【作者】 梁颂军；

【导师】 谢水波；

【作者基本信息】 南华大学 ， 市政工程， 2010， 硕士

【摘要】 含编码非特异性酸性磷酸酶基因(phoN)的细菌如柠檬酸杆菌(Citrobacter freudii)等能有效地吸附去除废水中的重金属,但这类微生物对辐射敏感。耐辐射奇球菌(Deinoeoeeus radiodurans)是最抗辐射的生物之一,存活的辐射剂量高达15kGy。利用基因工程技术将phoN克隆到D. radiodurans中,使之能在辐射环境下更大容量富集水中的U(Ⅵ),实现铀等放射性重金属污染的有效生物修复及再资源化,具有重要的科学意义。本研究内容包括目的基因的扩增,重组穿梭表达载体的构建、重组菌的筛选、PhoN蛋白的表达及重组菌D. radiodurans富集U(Ⅵ)性能的研究等。以Salmonella enterica serovar Typhimurium基因组DNA为模板,PCR扩增得到phoN,将其克隆到pMD18T-Vector中。用SpeⅠ、NdeⅠ限制性内切酶对重组转移载体T-VectorphoN与穿梭载体pRADZ3分别进行双酶切,再将phoN片段和穿梭载体pRADZ3中的大片段通过T4DNA连接酶连接。经PCR与双酶切双重鉴定,证实重组穿梭载体pRADZ3phoN构建成功。转化E. coli DH5α与D.radiodurans感受态细胞,使其在正常情况下(不需诱导剂)表达PhoN蛋白,经Western blot证实phoN基因在E.coli DH5α与D. radiodurans中成功表达。重组菌E. coli与重组菌D. radiodurans富集U(Ⅵ)的实验表明,重组菌对U(Ⅵ)的去除率较相应的宿主菌均有较大的提高,其中重组菌D. radiodurans对U(Ⅵ)的富集量较宿主菌D. radiodurans的增加约2倍,去除率达85.64%。比较重组菌D. radiodurans与宿主菌D. radiodurans的生长曲线,表明重组穿梭质粒的导入使得重组菌的生长受到抑制。pH对重组菌D. radiodurans富集U(Ⅵ)的过程有显著影响。当pH为6时去除率达到最大值88.32%,在低pH下,去除率极低。在60min时基本达到了吸附平衡。其富集量与菌体浓度负相关,与铀起始浓度正相关。更多还原

【Abstract】 Bacteria such as Citrobacter sp. harboring phoN, a gene encoding a nonspecific acid phosphatase, have been reported to precipitate heavy metals efficiently. However, the sensitivity of such bacteria to the adverse effects of radiation makes them unsuitable candidates for remediating radioactive waste. The most radiation resistant organism yet discovered is the bacterium Deinococcus radiodurans, which have the extraordinary ability to withstand radiation doses of up to 15 kG.Cloning the phoN gene into D. radiodurans by genetic engineering technology could improve host bacteria’s bioaccumulation capacity in radiation environment, and achieve effective biological repair and reutilization, which has a very important scientific significance. Amplification of phoN gene, construction of recombined shuttle vector, identification of recombinant bacteria, expression of PhoN protein, and U(Ⅵ) bioaccumulation performance of recombinant bacteria D. radiodurans were included in this paper.PhoN gene that was amplified from Salmonella enterica serovar Typhimurium genomic DNA by PCR, was cloned into pMD18T-Vector. Recombined transfer vector T-VectorphoN was digested by restriction enzymes of Spe I and Nde I, and then the purified phoN gene was inserted into shuttle vector pRADZ3 which was digested by the same restriction enzymes. The recombined shuttle vector pRADZ3phoN was identified by PCR and restriction analysis, and transformed into E. coli DH5a and D. radiodurans competent cell. A recombinant fusion PhoN protein was expressed in normal growth condition without induction. The expression of PhoN protein in E. coli DH5a and D. radiodurans was confirmed by Western blot.The U(Ⅵ) bioaccumulation performance of recombinant E. coli and recombinant D. radiodurans was evaluated. The results showed that the maximum U(VI) bioaccumulation capacity of recombinant D. radiodurans increased about two times compared to original D. radiodurans, and the removal rate of U(VI) was 85.64%.It indicated that the growth of recombinant D. radiodurans was inhibited due to the inserted recombinant shuttle plasmid, compared with growth curve of recombinant D. radiodurans and original D. radiodurans. The pH value had a significant effect on U(VI) bioaccumulation performance of recombinant D. radiodurans. The removal efficiency increased with the increased of pH, and reached maximum 88.32% at pH 6. Its bioaccumulation balance time was about 60 minutes. The U(VI) bioaccumulation capacity of recombinant D. radiodurans increased with a decrease of bacteria concentration, and increased with an increase of the initial concentration of uranium.更多还原