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土壤中铁铝氧化物对Bt毒素吸附的影响.pdf

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华中农业大学2010届硕士研究生学位论文目录摘要I√6L】3STRACT..IIIl前言.11.1土壤中Bt毒素的来源和积累残留21.1.1土壤中Bt毒素的来源21.1.2土壤中Bt毒素的积累残留21.2 Bt毒素与土壤的相互作用..31.2.1 Bt毒素与土壤活性颗粒的吸附结合..31.2.2 Bt毒素在土壤颗粒表面吸附的机理..51.2.2.1 Bt毒素吸附过程51.2.2.2 Bt毒素与土壤颗粒间的作用51.3 Bt毒素对土壤的影响71.3.1 Bt毒素对土壤理化性质的影响..71.3.2 Bt毒素对土壤生态系统的影响一81.3.2.1对土壤酶的影响81.3.2.2对土壤微生物和动物的影响81.4土壤中的铁铝氧化物91.5本研究的内容及意义.102材料和方法.112.1实验材料.1l2.1.1苏云金芽胞杆菌毒素制备.112.1.2 Bt毒素溶液的配制112.1.3土壤样品..112.1.5土壤基本性质的测定..122.1.6供试土壤去铁铝的处理1 32.1.7各种形态铁铝含量的测定142.1.8除去不同形态铁铝氧化物后土壤比表面积的测定一142.2 Bt毒素浓度的测定..142.2.1 ELISA法.1zI2.2.2紫外吸收法1 42.3实验方法..1 5土壤中铁铝氧化物对Bt毒素吸附的影响2.3.1供试土样中Bt毒素本底含量的测定..152.3.2各种形态铁铝氧化物对Bt毒素吸附的影响152.3.3除去不同形态铁铝氧化物的土壤对Bt毒素的等温吸附。152.4数据统计分析162.5 Bt毒素在土壤中的吸附模型1 63结果与分析1 73.1供试土样基本性质及Bt毒素本底含量..1 73.1.1供试土样的理化性质1 73.1.2单独提取的土壤中各形态铁铝的含量1 73.1.3供试土样Bt毒素本底含量..193.1.4除去各种形态铁铝氧化物的土壤比表面积193.1.5土壤除去不同形态铁铝氧化物的有机质含量变化203.2各种形态铁铝氧化物对Bt毒素吸附的影响..203.2.1有机络合态铁铝对Bt毒素吸附的影响.203.2.2游离态铁铝对Bt毒素吸附的影响。213.2.3非晶形铁铝对Bt毒素吸附的影响一223.2.4层间态铁铝对Bt毒素吸附的影响一233.2.5吸附态铁铝对Bt毒素吸附的影响.243.2.6交换态铁铝对Bt毒素吸附的影响.253.3 Bt毒素在除去铁铝氧化物的土壤上的等温吸附263.3.1除去铁铝氧化物的砖红壤上的等温吸附.263.3.2 Bt毒素在除去铁铝氧化物的赤红壤上的等温吸附..283.3.3 Bt毒素在除去铁铝氧化物的红壤上的等温吸附303.3.4 Bt毒素在除去铁铝氧化物的黄壤上的等温吸附323.3.5 Bt毒素在除去铁铝氧化物的水稻土上的等温吸附343.4 Bt毒素在矿物表面的等温吸附364讨论~385结j沧~40参考文献42致谢.49华中农业大学2010届硕士研究生学位论文摘要转Bt基因作物发展迅速,其释放的毒素可能在土壤中残留和累积,影响土壤生态系统的功能和生物多样性,引起人们对环境安全问题的关注。本课题以海南澄迈县砖红壤、广西南宁赤红壤、湖北咸宁红壤、贵阳花溪桐木岭黄壤、江西南昌水稻土为对象,研究了有机络合态、游离态、非晶形、层间态、吸附态、交换态铁铝氧化物对Bt毒素吸附的影响,着重比较了Bt毒素在除去不同形态铁铝氧化物土壤和原土壤上的等温吸附特征,同时还研究了Bt毒素在针铁矿、赤铁矿、三水铝石上的吸附特性,为明确各形态铁铝氧化物对Bt毒素吸附的影响提供参考,有助于了解转Bt基因作物释放的毒素在土壤中的去向与积累规律,阐明Bt原毒素对生态环境的影响。主要结果如下15种土壤除去有机络合态铁铝F印-Alp后,对Bt毒素的吸附量都有所增加。与原砖红壤、赤红壤、红壤、黄壤、水稻土相比,毒素的吸附量分别增加了18.5%、3.5%、12.4%、15.5%、52.9%。毒素吸附量的增加与去除有机络合态铁Fep的含量正相关,与去除有机络合态铝Alplj勺含量负相关,且与有机质的减少量有关。2Bt毒素在除去游离态铁铝Fed.Ald的砖红壤、赤红壤、红壤、黄壤、水稻土表面的吸附量都显著降低,分别下降了55.6%、37.O%、70.5%、21.8%、45.8%。除去游离态铁铝,土壤表面负电荷增加,比表面积下降,负电荷和外表面积之比升高,促使Bt毒素的吸附量显著下降。3与原土壤相比,在除去非晶形铁铝Feo.Alo的砖红壤、赤红壤和水稻土上Bt毒素的吸附量差异显著,分别下降了27.7%、34.6%和14.4%,而在除去非晶形铁铝的红壤和黄壤上差异不显著,吸附量分别下降了6.3%和12.4%。除去非晶形铁铝后,土壤的PZC降低,永久负电荷增加,Bt毒素吸附量下降。4Bt毒素在除去层间态铁铝InFe-tn剐的5种土壤上吸附量都显著下降。与原砖红壤、赤红壤、红壤、黄壤、水稻土相比吸附量下降了37.5%、39.3%、38.2%、14.O%、18.8%。土壤除去层间态铁铝后,层间负电荷量增加,与带负电荷的Bt毒素之间的静电斥力增强,Bt毒素的吸附量降低。同时,层间态铁铝的减少,使可与毒素的羧基和羟基发生配位交换的羟基减少,对Bt毒素的吸附量降低。土壤中铁铝氧化物对Bt毒素吸附的影响5与原土壤相比,在除去吸附态铁铝HyFe.HyAl的砖红壤、赤红壤和红壤上Bt毒素的吸附量差异显著,分别从原来的182.1、210.1和157.8mg g-1降至124.3、156.7和116,7mg g-1,下降幅度分别为31.7%、25.4%和26.1%;而在除去非晶形铁铝的红壤和黄壤上差异不显著,吸附量分别下降了3.1%和6.3%。5种土壤的吸附态铁的含量均很低,它对Bt毒素吸附的影响可以忽略。毒素吸附量降低的主要原因是除去了吸附态铝。6Bt毒素在除去交换态铁铝ExFe.ExAl的5种土壤上的吸附量都显著降低。