转Bt基因作物Bt毒素在土壤中的环境去向及其生态效应

综述了转Bt基因作物的Bt毒素在土壤中的环境去向及其生态效应的研究进展。重点阐述了：①Bt毒素与土壤表面活性颗粒结合及其与土壤理化性质的关系;②Bt毒素微生物利用与降解;③Bt毒素的杀虫活性;④后茬作物和土壤动物对Bt毒素的吸收与利用;⑤Bt毒素的垂直运移;⑥Bt毒素对土壤生物和生态过程的影响。转Bt基因作物的Bt毒素对土壤生态系统的影响急需在生态系统水平进行深入细致的长期定位研究。     

转Bt基因作物杀虫蛋白土壤残留及检测研究进展

随着转Bt基因作物的大面积推广和应用,其释放的毒蛋白在土壤中的残留及对土壤生态系统的影响等问题已经成为人们关注的热点。国内外学者们通过室内构建大田模拟模型的方法对土壤中残留的Bt毒蛋白进行了研究,并取得了显著的进展。土壤是生态系统中物质循环和能量转化过程的主要场所,转Bt基因植物的外源基因表达的Bt毒蛋白可以通过植株残体、根及根系分泌物和花粉的散播等途径进入土壤生态系统,这些高度特化了的Bt毒素蛋白一旦在土壤中积累,将会导致土壤特异生物功能类群以及土壤多样性发生改变,甚至产生级联效应。大量研究表明,苏云金芽孢杆菌产生的Bt毒蛋白进入土壤后,可与土壤粘粒和腐质酸迅速结合,不易分离,而且较之游离态,更难被土壤微生物降解。纯化的Bt毒蛋白与无菌土壤中活性颗粒紧密结合后,存留时间至少可达234d。虽然结合态的Bt蛋白用酶联免疫法(ELISA)方法检测不到,但生物测定表明其仍保持杀虫活性。对转Bt基因作物的研究表明,Bt棉组织埋入土壤7d内,土壤中可提取的杀虫晶体蛋白浓度快速下降,之后下降速度比较稳定,甚至维持数周不变。而Bt玉米根系分泌物和植株残体释放的杀虫晶体蛋白在土壤中至少保持180d杀虫活性。虽然关于Bt毒蛋白在土壤中存留时间的长短,可能因实验材料、试验方法和条件的不同而不同。但是总之,如果长期种植转Bt基因作物,很可能会造成Bt毒蛋白在土壤中的积累,并最终威胁到整个土壤生态系统的平衡。目前对土壤中Bt毒蛋白定性和定量检测的方法主要有印迹分析法(Western-blotting)、SDS-PAGE法、斑点印迹酶联免疫吸附法(dot-blotELISA)、流式细胞仪法(Flowcytometer,FCM)、ELISA平板试剂盒及试剂条和生物测定法。其中最直接、简易、准确的方法是ELISA平板试剂盒及试纸条快速检测法和生物测定法。但检测土壤残留Bt毒蛋白时,采用的方法不同,检测的结果也有差异。     

转Bt基因作物释放杀虫晶体蛋白对土壤生态安全的影响

随着转Bt基因抗虫作物的大面积推广种植,其环境安全性问题日益引起关注。转Bt基因作物在生长期内持续不断地向环境释放杀虫晶体蛋白,这些杀虫晶体蛋白积累一旦超过了昆虫的消耗及环境因子对其的钝化,就可能对非靶标昆虫或土壤微生物造成伤害。转Bt基因作物向土壤中释放杀虫晶体蛋白的途径主要有3种:根系分泌、花粉飘落和秸秆还田。释放到土壤中的Bt杀虫晶体蛋白能够迅速被土壤活性颗粒吸附,1～3 h就能达到吸附平衡。吸附态Bt杀虫晶体蛋白不易被土壤微生物或酶降解,导致杀虫活性持续时间显著延长。土壤微生物种群变化是衡量Bt作物对土壤生态影响的重要指标。研究表明,Bt作物根系分泌物或Bt生物体降解释放的杀虫晶体蛋白对于土壤蚯蚓、线虫、原生动物、细菌和真菌没有毒性,但可使丛枝菌根真菌(AMF)菌丝长度减小,不能形成侵染单元。Bt杀虫晶体蛋白对土壤酶活性的影响程度依这类蛋白的导入方式或Bt作物生育期的不同而呈现差异。土壤中Bt Cry1Ab蛋白能被部分后茬作物吸收,但不同的商品试剂盒检测结果存在差异。文章综述了Bt杀虫晶体蛋白在土壤中释放、吸附、残留特性及其对土壤动物、土壤微生物、土壤酶活性和后茬作物的影响,旨在为转Bt基因作物释放杀虫晶体蛋白的土壤生态安全评价提供参考依据。     

