高效汞降解菌株的筛选及耐汞基因的克隆
|
摘要
【目的】利用氯化汞(HgCl2)进行选择性培养,从自然界的土壤中分离耐汞菌。从中筛选出可以高效还原无机汞离子的优良菌株进行研究,以实际应用于工业汞污水的处理和汞污染土壤和水体的修复。
【方法】①耐汞菌株的分离:采集日光灯厂厂区汞污染的土壤,用常规方法在含HgCl2的培养基上培养,长出的单菌落进一步分离纯化、观察菌落的形态、革兰氏染色、细菌生化分类鉴定及抗生素耐药性分析;
②耐汞水平测定:用含Hg2+浓度不同的培养液进行培养,观察菌株的生长情况;
③高耐菌株生长能力的研究:包括观察菌株在含汞培养液中的生存趋势;在不同pH条件下的生长情况;在灭菌自来水及农田土中的生存能力;
④高耐菌株对Hg2+的还原能力:将待测菌接种于含汞浓度不同的LB培养液中,培养48h后取上清,用双道原子荧光光度计测定残余的汞含量,得出汞还原率;
⑤16SrRNA分类鉴定:提取待测菌的基因组DNA,根据细菌16SrRNA基因中的保守序列设计并合成引物,PCR扩增产物,测序结果通过BLASTN数据库及同源性比较分析而明确待测菌的分类鉴定。
⑥merA基因片段的克隆和鉴定:根据Gene Bank数据库提供的已知的merA基因序列及同源性比较分析,在高度保守的DNA序列区设计PCR引物,对待测菌的基因组DNA进行扩增,测序结果通过BLASTN进行同源性比较分析,鉴定待测菌是否含有merA基因以及其与已知的merA基因的同源性。同时尝试将所扩增的基因片段转化合适的受体菌。
【Objective】: Through HgCl2-selected media, some special bacterial stains would be isolated from mercury- polluted soil, and then the best one should be selected on the basis of the phenotypic, physiological, biochemical tests and genic sequence analysis. All these results will help to apply the selected one into bioremediation of mercury-contaminated soil and water actually.
【Methods】:①Isolation of high mercury-resistant bacteria: use HgCl2-selected media to incubate the bacteria out of the soil which we collect from fluorescent lamp’s manufactory by normal process. Different colonies would be incubated alone, and then we will discriminate them by tests including: the Gram's staining; phenotypic, phy-siological and biochemical test; antibiotic resistance test, and so on.
②Hg2+ toleration test: incubate the bacteria by using the media that have different Hg2+ concentrations, and then observe the growing of those bacteria.
③Researches on the growing of the selected high mercury- degrading bacterium: including monitor the bacterium’s growth under different Hg2+ concentrations or pH conditions; observing its ability of living in the soil and natural water.
④Testing the level of transforming Hg2+ into Hg0: inoculate the growing bacteria which are just on logarithmic growth phase into several LB liquid media that contain different definite Hg2+ concentrations. After 48 hours, collect the liquid by centrifugation to determine the rudimental Hg2+ concentration on AFS-230a double-tracts atomic fluorescence absorptiometer.
⑤Classification and identification: according to the reserved 16SrRNA sequences by comparing the same bacterial strains in
【方法】①耐汞菌株的分离:采集日光灯厂厂区汞污染的土壤,用常规方法在含HgCl2的培养基上培养,长出的单菌落进一步分离纯化、观察菌落的形态、革兰氏染色、细菌生化分类鉴定及抗生素耐药性分析;
②耐汞水平测定:用含Hg2+浓度不同的培养液进行培养,观察菌株的生长情况;
③高耐菌株生长能力的研究:包括观察菌株在含汞培养液中的生存趋势;在不同pH条件下的生长情况;在灭菌自来水及农田土中的生存能力;
④高耐菌株对Hg2+的还原能力:将待测菌接种于含汞浓度不同的LB培养液中,培养48h后取上清,用双道原子荧光光度计测定残余的汞含量,得出汞还原率;
⑤16SrRNA分类鉴定:提取待测菌的基因组DNA,根据细菌16SrRNA基因中的保守序列设计并合成引物,PCR扩增产物,测序结果通过BLASTN数据库及同源性比较分析而明确待测菌的分类鉴定。
⑥merA基因片段的克隆和鉴定:根据Gene Bank数据库提供的已知的merA基因序列及同源性比较分析,在高度保守的DNA序列区设计PCR引物,对待测菌的基因组DNA进行扩增,测序结果通过BLASTN进行同源性比较分析,鉴定待测菌是否含有merA基因以及其与已知的merA基因的同源性。同时尝试将所扩增的基因片段转化合适的受体菌。
【Objective】: Through HgCl2-selected media, some special bacterial stains would be isolated from mercury- polluted soil, and then the best one should be selected on the basis of the phenotypic, physiological, biochemical tests and genic sequence analysis. All these results will help to apply the selected one into bioremediation of mercury-contaminated soil and water actually.
