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1 微生物学通报 Microbiology China Feb. 20, 2017, 44(2): DOI: /j.microbiol.china 研究报告基于 PCR-DGGE 和高通量测序分析白云边酒窖泥细菌群落结构与多样性黄莹娜 1 熊小毛 2 胡远亮 3 梁运祥 1 赵述淼 ( ) ( ) ( ) 1* 摘要 : 目的探索白云边酒不同窖龄窖泥细菌群落结构及其多样性, 分析窖泥细菌群落特征对兼香型白酒风格形成的影响方法分别提取 2 年和 23 年窖泥样品总 DNA, 采用 PCR-DGGE 基因克隆以及高通量测序技术, 研究窖泥细菌的分布情况结果白云边窖泥细菌归属于变形菌门 (Proteobacteria) 拟杆菌门(Bacteroidetes) 厚壁菌门(Firmicutes) 放线菌门 (Actinobacteria) 4 个菌门 2 23 年窖泥共同的优势菌属 ( 1.0%) 包括棒状杆菌属 (Corynebacterium) 香味菌属(Myroides) 鞘氨醇杆菌属(Sphingobacterium) 乳杆菌属 (Lactobacillus) 梭菌属(Clostridium) 醋杆菌属(Acetobacter) 产碱杆菌属(Alcaligenes) 肠杆菌属 (Enterobacter) 和不动杆菌属 (Acinetobacter) 2 年窖泥特有优势菌属为 Dysgonomonas Fluviicola 变形杆菌属(Proteus) 和 Wohlfahrtiimonas, 而 23 年窖泥特有优势菌属为葡萄球菌属 (Staphylococcus) Hazenella 魏斯氏菌属(Weissella) 葡糖醋杆菌属(Gluconacetobacter) 和摩根氏菌属 (Morganella) 结论 2 年窖泥细菌群落多样性高于 23 年窖泥比较了白云边两种窖龄窖泥主要细菌组成情况, 为研究微生物对白云边酒浓酱兼香型独特风味形成的影响提供依据关键词 :PCR-DGGE, 高通量测序, 窖泥,16S rrna 基因, 细菌群落 Foundation item: The Fundamental Research Funds for the Central Universities (No. 2014JC004); Natural Science Foundation of Hubei Province (No. 2015CFC797) *Corresponding author: Tel: ; shumiaozhao@mail.hzau.edu.cn Received: January 21, 2016; Accepted: May 30, 2016; Published online ( October 09, 2016 基金项目 : (No. 2014JC004) (No. 2015CFC797) * 通讯作者 :Tel shumiaozhao@mail.hzau.edu.cn 收稿日期 : 接受日期 : 优先数字出版日期 (

2 376 微生物学通报 Microbiol. China 2017, Vol.44, No.2 Bacterial community and diversity in pit mud of Baiyunbian liquor analyzed by PCR-DGGE and high-throughput sequencing HUANG Ying-Na 1 XIONG Xiao-Mao 2 HU Yuan-Liang 3 LIANG Yun-Xiang 1 ZHAO Shu-Miao 1* (1. College of Life Science and Technology, Huazhong Agricultural University, Wuhan, Hubei , China) (2. Baiyunbian Company Limited of Hubei Province, Songzi, Hubei , China) (3. College of Life Science Hubei Normal University, Huangshi, Hubei , China) Abstract: [Objective] To study bacterial community and diversity in pit mud with different-aged cellars used to produce Baiyunbian liquor. [Methods] Total DNA of microbes in pit mud (2 and 23 year) were extracted, then PCR-DGGE and high-throughput sequencing technology were used to analyze bacterial communities. [Results] Bacteria in the pit mud fell into 4 phyla: Proteobacteria, Bacteroidetes, Firmicutes and Actinobacteria, with dominant genera ( 1.0%) including Corynebacterium, Myroides, Sphingobacterium, Lactobacillus, Clostridium, Acetobacter, Alcaligenes, Enterobacter, and Acinetobacter. Furthermore, Dysgonomonas, Fluviicola, Proteus and Wohlfahrtiimonas were present in 2-year uniquely, whereas Staphylococcus, Hazenella, Weissella, Gluconacetobacter and Morganella were especially predominant in 23-year pit mud. [Conclusion] Bacterial diversity in pit mud of 2-year was higher than in that of 23-year. Keywords: PCR-DGGE, High-throughput sequencing, Pit mud, 16S rrna gene, Bacterial community [1] PCR-DGGE [2-5] [6] DGGE [7] [8-9] [10-11] [12] [13] [14-15] PCR-DGGE 材料与方法 1.1 材料窖泥 : 2 ( ) 23 ( ) g 20 C 主要试剂和仪器 :Taq DNA TEMED APPLICHEM MagExtractor (PCR & Gel Clean Up) ( ) Gel Extraction Kit OMEGA pmd18-t TaKaRa 40% Mycycle PCR DCode DGGE Bio-Rad