转Bt基因作物对土壤生态影响的研究进展

将Bt基因加以修饰改造后转入农作物中进行表达,使其成为具有抵抗特异害虫能力的转Bt基因作物。转Bt基因作物的产物Bt蛋白通过作物残体、根系分泌物和花粉3种方式进入土壤。Bt蛋白在土壤中会发生富集作用,其含量在作物的不同发育期有所不同。Bt蛋白会对土壤蛋白酶、脲酶、蔗糖酶、磷酸酶、脱氢酶等土壤酶活性和土壤细菌、真菌、放线菌等土壤微生物以及土壤线虫、环节动物、昆虫和蜘蛛等土壤动物产生影响。     

转Bt基因作物对蚯蚓影响研究进展

转苏云金杆菌杀虫蛋白(Bt)基因作物的商品化种植可能对土壤生态系统产生不利影响是近10年来颇有争议的问题.转Bt基因作物可通过多种方式向土壤中释放苏云金杆菌杀虫蛋白即Bt蛋白,从而引起土壤生物和生态系统基本功能的变化;蚯蚓可加快动植物残体的降解,促进有机质的分解和矿化,与其他土壤生物相比,蚯蚓对某些污染物更敏感.本文从研究中用到的蚯蚓种类、采用的实验方式、研究的科学问题等方面综述了转Bt基因作物对土壤动物蚯蚓影响的研究进展,并对转Bt基因作物对土壤动物蚯蚓影响研究的发展趋势进行了展望,旨在为转Bt基因作物对非靶标土壤动物的影响提供参考,进而为全面评价转Bt基因作物对土壤生态系统的影响提供依据.     

转Bt基因水稻对土壤微生态系统的潜在影响

随着转基因作物商品化应用的增多,对其进行生态风险性评价尤为重要.国内外对转基因作物中外源基因向野生亲缘物种漂移的可能性、昆虫对抗虫转基因作物的耐受性以及转基因作物对生物多样性的潜在影响等问题进行了广泛的研究.文中从Bt杀虫结晶蛋白在土壤中的残留特性、Bt杀虫晶体蛋白对土壤微生物可培养类群和土壤酶活性的影响等方面对转Bt基因抗虫水稻的潜在生态风险性进行了简要综述,以期为同类研究提供有益的信息.     

转Bt基因玉米的生态安全性研究进展

随着转基因作物的应用和推广 ,转 Bt基因作物释放后对生态环境及其它方面产生的潜在影响越来越受到重视。分别从生物活性杀虫晶体蛋白在土壤中的残留特性、杀虫晶体蛋白对土壤中非目标生物的影响、转 Bt基因玉米植株体成分的变化、转Bt基因玉米花粉中杀虫晶体蛋白的表达特性及其在田间和马力筋叶片上的散积状况、花粉中表达的杀虫晶体蛋白对君主斑蝶的毒性、君主斑蝶幼虫暴露在 Bt花粉中的概率及综合风险评价估算等方面对转 Bt基因玉米产生的杀虫晶体蛋白与土壤生态环境的相互作用、花粉对非目标生物影响的研究现状进行了综述。通过对转 Bt基因作物生态安全性的科学评价和广泛宣传 ,以确保生物技术的健康发展。     

转Bt基因作物对非靶标土壤动物的影响

转Bt基因作物已经在世界范围内广泛种植.随着转基因作物的快速发展与推广,有必要深入研究其对土壤生态系统的影响.本文概述了转Bt作物对土壤动物群落以及蚯蚓、线虫、虫兆虫、螨类和甲虫等重要类群的种群动态影响的研究进展,介绍了转Bt基因作物的发展历史,分析了Bt蛋白进入土壤的途径及其在土壤环境中的残留与降解的动态,阐述了未来转Bt基因作物对非靶标土壤动物影响的生态风险分析的重要领域,旨在为研究转Bt基因作物对非靶标土壤动物影响提供参考.     

