基于稳定同位素示踪研究Bt蛋白碳素和氮素在土壤中转化行为及微生物驱动机制

Bt蛋白的环境行为和生态效应是转Bt基因植物环境安全评价的重要内容，而外源Bt蛋白C、N在不同类型土壤中转化过程以及微生物菌群驱动机制尚缺乏系统研究。本项目前期通过大肠杆菌原核表达制备了具有杀虫活性、高纯度13C和15N标记Bt蛋白；在此基础上：1）应用稳定同位素质谱技术示踪外源Bt蛋白在不同类型土壤中归趋，探讨土壤理化因素对C、N转化行为的影响；2）利用qPCR分析C、N转化过程中微生物功能基因丰度变化；3）采用DNA-SIP和核糖体RNA高通量测序结合纯培养平板分离法，对参与Bt蛋白降解和C、N转化的可培养微生物进行分离鉴定和功能分析；4）应用RNA-SIP结合宏转录组测序分析活跃微生物组成及C、N转化功能基因相对表达丰度差异，确定其对Bt蛋白C、N转化行为贡献率。综合上述结果解析Bt蛋白转化的关键微生物过程及基因表达驱动机制，为转Bt基因植物及其表达产物的生态安全评价提供科学依据。

The environmental behaviors and ecological effects of Bt protein are important aspects of the environmental safety assessment of Bt plants. However, the carbon and nitrogen transformation process of exogenous Bt protein in different types of soils and the driving mechanisms of microbes has not been studied systematically. The project has already produced 13C/15N labeled Bt protein with high purity and insecticidal activity using prokaryotic expression system of E. coli. Based on the above-mentioned work, subsequent studies will be conducted as follows: 1) to determine the fate of exogenous Bt protein in different types of soils using 13C/15N stable isotope mass spectrometry technique, and investigate the effects of soil physical and chemical factors on the carbon and nitrogen transformation behaviors ; 2) to analyze variation of the abundance of microbial functional genes involved in the C and N transformation by quantitative PCR (qPCR); 3) to perform the isolation, identification and functional analysis of cultured microbes involved in Bt protein degradation and C or N element transformation using DNA-SIP and ribosomal RNA sequencing, combined with plate separation culture method; 4) to clarify the composition of active microbes and the relative expression abundance difference of functional genes involved in the C or N transformation, and thus determine its contribution to the C or N transformation behaviors of Bt protein using RNA-SIP and metatranscriptomic sequencing. In conclusion, the project will elucidate the important key microbial processes and uncover the gene expression driving mechanisms during the transformation processes of Bt protein in soils, which would provide essential scientific basis for the ecological safety assessment of Bt plants and expressed products.