海洋沉积物中微生物基因组DNA提取方法的比较研究  

Comparison Study on Extraction Methods of Microbial DNA in Marine Sediments

作　　者：贺惠[1,2,3] 张玉[4,2,3] 甄毓[4,2,3] 米铁柱[4,2,3] 陈阳阳[4,2,3] 于志刚[3,5] 

机构地区：[1]中国海洋大学海洋生命学院,山东青岛266003 [2]中国海洋大学海洋环境与生态教育部重点实验室,山东青岛266100 [3]青岛海洋科学与技术国家实验室海洋生态与环境科学功能实验室,山东青岛266071 [4]中国海洋大学环境科学与工程学院,山东青岛266100 [5]中国海洋大学海洋化学理论与工程技术教育部重点实验室,山东青岛266100

出　　处：《中国海洋大学学报（自然科学版）》2017年第6期17-24,共8页Periodical of Ocean University of China

基　　金：国家自然科学基金项目(41521064;41376093);中国科学院海洋生态与环境科学重点实验室;青岛海洋科学与技术国家实验室海洋生态与环境科学开放课题项目(KLMEES201601)资助~~

摘　　要：沉积物成分复杂,腐殖酸等物质含量较高,这些杂质在DNA提取过程中不易除去,可能会对后续的PCR扩增等产生抑制作用。因此,高质量DNA的获得是海洋微生物分子生态学研究的基础和前提。本研究采用6种不同的方法,分别提取同一来源海洋沉积物样品中的微生物基因组DNA。对DNA纯度、得率、16SrRNA基因拷贝数和微生物群落特征等多方面进行比较,结果表明:方法1(QIAamp DNA Stool Mini Kit)、方法2(PowerSoil~DNA Isolation Kit)和方法3(RNA PowerSoil~DNA Elution Accessory Kit)得到的DNA产量较低,纯度却较高,可以直接用于后续分子生物学分析;与方法2相比,方法3得到的细菌16SrRNA基因拷贝数和单位质量DNA中可扩增细菌16SrRNA基因模板量较低;方法4(SDS高盐法)得到的DNA虽然产量高,但杂质含量也高,抑制了后续PCR反应的进行;方法5(方法4得到的DNA经QIAamp DNA Stool Mini Kit纯化)和方法6(方法4得到的DNA经PowerSoil~DNA Isolation Kit纯化),虽然纯度有所提高,但DNA大量损失,且耗时长,花费高。另外,方法2在提取过程中可以最大程度裂解菌体细胞壁,能更好反映微生物群落特征。综合以上结果,方法2(PowerSoil~DNA Isolation Kit)提取海洋沉积物微生物基因组DNA效果最佳。本研究可为海洋微生物分子生态学研究提供一种有效的技术手段。The composition of sediments is complex, and the impurities, such as humic acid, are difficult to remove when DNA extraction. Because of the inhibition on PCR amplification, high quality DNA is the basis and prerequisite for the molecular ecology of marine microorganisms. In this study, six different methods were used to extract microbial DNA from marine sediments. Comparisons of the purity and yield of microbial DNA, 16S rRNA gene copy numbers and the characteristics of microbial community were used to analyze the quality and quantity of the genomic DNA. The results showed that DNA purity obtained by method 1, method 2 and method 3 were high despite low in yield, which could be directly used in the following microbial ecology analysis. Compared to method 2, the bacterial 16S rRNA gene copy numbers and template amount for bacterial 16S rRNA gene in per nanogramme DNA obtained by method 3 was low. DNA obtained by method 4 was high in yield but low in purity. After purification （method 5 and method 6）, DNA samples were high in purity hut there were massive losses. Moreover, method 2 could fully break up cells, reflecting the characteristics of microbial community better. Synthesizing the above results, the method 2 （PowerSoil DNA Isolation Kit） was the best one to marine sediment microbial DNA extraction. This study provides an effective technique for the molecular ecology of marine microorganisms.

关 键 词：沉积物 微生物 DNA提取 

分 类 号：Q93-3[生物学—微生物学]