3 : PCR-DGGE 377 ST 16R ND-2000 Thermo 引物 :16S rrna V3 P2 (5 -AT TACCGCGGCTGCTGG-3 ) P3 (5 -CCTACGGGA GGCAGCAG-3 ) GC-P3 (GC 5 -CGCCCG CCGCGCGCGGCGGGCGGGGCGGGGGCACGGG GGG-3 ) 1.2 方法窖泥总 DNA 提取及纯化 : CTAB 2 23 DNA 20 C S rrna 基因 V3 区 PCR 扩增 :PCR (50.0 μl) 5 U/μL Taq 0.5 μl 10 Buffer 5.0 μl 2.5 mmol/l dntps 4.0 μl 10 μmol/l P2/GC-P3 1.0 μl 100 ng/μl DNA 1.0 μl 37.5 μl PCR 94 C 4 min 94 C 1 min 50 C 1 min 72 C 1 min C 10 min PCR 1% MagExtractor (PCR & Gel Clean Up) DNA PCR 产物 DGGE 检测 : 8% 46% 54% (100% 7 mol/l 40% ) PCR 400 ng 60 C 0.5 TAE 20 V 10 min 80 V 828 min Quantity One Match Trace Int Bio-Dap DGGE 主条带基因克隆测序 : 25 μl 4 C 16 h 3 μl P2/P3 PCR PCR 1% Gel Extraction Kit 4.5 μl pmd18-t 100 μl DH5α 900 μl LB 37 C 1 h r/min 1 min 900 μl 100 mg/l LB 37 C 12 h P2/P3 PCR ( ) NCBI GenBank 窖泥总 DNA 高通量测序 : DNA MagExtractor (PCR & Gel Clean Up) Illumina (MiSeq) OTU 聚类分析 : QIIME Usearch 0.97 OTU (Operational taxonomic units) [16] 物种注释和丰度分析 : QIIME OTU Tag RDP 16S (GreenGenes OTU OTU OTU [17] Alpha 多样性分析 : QIIME Alpha [18] 2 结果与分析 2.1 细菌 DGGE 图谱分析及主条带基因克隆测序 CTAB DNA 23 kb P2/GC-P DNA 16S rrna V3 PCR 250 bp DGGE 2 23 ( 1) Quantity One Bio-Dap Shannon s H H (3.29) 2 Simpson s

378 微生物学通报 Microbiol. China 2017, Vol.44, No.2 图 1 窖泥细菌 16S rrna 基因 V3 区 DGGE 图谱 Figure 1 DGGE of V3 regions of the bacterial 16S rrna genes from pit mud samples A 2 B 23.