【Methods】:①Isolation of high mercury-resistant bacteria: use HgCl2-selected media to incubate the bacteria out of the soil which we collect from fluorescent lamp’s manufactory by normal process. Different colonies would be incubated alone, and then we will discriminate them by tests including: the Gram's staining; phenotypic, phy-siological and biochemical test; antibiotic resistance test, and so on.
②Hg2+ toleration test: incubate the bacteria by using the media that have different Hg2+ concentrations, and then observe the growing of those bacteria.
③Researches on the growing of the selected high mercury- degrading bacterium: including monitor the bacterium’s growth under different Hg2+ concentrations or pH conditions; observing its ability of living in the soil and natural water.
④Testing the level of transforming Hg2+ into Hg0: inoculate the growing bacteria which are just on logarithmic growth phase into several LB liquid media that contain different definite Hg2+ concentrations. After 48 hours, collect the liquid by centrifugation to determine the rudimental Hg2+ concentration on AFS-230a double-tracts atomic fluorescence absorptiometer.
⑤Classification and identification: according to the reserved 16SrRNA sequences by comparing the same bacterial strains in
引文
[1] 费云芸,刘代成.低剂量汞元素的毒性作用机理[J].山东师范大学学报,2003,18(1):88—90;
[2] 钱浩骏,叶细标,傅华. 汞及其化合物的慢性神经毒性[J]. 环境与职业医学,2005,4,22(2):160-166;
[3] 江刚. 格陵兰汞污染增加[J]。中国环境科学,2001,21(6):552;
[4] 徐小清,邓冠强,丘昌强,张晓华,惠嘉玉.长江三峡库区江段沉积物的重金属污染特征[J].水生生物学报,1999,23(1):1-10;
[5] 中国环境年鉴.中国环境年鉴编辑委员会.中国环境年鉴出版社,2000.714- 720;
[6] 中国科学院生态环境研究中心环境化学与生态毒理学重点实验室,中加环境汞污染研讨会。北京,2004.12.3;
[7] 党民团,刘娟. 中国汞污染的现状及防治对策[J]. 应用化工 ,2005,34(7):394-396;
[8] 张燕萍,颜崇淮,沈晓明. 环境中汞污染来源、人体暴露途径及其检测方法[J]. 广东微量元素科学,2004,11( 6):11-15;
[9] 邓旭, 李清彪, 孙道华等. 具超强汞富集能力基因工程菌生长情况的研究[J]. 工业微生物,2003,33(3):11-15;
[10]中国科学院微生物研究所菌种分类组.一般细菌鉴定手册.北京:科学出版社, 1978: 65- 66;
[11]陈宏伟.抗汞菌株的分离鉴定及特性[J].黑龙江大学自然科学学报,2000,17(3):86-88;
[12]陈亚丽,张先恩,刘虹等.甲基对硫磷降解菌假单胞菌WBC-3的筛选及其降解性能的研究[J].2002,42(4):490-497;
[13] N. Ramaiah, J. De. Unusual Rise in Mercury-Resistant Bacteria in Coastal Environs [J]. Microb Ecol (2003) 45:444–454;
[14]包幼迪,俞树高,代庚孙等。痢疾杆菌耐药性变异的研究 I.478 株痢菌传递性 R 质粒的检测[J].中华微生物学和免疫学杂志,1982,2(5):304-307;
[15]刘晓松,刘桂明,梁志坚.氢化物发生一原子荧光光度法测定饮用水中的微量汞[J]. 中国卫生检验杂志.2005,15(7):811-812;
[16]Nancy M. Duteau, Julia D. Rogers and Christian T. Bartholomay etal. Species-specific oligonucleotides for enumeration of Pseudomonas putida F1, Burkholderia sp. Strain JS150, and Bacillus subtilis ATCC7003 in biodegradation experiments [J]. Appl. Environ. Mcrobiol. 1998, 64(12):4994-4999;
[17]周桢林,陈琦.假单胞菌质粒抗汞基因的克隆[J];中山大学学报自然科学版.1990,29(3):129-135;
[18]J.萨姆布鲁克,D.W.拉塞尔著,黄培堂等译.分子克隆实验指南(第三册).北京:科学出版社。2002;
[19]J.L.Means and R.E.Hinchee,et al. Emerging technology for bioremediation of metals. CRC press, Boca raton, Fla. 1994.p99-105;
