3种试剂盒对烟丝DNA提取及其微生物群落多样性分析效果的比较

Three Kits on Extracting Cut Tobacco DNA and Their Analysis Effect on Microbial Community Diversity

筛选能高效提取烟丝DNA,并能获取其微生物群落多样性较高的试剂盒。选择3种不同的试剂盒提取烟丝DNA,比较DNA的纯度和浓度,并将提取的DNA进行原核生物16S rRNA基因V3~V4区和真菌ITS2区的PCR扩增及高通量扩增子测序,进而比较烟丝样品微生物群落多样性和结构差异。结果表明:(1)DNeasy■PowerPlant■Pro Kit植物基因组提取试剂盒(P)提取的烟丝DNA浓度为(103.67±1.7)ng/μL,显著高于其他试剂盒(P<0.01),且3种试剂盒提取的DNA纯度均达到合格水平。(2)3种试剂盒提取的DNA样本均能反映样本中原核生物群落多样性与结构,从门分类水平看,试剂盒P检测到11个门类,E.Z.N.A.TM HP Plant DNA Kit植物基因组提取试剂盒(M)检测到8个门类,DNeasy■PowerSoil■Kit土壤基因组提取试剂盒(S)检测到5个门类;从属分类水平看,试剂盒P检测到83个属,试剂盒S检测到71个属,试剂盒M检测到69个属。(3)3种试剂盒均能检测到Unclassified类群、子囊菌门Ascomycota、接合菌门Zygomycota和Others等4个真菌门类,但试剂盒P检测到的类群比例最高;在属分类水平上,试剂盒P检测出30个属,试剂盒S检测出20个属,试剂盒M检测出11个属。DNeasy■PowerPlant■Pro Kit植物基因组提取试剂盒(P)能有效提取烟丝及其所含微生物的总DNA,并能检测出更高的烟丝微生物群落多样性,推荐该试剂盒用于烟丝样品中微生物群落多样性与结构的分析。

The paper aims to select a kit which could efficiently extract cut tobacco DNA and obtain relatively high diversity of the microbial community.The cut tobacco DNA was extracted by using three different kits,and these extracted DNA was used for amplicon high-throughput sequencing of the prokaryotic 16S rRNA gene V3-V4 region and the fungal ITS2 region.We compared the purity and concentration and the microbial community diversity and structure.The results showed that:(1)the concentration of cut tobacco DNA from DNeasy■PowerPlant■Pro Kit(P)was(103.67±1.7)ng/μL,significantly higher than that of other kits(P<0.01),and the DNA purity from three different kits reached the qualified level;(2)all DNA samples extracted by using the three different kits could reflect the prokaryotic community diversity and structure;at the phylum level,the kit P detected 11 phyla,E.Z.N.A.TM HP Plant Kit(M)detected 8 phyla,and DNeasy■PowerSoil■Kit(S)detected 5 phyla;at the genus level,83 genera were detected by kit P,71 genera were detected by kit S,and 69 genera were detected by kit M;(3)the three kits could detect four phyla of fungi such as Unclassified group,Ascomycota,Zygomycota and Others,but kit P detected the highest diversity of microbial community at the phylum level;and at the genus level of fungi,30 genera were detected by kit P,20 genera were detected by kit S,and 11 genera detected by kit M.DNeasy■PowerPlant■Pro Kit(P)can effectively extract the total DNA of in cut tobacco and cut tobacco microbes,and it can detect more microbial community diversity,so it is recommended for the analysis of tobacco microbial community diversity and structure.