DNA提取方法对苎麻脱胶菌群PCR-DGGE结果偏移的影响  

On the Influence of DNA Extraction from Ramie Retting Microbial Community for Bias of PCR-DGGE Result

作　　者：黄鹏[1] 陈洪高[1] 饶维桥[1] 曾庆福[1] 

机构地区：[1]武汉纺织大学纺织印染清洁生产教育部工程中心,武汉430071

出　　处：《基因组学与应用生物学》2010年第6期1184-1191,共8页Genomics and Applied Biology

基　　金："十一五"国家科技支撑计划项目(2006BAC02A00);湖北省教育厅重点项目(D20081703)共同资助

摘　　要：为筛选和优化出较适宜的苎麻脱胶菌群DNA提取方法,本文分别以来自苎麻沤麻环境的6种纯培养菌等丰度混合物和苎麻自然沤麻菌群为材料,研究"溶菌酶-SDS"法、"超声波-溶菌酶-SDS"法、"蛋白酶K-SDS"法以及"冻融-蛋白酶K-SDS"法4种DNA提取方法对菌群16Sr DNA基因PCR-DGGE偏移结果的影响。结果表明,4种方法均能从2类材料中提取出了超过1600ng/μL的DNA,不同方法之间DNA产率略有差异,经过超声波处理或反复冻融处理的DNA有明显的降解,但4种方法提供的DNA模板均扩增出了450bp的16Sr DNA基因片段。4种DNA提取方法对DGGE结果有明显影响,且只有"冻融-蛋白酶K-SDS"法检测到了纯培养菌混合物中的全部6种细菌,4种方法获得的自然沤麻菌群的DGGE指纹图谱也明显不同,最多产生17条带("冻融-蛋白酶K-SDS"法),最少只有9条带("溶菌酶-SDS"法),增加超声波或冻融等物理处理可以使部分弱带变强。因此,组合应用物理、生物和化学等细胞裂解方法可以提供更有代表性的DNA,可减少PCR-DGGE结果的偏移。In order to screen and optimize better DNA extraction methods for ramie retting community, in this paper, we collected a composite bacterial group mixed by six bacterial species and a natural microbial group from ramie retting environment to investigate the possible bias of the PCR-DGGE fingerprint in 16S rDNA by 4 DNA extraction methods including ＂lysozyme/SDS＂, ＂ultrasound/lysozyme/SDS＂, ＂proteinase K/SDS＂ and ＂freezing-thawing/proteinase K/SDS＂, respectively. The results showed that the concentrations of extracted DNA by four methods from two materials were all more than 1 600 ng/μL with a little discrepancy for DNA yield among the four methods. However, DNA tails were observed obviously when ultrasonic or repeated freezing-thawing were used in DNA extracting process, and the expected fragments about 450 bp were obtained from DNA models extracted by four methods from the two materials. The results also displayed that there was significant effect on DGGE by the four DNA extraction methods, but only one composite bacterial group DGGE fingerprints generated from genomic DNA model provided by ＂freezing-thawing/proteinase K/SDS＂ method showed 6 bands expected, and the DGGE fingerprints of natural ramie retting microbial group acquired by four methods were also substaintially different, such as, 17 bands was the most by ＂freezing-thawing/proteinase K/SDS＂ method, while the least one was 9 bands generated by ＂lysozyme/SDS＂ method. Moreover, some weak bands could become legible by increasing the treatment of ultrasonic or repeated freezing-thawing. In conclusion, the bias of DGGE fingerprint could be avoided in great extent through combining and using cell disruption method such as physical, biological and chemical.

关 键 词：苎麻脱胶菌群 DNA提取方法 DGGE指纹 偏移 

分 类 号：S563.1[农业科学—作物学] Q943.2[生物学—植物学]