DNA指纹分析技术在群落级生命系统应用的可能性
THE FEASIBILITY FOR APPLICATION OF DNA FINGERPRINTING TO COMMUNITY-LEVEL LIFE SYSTEM
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摘要: DNA指纹分析是一种关于整体遗传结构分析的强有力的分子生物学技术,广泛应用于种群及个体水平层次生命系统.本文以东湖浮游生物为对象,用随机引物和特异引物进行扩增探讨了DNA指纹分析技术在群落级生命系统应用的可能性.实验表明:取自分别处于超富营养水平、富营养水平和中营养水平的Ⅰ、Ⅱ、Ⅲ站样本的模板DNA,无论是以随机引物M-01、M-02、M-03、M-18及M-19还是特异引物CW15946/4、EGMS6、EGMS4、ITS1及HSP扩增均获得清晰且稳定的图谱;各站间浮游生物群落DNA指纹拓扑结构存在明显的表观差异:居中营养水平的Ⅲ站谱带较多,而分别居富营养水平、超富营养水平的Ⅱ站和Ⅰ站较少;各站既有特异性谱带也有共有的谱带,Ⅲ站包含了Ⅱ站和Ⅰ站33%-100%的谱带.本文还结合现有有关物种生物多样性及理化方面的资料,对所获得群落DNA指纹图谱的生物学意义进行尝试性的、定性的讨论.Abstract: PCR-based DNA fingerprinting techniques such as random amplified polymorphic DNA analysis(RAPD)represents a very informative and cost-effective approach for assessing genetic diversity of a wide range of organisms. As a powerful molecular biology technology,DNA fingerprinting is widely applied on the study of integral genetic structure. All DNA fingerprinting techniques can discover the genetic diversity. For a individual or cell,they can discover the diversity of one genome; For population,they can discover the diversity of total DNA including many individuals. If for community, more information for different species can be discovered from many kinds of DNA templates. However,it is restricted to population and individual-level life system. This paper first explored the feasibility for application of DNA fingerprinting to community-level life system with plankton community from three stations of different eutrophic status in Lake Donghu as samples. Total community DNAs were extracted. PCR fingerprinting techniques based on the use of arbitrary primers(RAPD-PCR)have been developed and compared for their ability to generate“fingerprint”patterns characteristics. 20 RAPD primers were screened. 5 primers which amplified clear and sharp bands were used to discuss from these total RAPD primers. The results show that(1)we constructed a DNA extraction method adapating to plankton community through a series of getting rid of impurities. The high quality of template DNA is a precondition for DNA fingerprinting analysis. The composition and their concentration are key factors for DNA extraction. The template DNAs extracted from Station Ⅰ of hypertrophication,Station Ⅱ of eutrophication and Station Ⅲ of mesotrophication,respectively,were amplified repreduciblely;(2)the resulting amplified maps are clear and stable in both RAPD by random primers M-01,M-02,M-03,M-18 and M-19,and PCR by specific primers CW 15946/47,EGMS6,EGMS4,ITS1 and HSP,and there were some relationship between the topological structure and trophic status. From RAPD and specific primers PCR amplification, there are many variations among three stations. Station Ⅲ comprising 33%—100% bands of Station Ⅰ or Station Ⅱ. This indicats species biodiversity of Station Ⅲ is the highest. In addition,the DNA fingerprinting maps obtained were tentatively and qualitatively discussed with the available data of species biodiversity and physical chemistry. In the present work,the results of this study will contribute to our understanding of the feasibility for application of DNA fingerprinting to community-level life system in plankton.