[20]Brunke, M., Deckwer,W.D.,Frischmuth,A.,et al. FEMS Microbiol. 1993.Rev. 11, 145±152;
[21]Von Canstein H, Li Y and Timmis KN et.al. Removal of mercury from chlorakali electrolysis wastewater by a mercury-resistant Pseudomonas putida strain. Appl. Environ. Microbiol[J]. 1999, 65(12):5279-5284;
[22] Jacques Ravel, Hildgund Schrempf, and Russsell T. HILL. Mercury Resistance Is Encoded by Transferable Giant Linear Plasmids in Two Chesapeake Bay Streptomyces Strains?. Appled and Environmental Microbiology[J]. 1998. Sept. Vol. 64, No.9.p.3383–3388;
[23] Milkman,R. and McKane,M. DNA sequence variation and recombination in E. coli. In The Population Genetics of Bacteria .Society for General Microbiology Symposium. Edited by S.Baumberg,J.P.W.Young, E.M.H. Wellington & J.R. Saunders. Cambridge: Cambridge University Press. 1995.vol.52 pp.127-142;
[24] Bizily S P. Rugh C L, Summers A O, et al. Phytoremediation of methylmercury pollution: merB expression in Arabidopsis thaliana confers resistance to organomercurials. Proc Natl Acad Sci USA.1999.96:6808-6813。
[1] Andréa M.A. Nascimento and Edmar Chartone-Souza. Operon mer: Bacterial resistance to mercury and potential for bioremediation of contaminated environments. [J]Genet. Mol. Res. 2003,2 (1): 92-101;
[2] Hongi Yu,Lien Chu,and Tapan K.Misra.Intracellular Inducer Hg2+ Concentration Is Rate Determining for the Expression of the Mercury-Resistance Operon in Cells.Journal Of Bacteriology.May .[J]Appl Microbiol Biotechnol 1996,178(9): 2172 – 2714;
[3] J effrak . Schaefer , J aneyagi Johnr. R einfelder T amaracardona Kristiem. E llickson Shoshanatel-or and Tamerbarkay. Role of the Bacterial Organomercury Lyase (MerB) in Controlling Methylmercury Accumulation in Mercury-Contaminated Natural Waters. [J]Environ.Sci.Technol.2004.38:4304-4311;
[4] K.Iohara,R.Iiyama,K. Nakamura and S.Silver。The mer operon of a mercury-resistant Pseudoalteromonas haloplanktisstrain isolated from Minamata Bay, Japan。[J] Appl Microbiol Biotechnol.2001,56:736-741;
[5] Milkman,R. McKane,M. DNA sequence variation and recombination in E. coli. In The Population Genetics of Bacteria. Society for General Microbiology Symposium. Cambridge: Cambridge University Press 1995,52:127-142;
[6] von Canstein, H., Timmis, K. N.Removal of Mercury from Chloralkali Electrolysis Wastewater by a Mercury-Resistant Pseudomonas putida Strain Appl. Environ. [J]Microbiol. 1999.65,5279-5284;
[7] 中国科学院生态环境研究中心环境化学与生态毒理学重点实验室,中加环境汞污染研讨会。北京,2004.12.3。
[8] Xiu xq(徐小清),Deng gq (邓冠强), Qiu cq (丘昌强),Zhang xh (张晓华),Hui jy (惠嘉玉). HEAVY METAL POLLUTION IN SEDIMENTS FROMTHE THREE GORGE RESERVOIR AREA(长江三峡库区江段沉积物的重金属污染特征).Acta Hydrobiologica Sinica (水生生物学报)。1999,23(1):1-10.