与原土壤相比,在除去交换态的砖红壤、赤红壤、红壤、黄壤、水稻土上Bt毒素的吸附差异均显著,下降幅度相似,分别下降了22.4%、23.1%、24.5%、23.3%、21.3%,主要是受除去土壤交换态铝的影响。除去交换态铁铝,土壤表面部分离子的水合半径增大,供给吸附的能量减少,毒素的吸附量降低。7在供试5种土壤中,6种形态的铁铝氧化物对Bt毒素吸附的影响程度不同。在砖红壤上,不同形态的铁铝氧化物影响作用的强弱为游离态层间态非晶形态、吸附态、有机络合态交换态;在赤红壤上,影响的强弱顺序为游离态、层间态铁铝有机络合态、非晶形、吸附态铁铝交换态铁铝;在红壤上,影响的强弱顺序为游离态、层间态铁铝有机络合态、非晶形铁铝交换态、吸附态铁铝;在黄壤上,顺序为游离态、交换态铁铝有机络合态铁铝非晶形、层间态、吸附态铁铝;在水稻土中的影响强弱顺序为游离态铁铝非晶型态、层间态、有机络合态铁铝交换态、吸附态铁铝。各种形态的铁铝氧化物对Bt毒素在5种供试土壤吸附的影响程度不尽相同,这与供试土壤自身的理化性质、各形态铁铝氧化物含量以及其对毒素吸附影响因素的不同有关。8Bt毒素易在土壤和矿物表面吸附,其等温吸附曲线均为L型,用Freundlich和l_angrnuir方程拟合结果表明Bt毒素在去除有机络合态铁铝的土壤表面,吸附亲和力最大,在去除游离态铁铝的供试土壤表面亲和力最小。Bt毒素在不同矿物表面的等温吸附差异较大,总体趋势是针铁矿赤铁矿三水铝石。关键词苏云金芽孢杆菌;Bt毒素;吸附铁铝氧化物Ⅱ华中农业大学2010届硕士研究生学位论文AB STRACTSince the fast developing transgenic Bt plants can impact ecosystem operation andbiodiversity by retaining and accumulating the released toxins,broad concerns aboutenvironmental security arose among peoples.Taking the latosol of Chengrnai County,Hainan province,latosolic red soil of Nanning City,Guangxi province,red soil ofXianning City,Hubei province,yellow soil of Huaxitongmuling in Guiyang City,Guizhou province and paddy soil of Nanchang city,Jiangxi province as researchtargets,the effect on Bt toxin adsorption by different s of iron and aluminiumoxides,such as organic complex state,free state,amouphous state,interlayer state,adsorption state and exchangeable state,Was studied and the comparison of isothermaladsorption characteristics of Bt toxins in all the selected soils whose iron andaluminium oxides of different s being removed and unremoved previously Wasfocused on in particular.Besides,the research covers isothermal adsorptioncharacteristics of Bt toxins in goethite,hematite and gibbsite.The whole study willprovide reference for defining the effect of different s of iron and aluminiumoxides on Bt toxins adsorption,facilitate exploration of characteristics of migrationand accmul.ation for the toxins in soil released by transgenic Bt plants,and is of farreaching importance on in-depth study of effect on ecological environment brought byoriginal Bt toxins.The main results are showed below.1The adsorption amount of Bt toxin in the selected 5 kinds of soils increased afterthe Fep-Alp being removed.Comparing with Fep-Alp·unremoved soil,the toxinadsorption amount increse of 1 8.5%、3.5%、12.4%、15.5%、52.9%goes for latosol,latosolic red soil,red soil,yellow soil and paddy soil respectively.The increasingtoxin adsorption amount Was in positive correlation with the content of removed Fep,and in negative correlation with that of removed Alp.Besides,it is related with thedecreased organic