含phoN重组菌D.radiodurans的构建及其富集U（Ⅵ）性能的试验研究
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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。
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.
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。
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.
引文
[1]Khani MH, Keshtkar AR, Ghannadi M, et al. Equilibrium, kinetic and thermodynamic study of the biosorption of uranium onto Cystoseria indica algae. Journal of Hazardous Materials,2008,150:612-618.
[2]谢水波,王水云,张浩江,等.硫酸盐还原菌还原U(Ⅵ)的影响因素与机制.环境科学,2009,30(7)：1962-1967.
[3]魏广芝,徐乐昌.低浓度含铀废水的处理技术及其研究进展[J].铀矿冶,2007,26(2)：90-95
[4]Xie S B, Yang J, Chen C, et al. Study on biosorption kinetics and thermodynamics of uranium by Citrobacter freudii [J]. Journal of Environmental Radioactivity,99(2008):126-133
[5]唐志坚,张平,左社强.低浓度含铀废水处理技术的研究进展[J].工业用水与废水,2003,34(4)：9-12
[6]李合莲,陈家军.铀尾矿对地下水的环境影响研究[J].环境污染治理技术与设备,2000,1(3)：82-88
[7]郑大中.铀的迁移富集机理新探索[J].四川地质学报,2003,23(2)：77-86
[8]池翠萍,王仲文.贫铀武器对人体健康的影响[J].中华放射医学与防护杂志,2002,22(2)：137-139
[9]胡恋,谢水波,张晓健,等.微生物吸附处理低浓度含铀废水的效能[J].安全与环境学报,2007,7(2)：57-60
[10]柏云,张静,冯易君.生物吸附法处理含铀废水研究进展[J].四川环境,2003,22(2)：9-16
[11]杨晶,谢水波,王清良,等.微生物吸附铀的机理研究现状[J].铀矿冶,2006,25(4)：192-195
[12]闵茂中,Xu H F, Barton L L,等.厌氧菌Shewcenella putrefaciens还原U(Ⅵ)的实验研究：应用于中国层间氧化带砂岩型铀矿[J].中国科学D辑,地球科学,2004,34(2)：125-129
[13]闵茂中,王汝成,边立曾,等.层间氧化带砂岩型铀矿中的生物成矿作用[J].自然科学进展,2003,13(2)：164-168
[14]Yang J B, Bohumil V. Biosorption of Uranium on Sargassum Biomass [J]. Wat. Res.,1999,33(15):3357-3363
[15]黄民生,郑乐平,朱锦良.微生物对水中铀的富集与还原[J].核技术,2002,25(2)：123-131
[16]刘文娟,徐伟,王宝娥.溶液中其他离子对微生物吸附铀的影响[J].铀矿冶,2004,23(3)：143-146
[17]Brady D, Rose P D. The use of hollow Fiber cross-flow microfitration in bioaccumulation and continues removal of heavy metals from solution by Saccharomyces cerevisiae [J]. Biotechnol Biong,1994,44:1362-1366
[18]吴娟,李清彪,邓晓,等.假单胞菌EPS-5028富集铀[J].微生物学报,1999,39(1)：87-90
[19]M Tsezos. Recovery of uranium from biological adsorbent-desorption equilibrium [J]. Biotechnol Bioeng.1984,26:973-981
[20]张兰英,刘娜,孙立波等.现代环境微生物技术[M].北京：清华大学出版社,2005.
