不同DNA提取方法对大曲基因组DNA提取效果及扩增子文库的影响  

Effects of different DNA extraction methods on genomic DNA extraction and amplicon libraries of Daqu

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作  者:叶光斌 幸洪静 杨志阳 宗绪岩 YE Guangbin;XING Hongjing;YANG Zhiyang;ZONG Xuyan(College of Bioengineering,Sichuan University of Science&Engineering,Yibin 644000,China;Key Laboratory of Brewing Biotechnology and Application of Sichuan Province,Sichuan University of Science&Engineering,Yibin 644000,China)

机构地区:[1]四川轻化工大学生物工程学院,四川宜宾644000 [2]四川轻化工大学,酿酒生物技术及应用四川省重点实验室,四川宜宾644000

出  处:《食品与发酵工业》2024年第12期118-126,共9页Food and Fermentation Industries

基  金:四川省科技厅重点研发项目(2022YFS0547);酿酒生物技术及应用四川省重点实验室开放基金项目(NJ2014-09)。

摘  要:大曲是中国传统白酒典型的“多酶多菌”的发酵剂。免培养的分子生物学方法已经成为大曲微生物群落研究的常规方法。通过评估不同DNA提取方法对大曲基因组DNA提取效果及扩增子文库的影响,选择适合用于大曲DNA提取的最佳方法。该研究选择4种DNA提取方法分别对2种大曲进行大曲微生物DNA的提取,结合DNA提取质量、qPCR定量结果、细菌16S rRNA基因和真菌ITS基因扩增子文库的Illumina Miseq测序结果,评估大曲基因组DNA不同提取方法的效果。从DNA得率和生物量的综合分析表明,SDS-based提取法的DNA得率高于试剂盒提取法,其中原位SDS-based提取法DNA得率高达5.46×10^(4)ng/g,但DNA纯度较低;SDS-based提取法的qPCR定量结果比试剂盒提取法高10^(1)~10^(6)量级,其中洗脱加SDS-based提取法的微生物生物量最高,比原位SDS-based提取法的生物量高10^(1)~10^(3)量级;试剂盒提取法的微生物生物量最低,细菌和真菌的生物量只有10^(2)~10^(4)copies/g。扩增子文库间Shannon多样性指数的比较结果表明,不同DNA提取方法在真菌ITS扩增子文库间存在显著差异,而在细菌16S rDNA扩增子文库间差异不显著。洗脱处理样本的真菌扩增子文库Shannon指数要高于原位处理的样本。通过对扩增子文库间存在显著性差异操作分类单元(operational taxonomic units,OTU)的比较可以看出,在细菌16S rDNA扩增子文库中,多数OTUs存在丰度差异,但它们大多数能在不同DNA提取方法中检测到;然而在真菌ITS扩增子文库间,少数OTUs在不同DNA提取方法中检测不到,说明DNA提取方法对部分真菌微生物基因组的提取存在显著影响,且洗脱处理有利于真菌基因组DNA的提取。综合对比4种DNA提取方法,洗脱加SDS-based提取法最适合用于大曲微生物的定量和群落结构分析。Daqu is a typical multi-enzyme and multi-microbes fermentation starter of traditional Chinese liquor.The culture-independent molecular method has become a general method for the study of the Daqu microbial community.To determine the most suitable DNA extraction method of Daqu samples,the effects of different DNA extraction methods on Daqu genomic DNA extraction and amplicon libraries were evaluated.In this study,four DNA extraction methods were selected for the extraction of microbial DNA from two kinds of Daqu.Combined with DNA extraction quality,qPCR quantitative results,Illumina Miseq sequencing results of bacterial 16S rRNA gene,and fungal ITS gene amplicon library,the effects of different extraction methods of Daqu genomic DNA were evaluated.The comprehensive analysis of DNA yield and microbial biomass showed that the DNA yield of the SDS-based extraction method was higher than that of the kit extraction method,and the DNA yield of the in-situ SDS-based extraction method was as high as 5.46×10^(4)ng/g,but the DNA purity was low.The qPCR quantitative results of the SDS-based extraction method were 10^(1)-6 orders of magnitude higher than those of the kit extraction method,and the microbial biomass of the washing-pretreatment plus SDS-based extraction method was the highest,which was 10^(1)-3 orders of magnitude higher than that of the in-situ SDS-based extraction method.Whereas the microbial biomass of the DNA extraction kit was the lowest,with only 10^(2)-4 copies/g of bacteria and fungi.The comparison of the Shannon diversity index among amplicon libraries showed that different DNA extraction methods had significant differences among fungal ITS amplicon libraries,but no significant differences among bacterial 16S rDNA amplicon libraries.The Shannon index of the fungal amplicon library of the washing-pretreated samples were higher than that of the in-situ treated samples.By comparing the operational taxonomic units(OTU)with significant differences among the amplicon libraries,it could be seen that most of t

关 键 词:DNA提取方法 大曲 扩增子文库 微生物群落 qPCR Illumina Miseq测序 

分 类 号:TS261.11[轻工技术与工程—发酵工程] Q78[轻工技术与工程—食品科学与工程]

 

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