山羊瘤胃微生物基因组DNA提取方法比较  

Methods Comparison of Genomic DNA Extraction for Rumen Microbiology in Goats(Capra hircus)

作　　者：曾波[1] 康劲翮[1] 汤少勋[1] 王敏[1] 韩雪峰[1] 周传社[1] 颜琼娴[1] 贺志雄[1] 谭支良[1] 

机构地区：[1]中国科学院亚热带农业生态过程重点实验室/湖南省农业生态工程重点实验室/中国科学院亚热带农业生态研究所,长沙410125

出　　处：《农业生物技术学报》2015年第6期823-830,共8页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金项目(No.31201826和No.31320103917);中央驻湘科研机构技术创新发展专项(No.2013TF3006)

摘　　要：为了获得高质量的山羊(Capra hircus)瘤胃微生物基因组DNA,用于分析瘤胃微生物多样性,本研究采用珠磨+Tris-饱和酚法、珠磨+十二烷基磺酸钠(sodium dodecyl sulfate,SDS)法、珠磨+SDS/异硫氰酸胍(guanidine thiocyanate,GITC)法、珠磨+SDS/醋酸铵(NH4Ac)法、珠磨+SDS/GITC/NH4Ac法和SDS/GITC法等6种DNA提取法提取山羊瘤胃微生物基因组DNA。通过DNA浓度和纯度的测定、聚合酶链式反应(polymerase chain reaction,PCR)、变性梯度凝胶电泳技术(denaturing gradient gel electrophoresis,DGGE)对提取效果进行比较,以找到适合于山羊瘤胃微生物基因组DNA的提取方法。结果表明,应用珠磨+SDS/GITC/NH4Ac法提取的瘤胃微生物基因组DNA纯度较好,浓度高达96.4 ng/μL,并可同时扩增出瘤胃细菌和产甲烷古菌的16S r DNA片段,且细菌和产甲烷古菌的多样性指数均最高,更适合于瘤胃微生物菌群后续分子生物学研究。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 ofruminal microflora.

关 键 词：山羊 瘤胃微生物 基因组DNA 变性梯度凝胶电泳技术(DGGE) 

分 类 号：S792.14[农业科学—林木遗传育种]