DNA指纹技术在污染土壤生态毒理诊断中的应用

摘要/Abstract

摘要： 生物标记物能在细胞或分子水平上指示暴露-效应关系,是进行污染土壤生态毒理诊断的主要技术手段之一。随着分子生物学技术的飞速发展,出现了一系列以聚合酶链式反应为基础的、在分子水平上检测污染物质导致的生物体DNA损伤的DNA指纹技术。DNA指纹技术的主要类型有:随机扩增多态性DNA(RAPD)、聚合酶链式反应-单链构象多态性(PCR-SSCP)、扩增片段长度多态性(AFLP)、任意引物聚合酶链式反应(AP-PCR)、差异显示反转录聚合酶链式反应(DDRT)、短DNA重复序列(SSR)及限制片段长度多态性(RFLP)等。这些技术与检测基因突变、染色体畸变和损伤为主的一系列经典研究方法如彗星分析、微核实验等相比具有简便、快速、灵敏等优点。本文着重介绍了随机扩增多态性DNA、聚合酶链式反应-单链构象多态性、扩增片段长度多态性3种重要的DNA指纹技术在污染土壤诊断中的应用。

关键词: 林隙更新, 阔叶红松林, 树种多样性, 林隙结构, 林分动态

Abstract: Biomarker can show the exposure-effect relationships at molecular and cellular levels,and is one of the most effective techniques in eco-toxicological diagnosis of contaminated soil.With the progress of molecular biology,a number of selective and sensitive PCR-based DNA fingerprint assays were developed to detect the DNA lesions caused by pollutants.The DNA fingerprint techniques mainly include random amplified polymorphic DNA(RAPD),polymerase chain reaction-single strand conformation polymorphism,amplified fragment length polymorphism,arbitrary primed-polymerase chain reaction,differential display reverse transcription,short-sequence DNA repeat,and restriction fragment length polymorphism.These techniques possess the characters of simplicity,reproducibility and sensitivity,compared with classical methods such as comet assay and micronucleus test.In this paper,the application of three main DNA fingerprints in eco-toxicological diagnosis of contaminated soil was discussed emphatically.

Key words: Gap regeneration, Mixed-broad-leaved Korean pine forests, Tree diversity, Gap structure, Stand dynamics

中图分类号:

齐雪梅, 李培军, 刘宛, 张海荣, 许华夏, 韩春梅. DNA指纹技术在污染土壤生态毒理诊断中的应用[J]. 生态学杂志, 2005, (11): 1351-1356.

QI Xuemei, LI Peijun, LIU Wan, ZHANG Hairong, XU Huaxia, HAN Chunmei. Application of DNA fingerprint in eco-toxicological diagnosis of contaminated soil[J]. cje, 2005, (11): 1351-1356.

