检测环境中重金属镉的特异性发光工程菌株的研究
摘要
在自然环境中,含镉的化合物具有较高的脂溶性和生物富集性,毒性强、危害大。传统的物理化学方法并不能正确反映出镉化合物对生物体引发的生物毒性效应,而生物方法可以直接检测镉化合物对生物体的毒理作用,更能够体现镉化合物在环境中的实际污染程度。本文以全套细菌荧光素酶基因( luxCDABE )为报告基因,以含有特异性镉启动子PcadA的质粒(pPcadA-gfp)为载体,构建由Cd2+诱导的含报告基因luxCDABE的重组质粒(pPcadA-luxCDABE-gfp),感受态转化法将其导入枯草杆菌B.subtilis受体菌,构建特异性检测Cd2+的工程菌,并应用于环境中痕量重金属镉的检测。研究方法如下:
(1)鉴定实验所需的两个菌种:含有质粒pMD20-T-luxCDABE的大肠杆菌E.coli HB101和含有质粒pPcadA-gfp的枯草杆菌B.subtilis 1A751。E.coli HB101采用蓝白斑筛选法鉴定。将B.subtilis 1A751菌悬液制成载玻片,在荧光显微镜下观察菌体特征,提取质粒pPcadA-gfp,BamHI/HindIII和BamHI/EcoRI双酶切后,用琼脂糖凝胶电泳分析鉴定;
(2)构建重组质粒(pPcadA-luxCDABE-gfp):利用基因工程技术,用BamHI单酶切获得目的基因luxCDABE及载体质粒pPcadA-gfp;在T4连接酶的作用下,将luxCDABE插入到PcadA的下游,构建重组质粒pPcadA-luxCDABE-gfp。将重组质粒(pPcadA-luxCDABE-gfp)导入E.coli进行增殖,通过Cm抗性平板筛选获得克隆并进行酶切鉴定。筛选的重组质粒导入耐受重金属镉的B.subtilis,做部分研究。
实验结果:通过蓝白斑筛选E.coli HB101菌株,Amp抗性平板上生长白色菌落表示菌体中含有质粒pMD20-T-luxCDABE;B.subtilis 1A751菌悬液在蓝光激发下发出绿色荧光,说明菌体中含有质粒pPcadA-gfp,并能表达出GFP。从宿主菌中提取重组质粒pPcadA-luxCDABE-gfp,经酶切初步鉴定,pPcadA-luxCDABE-gfp构建成功,为进一步研究Cd2+是否诱导luxCDABE的表达及获得监测Cd2+的工程菌株打下基础。
In the nature, cadmium compounds have a high liposolubility and bio-concentration, it’s very harmful and toxic. Traditional physical and chemical methods cannot correctly reply the biological toxic effects that cadmium compounds caused to organisms. But biological methods can directly detect the toxicological effect of cadmium compounds on the organisms, more reflect the actual pollution level. In my paper,a full set of bacterial luciferase gene(luxCDABE) serves as reporter gene, and the pPcadA-gfp plasmid containing the cadmium-specific promoter(PcadA) acts as vector to construct the recombinant DNA(pPcadA-luxCDABE-gfp) whose expression is induced by Cd2+. B.subtilis was employed as the host bacteria, and the recombinant DNA was transformed into B.subtilis in order to obtain the specific engineering strains which can detect trace cadmium in the environment. Research methods are as follows:
(1)Identification of two laboratory keeping strains: E.coli HB101 containing pMD20-T-luxCDABE and B.subtilis 1A751 containing plasmid pPcadA-gfp. Blue-write spot screening was used to identify E.coli HB101.Making B.subtilis 1A751 solution into a glass slide, and observe the cell characteristics under the fluorescence microscope, Plasmid pPcadA-gfp was extracted and identified by BamHI/HindIII and BamHI/EcoRI double enzyme digestion and agarose gel electrophoresis analysis;
(2)Construction of recombinant pPcadA-luxCDABE-gfp: Through the genetic engineering methods,BamHI was used for isolating the target gene luxCDABE and circular vector plasmid pPcadA-gfp, and then transformation and screening was performed. T4 ligase was applied to inserting luxCDABE into the downstream of PcadA to generate recombinant plasmid DNA pPcadA-luxCDABE-gfp. Recombinant plasmid DNA was induced into E.coli for growth, clones was screened by Cm resistance plate and the plasmid DNA was extracted and identified by restriction enzyme digestion. Screened recombinant DNA was induced into B.subtilis tolerancing the heavy metal cadmium, did some research.