[21]张迎明.镍吸附基因工程菌的构建及吸附性能研究[D].暨南大学,2006
[22]XIE S B, HU L, ZHANG X J, et al. Study on biosorption efficiency and mechanism of uranium by Bacillus subtilis:progress in environmental science and technology Vol. I, Beijing, China, November 13-16,2007[C].Beijing: Science Press & Science Press USA Inc.,2007:293-298
[23]Chen S, Wilson D B. Construction and characterization of Escherichia coli genetically engineered for bioremediation of Hg2+-contaminated environments [J]. Appl Environ Microbiol,1997,63(6):2442-2445
[24]Deng X, Li Q B, Lu Y H, et al. Genetic engineering of E. coli SE5000 and its potential for Ni2+ bioremediation [J]. Process Biochemistry,2005,40(1): 425-430
[25]Gregor G, Bin F, Barry P R, et al. ZitB (YbgR), a Member of the Cation Diffusion Facilitator Family, Is an Additional Zinc Transporter in Escherichia coli [J]. Journal of Bacteriology,2001,183(15):4664-4667
[26]Stephan C, Tanja B, Christoph V, et al. A Transporter in the Endoplasmic Reticulum of Schizosaccharomyces pombe Cells Mediates Zinc Storage and Differentially Affects Transition Metal Tolerance [J]. J Biol Chem,2002,277(20): 18215-18221
[27]Bert J. van der Zaal, Leon W. Neuteboom, Johan E. Pinas, et al. Overexpression of a Novel Arabidopsis Gene Related to Putative Zinc-Transporter Genes from Animals Can Lead to Enhanced Zinc Resistance and Accumulation [J]. Plant Physiology,1999,199:1047-1055
[28]Nicole S P, Paul B L, Stephen D E, et al. The molecular physiology of heavy metal transport in the Zn/Cd hyperac cumulator Thlaspi caerulescens [J]. Plant Biology,2000,97(9):4956-4960
[29]Nobuaki S, Yube Y, Nozomu K, et al. Functional characterization of a heavy metal binding protein CdI19 from Arabidopsis [J]. The Plant Journal,2002,32(2): 165-173
[30]Chai T, Chen Q, Zhang Y, Dong J, et al. Cadmium resistance in transgenic tobacco plants enhanced by expressing bean heavy metal-responsive gene PvSR2 [J]. Science in China Series C:Life Sciences.2003,46(6):23-30.
[31]Hao Z Q, Chen S L, Wilson D B. Cloning, Expression, and Characterization of Cadmium and Manganese Uptake Genes from Lactobacillus plantarum [J]. Applied and Environmental Microbiology,1999,65(11):4746-4752.
[32]Stephan C, Kim E J, Dieter N, et al. Tolerance to toxic metals by a gene family of phytochelatin synthases from plants and yeast [J]. The EMBO Journal,1999, 18(12):3325-3333
[33]Carolina S, Pavel K, Tomas R, et al. Metalloadsorption by Escherichia coil Cells Displaying Yeast and Mammalian Metalothioneins Anchored to the Outer Membrane Protein LamB [J]. Journal of Bacteriology,1998,180(9):2280-2284
[34]Deepti A, Amara S R, Shree K A. Engineering of Deinococcus radiodurans R1 for Bioprecipitation of Uranium from Dilute Nuclear Waste [J]. Applied and Environmental Microbiology,2006,72(12):7873-7878
[35]Marc Vallsa, Victor de Lorenzob, Roser Gonzalez-Duartea, et al. Engineering Outer-Membrane Proteins in Pseudomonas putida for Enhanced Heavy-Metal Bioadsorption [J]. Journal of Inorganic Biochemistry,2000,79(1/2/3/4):219-223.
[36]Weon Bae, Rajesh K. Mehra, Ashok Mulchandani, et al. Genetic Engineering of Escherichia coli for Enhanced Uptake and Bio accumulation of Mercury [J]. Applied and Environmental Microbiology,2001,67(11):5335-5338.
[37]Pavel Kotrba, Lucie Dole c kova, Victor de Lorenzo, et al. Enhanced Bioaccumulation of Heavy Metal Ions by Bacterial Cells Due to Surface Display of Short Metal Binding Peptides [J]. Applied and Environmental Microbiology, 1999,65(3):1092-1098
[38]Kuroda K, Shibasaki S, Ueda M, et al. Cell surface-engineered yeast displaying a histidine oligopeptide (hexa-His) has enhanced adsorption of and tolerance to heavy metal ions [J]. Applied Microbiology and Biotechnology,2001,57(5-6): 697-701
[39]Sriprang R, Makoto H, Hisayo O, et al. Enhanced Accumulation of Cd2+ by a Mesorhizobium sp. Transformed with a Gene from Arabidopsis thaliana Coding for Phytochelatin Synthase [J]. Applied and Environmental Microbiology,2003, 69(3):1791-1796