055 2 23 ( 1) DGGE 20 2 表 1 窖泥细菌 16S rrna 基因 V3 区 DGGE 图谱多样性指数 Table 1 Diversity index in DGGE profiles of V3 regions of the bacterial 16S rrna genes Index 2-year 23-year Species richness (S) 32 28

4 378 微生物学通报 Microbiol. China 2017, Vol.44, No.2 图 1 窖泥细菌 16S rrna 基因 V3 区 DGGE 图谱 Figure 1 DGGE of V3 regions of the bacterial 16S rrna genes from pit mud samples A 2 B 23. Note: A: Pit mud at 2 years; B: Pit mud at 23 years. (1/D) H Berger-Parker index (d) ( 1) DGGE 20 2 表 1 窖泥细菌 16S rrna 基因 V3 区 DGGE 图谱多样性指数 Table 1 Diversity index in DGGE profiles of V3 regions of the bacterial 16S rrna genes Index 2-year 23-year Species richness (S) Shannon s index (H ) Simpson s index (1/D) Berger-Parker index (d) (Myroides sp.) (Flavobacterium sp.) (Lactobacillus sp.) (Acetobacter sp.) (Proteus hauseri) (Sphingobacterrium sp.) (Clostridium sp.) (Acinetobacter sp.) 23 (Pseudoalteromonas sp.) (Clostridium tyrobutyricum) (Proteus hauseri) (Sphingobacterrium sp.) (Bacillus sp.) (Morganella sp.) (Proteus vulgaris) (Proteus hauseri) (Sphingobacterrium sp.) (Myroides sp.) (Flavobacterium sp.) (Lactobacillus sp.) (Bacillus sp.) (Proteus vulgaris) (Acinetobacter sp.) (Acetobacter sp.) (Pseudoalteromonas sp.) (Clostridium tyrobutyricum) (Morganella sp.) 23 ( 2) 2.2 高通量测序分析窖泥细菌多样性 bp OTU Chao Coverage 95.6% 96.4% Chao 1 Coverage Shannon ( ) PD whole tree ( ) DGGE

5 : PCR-DGGE 379 表 2 DGGE 主条带 NCBI BLAST 比对结果 Table 2 NCBI BLAST of the main bands from DGGE Band No, Closest genera Chinese name Similarity (%) Accession No. 1 Myroides sp. 100 KP Flavobacterium sp. 100 HQ Acinetobacter sp. 100 AB Myroides sp. 100 KF Pseudoalteromonas sp. 94 JX Nitrospina sp. 90 JX Clostridium tyrobutyricum 100 KF Lactobacillus acetotolerans 99 NR_ Lactobacillus sp. 100 KP Corynebacterium sp. 99 KF Acetobacter sp. 100 KM Proteus hauseri 100 KM Sphingobacterrium sp. 100 HQ Bacillus sp. 100 HM Morganella sp. 100 KJ Myroides sp. 99 KC Proteus vulgaris 100 KT Clostridium sp. 98 DQ Acinetobacter sp. 100 KJ Bacillus sp. 99 KC Sample name Amplicon type Table 3 Clean read 表 3 窖泥样品扩增子高通量测序数据统计 Data statistics of amplicon sequencing of pit mud samples Average length Mapped reads OTUs Chao 1 Shannon PD whole tree Coverage (%) 2-year 16S year 16S Clean read Tag Average length Tag Mapped reads OTUs OTU. Note: Clean read: The number of reads after quality control; Average length: The average length of clean reads; Mapped reads: The number of reads be used to clustering analysis; OTUs: The number of OTU in sample. 2.3 高通量测序分析窖泥细菌群落结构组成 (Proteobacteria) (Bacteroidetes) (Firmicutes) (Actinobacteria) (Proteobacteria) (Firmicutes) % 41.1% 26.8% 26.7% (Bacteroidetes 32.2% 20.5%) 2 (Actinobacteria 1.8% 11.8%) 23 ( 2A) 2B % 81.8%

