基于铀酰高效富集的双抗性功能基因工程菌及氟铀胁迫下铀富集机理

Wastewater containing Uranium would threaten human health and cause certain environmental security during the Uranium mining and Uranium hydrometallurgy process, if it was not treatmented in time. Therefore, studies on the new method for wastewater containing Uranium treatment, enriching Uranium high efficient from the wastewater containing low concentration of uranium, have important strategic significance for Uranium resource and harmful radiation protection. Based on special experimental installations and prophase research achievement of biological treating Uranium contaminated water in our school, many bio-adsorbents were lack of ideal resistance for some Uranium and fluorine ions, and limited to be used in actual application. So a novel double resistances function genetic engineering bacteria would be constructed, using DsrA、flr gene into Deinococcus radiodurans(DR) at the same time using CRISPR/Cas9 gene editing techniques, which is high enriching capacity, radiation resistance and better stress activity on fluorine and Uranium. The theory evidence of biology treating wastewater containing Uranium would be builded, which would be completed by analyzing the biological characteristics, the function genetic of the resistance to radiation and fluorine, and study on the genetic characteristics, the behavior and mechanism of enriching Uranium high efficient,and the response mechanism on fluorine and Uranium stress.At the same time, it would be supported to the safe produce in the Uranium mines and remediation of the entironment around tailings areas of uranium mines. This work is feasible with brilliant prospected and scientific significance.

铀矿采冶过程中所产生的含铀废水，若不进行及时治理，将威胁人类健康，造成一定的环境安全问题。研究含铀废水治理新方法并实现铀的高效富集，对铀资源化和有害辐射防护均具有重要的战略意义。本项目组大量的前期研究，发现普通生物吸附剂对铀、氟等一些离子缺乏理想抗性，导致实际应用受限。针对目前含铀废水治理中的瓶颈问题，本项目拟采用CRISPR/Cas9基因编辑技术，将还原基因DsrA、耐氟基因flr，同时转入耐辐射奇球菌DR基因组中稳定表达，构建一种兼备高富集容量以及氟铀高耐受力等优势的高效富集双抗性功能基因工程菌。通过分析其生物学特性、耐辐射与氟的功能基因分析，采用实时荧光定量PCR、Solexa高通量测序以及基因转录技术研究其遗传特点、高效富集铀的行为与机理以及氟铀胁迫响应机制，为铀矿冶含铀废水微生物治理提供理论基础。本项目对指导铀厂矿安全生产和尾矿库辐射防护等方面具有重要的应用价值。

铀矿开采和水冶过程中所产生的含铀废水威胁人类健康并造成一定的环境安全问题，研究含铀废水治理新方法并实现铀的高效富集，对铀资源化和有害辐射防护具有重要的战略意义。本项目将硫酸盐还原菌的关键还原基因dsrA转入大肠杆菌中构建含还原基因的重组大肠杆菌DH5α-dsrA，经多因素考察对铀的富集效率，证实还原基因dsrA可增强异源微生物的铀富集性能，但大肠杆菌在铀中的耐受性不强；进一步将还原基因dsrA转入转入到耐辐射奇球菌中，构建抗辐射、高还原性能的多效基因工程菌Deino-dsrA，在铀溶液中生存周期较长，具备理想的极端抗性，其还原富集效率优于野生型DR菌；在酸性条件下，也优于文献报道硫酸盐还原菌的富集率。将秀丽隐杆线虫的关键耐氟基因flr-2，转入耐辐射奇球菌DR基因组中并正常表达，成功构建抗辐射、高抗氟性能的双抗性基因工程菌DR-flr-2，经多因素考察在氟铀溶液中的生存率及铀富集效率，证实抗氟基因flr-2可增强异源微生物的抗氟性能，从而提升其在含氟铀溶液中的铀富集性能。将还原基因dsrA和抗氟基因flr-2转入到耐辐射奇球菌中并正常表达，构建一种兼备高富集容量以及氟铀高耐受力等优势的高效富集双抗性功能基因工程菌Deino-dsrA-flr-2，生存活性实验证实新构建的基因工程菌存活率提高，经多因素考察铀富集效率，结果显示其还原富集效率比野生型菌株显著提升，经红外光谱、扫描电镜和能谱研究了工程菌高效富集铀的行为与机理，转录组测序探明了氟铀胁迫响应的几个关键基因及涉及的主要通路，该富铀机理与耐氟铀机制为铀矿冶含铀废水微生物治理提供理论基础。在项目研究期间，本项目组共发表论文6篇(其中SCI收录5篇，含在线发表SCI 1篇），申请国家发明专利 3 项（其中授权2项），培养硕士研究生7位，圆满完成了工作内容，达到了预期目标。同时，本项目对指导铀矿安全生产和尾矿库有害辐射防护等方面具有有重要的应用价值。