[40]胡章立,邢苗,吴玉荷,等.转MT-like基因衣藻的重金属结合能力与抗性特征分析[J].湖泊科学,2002,14(3)：247-252
[41]Serre L, Rossy E, Eva P P, et al. Crystal Structure of the Oxidized Form of the Periplasmic Mercury-binding Protein MerP from Ralstonia metallidurans CH3 [J]. Journal of Molecular Biology,2004,339, (1):161-171
[42]Tamar B, Susan M M, Anne O S. Bacterial mercury resistance from atoms to ecosystems [J]. FEMS Microbiology Reviews,2003,27(2-3):355-384
[43]Bang S W, Clark D S, Jay D K. Engineering Hydrogen Sulfide Production and Cadmium Removal by Expression of the Thiosulfate Reductase Gene (phsABC) from Salmonella enterica Serovar Typhimurium in Escherichia coli [J]. Applied and Environmental Microbiology,2000,66(9):3939-3944
[44]Brim H, Sara C M, James K F, et al. Engineering Deinococcus radiodurans for metal remediation in radioactive mixed waste environments [J]. Nature Biotechnology,2000,18:85-90
[45]Basnakova G, Stephens E R, Thaller M C, et al. The use of Escherichia coli bearing a phoN for the removal of uranium and nickel from aqueous flows [J]. Appl Microbiol Biotechnol,1998,50:266-272
[46]Deng X, Li Q B, Lu Y H et al. Uptake of nickel from industrial wastewater by genetically engineered Escherichia coli JM109 [J]. Water Res,2003, 37(10):2505-2511
[47]袁建军,卢英华.高选择性重组基因工程菌治理含汞废水的研究[J].泉州师范学院学报,2003,21(6)：71-75
[48]赵肖为,李清彪,卢英华,等.高选择性基因工程菌E.coli SE5000生物富集水体中的镍离子[J].环境科学学报,2004,24(2)：231-236
[49]蔡颖,赵肖为,邓旭,等.基因工程菌生物富集废水中重金属镉[J].水处理技术,2006,32(1)：26-29
[50]郑杨春,邓旭,李清彪,等.高选择性重组菌在含Hg2+环境中的生长富集Hg2+耦合及其连续处理Hg2+废水[J].厦门大学学报,2005,44：98-101
[51]邓旭,李清彪,卢英华,等.基因工程菌大肠杆菌JM109富集废水中镍离子的研究[J].生物工程学报,2003,19(3)：343-348
[52]王建龙.耐辐射基因工程菌Deinococcus radiodurans及其在环境修复中的应用[J].辐射研究与辐射工艺学报,2004,22(5)：257-260
[53]华跃进,高冠军.耐辐射异常球菌DNA损伤与修复相关基因的比较基因组研究[J].微生物学报,2003,43(1)：120-126
[54]宋道军,余增亮.抗辐射菌抗辐射机理的研究新进展[J].生命科学,1999,11(5)：221-224
[55]孙翠凤,刘芬菊,汪涛.耐辐射球菌Deinococcus radiodurans辐射抗性的研究[J].辐射研究与辐射工艺学报,2002,20(3)：161-164
[56]杜泽吉,高桥佳子,鸣海一成.抗辐射菌Deinococcus radiodurans的生长特征及形态特征[J].苏州医学院学报.1998,18(11)：1131-1132
[57]刘芬菊,孔向蓉,孙翠凤,等.抗辐射菌形态及敏感性研究[J].工业卫生与职业病,2002,28(4)：203-205
[58]宋道军,余增亮.三种辐射源对耐辐射微球菌作用机理的比较研究[J].生物物理学报,1998,14(1)：185-188
[59]Coxm M, Battista J R. Deinococcus radiodurans the consummate survivor [J]. Nat RevMicrobiol,2005,3 (11):882-892
[60]Dalym J, Mintonl K W. Resistance to radiation [J]. Science,1995,270:1318
[61]White O, Eisen J A, Heidelberg J F, et al. Genome sequence of the radio-resistant bacterium Deinococcus radiodurans R1 [J]. Science,1999,286:1571-1577
[62]Kolodner R D, Marsischky G T. Eukaryotic DNA mismatch repair [J]. Curr Opin Genet Dev,1999,9 (1):89-96
[63]黄新,杨天佑,常胜合,等.耐辐射奇球菌抗辐射机制的研究进展[J].安徽农业科学,2008,36(20)：8410-8412
[64]Fredrickson J K, Kostandarithes H M, Li S W, et al. Reduction of Fe(Ⅲ), Cr(Ⅵ), U(Ⅵ), and Tc(Ⅶ) by Deinococcus radiodurans Rl [J]. Appl Environ Microbiol, 2000,66(5):2006-2011
[65]V enkateswaran K, Moser D P, Dollhopf M E, et al. Polyphasic taxonomy of the genus Shewanella and description of Shewanella oneidensis sp. nov. [J]. International Journal of Systematic Bacteriology,1999,49(4):705-724
[66]周绪斌,邢瑞云,吕星.耐辐射奇球菌在放射性环境中的生物修复作用[J].微生物学通报,2004,31(1)：118-122
[67]Lange C C, Wackett L P, Minton K W, et al. Engineering a recombinant Deinococcus radiodurans for organopollutant degradation in radioactive mixed waste environments[J]. Nature Biotechnology,1998,16:929-933
[68]Macaskie L E, Bonthrone K M, Rouch D A. Phosphatase-mediated heavy metal accumulation by a Citrobacter sp. and related enterobacteria[J]. FEMS Microbiology Letters,1994,121(2):141-146
[69]EILEEN L, KENNETH W M, Gene Fusions with lacZ by Duplication Insertion in the Radioresistant Bacterium Deinococcus radiodurans [J]. JOURNAL OF BACTERIOLOGY,1990,172(6):2955-2961
[70]J.萨姆布鲁克,D.W.拉塞尔.分子克隆实验指南(第3版)[M].北京：科学出版社,2002,1493-1495.
