羔羊瘤胃内容物微生物基因组DNA的提取方法比较  

Comparison of extraction methods of microbial genomic DNA from rumen contents of lambs

作　　者：翁秀秀[1] 刘婷[2] WENG Xiuxiu;LIU Ting(College of Pastoral Agriculture Science and Technology,Lanzhou University,Lanzhou 730020,China;College of Animal Science and Technology,Gansu Agricultural University,Lanzhou 730070,China)

机构地区：[1]兰州大学草地农业科技学院,甘肃兰州730020 [2]甘肃农业大学动物科学技术学院,甘肃兰州730070

出　　处：《甘肃农业大学学报》2021年第6期10-18,共9页Journal of Gansu Agricultural University

基　　金：国家自然科学基金项目(31860656)。

摘　　要：【目的】旨在筛选稳定可靠的瘤胃内容物微生物基因组DNA的提取方法,获得高质量瘤胃微生物基因组DNA,用于羔羊瘤胃内容物微生物多样性的研究.【方法】采用液氮研磨+珠磨+CTAB法、珠磨+CTAB法、液氮研磨+珠磨+SDS+GITC法和液氮研磨+CTAB+SDS法4种方法提取羔羊瘤胃内容物微生物基因组DNA.通过DNA浓度和纯度、琼脂糖凝胶电泳检测和q-PCR对提取效果进行比较,分析各提取方法对羔羊瘤胃内容物微生物基因组DNA的影响.【结果】4种方法提取到的基因组DNA经q-PCR分析,瘤胃细菌和产甲烷菌的数量存在显著差异(P<0.001),其中琥珀酸拟杆菌(Fibrobacter succinogenes)数量最高,总细菌(total bacteria)次之,白色瘤胃球菌(Ruminococcus albus)数量最少.液氮研磨+珠磨+SDS+GITC法所获得的羔羊瘤胃内容物微生物DNA样品浓度和纯度较高;瘤胃细菌和产甲烷菌数量显著高于其他3种方法(P<0.001).【结论】液氮研磨+珠磨+SDS+GITC法能够得到高质量的羔羊瘤胃内容物微生物基因组DNA,更适合于羔羊瘤胃内容物微生物分子生物学研究.【Objective】The study aimed to screen the stable and reliable method of DNA extraction of rumen microbe from rumen content of lambs for the purpose of providing high-quality genomic DNA for microbial diversity research on rumen content of lambs.【Method】Four methods,i.e.,liquid-nitrogen grinding+bead milling+CTAB,bead milling+CTAB,liquid-nitrogen grinding+bead milling+SDS+GITC and liquid-nitrogen grinding+CTAB+SDS,were used to extract rumen microbial genomic DNA from rumen content of lambs.The quality of DNA was evaluated by determining DNA concentration and purity and comparing the results of agarose-gel electrophoresis and q-PCR,followed by analyzing the effects of different methods on microbial genomic DNA of rumen contents.【Result】It was showed that the number of rumen bacteria and methanogens was significantly different among the four methods through analyzing genomic DNA with the method of by Q-PCR(P<0.001).Of these microbes,the number of Fibrobacter succinogenes was the highest,followed by total bacteria and Ruminococcus albus.Both concentration and purity of DNA extracted by liquid nitrogen milling+bead milling+SDS+GITC was much higher than that by other methods,and also the numbers of both rumen bacteria and methanogens were the highest among the four methods(P<0.001).【Conclusion】The high-quality rumen microbial genomic DNA could be extracted by the method of liquid-nitrogen milling+bead milling+SDS+GITC,which was suitable for the molecular biology research on rumen microbes of lambs.

关 键 词：羔羊 瘤胃内容物 提取方法 基因组DNA 

分 类 号：Q78[生物学—分子生物学]