食品与发酵工业 >

2024 , Vol. 50 >Issue 12: 118 - 126

DOI: https://doi.org/10.13995/j.cnki.11-1802/ts.038832

研究报告

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

  • 叶光斌 ,
  • 幸洪静 ,
  • 杨志阳 ,
  • 宗绪岩
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  • 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)
第一作者：博士,副教授(通信作者,E-mail:guangbin1980@126.com)

收稿日期: 2024-02-04

  修回日期: 2024-02-24

  网络出版日期: 2024-07-11

基金资助

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)

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

  • YE Guangbin ,
  • XING Hongjing ,
  • YANG Zhiyang ,
  • ZONG Xuyan
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  • 1(College of Bioengineering, Sichuan University of Science & Engineering, Yibin 644000, China)
    2(Key Laboratory of Brewing Biotechnology and Application of Sichuan Province, Sichuan University of Science & Engineering, Yibin 644000, China)

Received date: 2024-02-04

  Revised date: 2024-02-24

  Online published: 2024-07-11

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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5.46×104 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101~106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101~103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102~104 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。

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

本文引用格式

叶光斌 , 幸洪静 , 杨志阳 , 宗绪岩 . 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[J]. 食品与发酵工业, 2024 , 50(12) : 118 -126 . DOI: 10.13995/j.cnki.11-1802/ts.038832

Abstract

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 × 104 ng/g, but the DNA purity was low.The qPCR quantitative results of the SDS-based extraction method were 101-6 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 101-3 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 102-4 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 the OTUs in the bacterial 16S rDNA amplicon library had differences in abundance, but most of them could be detected in different DNA extraction methods.However, among the fungal ITS amplicon libraries, most OTUs showed a similar trend between different DNA extraction methods, indicating that the DNA extraction method had a significant effect on the extraction of some fungal microbial genomes, and the washing-pretreatment was beneficial to the extraction of fungal genomic DNA.Compared with the four DNA extraction methods, the washing-pretreatment plus SDS-based extraction method was the most suitable for the quantitative and community structure analysis of Daqu microorganisms.

Key words： DNA extraction method; Daqu; amplicon library; microbial community; qPCR; Illumina Miseq sequencing

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