[71]Macaskie L E, Bonthrone K M, Rouch D A. Phosphatase-mediated heavy metal accumulation by a Citrobacter sp. and related enterobacteria[J]. FEMS Microbiology Letters,1994,121(2):141-146
[72]Lynne E M, Karen M B, Ping Y, et al. Enzymically mediated bioprecipitation of uranium by a Citrobacter sp.:a concerted role for exocellular lipopolysaccharide and associated phosphatase in biomineral formation[J]. Microbiology,2000, 146(8):1855-1867
[73]John A F, Victoria J M A, Alex C, et al. PhoN-type acid phosphatases of a heavy metal-accumulating Citrobacter sp.:resistance to heavy metals and affinity towards phosphomonoester substrates[J]. FEMS Microbiology Letters,1995, 130(2-3):87-97.
[74]Megumi K, Atsuo N, Hideo H. Molecular Analysis of the Salmonella typhimurium phoN Gene, Which Encodes Nonspecific Acid Phosphatase [J]. JOURNAL OF BACTERIOLOGY,1991,173(21):6760-6765
[75]孙晗笑,陆大祥,刘飞鹏.转基因技术理论与应用[M].郑州：河南医科大学出版社,2000,180-183
[2]谢水波,王水云,张浩江,等.硫酸盐还原菌还原U(Ⅵ)的影响因素与机制.环境科学,2009,30(7)：1962-1967.
[3]魏广芝,徐乐昌.低浓度含铀废水的处理技术及其研究进展[J].铀矿冶,2007,26(2)：90-95
[4]Xie S B, Yang J, Chen C, et al. Study on biosorption kinetics and thermodynamics of uranium by Citrobacter freudii [J]. Journal of Environmental Radioactivity,99(2008):126-133
[5]唐志坚,张平,左社强.低浓度含铀废水处理技术的研究进展[J].工业用水与废水,2003,34(4)：9-12
[6]李合莲,陈家军.铀尾矿对地下水的环境影响研究[J].环境污染治理技术与设备,2000,1(3)：82-88
[7]郑大中.铀的迁移富集机理新探索[J].四川地质学报,2003,23(2)：77-86
[8]池翠萍,王仲文.贫铀武器对人体健康的影响[J].中华放射医学与防护杂志,2002,22(2)：137-139
[9]胡恋,谢水波,张晓健,等.微生物吸附处理低浓度含铀废水的效能[J].安全与环境学报,2007,7(2)：57-60
[10]柏云,张静,冯易君.生物吸附法处理含铀废水研究进展[J].四川环境,2003,22(2)：9-16
[11]杨晶,谢水波,王清良,等.微生物吸附铀的机理研究现状[J].铀矿冶,2006,25(4)：192-195
[12]闵茂中,Xu H F, Barton L L,等.厌氧菌Shewcenella putrefaciens还原U(Ⅵ)的实验研究：应用于中国层间氧化带砂岩型铀矿[J].中国科学D辑,地球科学,2004,34(2)：125-129
[13]闵茂中,王汝成,边立曾,等.层间氧化带砂岩型铀矿中的生物成矿作用[J].自然科学进展,2003,13(2)：164-168
[14]Yang J B, Bohumil V. Biosorption of Uranium on Sargassum Biomass [J]. Wat. Res.,1999,33(15):3357-3363
[15]黄民生,郑乐平,朱锦良.微生物对水中铀的富集与还原[J].核技术,2002,25(2)：123-131
[16]刘文娟,徐伟,王宝娥.溶液中其他离子对微生物吸附铀的影响[J].铀矿冶,2004,23(3)：143-146
[17]Brady D, Rose P D. The use of hollow Fiber cross-flow microfitration in bioaccumulation and continues removal of heavy metals from solution by Saccharomyces cerevisiae [J]. Biotechnol Biong,1994,44:1362-1366
[18]吴娟,李清彪,邓晓,等.假单胞菌EPS-5028富集铀[J].微生物学报,1999,39(1)：87-90
[19]M Tsezos. Recovery of uranium from biological adsorbent-desorption equilibrium [J]. Biotechnol Bioeng.1984,26:973-981
[20]张兰英,刘娜,孙立波等.现代环境微生物技术[M].北京：清华大学出版社,2005.
