甲烷囊菌属特异性引物的设计及其在古井贡酒窖泥质量评价中的初步应用
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摘要
甲烷囊菌(Methanoculleus)作为优势甲烷菌存在于浓香型白酒的窖泥中,该研究对古井贡酒窖泥中含量最多的30个属的16S rDNA全长代表序列进行傅里叶转换多序列比对(MAFFT),得到114个可能的甲烷囊菌特异性单核苷酸多态性(SNP)位点。对SNP位点在甲烷囊菌属内和属外5000多条序列进一步甄别,找出了3个效果最好的SNP位点,利用其中1个位点和半随机引物-聚合酶链反应(SAP-PCR)技术原理设计了两对特异性引物。以窖泥宏基因组为模板,成功扩增出了甲烷囊菌的相应序列。将引物应用于18个古井窖泥样本的实时定量聚合酶链反应(RT-QPCR)分析,结果表明,老厂优质窖泥中甲烷囊菌的相对含量远远高于新厂普通窖泥(最高达4000多倍)。利用本研究的方法原则上可以设计任意其他菌属在复杂菌群背景下的特异性引物。
Methanoculleus was found as dominant methanogen in pit mud of strong-flavor Baijiu. The representative full-length 16 S rDNA sequences of 30 dominant genera in Gujinggong liquor pit mud were aligned by the multiple alignment using fast Fourier transform(MAFFT), and 114 possible specific single nucleic polymorphism(SNP) sites of Methanoculleus were obtained, among them, 3 optimal SNP sites were founded by comparing more than 5000 sequences of intra-genus and extra-genus of Methanoculleus. Two pairs of specific primers were designed by one SNP site and semi random-primer polymerase chain reaction(SAP-PCR) design principle. The corresponding sequences of Methanoculleus were successfully amplified by using pit mud genome as template. The primers were applied to the real-time quantitative PCR(RT-QPCR) analysis, and the results indicated that the relative content of Methanoculleus in the high-quality pit mud from old factory was much higher than that in the low-quality pit mud from the new factory(up to more than 4000 times). In principle, the study also provided a useful protocol to design specific primers for any taxonomic level in a complex microbial community.
Methanoculleus was found as dominant methanogen in pit mud of strong-flavor Baijiu. The representative full-length 16 S rDNA sequences of 30 dominant genera in Gujinggong liquor pit mud were aligned by the multiple alignment using fast Fourier transform(MAFFT), and 114 possible specific single nucleic polymorphism(SNP) sites of Methanoculleus were obtained, among them, 3 optimal SNP sites were founded by comparing more than 5000 sequences of intra-genus and extra-genus of Methanoculleus. Two pairs of specific primers were designed by one SNP site and semi random-primer polymerase chain reaction(SAP-PCR) design principle. The corresponding sequences of Methanoculleus were successfully amplified by using pit mud genome as template. The primers were applied to the real-time quantitative PCR(RT-QPCR) analysis, and the results indicated that the relative content of Methanoculleus in the high-quality pit mud from old factory was much higher than that in the low-quality pit mud from the new factory(up to more than 4000 times). In principle, the study also provided a useful protocol to design specific primers for any taxonomic level in a complex microbial community.
引文
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[13]兰阿峰,杨曼,郭素芬,等.野生秦岭羚牛瘤胃产甲烷古菌多样性研究[J].安徽农业科学,2018,46(8):88-91.
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[17]胡贝,陈瑶,张文学,等.几种主要窖泥微生物丰度与窖龄的关系[J].应用与环境生物学报,2014,20(3):389-394.
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[20]BUI M,LIU Z.Simple allele-discriminating PCR for cost-effective and rapid genotyping and mapping[J].Plant Meth,2009,5(1):1.
[21]LIU Z L,HAN Y,LI J W,et al.Analysis of microbial community structure of pit mud for Chinese strong-flavor liquor fermentation using next generation DNA sequencing of full-length 16S r RNA[J].Bio Rxiv,2018,doi:https://doi.org/10.1101/380949.
[22]唐立群,肖层林,王伟平,等.SNP分子标记的研究及其应用进展[J].中国农学通报,2012,28(12):154-158.
[23]PEARSON T,FALKOW S.Phylogenetic discovery bias in Bacillus anthracis using single-nucleotide polymorphisms from whole-genome sequencing[J].Proceed Natl Acad Sci USA,2004,101(37):13536-13541.
[24]WANG H T,SUN H,KWON W S,et al.A PCR-based SNP marker for specific authentication of Korean ginseng(Panax ginseng)cultivar"Chunpoong"[J].Molecul Biol Report,2010,37(2):1053-1057.
[25]FERNáNDEZ-NO I C,BOHME K,CAAMANO-ANTELO S,et al.Identification of single nucleotide polymorphisms(SNPs)in the 16S r RNAgene of foodborne Bacillus spp[J].Food Microbiol,2015,46(4):239-245.