Results: E.coli HB101 strains containing plasmid pMD20-T-luxCDABE was screened by blue-white screening method, if white colonies growed on the Amp resistance plate, the phenomenon shows the successful transformation of the plasmid pMD20-T-luxCDABE; B.subtilis 1A751 bacteria solution in glass slide emited green fluorescent under blue light excitation, indicated the successful plasmid transformation and the expression of GFP. The plamid pPcadA-luxCDABE-gfp was extracted from the engineering strains, preliminary study has identified that the target gene had been inserted into the vector DNA. It provide the basis for further study on the luxCDABE expression induced by Cd2+ and obtianing the engineered stains for monitoring Cd2+ existing in the environment .
(1)鉴定实验所需的两个菌种:含有质粒pMD20-T-luxCDABE的大肠杆菌E.coli HB101和含有质粒pPcadA-gfp的枯草杆菌B.subtilis 1A751。E.coli HB101采用蓝白斑筛选法鉴定。将B.subtilis 1A751菌悬液制成载玻片,在荧光显微镜下观察菌体特征,提取质粒pPcadA-gfp,BamHI/HindIII和BamHI/EcoRI双酶切后,用琼脂糖凝胶电泳分析鉴定;
(2)构建重组质粒(pPcadA-luxCDABE-gfp):利用基因工程技术,用BamHI单酶切获得目的基因luxCDABE及载体质粒pPcadA-gfp;在T4连接酶的作用下,将luxCDABE插入到PcadA的下游,构建重组质粒pPcadA-luxCDABE-gfp。将重组质粒(pPcadA-luxCDABE-gfp)导入E.coli进行增殖,通过Cm抗性平板筛选获得克隆并进行酶切鉴定。筛选的重组质粒导入耐受重金属镉的B.subtilis,做部分研究。
实验结果:通过蓝白斑筛选E.coli HB101菌株,Amp抗性平板上生长白色菌落表示菌体中含有质粒pMD20-T-luxCDABE;B.subtilis 1A751菌悬液在蓝光激发下发出绿色荧光,说明菌体中含有质粒pPcadA-gfp,并能表达出GFP。从宿主菌中提取重组质粒pPcadA-luxCDABE-gfp,经酶切初步鉴定,pPcadA-luxCDABE-gfp构建成功,为进一步研究Cd2+是否诱导luxCDABE的表达及获得监测Cd2+的工程菌株打下基础。
In the nature, cadmium compounds have a high liposolubility and bio-concentration, it’s very harmful and toxic. Traditional physical and chemical methods cannot correctly reply the biological toxic effects that cadmium compounds caused to organisms. But biological methods can directly detect the toxicological effect of cadmium compounds on the organisms, more reflect the actual pollution level. In my paper,a full set of bacterial luciferase gene(luxCDABE) serves as reporter gene, and the pPcadA-gfp plasmid containing the cadmium-specific promoter(PcadA) acts as vector to construct the recombinant DNA(pPcadA-luxCDABE-gfp) whose expression is induced by Cd2+. B.subtilis was employed as the host bacteria, and the recombinant DNA was transformed into B.subtilis in order to obtain the specific engineering strains which can detect trace cadmium in the environment. Research methods are as follows:
(1)Identification of two laboratory keeping strains: E.coli HB101 containing pMD20-T-luxCDABE and B.subtilis 1A751 containing plasmid pPcadA-gfp. Blue-write spot screening was used to identify E.coli HB101.Making B.subtilis 1A751 solution into a glass slide, and observe the cell characteristics under the fluorescence microscope, Plasmid pPcadA-gfp was extracted and identified by BamHI/HindIII and BamHI/EcoRI double enzyme digestion and agarose gel electrophoresis analysis;
(2)Construction of recombinant pPcadA-luxCDABE-gfp: Through the genetic engineering methods,BamHI was used for isolating the target gene luxCDABE and circular vector plasmid pPcadA-gfp, and then transformation and screening was performed. T4 ligase was applied to inserting luxCDABE into the downstream of PcadA to generate recombinant plasmid DNA pPcadA-luxCDABE-gfp. Recombinant plasmid DNA was induced into E.coli for growth, clones was screened by Cm resistance plate and the plasmid DNA was extracted and identified by restriction enzyme digestion. Screened recombinant DNA was induced into B.subtilis tolerancing the heavy metal cadmium, did some research.