6 380 微生物学通报 Microbiol. China 2017, Vol.44, No.2 Figure 2 图 2 不同窖泥样品门 (A) 科(B) 水平细菌丰度比例 Relative abundance of bacterial 16S rrna genes from pit mud samples at the phylum level (A) and family level (B) identified with a modified 16S rrna gene database (Flavobacteriaceae 21.3% 3.0%) (Enterobacteriaceae 12.1% 14.7%) (Sphingobacteriaceae 6.9% 16.9%) (Clostridiaceae 4.2% 2.4%) (Alcaligenaceae 3.4% 4.4%) (Brucellaceae 2.8% 4.7%) 2 23 (Moraxellaceae) (Lachnospiraceae) (Lactobacillaceae) (Xanthomonadaceae) 23 2 (Corynebacteriaceae) (Bacillaceae) (Staphylococcaceae) ( 4) 5 2 ( Myroides) (Acinetobacter) (Clostridium) (Lactobacillus) (Sphingobacterium) (Enterobacter) Wohlfahrtiimonas (Proteus) 1% 74.1% 23 (Sphingobacterium) (Acetobacter) ( Corynebacterium) (Morganella) (Bacillus) (Ochrobactrum) (Clostridium) (Staphylococcus) 1% 87.0% 2 Dysgonomonas Fluviicola (Proteus) Wohlfahrtiimonas 23 (Staphylococcus) Hazenella 表 4 窖泥中主要细菌菌科的相对丰度 ( 2.5%) Table 4 Relative abundance (%) of 14 major families ( 2.5%) from each pit mud sample Taxon name 2-year (%) 23-year (%) Flavobacteriaceae Enterobacteriaceae Moraxellaceae Lachnospiraceae Lactobacillaceae Sphingobacteriaceae Clostridiaceae Xanthomonadaceae Alcaligenaceae Brucellaceae Acetobacteraceae Corynebacteriaceae Bacillaceae Staphylococcaceae

: PCR-DGGE 381 表 5 Table 5 窖泥中主要细菌菌属的相对丰度 ( 1%) Relative abundance (%) of 25 major genera ( 1%) from each pit mud sample Taxon name 2-year (%) 23-year (%) Corynebacterium 1.4 9.6 Dysgonomonas 2.1 0.

7 : PCR-DGGE 381 表 5 Table 5 窖泥中主要细菌菌属的相对丰度 ( 1%) Relative abundance (%) of 25 major genera ( 1%) from each pit mud sample Taxon name 2-year (%) 23-year (%) Corynebacterium Dysgonomonas Fluviicola Myroides Sphingobacterium Bacillus Oceanobacillus Staphylococcus Hazenella Lactobacillus Weissella Clostridium Ochrobactrum Acetobacter Gluconacetobacter Advenella Alcaligenes Comamonas Enterobacter Morganella Proteus Providencia Serratia Acinetobacter Wohlfahrtiimonas (Weissella) (Gluconacetobacter) (Morganella) ( 1.0%) (Corynebacterium) (Myroides) (Sphingobacterium) (Lactobacillus) (Clostridium) (Acetobacter) (Alcaligenes) (Enterobacter) (Acinetobacter) ( 3) DGGE 2.4 高通量测序分析窖泥细菌群落波动情况 2 23 (Myroides 19.8% 2.0%) 图 3 不同窖泥样品细菌属水平 Venn 图 Figure 3 Venn diagram of bacteria from different pit mud samples (Sphingobacterium 6.4% 16.7%) (Acetobacter 2.5% 13.0%) (Acinetobacter 9.7% 1.0%) (Corynebacterium 1.4% 9.6%) (Clostridium 9.2% 3.4%) (Morganella 0.1% 8.9%) (Bacillus 0.6% 7.7%) (Lactobacillus 7.1% 1.6%) (Ochrobactrum 0.9% 4.2%) ( 4) DGGE 3 结论与讨论 DGGE ( Corynebacterium) (Myroides) (Sphingobacterium) (Lactobacillus) (Clostridium) 图 4 窖泥样品间丰度波动前 10 的细菌菌属 Figure 4 Top 10 fluctuations in the composition of bacteria between the pit mud samples

