转Bt基因作物杀虫蛋白土壤残留及检测研究进展

Research Advances in Soil Remaining and Monitor of Toxins Protein Released by Transgenic Bt Crops

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。

With the large area popularization and application of transgenic Bt crops, such questions as its toxin protein residue in the soil and impact on soil ecosystem released, etc. have already become concerned focuses of people. Domestic and international scholar structure land for growing field crops method, simulation of model Bt toxin protein that remain carry on research to soil in room, make remarkable progress. The soil is a main place that the material circulation and energy transform course in the ecosystem, transfer to Bt other source Bt toxin protein of gene expression, gene of plant can enter the soil ecosystem through plant incomplete body, root, root exudates and way of disseminating etc. of pollen, once Bt toxin protein that height take specially the accumulate among soil, cause soil peculiar biological function group and soil variety change, even produce the effect of one grade of antithetical couplets. A large number of research indicate, Bt toxin protein of Bacillus thuringiensis that produce enter after the soil, can glue grain sour to combine rapidly with rotten quality, difficult to separate, and compared with the attitude of dissociating with soil, difficult the little biodegradation of soil. After toxin protein of Bt of the purification is combined closely with active particle in the aseptic soil, it is can be up to 234 days at least to retain time. Though the Bt protein which combines can not measure with ELISA method, but it indicates it still keeps insecticidal activity that the living beings determine. To transgenic Bt crops research indicate Bt cotton organization imbed soil in the 7d , whom soil can draw insecticidal crystal protein density drop fast, later the decrease speed was more steady, had even kept not changing for several weeks. And Bt maize root exudates and plant that incomplete body release to kill worm crystal albumen keep 180d abating worm＇ s activation at least of the soil. Though retain size of time among soil by toxin protein about Bt, may be different because experiment material, test method are different from condition. But in a word,if plant and transfer to the gene crop of Bt for a long time, will possibly cause the accumulation in the soil of Bt toxin protein, and threaten the balance of the whole soil ecosystem finally. Bt toxin protein determine the nature to soil at present and for analytic approach of a trace method that ration measure （Western-blotting） , SDS-PAGE law, spot trace enzyme unite the immune adsorption method （ dot-blot ELISA）, flowing type cell＇ s appearance law （ FCM）, ELISA dull and stereotyped reagent box and reagent article and living beings determine the law. Among them most direct simple and easy, accurate method whether ELISA dull and stereotyped reagent box try on note measure law and living beings determine law fast. But while measuring the soil and remaining Bt toxin protein, methods to adopt are different, the result measured has a difference.