Results: E.coli HB101 strains containing plasmid pMD20-T-luxCDABE was screened by blue-white screening method, if white colonies growed on the Amp resistance plate, the phenomenon shows the successful transformation of the plasmid pMD20-T-luxCDABE; B.subtilis 1A751 bacteria solution in glass slide emited green fluorescent under blue light excitation, indicated the successful plasmid transformation and the expression of GFP. The plamid pPcadA-luxCDABE-gfp was extracted from the engineering strains, preliminary study has identified that the target gene had been inserted into the vector DNA. It provide the basis for further study on the luxCDABE expression induced by Cd2+ and obtianing the engineered stains for monitoring Cd2+ existing in the environment .
引文
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[2]刘发欣,高怀友.镉的食物链迁移及其污染防治对策研究[J].全国耕地土壤污染监测与评价技术研讨会. 2006(7). 176~180
[3]李然,李嘉,赵文谦.水环境中重金属污染研究概述[J].四川环境. 1997, 1:18~22
[4] Pongratz R. Arsenic speciation in environmental samples of contaminated soil[J]. The Science of the Total Environment. 1998, 224: 133~141
[5]曾军.基于luxAB标记菌株的发光定量分析及其在镉毒性检测中的应用[D]. 2006: 4~16
[6] Leo Yen-Cheng Lin, Edward A. Meighen. The Biochemistry and Molecular Biology of Bacterial Bioluminescence. 2004(1):1~40
[7]黄正,王家玲.发光细菌的生理特性及其在环境监测中的应用[J].环境科学. 1994, 16(3): 87~90
[8]陈声明,陈伟方,许灵群.发光细菌与环境监测[J].环境污染与防治. 1992, 14(3): 26~29
[9] Bauman P et al.. Ann. Rev. Microbio.. 1983, 37, 369
[10]张促伦.微缩发光分析技术应用实例(五)—环境污染的监测[J].生物化学与生物物理进展. 1999, 27(4).
[11]张进兴,逄爱梅,孙修勤等.海洋发光细菌的发光及其应用[J].发光学报. 2007, 28(2):167~171
[12] P.J.Herring. The spectral characteristics of luminous marine organisms[J]. Printed in Great Britain. 1983(3), Proc. R. Soc. Lond. B. 220: 183~217
[13]夏铁骑,常慧萍.发光细菌Lux基因系统的遗传学研究及应用.濮阳职业技术学院学报[J]. 2008, 21(4): 1~3
[14]程英,裴宗平,邓霞等.生物监测在水环境中的应用及存在问题探讨[J].环境科学与管理. 2008, 33(2). 111-114
[15] Bulich A A, Isenberg D L. Use of the luminescent bacteria system for the rapid assessment of aqustic toxicity[J]. ISA Transactions, 1981, 20(1): 29~32
[16] KenRealTM.生物发光毒性测试分析[N]. Chemi&BioLuminescence : 1~3
[17]黄正,王家玲.细菌发光试验及Ames试验检测工业废水急性毒性及致突变性的研究[J].环境科学. 1994, 15(6):70~71
[18] Alison M. Horsburgh, D. P. Mardlin, N. L. Turner, et a1. On-line microbial biosensing and fingerprinting of water pollutants[J]. Biosensors and Bioelectronics. 2002, 17: 495~501
[19]张敏,任慧霞.报告基因的应用研究进展[J]. Food and Drug. 2007, 9(09A): 46
[20]杜宗军,王祥红,李海峰.发光细菌的研究和应用[J].高技术通讯. 2003, 12: 103~106
[21] Man Bock Gu, Robert J. Mitchell, Byoung Chan Kim. Whole-Cell-Based Biosensors for Environmental Biomonitoring and Application[B]. Adv Biochem Engin/Biotechnol. 2004, 87: 269~305
[22] Lewis J C, Feltus A, Ensor C M, Ramanathan S, Daunert S. Application of reporter genes[J]. Analytical chemistry. 1998, 70(17): 579A~585A
[23] Silver S. Genes for all metals-a bacterial view of the Periodic Table[J]. Industrial Microbiology and Biotechnology.1998, 20: 1~12
[24] Ranjit S.Shetty, Sapna K. Deo, Puja Shah, et a1.Luminescence-based whole-cell-sensing systems for cadmium and lead using genetically engineered bacteria[J]. Anal Bioanal Chem. 2003, 376:11~17
[25] Yan Sun, Marco D. Wong, Barry P. Rosen. Role of Cysteinyl Residues in Sensing Pb(II), Cd(II), and Zn(II) by the Plasmid pI258 CadC Repressor[J]. Biological Chemistry. Printed in U.S.A.. 2001, 276(18): 14955~14960
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