8 382 微生物学通报 Microbiol. China 2017, Vol.44, No.2 (Acetobacter) (Alcaligenes) (Enterobacter) (Acinetobacter) 2 23 [7] (Sphingobacterium) (Acetobacter) (Corynebacterium) (Morganella) (Bacillus) (Ochrobactrum) (Staphylococcus) Hazenella (Weissella) (Gluconacetobacter) 23 2 (Acetobacter) (Weissella) (Gluconacetobacter) [6] [19] Guo [20] (Bacteroidetes) (Firmicutes) [7,21] (Lactobacillus) (Bacillus) (Clostridium) (Petrimonas) [2] Ding [22] DGGE (Clostridium) Liang [23] (Firmicutes) (Actinobacteria) (Proteobacteria) (Synergistetes) (Clostridium) (Lactobacillus) (Bacillus) [24] (Clostridiaceae) (Ruminococcaceae) (Thermoanaerobacteriaceae) [25] [26] (Bacillus) (Proteobacteria) (Bacteroidetes) (Firmicutes) (Actinobacteria) (Lactobacillus) (Bacillus) (Clostridium) 3 3 (Lactobacillus) (Bacillus) (Clostridium) (Corynebacterium) (Myroides) (Sphingobacterium) (Acetobacter) (Alcaligenes) (Enterobacter) (Acinetobacter) DGGE DGGE DGGE DNA 1.5% [12]

