收稿日期: 2007-12-15
修回日期: 2008-03-17
网络出版日期: 2008-04-10
基金资助
国家科技部重大基础研究前期研究专项“长江三角洲地区湖泊环境演变的人与自然相互作用过程研究”（编号：2004CCA02900）；中国科学院知识创新工程重大项目“人类活动驱动下的长江中下游湖泊环境变化研究”（编号：KZCX1-SW-12-1）联合资助.
Extraction and PCR Amplification of DNA from Lake Sediments
Received date: 2007-12-15
Revised date: 2008-03-17
Online published: 2008-04-10
对湖泊沉积物中保存的生物大分子—DNA进行研究分析，以探讨湖泊生态系统随环境改变而演变的过程。根据前人对土壤和海洋沉积物DNA的提取方法，针对湖泊沉积物的特点进行改进，在此基础上对太湖梅梁湾湖泊沉积岩芯进行DNA提取和扩增。结果表明：不同深度湖泊沉积物均能获得DNA，且纯度较高，OD260/ OD280 均大于1.4、OD260/ OD230大于1.1，可直接用于PCR（polymerase chain reaction ）扩增。对微囊藻的16S rRNA基因进行PCR扩增，结果发现在前5个沉积物样品中，均得到了200 bp左右的微囊藻16S rRNA基因片段，表明微囊藻或已降解的微囊藻基因片段存在于这5个层位的湖泊沉积物中。这为利用湖泊沉积物中的生物分子来推断历史湖泊生物群落结构，判断湖泊生态演变的历史提供了新的研究思路。
申慧彦 , 李世杰 . 湖泊沉积物中DNA提取与PCR扩增[J]. 地球科学进展, 2008 , 23(4) : 433 -438 . DOI: 10.11867/j.issn.1001-8166.2008.04.0433
Biomacromolecules in lake sediments are very important in the indication of environmental information. DNA(Deoxyribonucleic acid) is an important biomacromolecule, and its genetic information which corresponds to organism to indicate information of biology and environmental change of lake sediments con be studied. In recent years, several approaches based on the extraction of DNA from environmental samples have been developed for the microbial diversity in soil and sea sediments.
On the basis of previous work, an efficient method to extract DNA from lake sediments is reported. Samples from location in Lake Taihu were collected, and their genomic DNA with the size of 23kb was obtained from all sediment samples of Meiliang bay. Purification and efficiency of extracted DNA were evaluated through ultraviolet spectrum OD260/ OD280 and OD260/ OD230 ratios. Experimental results show that all samples mentioned above obtain quite purified genomic DNA with OD260/ OD280 ratios from 1.41 to 1.72, and OD260/ OD280 ratios from 1.13 to1.54. The changed regulation of DNA in different sediment depth is elucidated, the distribution of organic carbon and DNA is consistent in 15cm surface sediments, and DNA keeps low contents below 15cm. The extracted DNA is sufficient enough to be served as a template for PCR amplification of specific genetic sequences. In particular, Microcystis 16S rRNA gene fragments (200bp gene fragments) were obtained from 5 samples.
Key words： Lake sediments; Microcystis; DNA extraction; PCR amplification.
[1] Wang Sumin，Zhang Zhenke. The new progress of the study of lake sediment and environmental evolution in China [J]. Chinese Science Bulletin，1999，44（6）:579-587.[王苏民，张振克. 中国湖泊沉积与环境演变研究的新进展[J]. 科学通报，1999，44（6）:579-587.]
[2] Wang Sumin，Dou Hongshen，eds. China Lakes Chorography [M]. Beijing: Science Press，1998:22-27.[王苏民，窦鸿身主编. 中国湖泊志[M]. 北京: 科学出版社，1998:22-27.]
[3] Chen Jing'an，Wan Guojiang，Wang Fushun，et al. Environmental records of carbon in recent lake sediments [J]. Science in China（Series D），2002，32（1）: 73-80.[陈敬安，万国江，汪富顺，等. 湖泊现代沉积物碳环境记录研究[J].中国科学:D辑，2002，32（1）:73-80.]
[4] Reuss N，Conley D J，Bianchi T S. Preservation conditions and the use of sediment pigments as a tool for recent ecological reconstruction in four Northern European estuaries [J]. Marine Chemistry，2005，95:283-302.
[5] Xiao Xiayun，Shen Ji，Wang Sumin，et al. Palynological evidence for vegetational and climatic changes from the HQ deep drilling core in Yunnan Province，China [J]. Science in China（ Series D），2007，50（8）:1 189-1 201.[肖霞云，沈吉，王苏民，等. 鹤庆深钻孢粉记录揭示的2.78Ma以来的植被演替与气候变迁[J].中国科学：D辑，2007，37（6）:778-788.]
[6] Zhou J Z，Bruns M A，Tiedje J M．DNA recovery from Soils of diverse composition [J]．Applied and Environmental Microbiology，1996，62:316-322.
[7] Li Tao，Wang Peng，Wang Pinxian. A preliminary study on the diversity of bacteria in the Xisha trough sediment，the south China sea [J]. Advances in Earth Science，2006，21（10）:1 058-1 062.[李涛，王鹏，汪品先.南海西沙海槽沉积物细菌多样性初步研究[J].地球科学进展，2006，21（10）:1 058-1 062.]
