铅锌污染对Bt蛋白在土壤中吸附的促进机制及其降解行为的变化特征

The effect of insecticidal proteins released from transgenic Bt crops on soil ecological security has been caused extensive attention. In the early research, the applicants of this project found that lead and zinc pollution enhanced the adsorption of Bt protein and reduced its desorption in soils. The result showed that lead and zinc pollution in soil had significant influence on the environmental behavior of Bt protein. In China, the planting area of Bt cotton reaches 4.2 million hectares at present, and the farmland polluted by heavy metals approaches to 1/5 of the total area. What are the environmental consequences of Bt crops if they are planted in heavy metal polluted farmland? There are no literature reports. The interaction between heavy metal ions (lead and zinc ions) and the Bt protein are chosen as the starting-point in this study. Three kinds of representative soils, such as red soil, brown soil and black soil have been collected. The combined mechanism between Bt protein and lead and zinc ions will be characterized by electrospray ionization-mass spectrometry, CD, FTIR and XPS. The hydrophobicity and surface charge of Bt protein will be determined by fluorescent probes and Zeta potentiometer, which can provide experimental evidence for the judgement of the adsorption driving force. The relationship between total content and bioavailability of heavy metal (lead and zinc) and Bt protein adsorption and degradation will be elucidated, providing a scientific basis for the risk control. This study will give a new perspective for risk assessment and management of Bt crops. In summary, this study has theoretical significance and application value.

转Bt基因作物释放杀虫蛋白对土壤生态安全影响问题已引起高度关注。申请人前期研究发现，当土壤受到铅、锌离子污染后，土壤对Bt蛋白的吸附能力明显提高，解吸率明显降低。这一结果表明，铅、锌污染对Bt蛋白的环境行为有重要影响。目前我国Bt棉花种植面积已达420万公顷，而受重金属污染的耕地面积已达1/5，在重金属污染区域种植Bt作物会有怎样的环境后果？尚无文献报道。 本课题以铅、锌离子与Bt蛋白之间的相互作用为切入点，选用红壤、棕壤和黑土三种代表性土壤，采用电喷雾质谱、CD、FTIR、XPS等表征Bt蛋白与铅、锌离子之间的结合机制，采用荧光探针和Zeta电位仪测定Bt蛋白疏水性和表面电荷，为吸附驱动力的判定提供实验证据。探明铅、锌总量及有效形态转变与Bt蛋白吸附、降解的关系，为风险调控提供科学依据。本课题为Bt作物风险评价与治理研究提供了新视角，具有重要的现实理论意义和应用价值。

本研究分析了铅锌污染对土壤和蛋白质表面电荷、蛋白疏水性以及构象的影响，采用差示扫描、X射线光电子能谱、圆二色性光谱、荧光探针和红外光谱分析吸附前后Bt蛋白功能团变化，ELISA法测定土壤中Bt蛋白降解。首次发现：（1）土壤铅锌污染引起Bt蛋白吸附能力变化与铅锌离子浓度密切相关。在低浓度范围 (0.025-0.1mmol/L) 铅锌离子对Bt蛋白吸附有抑制作用，在中高浓度范围 (>0.1mmol/L) 铅锌离子则对Bt蛋白吸附有促进作用；（2）铅锌污染能引起土壤和Bt蛋白表面电位升高，通过减小两者之间的斥力而促进吸附；（3）铅锌污染能够提高Bt蛋白的疏水性，但同时也能够引起部分Bt蛋白产生盐析沉淀，这些都是对Bt蛋白吸附有利的因素；（4）铅锌污染对Bt蛋白在土壤中吸附的促进机制包括三个方面，即静电吸引力、疏水作用力和盐析沉淀作用；（5）Bt蛋白与铅锌离子之间大体按1：1形成配合物；（6）在铅锌污染土壤中，Bt蛋白含量在最初阶段呈下降趋势，但在末期（90-120天）则呈上升趋势，表明铅锌离子对Bt蛋白有增强吸附和缓慢释放的作用；（7）土壤中铅锌总量及化学形态对Bt蛋白吸附和降解均有影响。上述结果揭示了铅锌污染促进Bt蛋白在土壤中吸附的作用机制，探明了铅锌总量及有效形态与Bt蛋白吸附、降解的关系，对Bt作物风险评价与和调控提供了科学依据。