不同DNA提取方法对高通量测序检测沉积物微生物多样性的影响  

Study on the Effect of Different DNA Extraction Methods on High-Throughput Sequencing to Detect Microbial Diversity in Sediments

作　　者：王志龙 李春筱 蔡一鸣 夏雪山[1] 刘丽[1] WANG Zhilong;LI Chunxiao;CAI Yiming;XIA Xueshan;LIU Li(Faculty of Life Science and Technology,Kunming University of Science and Technology,Kunming 650500)

机构地区：[1]昆明理工大学生命科学与技术学院,云南昆明650500

出　　处：《昆明理工大学学报（自然科学版）》2021年第5期89-99,共11页Journal of Kunming University of Science and Technology(Natural Science)

基　　金：国家自然科学基金项目(31860028,31360016)。

摘　　要：高通量测序技术是目前沉积物中微生物多样性的主要检测方法,该技术受到不同方法提取基因组DNA质量的影响,然而,不同DNA提取方法应用于高通量测序对沉积物中微生物多样性的比较研究报道较少.通过常用的8种DNA提取方法对滇池沉积物样本进行基因组DNA提取,检测DNA浓度、纯度,选择出提取效果较好的4种方法,进行原核微生物16SrRNA和真核微生物18SrRNA扩增子测序,比较不同DNA提取方法的微生物群落特征.研究结果表明:S-MOBIO试剂盒法(商品化土壤DNA基因组提取试剂盒1,MOBIO DNeasy■PowerSoil■Kit,S-MOBIO)、S-OMEGA试剂盒法(商品化土壤DNA基因组提取试剂盒2,OMEGAE.Z.N.A■SoilDNAKit,S-OMEGA)、蛋白酶K-SDSPEG二次沉淀法(蛋白酶K-十二烷基硫酸钠-聚乙二醇二次沉淀法,S-SDS-PEG)、去腐剂-CTAB-蛋白酶K-试剂盒法(去腐剂-十六烷基三甲基溴化铵-蛋白酶K结合试剂盒法,S-CTAB)四种方法所提取的DNA质量及得率较高;16S/18SrRNA扩增子测序分析表明S-MOBIO试剂盒法(S-MOBIO)和蛋白酶K-SDS-PEG二次沉淀法(S-SDS-PEG)获得的OTU数量多,Alpha多样性指数高;Bate多样性分析中S-MOBIO试剂盒法(S-MOBIO)和蛋白酶K-SDS-PEG二次沉淀法(SSDS-PEG)得到的物种组成结构相似度高;微生物群落特征分析在原核微生物的门水平和属水平上差异不大,S-MOBIO试剂盒法(S-MOBIO)和蛋白酶K-SDS-PEG二次沉淀法(S-SDS-PEG)获得的物种较多,物种组成相似度更高,在真核微生物门水平和属水平上,四种方法所得到的优势种群差异明显;综合比较,确定S-MOBIO试剂盒法(S-MOBIO)和蛋白酶K-SDS-PEG二次沉淀法(S-SDS-PEG)提取DNA是基于高通量测序探究云南滇池沉积物微生物多样性的较好方法.本研究可为湖泊沉积物微生物分子生态学研究奠定基础.High-throughput sequencing technology is the main method for detecting microbial diversity in sediments at present, which is affected by the quality of genomic DNA extracted by different methods. However, there are few comparative research reports on the microbial diversity in sediments by applying different DNA extraction methods to high-throughput sequencing. In this study, 8 different DNA extraction methods were used to extract genomic DNA from the sediment of Dianchi Lake, detecting DNA concentration and purity, selecting four methods in better extraction effect, sequencing amplicon of the 16 S rRNA of prokaryotic micro organism and 18 S rRNA and comparing microbial community characteristics of different DNA extraction methods.The study shows that the quality and yield of the extracted DNA in four methods, which are MOBIO kit method(S-MOBIO), OMEGA kit method(S-OMEGA), Proteinase K-SDS-PEG secondary precipitation method(S-SDS-PEG) and PVP-CTAB-Protease K-kit method(S-CTAB). The 16 S/18 S rRNA amplicon sequencing results showed that the number of OUT and alpha diversity index obtained by method S-MOBIO and method S-SDS-PEG were higher. Bate diversity indicated that the species composition structure obtained by method S-MOBIO and method S-SDS-PEG had high similarity. Microbial community characteristics were not significantly different at the phylum and genus levels of prokaryotes.Method S-MOBIO and method S-SDS-PEG had more species and higher similarity of species composition. At the phylum and genus level of eukaryotes, the dominant populations obtained by the four methods were significantly different. By comprehensive comparison, DNA extraction by method S-MOBIO and method S-SDS-PEG were good methods to explore microbial diversity of lake sediments in Yunnan Dianchi based on high-throughput sequencing. The study will lay a foundation for ecology of microbial molecules in lake sediments.

关 键 词：DNA提取 高通量测序 扩增子测序 微生物群落 滇池沉积物 物种组成 

分 类 号：X172[环境科学与工程—环境科学] Q523[生物学—生物化学]