用于分析土壤微生物结构变化规律的变性梯度凝胶电泳条件研究
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摘要
研究确定土壤微生物基因组DNA提取方法、PCR扩增条件、DGGE电泳条件,为进一步研究分析土壤中微生物结构变化规律提供理论依据。土壤微生物基因组DNA提取采用直接法和间接法进行比较; PCR扩增条件调整扩增体系、DGGE电泳条件调整变性剂范围,并对其结果进行比较分析。通过对DGGE电泳相关条件的研究,结果显示,土壤中粗基因组DNA采用直接法提取,然后进行纯化; PCR扩增体系中加入BSA,DGGE电泳系统组成中变性剂浓度范围为35%～55%。确定了土壤微生物基因组DNA提取方法、PCR扩增条件、DGGE电泳条件,为后续的相关研究提供理论依据。
Determination of soil microbial genome DNA extraction methods,PCR amplification conditions,DGGE( denaturing gradient gel electrophoresis) conditions were studied in order to provide reliable theoretical basis for further study on the analysis on the variation pattern of microbial community structure in soil. Soil microbial genomic DNA extraction were compared using direct method and indirect method; PCR amplification condition adjustment amplification system,DGGE conditions to adjust the denaturant range,and the results were compared with analyses. The results showed that by studying the whole conditions associated with DGGE,it was concluded that the crude genomic DNA was extracted by direct method and then carried out purification. In PCR amplification system BSA was added,and the concentration of denaturants in the components of DGGE system was at 35%-55%. Therefore,the soil microbial genome DNA extraction method,PCR amplification conditions,DGGE conditions were confirmed and provided real,reliable,accurate theoretical foundation for the follow up studies.
Determination of soil microbial genome DNA extraction methods,PCR amplification conditions,DGGE( denaturing gradient gel electrophoresis) conditions were studied in order to provide reliable theoretical basis for further study on the analysis on the variation pattern of microbial community structure in soil. Soil microbial genomic DNA extraction were compared using direct method and indirect method; PCR amplification condition adjustment amplification system,DGGE conditions to adjust the denaturant range,and the results were compared with analyses. The results showed that by studying the whole conditions associated with DGGE,it was concluded that the crude genomic DNA was extracted by direct method and then carried out purification. In PCR amplification system BSA was added,and the concentration of denaturants in the components of DGGE system was at 35%-55%. Therefore,the soil microbial genome DNA extraction method,PCR amplification conditions,DGGE conditions were confirmed and provided real,reliable,accurate theoretical foundation for the follow up studies.
引文
[1]刘娣,范丙全,龚明波.秸秆还田技术在中国生态农业发展中的作用[J].中国农学通报,2008,24(6):404-407.
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[3]周琳,张晓君,李国勋,等. DGGE/TGGE技术在土壤微生物分子生态学研究中的应用[J].生物技术通报,2006,(5):67-71.
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[6]张瑞福,曹慧,崔中利,土壤微生物总DNA的提取和纯化[J].微生物学报,2003,43(2):276-282.
[7]黄进勇,岳彩鹏,周伟.麦田土壤细菌群落16S r DNA V3片段PCR产物的DGGE分析[J].河南农业大学学报,2007,41(4):396-400.
[8]姚文,朱伟云,韩正康.应用变性梯度凝胶电泳和16S r DNA序列分析对山羊瘤胃细菌多样性的研究[J].中国农业科学,2004,37(9):1374-1378.
[9]沈洪,汪虹,赵永昌,等.采用变性梯度凝胶电泳研究羊肚菌土壤真菌群落结构[J].食用菌通报,2009,16(2):6-12.
[10]蔡燕飞,廖宗文.土壤微生物生态学研究方法进展[J].土壤与环境,2002,11(2):167-171.
[11] Amannri,Ludwigw,Schleiferk H. Phylogenetic identificationand in situ detection of individualmicrobialcells withoutcultivation[J]. Microbiological Reviews,1995,59(1):143-169.
[12] Merothcb,Walterj,Hertelc,et al. Monitoring the bacterialpopulation dynamics in sourdough fermentation processes by using PCR-denaturing gradient gel electrophoresis[J]. Appl Environ Microbiol,2003,69(1):475-482.
[13] Yeates C,Gillings MR,Davison AD,et al. PcR amplification of crudemicrobial DNA extractedfrom soil[J]. Leters in Applied Microbi-ology,1997,25:303-307.
[14]邢德峰,任南琪,宋业颖,等. DG. DGGE分析产氢发酵系统微生物群落动态及其种群多样性[J].生态学报,2005,(7):1818-1823.
[15] Ginige MP,Hugenhotz P,Daims H,et al. Use of stable-isotope probing,full-cycle rRNA analysis,and fluorescence in situ hybridization microautoradiography to study a Methanol fed denitrifying microbial community[J]. Appl Environ Microbiol,2004,70(1):588-596.
[2]宫曼丽,任南琪,邢德峰. DGGE/TGGE技术及其在微生物分子生态学中的应用[J].微生物学报,2004,44(6):845-848.
[3]周琳,张晓君,李国勋,等. DGGE/TGGE技术在土壤微生物分子生态学研究中的应用[J].生物技术通报,2006,(5):67-71.
[4] MACURAJ. Trendsanda dvancesinsoil microbiology from 1924to 1974[J]. Geodermll,1974,12:311-329.
[5]杜涛,黄小毛,侯明生,从土壤中提取DNA用于PCR扩增[J].微生物学通报,2003,30(6):1-5.
[6]张瑞福,曹慧,崔中利,土壤微生物总DNA的提取和纯化[J].微生物学报,2003,43(2):276-282.
[7]黄进勇,岳彩鹏,周伟.麦田土壤细菌群落16S r DNA V3片段PCR产物的DGGE分析[J].河南农业大学学报,2007,41(4):396-400.
[8]姚文,朱伟云,韩正康.应用变性梯度凝胶电泳和16S r DNA序列分析对山羊瘤胃细菌多样性的研究[J].中国农业科学,2004,37(9):1374-1378.
[9]沈洪,汪虹,赵永昌,等.采用变性梯度凝胶电泳研究羊肚菌土壤真菌群落结构[J].食用菌通报,2009,16(2):6-12.
[10]蔡燕飞,廖宗文.土壤微生物生态学研究方法进展[J].土壤与环境,2002,11(2):167-171.
[11] Amannri,Ludwigw,Schleiferk H. Phylogenetic identificationand in situ detection of individualmicrobialcells withoutcultivation[J]. Microbiological Reviews,1995,59(1):143-169.
[12] Merothcb,Walterj,Hertelc,et al. Monitoring the bacterialpopulation dynamics in sourdough fermentation processes by using PCR-denaturing gradient gel electrophoresis[J]. Appl Environ Microbiol,2003,69(1):475-482.
[13] Yeates C,Gillings MR,Davison AD,et al. PcR amplification of crudemicrobial DNA extractedfrom soil[J]. Leters in Applied Microbi-ology,1997,25:303-307.
[14]邢德峰,任南琪,宋业颖,等. DG. DGGE分析产氢发酵系统微生物群落动态及其种群多样性[J].生态学报,2005,(7):1818-1823.
[15] Ginige MP,Hugenhotz P,Daims H,et al. Use of stable-isotope probing,full-cycle rRNA analysis,and fluorescence in situ hybridization microautoradiography to study a Methanol fed denitrifying microbial community[J]. Appl Environ Microbiol,2004,70(1):588-596.