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Keywords:
- DNA fingerprinting /
- Community life system /
- Plankton /
- Lake Donghu
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PCR-based DNA fingerprinting indicates host-related genetic variation in the nematophagous fungus Pochonia chlamydosporia[J]. Mycol. Res.,2003,107(2):198-205[5] Russell R J,Festing M F W,Deeny A A,et al. DNA fingerprinting for genetic monitoring of inbred laboratory rats and mice [J]. Laboratory. Animal Science,1993,43:460-465[6] Sudo T,Goto N,Mannen H,et al. Identification and minisatellite linkage analysis of SMXA recombinant inbred strains of mice by DNA fingerprinting[J]. Experimental Animals,1995,44:87-93[7] Wang X. M.,Yang X. R.,Progress of Studies on the molecular markers[J]. Journal of Tianjin Agricultural College,2000,7(1):21-24[王晓梅,杨秀荣. DNA分子标记研究进展.天津农学院学报,2000,7(1):21-24][8] Deng Z N,Gentile A,Nicolosi E,et al. Identification of in vivo and in vitro lemon mutants by RAPD markers[J]. J. Hort. Sci,1995,70:117-125[9] Patrick J C,Susan K B,Norman F W. Radom amplified polymorphic DNA-based genetic linkage maps of three cultivars[J]. J. Amer. Hort. Sci,1997,122(3):350-399[10] Muyzer G. DGGE/TGGE a method for identifying genes from natural ecosystems[J]. Curr. Microbiol,1999,2:317-322[11] Luo H F,Qi H Y,Xue K.,et al. A preliminary application of PCR-DCCE to study microbial diversity in soil[J]. Acta Ecologica Sinica,2003. 23(8):1570-1575[罗海峰,齐鸿雁,薛凯,等.PCR-DGGE技术在农田土壤微生物多样性研究中的应用.生态学报,2003,23(8):1570-1575][12] Frank S,Christoph C T. A new approach to utilize PCR-single-strand-conformation polymorphism for 16S rRNA gene-based microbial community analysis[J]. Appl. Environ. Microbiol,1998,64:4870-4876[13] Vybiral D,Witte A,Velimirov B. Investigation of 0.2 μm filterable bacterial from the Western Mediteranean Sea using a molecular approach:dominance of potention starvation forms[J]. FEMS Microbiol. Ecol,2000,31(2):153-161[14] LUKOW T, Danfied P F, Liesack W. Use of the T-RFLP technique to assess spatial and tempora changes in the bacterial community structure within an agricultural soil planted with transgenic and no-transgenic potato plants[J]. FEMS Microbiol. Ecol,2000,32:241-247[15] Bozena Z,Robert K,Irenenz M. Genetic and morphological variability among clones of Euglena Pisciformis based on RAPD and biometric analysis[J]. Algological studies,1996,81:1-21[16] Wang J X.,Xie S,L,Zhong J,Y. et al.,A modified rapid effiecient DNA extraction method of Euglenoids[J]. Acta Hytrobiologica Sinica,1999,23(5):533-536. [谢树莲,钟家钰,潘卉,等.一种改进的快速有效的裸藻DNA抽提方法.水生生物学报,1999,23(5):533-536.][17] Xia M. Research progress of biodiversity[J]. Journal of Northeast Agricultural University,1999,30(1):94-100. [夏铭.生物多样性研究进展.东北农业大学学报,1999,30(1):94-100][18] Xia M. Research progress of genetic diversity[J],Chinese Journal of Ecology. 1999,18(1):59-65. [夏铭.遗传多样性研究进展.生态学杂志,1999,18(1):59-65][19] Xie P, Zhu G Y, Lu M, et al. Effectes of the water eutrophic on plankton community diversity[J]. Acta Hytrobiologica Sinica,1996,20(appl):30-37[谢平,诸葛燕,戴莽,等.水体富营养化对浮游生物群落多样性的影响.水生生物学报,1996,20(增刊):30-37][20] Lei A P, Shi Z X,Wei Y X. Diversity of the phytoplankton in Donghu lake,Wuhan[J]. Acta Hytrobiologica Sinica,2003,27(2):179-184[雷安平,施之新,魏印心.武汉东湖浮游藻类物种多样性的研究.水生生物学报,2003,27(2):179-184][21] Gong Z J,Xie P,Tang H J, et al. The influence of eutrophycation upon community structure and biodiversitv of macrozoobenthos[J]. Acta Hytrobiologica Sinica,2001,25(3):210-216. [龚志军,谢平,唐汇涓,等.水体富营养化对大型底栖动物群落结构及其多样性的影响.水生生物学报,2001,25(3):210-216.]
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