9 : PCR-DGGE 383 参考文献 [1] Xiong XM. Summary of the production techniques of Maotai-Luzhou-flavor Baiyunbian liquor[j]. Liquor-Making Science & Technology, 2007(9): (in Chinese). [J]., 2007(9): [2] Shi S, Wang HY, Zhang WX, et al. Analysis of microbial communities characteristics in different pit mud of Luzhou-flavor liquor[j]. Liquor-Making Science & Technology, 2011(5): (in Chinese),,,. [J]., 2011(5): [3] Deng B, Shen CH, Shan XH, et al. PCR-DGGE analysis on microbial communities in pit mud of cellars used for different periods of time[j]. Journal of the Institute of Brewing, 2012, 118(1): [4] Tang YR, Zhong FD, Zhang WX. Analysis of the diversity and system development of bacterial flora in Luzhou-flavor Xijiu pit mud[j]. Liquor-Making Science & Technology, 2011(12): (in Chinese),,. [J]., 2011(12): [5] Hu XL, Wang HY, Wu Q, et al. Development, validation and application of specific primers for analyzing the Clostridial diversity in dark fermentation pit mud by PCR-DGGE[J]. Bioresource Technology, 2014, 163(7): [6] Ye GB, Luo HB, Yang XD, et al. Community structure of prokaryotes in pit mud of Lu-flavor liquor from Luzhou prefecture based on culture-independent approach[j]. Food Science, 2013, 34(17): (in Chinese),,,. [J]., 2013, 34(17): [7] Tao Y, Rui JP, Li JB, et al. Microbial community compositions and diversity in pit mud of Chinese Luzhou-flavor liquor[j]. CIESC Journal, 2014, 65(5): (in Chinese),,,. [J]., 2014, 65(5): [8] Nam YD, Lee SY, Lim SI. Microbial community analysis of Korean soybean pastes by next-generation sequencing[j]. International Journal of Food Microbiology, 2012, 115(1/2): [9] Zhao M, Zhang DL, Su XQ, et al. An integrated Metagenomics/Metaproteomics investigation of the microbial communities and enzymes in solid-state fermentation of Pu-erh tea[j]. Scientific Reports, 2015, 5: [10] Lauber CL, Hamady M, Knight R, et al. Pyrosequencing-based assessment of soil ph as a predictor of soil bacterial community structure at the continental scale[j]. Applied and Environmental Microbiology, 2009, 75(15): [11] Ferrenberg S, O Neill SP, Knelman JE, et al. Changes in assembly processes in soil bacterial communities following a wildfire disturbance[j]. The ISME Journal, 2013, 7(6): [12] Serkebaeva YM, Kim Y, Liesack W, et al. Pyrosequencing-based assessment of the bacteria diversity in surface and subsurface peat layers of a northern wetland, with focus on poorly studied phyla and candidate divisions[j]. PLoS One, 2013, 8(5): e63994 [13] Huber JA, Mark Welch DB, Morrison HG, et al. Microbial population structures in the deep marine biosphere[j]. Science, 2007, 318(5847): [14] Rui JP, Li JJ, Li JB, et al. Prokaryotic community structures in biogas plants with swine manure[j]. CIESC Journal, 2014, 65(5): (in Chinese),,,. [J]., 2014, 65(5): [15] Stolze Y, Zakrzewski M, Maus I, et al. Comparative metagenomics of biogas-producing microbial communities from production-scale biogas plants operating under wet or dry fermentation conditions[j]. Biotechnology for Biofuels, 2015, 8: 14 [16] Edgar RC. UPARSE: Highly accurate OTU sequences from microbial amplicon reads[j]. Nature Methods, 2013, 10(10): [17] McDonald D, Price MN, Goodrich J, et al. An improved Greengenes taxonomy with explicit ranks for ecological and evolutionary analyses of bacteria and archaea[j]. The ISME Journal, 2012, 6(3): [18] Kemp PF, Aller JY. Bacterial diversity in aquatic and other environments: what 16S rdna libraries can tell us[j]. FEMS Microbiology Ecology, 2004, 47(2): [19] Wang MY, Zhang WX. Analysis of microbial community structure in pit mud from two Chinese Luzhou-flavor liquor producing areas[j]. Microbiology China, 2014, 41(8): (in Chinese),. [J]., 2014, 41(8): [20] Guo MY, Huo DQ, Chai R, et al. Metagenomics of ancient fermentation pits used for the production of Chinese Strong-aroma liquor[j]. Genome Announcements, 2014, 2(5): e [21] Tao Y, Li JB, Rui JP, et al. Prokaryotic communities in pit mud from different-aged cellars used for the production of Chinese strong-flavored liquor[j]. Applied and Environmental Microbiology, 2014, 80(7): [22] Ding XF, Wu CD, Zhang LQ, et al. Characterization of eubacterial and archaeal community diversity in the pit mud of Chinese Luzhou-flavor liquor by nested PCR-DGGE[J]. World Journal Microbiology Biotechnology, 2014, 30(2): [23] Liang HP, Li WF, Luo QC, et al. Analysis of the bacterial community in aged and aging pit mud of Chinese Luzhou-flavour liquor by combined PCR-DGGE and quantitative PCR assay[j]. Journal of the Science of Food and Agriculture, 2015, 95(13): [24] Wang L, Wang YY, Wang HY, et al. Microbial composition of bottom pit mud in Jiangxiang Baijiu (liquor) pit[j]. Liquor-Making Science & Technology, 2015(1): (in Chinese),,,. [J]., 2015(1): [25] Huang ZG, Deng J, Wei CH, et al. Separation and identification of bacteria strains from fermented grains of Jiang-xiang liquor[j]. Liquor-Making Science & Technology, 2014(6): (in Chinese),,,. [J]., 2014(6): [26] Zhang Q, Zhao H, Zhang MY, et al. Separation of bacteria strains related to producing Jiangxiang-flavor and its roles in the accumulation of fermented grains[j]. Liquor-Making Science & Technology, 2014(7): (in Chinese),,,. [J]., 2014(7): 54-56

Microsoft Word - 793-797 tb20150504赵宏宇s-高校教改纵横.doc

Microsoft Word - 793-797 tb20150504赵宏宇s-高校教改纵横.doc 微生物学通报 Microbiology China tongbao@im.ac.cn Apr. 20, 2016, 43(4): 793 797 http://journals.im.ac.cn/wswxtbcn DOI: 10.13344/j.microbiol.china.150504 高校教改纵横生物工程专业发酵课程群建设探索 * 赵宏宇