[9] Editorial Committee of China Environment Yearbook(中国环境年鉴编辑委员会).China Environmental Yearbook(中国环境年鉴).Beijing: China Environmental Yearbook Press(中国环境年鉴出版社),2000.714-720 附: 作者简介:林宇岚(1979。08)女,福建医科大学病原生物学系。 研究方向:微生物的遗传工程。 联系方式:Tel:0591-83569055 Email:christin57190@126.cn
[2] 钱浩骏,叶细标,傅华. 汞及其化合物的慢性神经毒性[J]. 环境与职业医学,2005,4,22(2):160-166;
[3] 江刚. 格陵兰汞污染增加[J]。中国环境科学,2001,21(6):552;
[4] 徐小清,邓冠强,丘昌强,张晓华,惠嘉玉.长江三峡库区江段沉积物的重金属污染特征[J].水生生物学报,1999,23(1):1-10;
[5] 中国环境年鉴.中国环境年鉴编辑委员会.中国环境年鉴出版社,2000.714- 720;
[6] 中国科学院生态环境研究中心环境化学与生态毒理学重点实验室,中加环境汞污染研讨会。北京,2004.12.3;
[7] 党民团,刘娟. 中国汞污染的现状及防治对策[J]. 应用化工 ,2005,34(7):394-396;
[8] 张燕萍,颜崇淮,沈晓明. 环境中汞污染来源、人体暴露途径及其检测方法[J]. 广东微量元素科学,2004,11( 6):11-15;
[9] 邓旭, 李清彪, 孙道华等. 具超强汞富集能力基因工程菌生长情况的研究[J]. 工业微生物,2003,33(3):11-15;
[10]中国科学院微生物研究所菌种分类组.一般细菌鉴定手册.北京:科学出版社, 1978: 65- 66;
[11]陈宏伟.抗汞菌株的分离鉴定及特性[J].黑龙江大学自然科学学报,2000,17(3):86-88;
[12]陈亚丽,张先恩,刘虹等.甲基对硫磷降解菌假单胞菌WBC-3的筛选及其降解性能的研究[J].2002,42(4):490-497;
[13] N. Ramaiah, J. De. Unusual Rise in Mercury-Resistant Bacteria in Coastal Environs [J]. Microb Ecol (2003) 45:444–454;
[14]包幼迪,俞树高,代庚孙等。痢疾杆菌耐药性变异的研究 I.478 株痢菌传递性 R 质粒的检测[J].中华微生物学和免疫学杂志,1982,2(5):304-307;
[15]刘晓松,刘桂明,梁志坚.氢化物发生一原子荧光光度法测定饮用水中的微量汞[J]. 中国卫生检验杂志.2005,15(7):811-812;
[16]Nancy M. Duteau, Julia D. Rogers and Christian T. Bartholomay etal. Species-specific oligonucleotides for enumeration of Pseudomonas putida F1, Burkholderia sp. Strain JS150, and Bacillus subtilis ATCC7003 in biodegradation experiments [J]. Appl. Environ. Mcrobiol. 1998, 64(12):4994-4999;
[17]周桢林,陈琦.假单胞菌质粒抗汞基因的克隆[J];中山大学学报自然科学版.1990,29(3):129-135;
[18]J.萨姆布鲁克,D.W.拉塞尔著,黄培堂等译.分子克隆实验指南(第三册).北京:科学出版社。2002;
[19]J.L.Means and R.E.Hinchee,et al. Emerging technology for bioremediation of metals. CRC press, Boca raton, Fla. 1994.p99-105;
[20]Brunke, M., Deckwer,W.D.,Frischmuth,A.,et al. FEMS Microbiol. 1993.Rev. 11, 145±152;
[21]Von Canstein H, Li Y and Timmis KN et.al. Removal of mercury from chlorakali electrolysis wastewater by a mercury-resistant Pseudomonas putida strain. Appl. Environ. Microbiol[J]. 1999, 65(12):5279-5284;
[22] Jacques Ravel, Hildgund Schrempf, and Russsell T. HILL. Mercury Resistance Is Encoded by Transferable Giant Linear Plasmids in Two Chesapeake Bay Streptomyces Strains?. Appled and Environmental Microbiology[J]. 1998. Sept. Vol. 64, No.9.p.3383–3388;
