Abstract:In order to obtain high quality genomic DNA of rumen microbes in goats(Capra hircus) and analyze the microbial diversity in the rumen, 6 kinds of DNA extraction methods, including bead mill+Tris-Phenol method, bead mill+Sodium dodecyl sulfate (SDS) method, bead mill+SDS/Guanidine thiocyanate (GITC) method, bead mill+SDS/NH4Ac method, bead mill+SDS/GITC/NH4Ac method and SDS/GITC methods, were used to extract microbial genomic DNA of goats in the present research. These extraction methods were compared through measuring DNA concentration and purity, polymerase chain reaction(PCR), denaturing gradient gel electrophoresis(DGGE) technique to find the suitable method for extracting microbial genomic DNA in the rumen of goats. Results showed that microbial genomic DNA extracted by using bead mill plus sodium dodecyl sulfate/guandine thiocyanate/ammonium acetate method had better purity and the concentration of it was 96.4 ng/μL. Meanwhile, ruminal bacterial and methanogenic 16S rDNA was amplified simultaneously, and the highest 16S rDNA diversity indexes of ruminal bacteria and methanogen were found when using this method. Therefore, bead mill plus sodium dodecyl sulfate/guandine thiocyanate/ammonium acetate method is more suitable for the subsequent molecular biology research of ruminal microflora.
[1]Bera-Maillet C, Mosoni P, Kwasiborski A, Suau F, Ribot Y and Forano E.Development of a RT-qPCR method for the quantification of Fibrobacter succinogenes S85 glycoside hydrolase transcripts in the rumen content of gnotobiotic and conventional sheep[J].Journal of microbiological methods, 2009, 77:8-16[2]Kleikemper J, Pombo S A, Schroth M H, Sigler W V, Pesaro M and Zeyer J.Activity and diversity of methanogens in a petroleum hydrocarbon-contaminated aquifer [J].Applied and environmental microbiology, 2005, 71:149-158[3]Krause D O, Nagaraja T G, Wright A D and Callaway T R.Board-invited review: Rumen microbiology: leading the way in microbial ecology[J].Journal of animal science, 2013, 91:331-341[4]Krause D O, Smith W J and McSweeney C S.Extraction of microbial DNA from rumen contents containing plant tannins[J].BioTechniques, 2001, 31:294-298[5]Leng J, Xie L, Zhu R, Yang S, Gou X, Li S and Mao H.Dominant bacterial communities in the rumen of Gayals (Bos frontalis), Yaks (Bos grunniens) and Yunnan Yellow Cattle (Bos taurs) revealed by denaturing gradient gel electrophoresis[J].Molecular biology reports, 2011, 38:4863-4872[6]Parmar N R, Solanki J V, Patel A B, Shah T M, Patel A K, Parnerkar S, Kumar J I N and Joshi C G, .Metagenome of Mehsani Buffalo Rumen Microbiota: An Assessment of Variation in Feed-Dependent Phylogenetic and Functional Classification[J].Journal of molecular microbiology and biotechnology, 2014, 24:249-261[7]Strathdee F and Free A.Denaturing gradient gel electrophoresis (DGGE)[J].Methods in molecular biology, 2013, 1054:145-157[8]Tsai Y L and Olson B H.Rapid method for separation of bacterial DNA from humic substances in sediments for polymerase chain reaction[J].Applied and environmental microbiology, 1992, 58:2292-2295[9]Yu Z and Morrison M.Improved extraction of PCR-quality community DNA from digesta and fecal samples[J].BioTechniques, 2004, 36:808-812[10]Zhou M, Hernandez-Sanabria E and Guan L L.Characterization of variation in rumen methanogenic communities under different dietary and host feed efficiency conditions, as determined by PCR-denaturing gradient gel electrophoresis analysis[J].Applied and environmental microbiology, 2010, 76:3776-3786[11]Zoetendal E G, Akkermans A D and De Vos W M.Temperature gradient gel electrophoresis analysis of 16S rRNA from human fecal samples reveals stable and host-specific communities of active bacteria[J].Applied and environmental microbiology, 1998, 64:3854-3859