转Bt基因作物Bt毒蛋白在土壤中的安全性研究

商业化的转Bt基因作物获准在田间大面积种植,使其释放的Bt毒蛋白对土壤生态系统的生态风险性问题成为人们关注的焦点,本文综述了转Bt抗虫作物以植株残体、根系分泌物、花粉等形式释放的Bt毒蛋白通过田间耕作等方式进入土壤后的一些安全性问题,包括土壤活性颗粒对Bt毒蛋白的吸附作用。Bt毒蛋白在土壤中的杀虫活性、存留,土壤微生物对Bt毒蛋白的降解作用以及Bt毒蛋白对土壤生物的影响等。     

Bt毒素在转基因棉花与土壤系统中的分布

研究了转Bt基因棉花与土壤系统中Bt毒素的分布.结果表明,两种转Bt基因棉花地上部(叶片、茎秆)的毒素表达量(103.5～134.1 ng·g^-1)显著高于地下部分(根系)(44.7～21.2 ng·g^-1),土壤中Bt毒素总量可通过转基因棉花地上部分秸秆的处理得到控制;Bt毒素在转Bt基因棉花根系分泌物中的含量极低,如果控制Bt毒素的其它导入来源,将显著降低转Bt基因作物释放中因Bt毒素导入而引发的对土壤生态系统的扰动.     

转基因棉种植对土壤水解酶活性的影响

转Bt基因棉和转Bt＋cpTI基因棉种植面积不断扩大,它们种植后杀虫晶体蛋白在土壤中的残留特性及对土壤水解酶活性的影响是环境风险评价的重要组成部分,本文采用盆栽实验的方法对此进行了初步研究。结果表明,转基因棉出苗后生长到30天时可向土壤中释放Bt杀虫晶体蛋白,而双抗棉种植时CpTI杀虫晶体蛋白的释放量与品种有关;转基因棉出苗后30天时,与等价基因系非转基因棉（各对照）相比,转Bt基因棉（“中30”）和双抗棉A（转Bt＋CpTI棉“中41”）的种植并未使脲酶、蛋白酶和磷酸单脂酶活性发生显著变化,而双抗棉B（转Bt＋CpTI棉“双抗321”）的种植使土壤磷酸单脂酶活性显著下降。从杀虫晶体蛋白的释放和对酶活性的影响来看,双抗棉A的种植对土壤的生物活性扰动更小。     

大田环境下转Bt基因玉米对土壤酶活性的影响

在大田自然条件下,比较研究了转Bt基因玉米和非转基因亲本玉米在种植和秸秆分解时对土壤酶活性影响的差异。结果表明,与亲本非转基因玉米相比,在各生育期内种植转Bt玉米对土壤蛋白酶和土壤脲酶活性均没有显著影响;在喇叭口期和抽雄期,土壤蔗糖酶和土壤酸性磷酸酶活性显著提高。在秸秆还田后,两种玉米秸秆对土壤酸性磷酸酶活性的影响没有显著差异,但使用转Bt玉米秸秆的土壤蔗糖酶、土壤脲酶和土壤蛋白酶的活性则有显著提高。与亲本玉米相比,在所有观测期内,种植Bt玉米及秸秆还田对土壤酶活性的影响,在影响的幅度及趋势上随玉米生育期和土壤酶种类的不同而产生差异,但没有观测到显著不利影响;商业化Bt玉米的环境释放仍有待长期定位观测和评价。     

Impact of Bt corn on rhizospheric and soil eubacterial communities and on beneficial mycorrhizal symbiosis in experimental microcosms

A polyphasic approach has been developed to gain knowledge of suitable key indicators for the evaluation of environmental impact of genetically modified Bt 11 and Bt 176 corn lines on soil ecosystems. We assessed the effects of Bt corn (which constitutively expresses the insecticidal toxin from Bacillus thuringiensis, encoded by the truncated Cry1Ab gene) and non-Bt corn plants and their residues on rhizospheric and bulk soil eubacterial communities by means of denaturing gradient gel electrophoresis analyses of 16S rRNA genes, on the nontarget mycorrhizal symbiont Glomus mosseae, and on soil respiration. Microcosm experiments showed differences in rhizospheric eubacterial communities associated with the three corn lines and a significantly lower level of mycorrhizal colonization in Bt 176 corn roots. In greenhouse experiments, differences between Bt and non-Bt corn plants were detected in rhizospheric eubacterial communities (both total and active), in culturable rhizospheric heterotrophic bacteria, and in mycorrhizal colonization. Plant residues of transgenic plants, plowed under at harvest and kept mixed with soil for up to 4 months, affected soil respiration, bacterial communities, and mycorrhizal establishment by indigenous endophytes. The multimodal approach utilized in our work may be applied in long-term field studies aimed at monitoring the real hazard of genetically modified crops and their residues on nontarget soil microbial communities.     