[23] Milkman,R. and McKane,M. DNA sequence variation and recombination in E. coli. In The Population Genetics of Bacteria .Society for General Microbiology Symposium. Edited by S.Baumberg,J.P.W.Young, E.M.H. Wellington & J.R. Saunders. Cambridge: Cambridge University Press. 1995.vol.52 pp.127-142;
[24] Bizily S P. Rugh C L, Summers A O, et al. Phytoremediation of methylmercury pollution: merB expression in Arabidopsis thaliana confers resistance to organomercurials. Proc Natl Acad Sci USA.1999.96:6808-6813。
[1] Andréa M.A. Nascimento and Edmar Chartone-Souza. Operon mer: Bacterial resistance to mercury and potential for bioremediation of contaminated environments. [J]Genet. Mol. Res. 2003,2 (1): 92-101;
[2] Hongi Yu,Lien Chu,and Tapan K.Misra.Intracellular Inducer Hg2+ Concentration Is Rate Determining for the Expression of the Mercury-Resistance Operon in Cells.Journal Of Bacteriology.May .[J]Appl Microbiol Biotechnol 1996,178(9): 2172 – 2714;
[3] J effrak . Schaefer , J aneyagi Johnr. R einfelder T amaracardona Kristiem. E llickson Shoshanatel-or and Tamerbarkay. Role of the Bacterial Organomercury Lyase (MerB) in Controlling Methylmercury Accumulation in Mercury-Contaminated Natural Waters. [J]Environ.Sci.Technol.2004.38:4304-4311;
[4] K.Iohara,R.Iiyama,K. Nakamura and S.Silver。The mer operon of a mercury-resistant Pseudoalteromonas haloplanktisstrain isolated from Minamata Bay, Japan。[J] Appl Microbiol Biotechnol.2001,56:736-741;
[5] Milkman,R. McKane,M. DNA sequence variation and recombination in E. coli. In The Population Genetics of Bacteria. Society for General Microbiology Symposium. Cambridge: Cambridge University Press 1995,52:127-142;
[6] von Canstein, H., Timmis, K. N.Removal of Mercury from Chloralkali Electrolysis Wastewater by a Mercury-Resistant Pseudomonas putida Strain Appl. Environ. [J]Microbiol. 1999.65,5279-5284;
[7] 中国科学院生态环境研究中心环境化学与生态毒理学重点实验室,中加环境汞污染研讨会。北京,2004.12.3。
[8] Xiu xq(徐小清),Deng gq (邓冠强), Qiu cq (丘昌强),Zhang xh (张晓华),Hui jy (惠嘉玉). HEAVY METAL POLLUTION IN SEDIMENTS FROMTHE THREE GORGE RESERVOIR AREA(长江三峡库区江段沉积物的重金属污染特征).Acta Hydrobiologica Sinica (水生生物学报)。1999,23(1):1-10.
[9] Editorial Committee of China Environment Yearbook(中国环境年鉴编辑委员会).China Environmental Yearbook(中国环境年鉴).Beijing: China Environmental Yearbook Press(中国环境年鉴出版社),2000.714-720 附: 作者简介:林宇岚(1979。08)女,福建医科大学病原生物学系。 研究方向:微生物的遗传工程。 联系方式:Tel:0591-83569055 Email:christin57190@126.cn