[26]TAKAJO I,YAMADA A,UMEKI K,et al.Development of a simple and practical method of discrimination between Vibrio furnissii and V.fluvialis based on single-nucleotide polymorphisms of 16S r RNA genes observed in V.furnissii but not in V.fluvialis[J].J Microbiol Meth,2018,144(1):22-28.
[2]李芳莉.浓香型白酒窖泥微生物群落结构分析及其对原酒风味物质的影响[D].郑州:郑州轻工业学院,2017.
[3]李付丽,吴鑫颖,王晓丹,等.微生物技术在浓香型白酒增香方面的应用[J].中国酿造,2014,33(1):9-13.
[4]姚万春,唐玉明,任道群,等.功能性人工窖泥培养配方筛选研究[J].中国酿造,2014,33(8):63-66.
[5]张会敏.古井贡酒微生物群落结构及其与主要风味物质的关联研究[D].哈尔滨:哈尔滨工业大学,2016.
[6]赵东,郑佳,彭志云,等.高通量测序技术解析五粮液窖泥原核微生物群落结构[J].食品与发酵工业,2017,43(9):1-8.
[7]刘茂柯,唐玉明,邹兰,等.浓香型白酒窖泥古菌群落结构及其多样性[J].食品与发酵工业,2013,39(10):22-28.
[8]邓杰,卫春会,罗惠波,等.基于高通量测序的浓香型白酒窖池古菌群落结构分析[J].现代食品科技,2015,31(8):205-210.
[9]邓杰,卫春会,边名鸿,等.浓香型白酒不同窖龄窖池窖泥中古菌群落结构分析[J].食品科学,2017,38(8):37-42.
[10]刘茂柯,唐玉明,邹兰,等.浓香型白酒窖泥古菌群落结构及其多样性[J].食品与发酵工业,2013,39(10):22-28.
[11]韦艳霞,郑纪伟,杨锋,等.根管感染中产甲烷古细菌基于mcrA序列的系统发育分析[J].口腔生物医学,2014,5(4):181-184.
[12]华金玲,王立克,杨芝,等.PCR技术在黄淮白山羊瘤胃产甲烷菌检测中的应用[J].东北林业大学学报,2013,41(7):134-136.
[13]兰阿峰,杨曼,郭素芬,等.野生秦岭羚牛瘤胃产甲烷古菌多样性研究[J].安徽农业科学,2018,46(8):88-91.
[14]罗青春,刘超兰,吴正云,等.不同年份窖泥中主要产甲烷菌的荧光定量PCR研究[J].酿酒科技,2013(12):17-20.
[15]YU Y,LEE C,KIM J,et al.Group-specific primer and probe sets to detect methanogenic communities using quantitative real-time polymerase chain reaction[J].Biotechnol Bioeng,2005,89(6):670-679.
[16]YU Y,KIM J,HWANG S.Use of real-time PCR for group-specific quantification of aceticlastic methanogens in anaerobic processes:population dynamics and community structures[J].Biotechnol Bioeng,2006,93(3):424-433.
[17]胡贝,陈瑶,张文学,等.几种主要窖泥微生物丰度与窖龄的关系[J].应用与环境生物学报,2014,20(3):389-394.
[18]罗锡梅.基于单碱基识别的qPCR微生物群落定量技术的研究[D].哈尔滨:哈尔滨工业大学,2015.
[19]高志磊.古井窖泥微生物群落纲层级定量识别的引物设计和应用[D].哈尔滨:哈尔滨工业大学,2015.
[20]BUI M,LIU Z.Simple allele-discriminating PCR for cost-effective and rapid genotyping and mapping[J].Plant Meth,2009,5(1):1.
[21]LIU Z L,HAN Y,LI J W,et al.Analysis of microbial community structure of pit mud for Chinese strong-flavor liquor fermentation using next generation DNA sequencing of full-length 16S r RNA[J].Bio Rxiv,2018,doi:https://doi.org/10.1101/380949.
[22]唐立群,肖层林,王伟平,等.SNP分子标记的研究及其应用进展[J].中国农学通报,2012,28(12):154-158.
[23]PEARSON T,FALKOW S.Phylogenetic discovery bias in Bacillus anthracis using single-nucleotide polymorphisms from whole-genome sequencing[J].Proceed Natl Acad Sci USA,2004,101(37):13536-13541.
[24]WANG H T,SUN H,KWON W S,et al.A PCR-based SNP marker for specific authentication of Korean ginseng(Panax ginseng)cultivar"Chunpoong"[J].Molecul Biol Report,2010,37(2):1053-1057.
[25]FERNáNDEZ-NO I C,BOHME K,CAAMANO-ANTELO S,et al.Identification of single nucleotide polymorphisms(SNPs)in the 16S r RNAgene of foodborne Bacillus spp[J].Food Microbiol,2015,46(4):239-245.
[26]TAKAJO I,YAMADA A,UMEKI K,et al.Development of a simple and practical method of discrimination between Vibrio furnissii and V.fluvialis based on single-nucleotide polymorphisms of 16S r RNA genes observed in V.furnissii but not in V.fluvialis[J].J Microbiol Meth,2018,144(1):22-28.