matters.2The adsorption amount of the Bt toxins on the surface of Fed·Aid-removedsoils.1atosol,latosolic red soil,red soil,yellow soil and paddy soil declinedsignificantly up to 55.6%、37.O%、70.5%、21.8%and 45.8%respectively.Moreover,being attributable to the Fed-Aid·removing treatment,the number of negative chargeson the soil surface Was raised while the superficial area was diminished,and theirratio grew up,promoting the Bt toxin adsorption capacity falled off.III土壤中铁铝氧化物对Bt毒素吸附的影响3The Bt toxins adsorption amount for FeoAlo-removed soils-latosol,latosolic redsoil and paddy soil significantly decreased comparing with that for unremoved soils,up to 27.7%、34.6%and 14.4%,in contrast to slight reduction of6.3%and 12.4%forFeoAlo-removed yellow soil and red soil.Besides,responding to theFeo-Alo-remoVing treatment,the soil point of zero changePZCwas lowered,theamount of permanent negative charges grew up,and Bt toxins adsorption contentdiminished.4The adsorption amount of Bt toxin in all the selected 5 kinds of soils declinedsharply after the InFe·InAl being removed,up to 37.5%、39.3%、38.2%、14.O%、1 8.8%comparing witll mat in untreated soils.What’S more,this treatment led to theincrease of negative charges between two layers,reinforcement of the electrostaticrepulsion among Bt toxins attached with negative charges,and reduction of Bt toxinsadsorption capacity.Besides,the dropping amount of hydroxyls which Canligand-exchange with carboxyls and hydroxyls of toxins also contributed to theadsorption amount of Bt toxins decline.5The Bt toxins adsorption amount for HyFe-HyAl一removed soilslatosol,latosolicred soil and paddy soil significantly dropped off from 1 82.1、2 1 0.1 and 1 57.8mg 91to 1 24.3、1 56.7 and 1 1 6.7mg百1 respectively comparing with that for unremoved soils,up to 3 1.7%、25.4%and 26.1%.However,the significant reduction did not apply tothe treatments with HyFeHyAl一removed red and yellow soils,instead with 3.1%and6.3%’S cutback.On the basis of low content of HyFe for all the 5 soils,the effect ofFed on Bt toxins adsorption could be ignored.Hence,the dropping of toxin adsorptioncontent Was primarily caused by removing of HyAl.6The Bt toxins adsorption content declined markably in all the 5 tested soils afterExFe-ExAl being removed.The close reductions for atosol,latosolic red soil,red soil,yellow soil and paddy soil reached up to 22.4%、23.1%、24.5%、23.3%and 21.3%respectively,due to the removal of Ale mainly.The removal of ExFe-ExAl led to therise of some ions’hydration radius and cutback of adsorption energy supply,resultingin Bt toxins adsorption