共表达AlnA和BphC转基因苜蓿联合降解菌修复PCBs污染土壤的效能及根际微生态响应机制

Bioremediation of PCBs contaminated soil are limited by poor bioavailability of PCBs and low remediation effect. This project plans to construct transgenic alfalfa plants that can sustainable secrete biosurfactant (emulsification protein - AlnA) to the rhizosphere, and combination use this plant with PCBs degradation bacteria (Pseudomonas sp DN2) for the rhizoremediation of PCBs contaminated soil to solve the above mentioned problem. Study the effect of environmental factors on the AlnA secretion; explore the interactions and symbiotic relationship between DN2 and the transgenic alfalfa; characterize the spatial distribution and quantity change features of DN2 and AlnA in the rhizosphere; parse the physical and chemical properties of rhizosphere soil and activity change process of soil enzyme; reveal the microbial biomass turnover and community structure succession law in Rhizosphere. The final purpose of this study is to clarify the remediation effectiveness of PCBs contaminated soil and response mechanism of rhizosphere microecology by combining transgenic alfalfa and degradation bacteria. This study could not only get new PCBs repair plant, but also expect to realize the effective coupling between biosurfactant - PCBs - degrading bacteria and plants, which achieve the goal of enhancing bioremediation efficiency of the PCBs-contaminated soil. The study has a significant theoretical and practical value, and would provide scientific basis and reference for ecological safety for the assessment of the use of transgenic plants for soil organic pollution remediation.

本项目基于PCBs污染土壤生物修复受限于PCBs生物可利用性差、修复效果低下的问题，拟构建可持续向根际分泌生物表面活性剂（乳化蛋白-AlnA）的转基因苜蓿植株，联合PCBs降解菌（Pseudomonas sp DN2)进行污染土壤的根际修复研究。考察环境因素对AlnA分泌的影响；明确DN2与转基因苜蓿间的互作及共生关系；分析修复过程中根际土壤内DN2和AlnA的空间分布与数量变化特征；解析根际土壤理化性质与土壤酶活性改变历程；揭示根际区系中微生物量周转及群落结构演替规律；最终阐明转基因苜蓿联合降解菌修复PCBs污染土壤的效能及根际微生态响应机制。本研究能获得新型PCBs修复植物，有望实现表面活性剂-PCBs-降解菌-植物之间的有效耦合，达到提升PCBs污染土壤生物修复效能的目的。具有积极的理论意义和实用价值，并可为评估转基因植物用于土壤有机污染修复的生态安全性提供科学依据和借鉴。

本研究基于提升多氯联苯（PCBs）污染土壤植物修复效能的目的，利用农杆菌介导法遗传转化了苜蓿和拟南芥。通过组织培养、筛选、无性繁殖等过程，建立了系列共表达AlnA-BphC的转基因株系。对选育的株系进行ELISA检测、Western blot测定和体内酶活分析，证明了转入的细菌乳化蛋白基因（ALnA）和2,3-二羟基联苯开环酶基因（BphC）整合在了植株基因组内并可高效表达，且不影响植株的正常生长发育。利用水培实验研究表明，转基因植株与野生型相比，对PCBs的耐受性显著提高。土壤修复效能分析表明，在PCB28浓度为150ug/kg、修复时间为60天的条件下，共表达ALnA-BphC拟南芥对PCBs28的去除率可达85.94%，而野生型拟南芥仅为27.11%。ALnA联合转BphC基因苜蓿修复混合PCBs污染土壤的结果表明，转BphC基因苜蓿/ALnA处理对三氯代PCBs（(PCB16/PCB 32 、 PCB 31/PCB 28)）和四氯代PCBs(PCB49)的去除率分别是转BphC基因苜蓿的 3.6,1.1, 和 2倍。在PCBs的胁迫下，转基因苜蓿种植土壤中的脱氢酶、过氧化氢酶和多酚氧化酶活性增强。高通量测序分析根际土壤微生物群落结构显示，Pseudomonas, Arthrobacter和Sphingomonas显著相关于PCBs的去除。.本研究获得了新型的修复植物，明确了对PCBs污染土壤的修复效能，初步阐明了修复过程中的根际微生态响应机制，实现了表面活性剂-PCBs-降解菌-植物的有效耦合，并为转基因植物用于疏水性有机污染土壤的生物修复提供了借鉴和参考。