转基因苜蓿联合茶皂甙根际修复PCBs污染土壤的效能及机制

Polychlorinated biphenyls (PCBs) are persistent organic pollutants in soil and bioremediation of the contaminated soil has become a research focus. Plant remediation of PCBs is a promising technology, however it was not usually efficient because PCBs have strong hydrophobicity, low bioavailability and the inhibitory effect on plant growth. Based on obtained transgenic alfalfa expressing bphC.B, construct co-expressing tfdb-JLu and bphC.B in alfalfa to form a degradation pathway of PCBs in Alfalfa, which is similar to microbes. The application of transgenic alfalfa and tea saponin remediate of PCBs contaminated soil. The aim is to investigate the removal of PCBs in transgenic alfalfa; induce the degradation gene expression of PCBs in indigenous microbial by tea saponin; analyze the dynamic variation of PCBs in the rhizosphere soil and reveal the regional microbial diversity and community structure succession. Therefore, rhizosphere remediation efficiency and biological response mechanism were clarified. This study may provide a new phytoremediation technology for PCBs contaminated soil to enhance remediation efficiency and reduce their ecological risk.

多氯联苯(PCBs)是土壤中持久性有机污染物，其污染土壤的生物修复技术已成为研究热点。但因PCBs具有强的疏水性，生物可利用性低及对植物生长的抑制作用，致使最有应用前景的植物修复效能达不到预期。在前期获得转bphC.B苜蓿的基础上，申请者拟构建共表达tfdb-JLu和bphC.B的转基因苜蓿，在苜蓿体内建立类似微生物降解PCBs的通路。利用转基因苜蓿-茶皂甙联合修复PCBs污染土壤，考察转基因苜蓿清除体内PCBs残留的能力，检测茶皂甙诱导土著微生物PCBs降解基因表达水平；分析根际土壤中PCBs的消减动态，揭示根际区域微生物多样性和群落结构演替，阐明转基因苜蓿-表面活性剂联合根际修复效能及生物响应机制。本研究可为修复PCBs污染土壤提供新的植物修复技术体系，以提高PCBs污染土壤的原位修复效能，降低PCBs的生态风险。

本项目以研究转基因苜蓿原位修复多氯联苯（PCBs）污染土壤效能及降低PCBs在修复植株体内残留为目的，利用农杆菌介导法遗传转化苜蓿和拟南芥。通过遗传转化和抗性筛选等过程，培育共表达TfdB+BphC、BphC和TfdB等转基因植物株系。对转基因株系进行分子鉴定、蛋白检测及体内酶活分析，证明了2,4-二氯苯酚羟化酶基因 TfdB和2,3-二羟基联苯开环酶基因（BphC）整合进入植株基因组并可高效表达，并且转基因植株可正常生长发育。利用水培实验和室内模拟盆栽修复实验，证实TfdB+BphC基因在植株体内的表达，能显著提高植物对PCB28的耐受性（95%）；共表达TfdB+BphC植物株系对PCBs28的去除率可达85.94%，而野生型植株仅为27.11%；并且转基因植物体内的PCB28显著低于表达BphC和TfdB的转基因株系。PCBs降解菌P11能以茶皂素为唯一碳源生长，并且降解菌P11与茶皂素共培养之后，对联苯的降解效能显著提升，因此证明茶皂素不仅能促进降解菌的生长，提高土壤中PCBs生物可利用性，还能提升降解菌的降解效能。高通量测序分析根际土壤微生物群落结构显示，土壤中PCBs去除率的提高与特异性降解菌的微生物量提升和降解基因表达量增强密切相关。本研究获得了新型的修复植物，验证其对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-土著降解菌-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。