content decrease because of the difficulty enhancement foradsorption.7For all the 5 tested soils,the 6 kinds of Fe-Al oxides of different s rteddistinctive degree of effect Oil Bt toxins adsorption.For latosol,the order of effectIV、 华中农业大学2010届硕士研究生学位论文degree brought by different s of Fe·A1 was free stateinterlayer stateamouphous state,adsorption state,organic complex stateexchangeable state;forlatosolic,it was free state,interlayer stateorganic complex state,amouphous state,adsorption stateexchangeable state;for red soil,it Was free state,interlayer stateorganic complex state,amouphous stateexchangeable state,adsorption state;foryellow soil,it was free state,exchangeable stateorganic complex stateamouphous state,interlayer state,adsorption state;for paddy soil,it Was free stateamouphous state,interlayer state,organic complex stateexchangeablestate,adsorption state.8The adsorption isotherms of Bt toxin were L-type either on the surface of soil ormineral,both of thcrn being prone to adsorb Bt toxins.The Freundlich and Langrnuirequation fitting results showed that the maximal adsorption affinity applied to theFep-Alp·removed soil,comparing with the minimal one for the Fed-Aid。removed soil.As Bt toxin concentration increased,the adsorption amounts of Bt toxin appeared toincrease rapidly.Besides,the drastic disparity of adsorption isotherms of Bt toxin,which decreased in the order GeothiteHematiteGibbsite,were observed amongdifferent mineral surfaces.KeyWordsBacillus thuringiensis;Bt toxin;adsorption;iron and aluminium oxidesV华中农业大学2010届硕士研究生学位论文1前言苏云金芽胞杆菌Bacillus thuringiensis,简称Bt是一种广泛存在于土壤中的革兰氏阳性细菌,它的种群多样性丰富,根据其鞭毛抗原的差异,目前分离得到的Bt有71个血清型,83个亚种。Bt毒素insecticidal crystalprotein包括a-夕l、毒素、p矽}.毒素、6.内毒素和虱因子,其中起主要杀虫作用的是6.内毒素,它在昆虫肠的碱性和还原性环境下被降解为60 KDa左右的活性肽,能与受体蛋白结合,嵌合于细胞膜,引起膜穿孔最终导致昆虫瘫痪或死亡Hofte et a1.,1989;Van et a1.,1990;Cummings et a1.,1994;Ferre et a1.,1995。由于6.内毒素对昆虫专一性强、毒力高、修饰后在植物体内表达水平高,越来越多地导入植物体中崔林开等,2006。目前国际上已命名的Cry和Cyt杀虫蛋白基因有130多个Crickmore et a1.,1998;Schnepfet a1.,1998,Bt基因已成为植物抗虫基因工程中应用最广泛的抗虫基因,它被转入棉花、玉米等农作物中并得到应用喻子牛等,1995;赵建周等,1996。1996年后转基因棉花、马铃薯、玉米、水稻、烟草、番茄等50余种作物先后进入大规模商业化种植欧阳立明等,2001。我国目前种植的转基因作物主要是转基因抗虫棉,1997年首先批准在河北省种植,很快就取得了显著的经济和社会效益,到2005年长江中下游、新疆和黄淮广大棉区己大面积种植,总面积己达340万hm2,占棉花总播种面积的68%。中国科学院农业政策中心研究发现,中国小农户种植抗虫Bt棉后,棉花的平均产量增加9.6%且杀虫剂施用量减少60%。2009年底,农业部批准转Bt基因水稻在湖北省和山东省生产应用,我国转Bt基因作物的种植面积将进一步增加。转Bt基因作物可以抵抗害虫的危害,提高作物产量,减少农药使用和环境污染等优点。但转Bt基因作物的外源基因表达产物Bt毒素,可通过根系分泌物或作物残茬进入土壤生态系统,导入土壤中的毒素能被结合到土壤颗粒表面而不易被微生物降解并仍然保持活性,将导致毒素的累积,从而使土壤的特异生物功能类群以及生物多样性都有可能改变Koskella and Stotzky,1 997。
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