[21]张迎明.镍吸附基因工程菌的构建及吸附性能研究[D].暨南大学,2006
[22]XIE S B, HU L, ZHANG X J, et al. Study on biosorption efficiency and mechanism of uranium by Bacillus subtilis:progress in environmental science and technology Vol. I, Beijing, China, November 13-16,2007[C].Beijing: Science Press & Science Press USA Inc.,2007:293-298
[23]Chen S, Wilson D B. Construction and characterization of Escherichia coli genetically engineered for bioremediation of Hg2+-contaminated environments [J]. Appl Environ Microbiol,1997,63(6):2442-2445
[24]Deng X, Li Q B, Lu Y H, et al. Genetic engineering of E. coli SE5000 and its potential for Ni2+ bioremediation [J]. Process Biochemistry,2005,40(1): 425-430
[25]Gregor G, Bin F, Barry P R, et al. ZitB (YbgR), a Member of the Cation Diffusion Facilitator Family, Is an Additional Zinc Transporter in Escherichia coli [J]. Journal of Bacteriology,2001,183(15):4664-4667
[26]Stephan C, Tanja B, Christoph V, et al. A Transporter in the Endoplasmic Reticulum of Schizosaccharomyces pombe Cells Mediates Zinc Storage and Differentially Affects Transition Metal Tolerance [J]. J Biol Chem,2002,277(20): 18215-18221
[27]Bert J. van der Zaal, Leon W. Neuteboom, Johan E. Pinas, et al. Overexpression of a Novel Arabidopsis Gene Related to Putative Zinc-Transporter Genes from Animals Can Lead to Enhanced Zinc Resistance and Accumulation [J]. Plant Physiology,1999,199:1047-1055
[28]Nicole S P, Paul B L, Stephen D E, et al. The molecular physiology of heavy metal transport in the Zn/Cd hyperac cumulator Thlaspi caerulescens [J]. Plant Biology,2000,97(9):4956-4960
[29]Nobuaki S, Yube Y, Nozomu K, et al. Functional characterization of a heavy metal binding protein CdI19 from Arabidopsis [J]. The Plant Journal,2002,32(2): 165-173
[30]Chai T, Chen Q, Zhang Y, Dong J, et al. Cadmium resistance in transgenic tobacco plants enhanced by expressing bean heavy metal-responsive gene PvSR2 [J]. Science in China Series C:Life Sciences.2003,46(6):23-30.
[31]Hao Z Q, Chen S L, Wilson D B. Cloning, Expression, and Characterization of Cadmium and Manganese Uptake Genes from Lactobacillus plantarum [J]. Applied and Environmental Microbiology,1999,65(11):4746-4752.
[32]Stephan C, Kim E J, Dieter N, et al. Tolerance to toxic metals by a gene family of phytochelatin synthases from plants and yeast [J]. The EMBO Journal,1999, 18(12):3325-3333
[33]Carolina S, Pavel K, Tomas R, et al. Metalloadsorption by Escherichia coil Cells Displaying Yeast and Mammalian Metalothioneins Anchored to the Outer Membrane Protein LamB [J]. Journal of Bacteriology,1998,180(9):2280-2284
[34]Deepti A, Amara S R, Shree K A. Engineering of Deinococcus radiodurans R1 for Bioprecipitation of Uranium from Dilute Nuclear Waste [J]. Applied and Environmental Microbiology,2006,72(12):7873-7878
[35]Marc Vallsa, Victor de Lorenzob, Roser Gonzalez-Duartea, et al. Engineering Outer-Membrane Proteins in Pseudomonas putida for Enhanced Heavy-Metal Bioadsorption [J]. Journal of Inorganic Biochemistry,2000,79(1/2/3/4):219-223.