[8] Xing Peng，Kong Fanxiang，Cao Huansheng，et al. Relationship between bacterioplankton and phytoplankton community dynamics during late spring and early summer in Lake Taihu [J]. Acta Ecologica Sinica，2007，27（5）:1 696-1 702.[邢鹏，孔繁翔，曹焕生，等.太湖浮游细菌与春末浮游藻类群落结构演替的相关分析[J ].生态学报，2007，27（5）:1 696-1 702.]
[9] Ouellette A J A，Handy S M，Wilhelm S W. Toxic Microcystis is widespread in Lake Erie: PCR detection of toxin genes and molecular characterization of associated cyanobacterial communities [J]. Microbial Ecology，2006，51:154-165.
[10] Li Huirong，Yu Yong，Zeng Yinxin，et al. Phylogenetic analysis of bacterial diversity in Pacific Arctic sediments [J]. Acta Microbiologica Sinica，2006，46（2）:177-183.[李会荣，俞勇，曾胤新，等. 北极太平洋扇区海洋沉积物细菌多样性的系统发育分析[J].微生物学报，2006，46（2）:177-183.]
[11] Liang Xiaobing. The study of biological macromolecules from lake sediments [D]. Guiyang:Institute of Geochemistry，CAS，2002:16-76.[梁小兵. 湖泊沉积物中生物大分子的研究[D].贵阳：中国科学院地球化学研究所，2002：16-76.]
[12] Zhao Xingqing，Yang Liuyan，Chen Can，et al. Study on the microbial diversity in lake sediments by the method of PCR-DGGE [J]. Acta Ecologica Sinica，2006，26（11）: 2 610-2 616.[赵兴青，杨柳燕，陈灿，等. PCR-DGGE技术用于湖泊沉积物中微生物群落结构多样性研究[J].生态学报，2006，26（11）: 2 610-2 616.]
[13] Xu Yao，Li Renhui，Wang Guoxiang，et al. Molecular detection of Microcystis （Cyanobacteria） from sediments by DNA amplification [J]. Journal of Lake Science，2007，19（4）:492-496.[徐瑶，李仁辉，王国祥，等.湖泊底泥中微囊藻DNA的分子检测[J].湖泊科学，2007，19（4）: 492-496.]
[14] Liang Xiaobing，Wan Guojiang，Huang Ronggui. Expectation and application of PCR-RFLP in environmental geochemistry [J]. Geology-geochemistry，2001，29（4）:94-98.[梁小兵，万国江，黄荣贵.PCR-RFLP技术在环境地球化学研究中的应用及展望[J].地质地球化学，2001，29（4）:94-98.]
[15] Liang Xiaobing，Wan Guojiang，Huang Ronggui，et al. Variation of protein and RNA in the sediments of Lake Aha [J]. Geochimica，2001，30（4）:342-346.[梁小兵，万国江，黄荣贵，等.蛋白质和核糖核酸在阿哈湖沉积物中的变化特征[J].地球化学，2001，30（4）:342-346.]
[16] Cai Qiming. The Study on Environment and Ecology of Lake Taihu [M]. Beijing: China Meteorological Press，1998.[蔡启铭主编.太湖环境生态研究[M].北京:气象出版社，1998.]
[17] Rin ta-K anto J M，Ouellette A J A，Twiss M R，et al. Quantification of Toxic Microcystis spp. during the 2003 and 2004 Blooms in Western Lake Erie using quantitative real-time PCR [J]. Environmental Science and Technology，2005，39: 4 198-4 205.
[18] Miller D N，Bryant J E，Madsen E L，et al. Evaluation and optimization of DNA extraction and purification procedures for soil and sediment samples [J]. Applied and Environmental Microbiology，1999，65（11）:4 715-4 724.
[19] Neilan B A，Jacobs D，DelDot T，et al. rRNA sequences and evolutionary relationships among toxic and nontoxic cyanobacteria of the genus Microcystis [J]. International Journal of Systeatic Bacteriology，1997，47（3）:693-697.
[20] Wei Zhongqing，Liu Chongqiang，Liang Xiaobing，et al. Degradation of organic matter in the sediments of Hongfeng Reservoir [J]. Chinese Science Bulletin，2005，50（20）: 2 377-2 380.[魏中青，刘丛强，梁小兵，等.贵州红枫湖沉积物中有机质的降解与微生物作用[J]. 科学通报，2005，50（14）:1 486-1 489.]
[21] Cullen D W，Hirseh P R. Simple and rapid method for direct extraction of microbial and from soil for PCR [J]. Soil Biol and Biochemistry，1998，30: 983-993.
[22] Krsek M，Wellington E M H. Comparison of different methods for the isolation and purification of total community DNA from soil [J]. Journal of Microbiological Methods，1999，39:1-16.
[23] Juniper S K，Cambon M A，Lesongeur F，et al. Extraction and purification of DNA from organic rich subsurface sediments [J]. Marine Geology，2001，174: 241-247.
[24] Guo N，Xie P. Development of tolerance against toxic Microcysits aeruginosa in three cladocerans and the ecological implications [J]. Environmental Pollution，2006，143（3）:513-518.
[25] William J D'Andrea，Melissa Lage，Jennifer B H Martiny，et al. Alkenone producers inferred from well-preserved 18S rDNA in Greenland lake sediments [J]. Journal of Geophysical Research，2006，111:1-8.
[26] Coolen M J L，Muyzer G，et al. Combined DNA and lipid analyses of sediments reveal changes in Holocene haptophyte and diatom populations in an Antarctic lake [J]. Earth and Planet Science Letters，2004，223:225-239.
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