16S rRNA高通量测序技术筛选牦牛瘤胃细菌基因组DNA提取方法及菌群结构

发布时间：2018-09-03 20:04

【摘要】：【目的】确定理想的牦牛瘤胃细菌基因组DNA提取方法及初步分析牦牛瘤胃细菌的群体结构。【方法】采用物理法(珠磨法、反复冻融法)、化学法(CTAB、SDS)及酶解法(溶菌酶、蛋白酶K)三者与瘤胃细菌特点相结合的方法,组合生成9种不同方法,即方法 1(CTAB+SDS+Lysozyme+无特殊物理处理法)、方法 2(CTAB+SDS+Lysozyme+反复冻融法)、方法 3(CTAB+SDS+Lysozyme+珠磨法)、方法 4(CTAB+Lysozyme+无特殊物理处理法)、方法 5(CTAB+Lysozyme+反复冻融法)、方法 6(CTAB+Lysozyme+珠磨法)、方法 7(SDS+Lysozyme+无特殊物理处理法)、方法 8(SDS+Lysozyme+反复冻融法)、方法 9(SDS+Lysozyme+珠磨法),同时以QIA amp DNA Stool Mini Kit(方法 10)为对照,以这10种方法来提取瘤胃微生物基因组DNA,并通过DNA浓度、纯度、DNA电泳图等基本性质及16S r RNA高通量测序结果对其进行分析比较,同时通过测序结果初步分析牦牛瘤胃细菌群体结构。【结果】不同DNA提取方法效率比对结果显示,同样的化学及生物酶裂解条件下,结合珠磨法及反复冻融法能够显著地增强细胞裂解效率,提高DNA产量。其中方法 3及方法 6提取的DNA具有较高的浓度及纯度,方法 7—10缺乏CTAB阳离子去污剂,提取的DNA量显著低于其他方法(P0.05),除方法 2和8所提取的样品经多次试验,PCR产物目的条带太弱或未检测到外,其余8种方法提取的样品PCR产物目的条带大小正确,浓度合适,符合高通量测序的要求。16S r RNA高通量测序共生成了191 349条原始数据,质控后得到有效序列171 231条。稀释性曲线分析表明,数据量合理并达到饱和,能够完整反映样品的菌群种类。OTU聚类数据统计、分类学和多样性指数分析显示方法 6和10包含的细菌较丰富。不同提取方法对革兰氏阳性菌提取效果比对结果表明方法 6裂解革兰氏阳性菌细胞壁的能力比其他方法相对较高。综上,方法 6(CTAB-Lysozyme-珠磨)提取的DNA产量、样品多样性指数及革兰氏阳性菌破壁能力均优于其他方法。菌群结构分析表明牦牛瘤胃细菌菌群结构包括21门、35纲、75科、112属,丰度较高的菌群依次是拟杆菌Bacteroidtes(64%)、厚壁菌Firmicutes(20%)、螺旋体Spirochaetae(2.3%)和变形菌Proteobacteri(1.8%),纤维杆菌Fibrobacter(1.7%)。牦牛瘤胃细菌群体结构与黄牛相比存在着一定的差异,这可能归因于饮食及环境的不同。【结论】利用16S r RNA高通量测序筛选出了牦牛瘤胃细菌基因组DNA理想提取方法,即方法 6(CTAB-Lysozyme-珠磨),并初步分析了牦牛瘤胃细菌群体结构,为研究牦牛瘤胃微生物群体特殊性及挖掘牦牛体内的基因资源奠定了基础。
[Abstract]:[Objective] To determine an ideal method for extracting genomic DNA from rumen bacteria of yak and to analyze the population structure of rumen bacteria. Methods: 1 (CTAB + SDS + Lysozyme + no special physical treatment), 2 (CTAB + SDS + Lysozyme + repeated freeze-thaw), 3 (CTAB + SDS + Lysozyme + bead milling), 4 (CTAB + Lysozyme + no special physical treatment), 5 (CTAB + Lysozyme + repeated freeze-thaw), 6 (CTAB + Lysozyme + bead milling), 7 (SDS + Lysozyme + no special substance). Methods 8 (SDS + Lysozyme + repeated freeze-thaw method), 9 (SDS + Lysozyme + bead milling method) and QIA amp DNA Stool Mini Kit (method 10) were used to extract genomic DNA of rumen microorganisms by these 10 methods. The genomic DNA of rumen microorganisms was analyzed by DNA concentration, purity, DNA electrophoresis and 16S R RNA high-throughput sequencing. The results showed that under the same conditions of chemical and biological enzymatic lysis, the combination of bead milling and repeated freeze-thaw method could significantly enhance the cell lysis efficiency and DNA production. Methods 7-10 lacked CTAB cationic detergent, and the amount of DNA extracted was significantly lower than that of other methods (P 0.05). Except for the samples extracted by methods 2 and 8, which were too weak or undetected after many tests, the bands of PCR products extracted by other eight methods were correct in size and proper in concentration. 16S R RNA high-throughput sequencing produced 191 349 original data, and 171 231 effective sequences were obtained after quality control. Dilution curve analysis showed that the amount of data was reasonable and saturated, which could fully reflect the species of bacteria. OTU cluster data statistics, taxonomy and diversity index analysis showed methods 6 and 6. The results showed that the ability of method 6 to lyse the cell wall of Gram-positive bacteria was higher than that of other methods. In summary, the DNA yield, diversity index of samples and the ability of Gram-positive bacteria to break the cell wall of method 6 (CTAB-Lysozyme-bead mill) were superior to other methods. Methods. The microflora structure analysis showed that the rumen microflora of yak included 21 phyla, 35 classes, 75 families and 112 genera. The higher abundance of the microflora were Bacteroides (64%), Firmicutes (20%), Spirochaetae (2.3%) and Proteobacteri (1.8%), Fibrobacter (1.7%). [Conclusion] An ideal method for genomic DNA extraction of yak rumen bacteria was screened out by 16S R RNA high-throughput sequencing, that is, method 6 (CTAB-Lysozyme-bead mill), and the structure of yak rumen bacterial population was preliminarily analyzed to study the rumen microbial population of yak. Particularity and digging the genetic resources of yak body laid the foundation.
【作者单位】：西南民族大学生命科学与技术学院;西南民族大学青藏高原研究院;
【基金】：西南民族大学中央高校基本科研业务费专项资金(2015NZYTD01) 西南民族大学研究生学位点建设项目(2014XWD-S0906) 四川省教育厅自然科学重点项目(15ZA0386) 西南民族大学研究生创新型科研项目(CX2016SZ079)
【分类号】：S823.85

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