[36]Weon Bae, Rajesh K. Mehra, Ashok Mulchandani, et al. Genetic Engineering of Escherichia coli for Enhanced Uptake and Bio accumulation of Mercury [J]. Applied and Environmental Microbiology,2001,67(11):5335-5338.
[37]Pavel Kotrba, Lucie Dole c kova, Victor de Lorenzo, et al. Enhanced Bioaccumulation of Heavy Metal Ions by Bacterial Cells Due to Surface Display of Short Metal Binding Peptides [J]. Applied and Environmental Microbiology, 1999,65(3):1092-1098
[38]Kuroda K, Shibasaki S, Ueda M, et al. Cell surface-engineered yeast displaying a histidine oligopeptide (hexa-His) has enhanced adsorption of and tolerance to heavy metal ions [J]. Applied Microbiology and Biotechnology,2001,57(5-6): 697-701
[39]Sriprang R, Makoto H, Hisayo O, et al. Enhanced Accumulation of Cd2+ by a Mesorhizobium sp. Transformed with a Gene from Arabidopsis thaliana Coding for Phytochelatin Synthase [J]. Applied and Environmental Microbiology,2003, 69(3):1791-1796
[40]胡章立,邢苗,吴玉荷,等.转MT-like基因衣藻的重金属结合能力与抗性特征分析[J].湖泊科学,2002,14(3)：247-252
[41]Serre L, Rossy E, Eva P P, et al. Crystal Structure of the Oxidized Form of the Periplasmic Mercury-binding Protein MerP from Ralstonia metallidurans CH3 [J]. Journal of Molecular Biology,2004,339, (1):161-171
[42]Tamar B, Susan M M, Anne O S. Bacterial mercury resistance from atoms to ecosystems [J]. FEMS Microbiology Reviews,2003,27(2-3):355-384
[43]Bang S W, Clark D S, Jay D K. Engineering Hydrogen Sulfide Production and Cadmium Removal by Expression of the Thiosulfate Reductase Gene (phsABC) from Salmonella enterica Serovar Typhimurium in Escherichia coli [J]. Applied and Environmental Microbiology,2000,66(9):3939-3944
[44]Brim H, Sara C M, James K F, et al. Engineering Deinococcus radiodurans for metal remediation in radioactive mixed waste environments [J]. Nature Biotechnology,2000,18:85-90
[45]Basnakova G, Stephens E R, Thaller M C, et al. The use of Escherichia coli bearing a phoN for the removal of uranium and nickel from aqueous flows [J]. Appl Microbiol Biotechnol,1998,50:266-272
[46]Deng X, Li Q B, Lu Y H et al. Uptake of nickel from industrial wastewater by genetically engineered Escherichia coli JM109 [J]. Water Res,2003, 37(10):2505-2511
[47]袁建军,卢英华.高选择性重组基因工程菌治理含汞废水的研究[J].泉州师范学院学报,2003,21(6)：71-75
[48]赵肖为,李清彪,卢英华,等.高选择性基因工程菌E.coli SE5000生物富集水体中的镍离子[J].环境科学学报,2004,24(2)：231-236
[49]蔡颖,赵肖为,邓旭,等.基因工程菌生物富集废水中重金属镉[J].水处理技术,2006,32(1)：26-29
[50]郑杨春,邓旭,李清彪,等.高选择性重组菌在含Hg2+环境中的生长富集Hg2+耦合及其连续处理Hg2+废水[J].厦门大学学报,2005,44：98-101
[51]邓旭,李清彪,卢英华,等.基因工程菌大肠杆菌JM109富集废水中镍离子的研究[J].生物工程学报,2003,19(3)：343-348
[52]王建龙.耐辐射基因工程菌Deinococcus radiodurans及其在环境修复中的应用[J].辐射研究与辐射工艺学报,2004,22(5)：257-260
[53]华跃进,高冠军.耐辐射异常球菌DNA损伤与修复相关基因的比较基因组研究[J].微生物学报,2003,43(1)：120-126
[54]宋道军,余增亮.抗辐射菌抗辐射机理的研究新进展[J].生命科学,1999,11(5)：221-224
[55]孙翠凤,刘芬菊,汪涛.耐辐射球菌Deinococcus radiodurans辐射抗性的研究[J].辐射研究与辐射工艺学报,2002,20(3)：161-164
