAb in the solution added and the adsorption or desorption of the protein by the soils, with coefficients being 0.86 and 0.99, respectively. Effects of organic matter content and pH of the soil on adsorption were positive and significant, with coefficients being 0.83 and 0.82, respectively. The correlation between the adsorption and the content of total nitrogen or readily available phosphorus was positive, while it was negative between the desorption and the content of total nitrogen, readily available phosphorus or readily available potash. Consequently the adsorption and desorption of CrylAb in the soils were controlled jointly by concentration of CrylAb protein and physical and chemical properties of the soil.The residue and degradation of CrylAb protein in soil were studied by putting crushed straws of transgenic sorghum in field and mixing purified CrylAb protein with 6 types of soils cultured in room. The highest content of CrylAb protein in field soil released from crushed straws of transgenic sorghum was detected at 22th day after the crushed straws were put into soil and the degradation period of the CrylAb protein was about 67 days. While under room conditions, the purified CrylAb protein in 6 types of soils degraded rapidly within 30 days, and at 60th day the amount of CrylAb protein was about 1/3 of the added originally, but then the degradation rate became slower and 6.88%-19.10% of CrylAb protein added still remained in different soils at about 240th day. The degradation rate varied with soils and the difference between powdery-muddy paddy soil and tipic podzolic soil was significant at 5% level.Effects of the CrylAb protein on the soil enzyme activities and themicroorganism populations in different periods were studied by using transgenic sorghum lines SRI 6, SR24 and their parent as materials. The Cryl Ab protein could be released into soil from the root of transgenic sorghum plant, but it was too low to reach the minimun detected by CrylAb/CrylAc Detection Kit. Effects of planting transgenic sorghum on the activities of dehydrogenase, invertase and urease in the rhizosphere soil varied with the developmental stage and significant differences were observed at seedling stage between transgenic sorghum and CK and between 2 transgenic lines, but no effect was observed on catalase in whole life cycle of the sorghum. In the rhizosphere soil, the CFU （colony-forming units） of fungal at seedling, jointing and heading stages, bacteria at seedling stage and actinomycetes at heading stage were significant different between transgenic sorghum and CK. Significant difference between 2 transgenic lines were also detected in number of culturable bacteria and fugual, but no difference in actinomycetes.The above results provided theoretical evendences and technology support to application and environmental safety evaluation of transgenic Bt plants.更多还原