【作者】 吴立成；

【导师】 叶庆富；

【作者基本信息】 浙江大学 ， 生物物理学， 2004， 硕士

【摘要】 本文以转Bt（cry1Ab）基因水稻-克螟稻（KMD）及其亲本水稻-秀水11（Xiushui 11）为试材，通过田间和实验室试验，研究了克螟稻生育期间Bt蛋白的表达、分泌、土壤中的残留及秸秆还土后Bt蛋白在土壤中的降解规律；同时从克螟稻秸秆中纯化制备出Bt纯蛋白，进而研究该蛋白在不同土壤中的吸附解吸与降解规律。试验中还对抗性选择标记基因（nptⅡ、hpt）水平转移到土壤细菌的可能性进行了初步探讨。主要结果如下： 1)Bt蛋白纯化制备和土壤中Bt蛋白提取方法的建立 采用0.1M Na₂CO₃-NaHCO₃+5.0mM DTT缓冲液作为提取剂以提取克螟稻秸秆中的Bt蛋白。提取液经冷冻干燥浓缩和截留分子量浓缩至较小体积，然后用（NH₄）₂SO₄分级沉淀（20％去杂蛋白，80％收集Bt粗蛋白）、离子交换色谱（A-50）、凝胶渗透色谱（G-150）等步骤后，得到了纯度较高的Bt蛋白（经SDS-PAGE电泳检测相对纯度80％以上）。生物活性测定表明，纯化制备所得的Bt纯蛋白仍保留杀虫活性。 国内外现有的方法难以有效地提取土壤中的Bt蛋白（提取效率4.6～35％），为研究Bt蛋白在土壤环境中的行为与归趋，建立适宜的提取方法尤为重要。本研究通过添加回收率试验（分别添加Bt纯蛋白和克螟稻秸秆），比较了3种提取方法对不同土壤中Bt蛋白的提取效率，得出了适用于从土壤中提取Bt蛋白的方法。利用新建的提取方法，土壤中Bt纯蛋白的添加回收率在46.18～81.73％之间；克螟稻秸秆还土后，秸秆中的Bt蛋白的提取回收效率在47.72～82.25％之间。新方法的提取效率明显高于Palm等法（提取效率7.25～34.15％）和Environlogic公司方法（提取效率4.61～21.32％）。某些土壤中Bt蛋白的提取效率相对较低，意味着Bt蛋白易与该土壤基质（粘土矿物和腐殖质等）形成难以提取的结合态残留，这进一步证实了Stotzky等人的研究结果。 2)Bt纯蛋白和克螟稻秸秆中的Bt蛋白在土壤中的降解规律 Bt纯蛋白的添加培养试验表明，在7种不同土壤中其降解趋势符合一级动力学反应方程Y=Y₀e^-λt，半减期为15.2～97.6d，培养前期（30d前）降解比较快，而后缓慢降低。Bt纯蛋白在粗砂土降解最慢，半减期为97.6d；而在滨海盐土和盐碱土中降解最快，半减期分别为19.6d和15.2d；当Bt纯蛋白的添加量为1.25μg／g时，粗砂土培养345d后仍然能够检测到Bt蛋白。试验结果还表明，所有培养土壤中，Bt纯蛋白的持留时间都大干150d。浙江大学硕士学位论文 克螟稻秸秆添加试验表明，克螟稻秸秆中的Bt蛋白在5种不同土壤中的降解趋势符合一级动力学反应方程Y=Yoe一“，半减期为10.7一32.ld，培养前期（20d前）降解比较快，而后缓慢降低。当克螟稻秸秆添加量为4%时，Bt蛋白在黄松土、小粉土、黄筋泥和红砂土中的半减期分别为15.2d、20.ld、32.ld和31.ld，其在后两种土壤中降解较慢，培养146d和138d后仍然能够检测到Bt蛋白存在。而在滨海盐土中，Bt蛋白降解最快，半减期仅为10.7d，培养60d后Bt蛋白浓度就低于检测限检测极限（0.5n岁g.air dried5011）。试验结果还表明，培养150d后，所有供试土壤中均检测不到Bt蛋白。3) Bt纯蛋白在土壤中的吸附解吸规律 Bt纯蛋白在土壤中的吸附率随着Bt蛋白加入浓度的降低而升高（浓度125一780n留mL）。在7种供试土壤中，小粉土的吸附率最高，Bt蛋白浓度为125和780ng/mL时，其吸附率分别为24.9%和40.8%;而黄松土和滨海盐土的吸附率相对最低，相应的吸附率分别为9.1%和31.7%，12.5%和30.8%。解吸附率随着吸附量的减少而降低。小粉土的解吸附率较低，分别为13.0%和5.9%。土壤对Bt纯蛋白的单位吸附量与Bt蛋白加入浓度和土壤的OM呈显著正相关（P<0.05）。4)生育期间克螟稻Bt蛋白的表达、分泌与根际土中的残留规律 分粟始期至成熟期，克螟稻地上部和根中Bt蛋白的表达量分别为3.23一8.22“g/s.FW和0.68一0.89协g/g.FW。克螟稻根系分泌的Bt蛋白量仅为1.66一48.02 ng/individual·d，根际土中的Bt蛋白的残留量低于检测限（0 .sn岁9 air-dried 5011）。生物测定还表明，克螟稻根际土及其提取液对棉铃虫（Heliochis armigera）初孵幼虫和3龄幼虫不产生致死性效应。因此，从Bt蛋白的空间转移角度分析，只要能防治克螟稻秸秆还田，生育期间根系分泌的Bt蛋白难以转移到土壤中，对敏感生物也不会造成危害。5)克螟稻生育期间和秸秆还土后抗性标记基因水平转移的可能性 对抽提土壤细菌DNA的方法进行了比较和改进，改良方法抽提的土壤细菌DNA的A26夕凡80为1.61，A260/A23。为0.86，在一定程度上优化了提取质量与得率。用该方法分别研究了连续多年种植克螟稻的田间土壤细菌和添加秸秆实验室培养的土壤细菌的DNA分子图谱，试验结果表明，在土壤细菌DNA中均没有扩增到抗性标记基因（nPtll、hPt）的片段，即未发现克螟稻中与目的基因（c尽]Ab）相串联的抗性标记基因（nPtll、如t）水平转移到土壤细菌中。明确的结论还有待于进一步的试验证实。更多还原

【Abstract】 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更多还原