[56]杜泽吉,高桥佳子,鸣海一成.抗辐射菌Deinococcus radiodurans的生长特征及形态特征[J].苏州医学院学报.1998,18(11)：1131-1132
[57]刘芬菊,孔向蓉,孙翠凤,等.抗辐射菌形态及敏感性研究[J].工业卫生与职业病,2002,28(4)：203-205
[58]宋道军,余增亮.三种辐射源对耐辐射微球菌作用机理的比较研究[J].生物物理学报,1998,14(1)：185-188
[59]Coxm M, Battista J R. Deinococcus radiodurans the consummate survivor [J]. Nat RevMicrobiol,2005,3 (11):882-892
[60]Dalym J, Mintonl K W. Resistance to radiation [J]. Science,1995,270:1318
[61]White O, Eisen J A, Heidelberg J F, et al. Genome sequence of the radio-resistant bacterium Deinococcus radiodurans R1 [J]. Science,1999,286:1571-1577
[62]Kolodner R D, Marsischky G T. Eukaryotic DNA mismatch repair [J]. Curr Opin Genet Dev,1999,9 (1):89-96
[63]黄新,杨天佑,常胜合,等.耐辐射奇球菌抗辐射机制的研究进展[J].安徽农业科学,2008,36(20)：8410-8412
[64]Fredrickson J K, Kostandarithes H M, Li S W, et al. Reduction of Fe(Ⅲ), Cr(Ⅵ), U(Ⅵ), and Tc(Ⅶ) by Deinococcus radiodurans Rl [J]. Appl Environ Microbiol, 2000,66(5):2006-2011
[65]V enkateswaran K, Moser D P, Dollhopf M E, et al. Polyphasic taxonomy of the genus Shewanella and description of Shewanella oneidensis sp. nov. [J]. International Journal of Systematic Bacteriology,1999,49(4):705-724
[66]周绪斌,邢瑞云,吕星.耐辐射奇球菌在放射性环境中的生物修复作用[J].微生物学通报,2004,31(1)：118-122
[67]Lange C C, Wackett L P, Minton K W, et al. Engineering a recombinant Deinococcus radiodurans for organopollutant degradation in radioactive mixed waste environments[J]. Nature Biotechnology,1998,16:929-933
[68]Macaskie L E, Bonthrone K M, Rouch D A. Phosphatase-mediated heavy metal accumulation by a Citrobacter sp. and related enterobacteria[J]. FEMS Microbiology Letters,1994,121(2):141-146
[69]EILEEN L, KENNETH W M, Gene Fusions with lacZ by Duplication Insertion in the Radioresistant Bacterium Deinococcus radiodurans [J]. JOURNAL OF BACTERIOLOGY,1990,172(6):2955-2961
[70]J.萨姆布鲁克,D.W.拉塞尔.分子克隆实验指南(第3版)[M].北京：科学出版社,2002,1493-1495.
[71]Macaskie L E, Bonthrone K M, Rouch D A. Phosphatase-mediated heavy metal accumulation by a Citrobacter sp. and related enterobacteria[J]. FEMS Microbiology Letters,1994,121(2):141-146
[72]Lynne E M, Karen M B, Ping Y, et al. Enzymically mediated bioprecipitation of uranium by a Citrobacter sp.:a concerted role for exocellular lipopolysaccharide and associated phosphatase in biomineral formation[J]. Microbiology,2000, 146(8):1855-1867
[73]John A F, Victoria J M A, Alex C, et al. PhoN-type acid phosphatases of a heavy metal-accumulating Citrobacter sp.:resistance to heavy metals and affinity towards phosphomonoester substrates[J]. FEMS Microbiology Letters,1995, 130(2-3):87-97.
[74]Megumi K, Atsuo N, Hideo H. Molecular Analysis of the Salmonella typhimurium phoN Gene, Which Encodes Nonspecific Acid Phosphatase [J]. JOURNAL OF BACTERIOLOGY,1991,173(21):6760-6765
[75]孙晗笑,陆大祥,刘飞鹏.转基因技术理论与应用[M].郑州：河南医科大学出版社,2000,180-183