Structure of Cry2Aa suggests an unexpected receptor binding epitope

BACKGROUND: Genetically modified (GM) crops that express insecticidal protein toxins are an integral part of modern agriculture. Proteins produced by Bacillus thuringiensis (Bt) during sporulation mediate the pathogenicity of Bt toward a spectrum of insect larvae whose breadth depends upon the Bt strain. These transmembrane channel-forming toxins are stored in Bt as crystalline inclusions called Cry proteins. These proteins are the active agents used in the majority of biorational pesticides and insect-resistant transgenic crops. Though Bt toxins are promising as a crop protection alternative and are ecologically friendlier than synthetic organic pesticides, resistance to Bt toxins by insects is recognized as a potential limitation to their application. RESULTS: We have determined the 2.2 A crystal structure of the Cry2Aa protoxin by multiple isomorphous replacement. This is the first crystal structure of a Cry toxin specific to Diptera (mosquitoes and flies) and the first structure of a Cry toxin with high activity against larvae from two insect orders, Lepidoptera (moths and butterflies) and Diptera. Cry2Aa also provides the first structure of the proregion of a Cry toxin that is cleaved to generate the membrane-active toxin in the larval gut. CONCLUSIONS: The crystal structure of Cry2Aa reported here, together with chimeric-scanning and domain-swapping mutagenesis, defines the putative receptor binding epitope on the toxin and so may allow for alteration of specificity to combat resistance or to minimize collateral effects on nontarget species. The putative receptor binding epitope of Cry2Aa identified in this study differs from that inferred from previous structural studies of other Cry toxins.     

Bacillus thuringiensis (Bt) toxin susceptibility and isolation of resistance mutants in the nematode Caenorhabditis elegans

The protein toxins produced by Bacillus thuringiensis (Bt) are the most widely used natural insecticides in agriculture. Despite successful and extensive use of these toxins in transgenic crops, little is known about toxicity and resistance pathways in target insects since these organisms are not ideal for molecular genetic studies. To address this limitation and to investigate the potential use of these toxins to control parasitic nematodes, we are studying Bt toxin action and resistance in Caenorhabditis elegans. We demonstrate for the first time that a single Bt toxin can target a nematode. When fed Bt toxin, C. elegans hermaphrodites undergo extensive damage to the gut, a decrease in fertility, and death, consistent with toxin effects in insects. We have screened for and isolated 10 recessive mutants that resist the toxin's effects on the intestine, on fertility, and on viability. These mutants define five genes, indicating that more components are required for Bt toxicity than previously known. We find that a second, unrelated nematicidal Bt toxin may utilize a different toxicity pathway. Our data indicate that C. elegans can be used to undertake detailed molecular genetic analysis of Bt toxin pathways and that Bt toxins hold promise as nematicides.     

Capturing the interaction types of two Bt toxins Cry1Ac and Cry2Ab on suppressing the cotton bollworm by using multi‐exponential equations

Transgenic crops are increasingly promoted for their practical effects on suppressing certain insect pests, but all transgenic crops are not equally successful. The insect pests can easily develop resistance against single Bacillus thuringiensis (Bt) toxin transgenic crops. Therefore, transgenic crops including two or more mixed Bt‐toxins can solve this problem by delaying the resistance development and killing the majority of targeted pests before the evolution of resistance. It is important to test the controlling effects of transgenic crops including multiple mixed toxins on a particular insect pest. Previous research has checked the cross‐resistance and interactions between Bt toxins Cry1Ac and Cry2Ab against one susceptible and four resistant strains of cotton bollworm. The results showed that independence was the main interaction type between two toxins for the susceptible strain, whereas synergism was the main interaction type for any one resistant strain. However, the optimal combinations of two toxins were not obtained. In the present study, we developed two multi‐exponential equations (namely bi‐ and tri‐exponential equations) to describe the combination effects of two Bt toxins. Importantly, the equations can provide predictions of combination effects of different continuous concentrations of two toxins. We compared these two multi‐exponential equations with the generalized linear model (GLM) in describing the combination effects, and found that the bi‐ and tri‐exponential equations are better than GLM. Moreover, the bi‐exponential equation can also provide the optimal dose combinations for two toxins.