糯米酒发酵过程中微生物基因组提取方法比较
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摘要
为了更加快速地获得糯米酒发酵过程中高质量的微生物基因组DNA,本实验通过测定基因组DNA浓度和进行聚合酶链式反应(PCR),利用溶菌酶-SDS-蛋白酶-K(A法),改良CTAB(B法)和改良CTAB-溶菌酶(C法)三种实验方法获得微生物基因组DNA,通过比较三种方法发现,C法提取效率最高,获得的基因组DNA浓度为1 573.97±711.51ng/μL;B法获得的基因组浓度为1 436.03±128.0ng/μL;A法效率最低,获得基因组浓度为411.67±133.29ng/μL.以基因组DNA为模板进行PCR,将扩增产物在0.6%琼脂糖凝胶中进行电泳检验.结果显示C法获得的条带最清楚明亮,表明DNA浓度最高、结构完整.基因组DNA浓度和PCR产物琼脂糖凝胶电泳条带的亮度表明,提取糯米酒发酵过程中微生物基因组DNA的最优方法为改良CTAB-溶菌酶法(C法).
In order to obtain higher quality and more rapid acquisition of microbial genomic DNA during fermentation of glutinous rice wine,the present study compared lysozyme-SDS-protease K method(A method)by determining genomic DNA concentration and performing polymerase chain reaction,Modified CTAB method(B method)and improved CTAB-lysozyme method(C method)three experimental methods.The experimental results showed that the highest extraction efficiency was C method,and the highest concentration of 1 573.97±711.51 ng/μL genomic DNA was obtained.The efficiency of B method was normal,obtaining a concentration of 1 436.03±128.0 ng/μL DNA.The method with the lowest efficiency was obtained with a DNA concentration of 411.67±133.29 ng/μL.The obtained genomic DNA was used as a template for the polymerase chain reaction,and the amplified product was subjected to electrophoresis in a 0.6%agarose gel for examination.The results showed that the band obtained by C-garnet gel electrophoresis was the most clear and bright,indicating that the DNA concentration was the highest and the structure was complete.According to the concentration of DNA and the brightness of the polymerase chain reaction product agarose gel electrophoresis band,the genomic DNA of the fermented glutinous rice wine should be selected using the C method(modified CTAB-lysozyme method).
In order to obtain higher quality and more rapid acquisition of microbial genomic DNA during fermentation of glutinous rice wine,the present study compared lysozyme-SDS-protease K method(A method)by determining genomic DNA concentration and performing polymerase chain reaction,Modified CTAB method(B method)and improved CTAB-lysozyme method(C method)three experimental methods.The experimental results showed that the highest extraction efficiency was C method,and the highest concentration of 1 573.97±711.51 ng/μL genomic DNA was obtained.The efficiency of B method was normal,obtaining a concentration of 1 436.03±128.0 ng/μL DNA.The method with the lowest efficiency was obtained with a DNA concentration of 411.67±133.29 ng/μL.The obtained genomic DNA was used as a template for the polymerase chain reaction,and the amplified product was subjected to electrophoresis in a 0.6%agarose gel for examination.The results showed that the band obtained by C-garnet gel electrophoresis was the most clear and bright,indicating that the DNA concentration was the highest and the structure was complete.According to the concentration of DNA and the brightness of the polymerase chain reaction product agarose gel electrophoresis band,the genomic DNA of the fermented glutinous rice wine should be selected using the C method(modified CTAB-lysozyme method).
引文
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[2]PATRICK R,RENAUD N,CARMELA C,et al.Extraction of DNA from soil[J].European Jouranl of Soil Biology,2003,39(4):183-190
[3]OGRAMA A,SAYLER G S,BARKAY T.The extraction and purification of micobial DNA from sediments[J].Journal of Microbiological Methods,1987,7(2):57-66
[4]赵裕栋,周俊,何璟.土壤微生物总DNA提取方法的优化[J].微生物学报,2012,52(9):1143-1150
[5]张英,柏干荣,黄明辉,等.植物基因组DNA提取方法学评析与验证[J].Drug Evaluation,2004.1(4):292-297
[6]邵劲松,浦牧野,顾祝军.3种土壤微生物基因组DNA提取方法的比较[J].南京晓庄学院学报,2013,05(3):60-62
[7]陈昆松,李方,徐昌杰,等.改良CTAB法用于多年生植物组织基因组DNA的大量提取[J].遗传,2004,26(4):529-531
[8]余志雄,欧高政,陈清西,等.火龙果总DNA提取方法比较研究[J].中国农学通报,2010,26(4):300-303
[9]陈艳秋.桦褐孔菌菌株遗传多样性及人工培养条件优化模式研究[D].长春:吉林农业大学,2007
[10]宋培勇.从土壤中提取DNA方法比较[J].微生物学杂志,2006,26(1):109-112
[11]王琨.糯米酒液态曲液态酿造工艺研究[D].柳州:广西科技大学,2015
[12]石发祥.糯米酒的酿造[J].农村新技术,2009,14:60
[13]陈邦,范代娣,王琰.土壤微生物DNA提取方法的研究[J].西北大学学报(自然科学版),2009,39(5):785-788
[14]李红,周友兵,陈炳耀,等.分子生物学技术在微生物多样性研究中的应用[J].河北大学学报(自然科学版),2004,24(3):450-454
[15]ZHAO X M,DUSZYNSKI D W,LOKER E S.A simple method of DNA extraction for Eimeria species[J].Journal of Microbiological Methods,2001,44(2):131-137
[16]薛建岗.内蒙古西部地区自然发酵酸粥化学成分及微生物组成分析[J].食品科技,2013,38(7):11-14
[17]许文涛,郭星,罗云波,等.微生物菌群多样性分析方法的研究进展[J].食品科学,2009,30(7):258-265
[18]刘小翠,李云波,赵思明.生米发酵食品的研究进展[J].食品科学,2006,26(10):616-619
[19]刘艳秋,孙广仁,杨奇,包怡红.轮叶党参糯米酒发酵特性的研究[J].中国酿造,2017,36(4):99-102
[20]朱英莲.雪梨糯米酒的研制[J].食品工业,2013,34(4):25-29
[21]常健,王琪.晋西北酸粥发酵过程中微生物基因组DNA提取方法比较[J].食品工业科技,2016,37(2):209-212
[22]WILMOTTE A,Van der Auwera G,De Wachter R.Structure of 16Sribosomal RNA of the theromophilic cyanobacterium Chlorogloeopsis HTF)(‘Mastigocladus laminosus HTF’)strain PCC7518,and phylogenetic analysis[J].Federation of European Biochemical Societies,1993,317(1-2):96-100
[23]RAY A A.SAS User’s Guide:Statistics[M].Cary,NC:SAS Institute,1985