参考文献

[1] 王海黎,陶澍.1999.生物标记物在水环境研究中的应用[J].中国环境科学,19(5):421～426.
[2] 孔繁祥.2002.环境生物学[M].北京:高等教育出版社.
[3] 刘宛,李培军,周启星,等.2004.污染土壤的生物标记物研究进展[J].生态学杂志,23(5):150～155.
[4] 宋玉芳,许华夏,任利萍,等.2001重金属对土壤中萝卜种子发芽与根伸长抑制的生态毒性[J].生态学杂志,20(3):4～8.
[5] 孙铁珩,周启星,李培军.2000.污染生态学[M].北京:科学出版社,305～328.
[6] 谭明家,王莉娥,李忠生,等.1997.应用PCR-SSCP检测转化细胞P53基因突变[J].中国药理学与毒理学杂志,11(2):159～161.
[7] 夏世钧,吴中亮.2001.分子毒理学基础[M].武汉:湖北科学技术出版社,305～306.
[8] 熊治廷.2002.环境生物学[M].武汉:湖北科学技术出版社,232～258.
[9] 赵永良,刘国廉.1998.PCR-SSCP技术检测辐射诱发的恶转细胞P53突变[J].中华放射医学与防护杂志,18(2):104～105.
[10] Angelis KJ, McGuffie M, Menke M, et al. 2000. Adaption to alkylation damage in DNA measured by the come assay[J]. Environ. Mol. Mutagen . , 36:146～ 150.
[11] Atienzar FA, Billinghurst Z, Depledge MH. 2002.4-n-Nonylphenol and 17-beta estradiol may induce common DNA effects in developing barnacle larvae [J]. Environ. Pollut., 120 (3): 735 ～738.
[12] Atienzar FA, Evenden A J, Jha AN. 2002. Use of the random amplified polymorphic DNA (RAPD) assay for the detection of DNA damage and mutations:possible implications of confounding factors[J]. Biomarkers, 7(1): 94～101.
[13] Bagley MJ, Anderson SL, May B. 2001. Choice of methodology for assessing genetic impacts of environmental stressors: Polymorphism and reproducibility of RAPD and AFLP fingerprints[J]. Ecotoxicology, 10 ( 4 ): 239 ～ 244.
[14] Conte C, Mutti I, Puglisi P. 1998. DNA fingerprinting analysis by a PCR-based method for monitoring the genotoxic effect of heavy metals pollution [J]. Chemosphere, 37 ( 14 ～ 15): 2739 ～ 2749.
[15] Ferrero M, Castano A. 1998. Characterization of RTG-2 fish cell line by random amplified polymorphic DNA[J]. Ecotoxicol. Environ. Saf., 40:56～64.
[16] Franck A. 1999. Quantitative assessment of genotoxicity using random amplied polymorphic DNA: Comparison of genomic template stability with key fitness parameters in daphnia magna exposed to benzo(a)pyrene[J]. Environ. Toxicol. Chem, 18:2275～ 2282.
[17] Grant WF. 1994. The present status of higher plant bioassay for the detection of environmental mutagens[J]. Mutat. Res., 310:175～ 185.
[18] Jeffreys AJ. 1985. Individual-specific" fingerprints " of human DNA[J]. Nature, 316:76～ 79.
[19] Junca H, Pieper DH. 2004. Functional gene diversity analysis in BTEX contaminated soils by means of PCR-SSCP DNA fingerprinting: Comparative diversity assessment against bacterial isolates and PCR-DNA clone libraries[J]. Environ. Microbiol., 6(2) :95～ 110.
[20] Kentc RH, Eady JJ, Ross GM. 1995. The comet moment at a measure of DNA damage in the comet assay[J]. Int .J. Radiat.Biol., 67 (6): 655 ～ 660.
[21] Lutz WK. 1986. Quantitative evaluation of DNA binding data for risk estimation for classification of direct and indirect carcinogens[J].J. Cancer Res. Clin . Oncol. , 112(2) :85～91.
[22] Maino G, Ventura A, Zuffi L. 1992. Algebraic treatment of giant dipole resonances built on excited states[J]. Phy. Rev. C. Nucl.Phys., 46(5): 1725 ～ 1734.
[23] Massimo L, Fabrizio G, Serena I, et al. 2004. Genetic and DNAmethylation change induced by potassium dichromate in Brassica napus L. [J]. Chemosphere , 54:1049～ 1058.
[24] Massimo L, Fabrizio G, Serena I, et al. 2003. AFLP analysis as biomarker of exposure to organic and inorganic genotoxic substances in plants[J]. Chemosphere, 52(7) : 1183 ～ 1188.
[25] Mengoni A, Gonnelli C, Galardi F. 2000. Genetic diversity and heavy metal tolerance in populations of Silene paradoxa L.(Caryophyllaceae): A random amplified polymorphic DNA analysis[J]. Mol. Ecol., 9(9): 1319 ～ 1324.
[26] Noumi T, Mosher ME, Natori S, et al. 1984. A phenylanine for serine substitution in the beta subunit of Escherichia coli F1-ATPase affects dependence of its activity on divalent cations[J]. Biol. Chem., 25(16) :259～268.
[27] Orita M. 1989. Detection of polymorphism of human DNA by gel electrophoresis as single strand conformation polymorphisms[J].Proc. Natl. Acad. Sci. USA, 86:2760～2770.
[28] Rong ZY, Yin HW. 2004. A method for genotoxicity detection using random amplified polymorphism DNA with Danio rerio[J]. Ecotoxicol. Environ. Saf., 58: 96～ 103.
[29] Sandra C, Roberta A, Massimo L, et al. 2002. Soil genotoxicity sssessment: A new strategy based on biomolecular tools and plant bioindicators[J]. Environ. Sci. Tech nol., 36 : 2748～ 2753 .
[30] Tanguy A, Boutet I, Bonhomme F, et al. 2002. Polymorphism of metallothionein genes in the Pacific oyster Crassostrea gigas as a biomarker of response to metal exposure[J]. Biomarkers, 7 (6):439～ 500.
[31] Theodorakis CW. 2001. Integration of genotoxic and population genetic endpoints in biomonitoring and risk assessment[J]. Ecotoxicology, 10(4): 245 ～ 256.
[32] Tuteja N, Mohan BS, Mithilesh KM. 2001. Molecular mechanisms of DNA damage and repair: Progress in plants [J]. Crit.Rev. Biochem . Mol. Biol., 36(4) :337～ 397.
[33] Welsh J. 1990. Fingerprinting genomes using PCR with arbitrary primers[J]. Nucleic Acid. Res., 18 : 7213 ～ 7218.
[34] Williams J. 1990. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers [J]. Nucleic Acid. Res.,18:6531～6535.
[35] Wozniak K, Blasiak J. 2003. In vitro genotoxicity of lead acatate:Induction of single and double DNA strand breaks and DNA-protein cross-links[J]. Mutat. Res., 535 : 127～ 139.