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

doi:10.3864/j.issn.0578-1752.2017.05.016

摘要/Abstract

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 rRNA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（P<0.05），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 rRNA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 rRNA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。

关键词: 牦牛, 瘤胃微生物, DNA, 提取方法, 菌群结构

Abstract: 【Objective】The objective of this study is to determine the optimal DNA extraction methods for yak rumen bacterial genome and preliminarily identify the bacterial community of yak rumen.【Method】Physical method (bead-beating, freeze-thaw), chemical method(CTAB, SDS) and enzymatic method (lysozyme, protease K) were combined with the characteristics of rumen bacteria, 9 different methods (method 1, CTAB-SDS-Lysozyme; method 2, CTAB-SDS-Lysozyme-freeze-thaw; method 3, CTAB-SDS-Lysozyme-bead-beating; method 4, CTAB-Lysozyme; method 5, CTAB-Lysozyme-freeze-thaw; method 6, CTAB- Lysozyme-bead-beating; method 7, SDS-lysozyme; method 8, SDS-lysozyme- freeze-thaw; method 9, SDS-lysozyme-bead-beating) were generated to extract the rumen bacterial DNA, besides, method 10, QIA amp DNA Stool Mini Kit, was also used as reference. The above 9 methods were compared and analyzed based on DNA concentration, purity, DNA electrophoretogram and 16S rRNA high throughput sequencing results, and the bacterial community of yak rumen was preliminarily identified based on the sequencing results.【Result】The comparison results of different DNA extraction methods efficiency showed that the combined beat-beating and freeze-thaw could significantly increase cell lysis efficiency and DNA yield under the same chemical and enzymatic conditions. Methods 3 and 6 obtained high DNA concentration and purity, methods 7, 8, 9, and 10 had lower volume of DNA than other methods because of the lack of CTAB. In addition to methods 2 and 8 as their PCR products were too weak or not detected, the PCR products of rest methods all had suitable objective strap size and concentration which met the requirements of 16S rRNA high throughput sequencing. After high throughput sequencing, a total of 191 349 raw reads were obtained, and 171 231 clean reads were obtained after quality control. Rare faction curves analysis showed that the amount of reads reached the saturation level, which could completely reflect the bacterial community species of the samples. OTU-based taxology and the diversity index analysis showed that methods 6 and 10 contained more abundant bacteria. The comparison of the effect of different extraction methods on Gram-positive bacteria showed that method 6 presented a comparatively higher capability in lysing cell wall of Gram-positive bacteria. Above all, the DNA yield, OTUs number, bacteria diversity, and excellent cell-breaking capability of method 6 (CTAB-Lysozyme- bead-beating) was better than that with other methods. The bacterial community structure analysis showed that the number of taxa in yak rumen contained approximately 21 phyla, 35 classes, 75 families, 112 genera. The bacteria community of higher abundance included Bacteroidtes (64%), Firmicutes (20%), Spirochaetae (2.3%), Proteobacteri (1.8%) and Fibrobacter (1.7%). Comparative analysis showed that there are some variation in the bacterial community between yaks and cattle, which may be attributed to different diets and habitats.【Conclusion】This study screened out an optimal DNA extraction method (method 6,CTAB-Lysozyme-Bead-beating) for yak rumen bacterial genome using 16S rRNA high-throughput sequencing, and the bacterial community structure of yak rumen was preliminarily evaluated. Results of the study provide a foundation for identifying the specificity of rumen microbial community and discovering distinct gene resource in yak.

Key words: yak, rumen microbe, DNA , extraction methods, bacteria community structure

杨琦玥，黄勇，陈亚冰，刘洛川，李键，兰道亮. 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[J]. 中国农业科学, 2017, 50(5): 932-941.

YANG QiYue, HUANG Yong, CHEN YaBing, LIU LuoChuan, LI Jian, LAN DaoLiang. Screening of Optimal DNA Extraction Methods and the Bacterial Community Composition of Yak Rumen Revealed by High 16S rRNA Throughput Sequencing[J]. Scientia Agricultura Sinica, 2017, 50(5): 932-941.

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