应用变性梯度凝胶电泳和16S rDNA序列分析对山羊瘤胃细菌多样性的研究

 以取自3头安装有永久性瘤胃瘘管的土种山羊的瘤胃内容物为材料, 经过DNA抽提和PCR扩增, 扩增产物利用变性梯度凝胶电泳技术(DGGE，一种DNA指纹技术)分析瘤胃细菌在两种日粮条件下的多样性。同时利用基因序列分析技术，分析了16个在DGGE胶上有匹配带的克隆的16S rDNA序列，并与现有的数据库进行了比较。结果表明，饲喂基础日粮时3头山羊瘤胃内容物的DGGE图谱有一定的相似性(43%～55%)；饲料中添加大豆黄酮一定程度上影响了瘤胃细菌的组成，DGGE谱带变化程度分别为1号36%、2号46%、3号30%。基因序列分析表明，DGGE图谱中优势条带的16S rDNA基因序列中有5个基因序列与基因数据库登录的相关序列的相似性大于97%，8个基因序列的相似性在90%～96%，余下的低于90%。相似性大于97%的5个克隆中，只有1个被鉴定为Prevotella sp.，其余4个都属于未被鉴定的瘤胃细菌。

Analysis of Rumen Bacterial Diversity of Goat by Denaturing Gradient Gel Electrophoresis and 16S rDNA Sequencing

Diversity of rumen bacteria of the rumen content of Chinese white goats was analyzed by PCR amplification, denaturing gradient gel electrophoresis (DGGE) and sequencing of 16S rDNA clone libraries. DNA was extracted from rumen contents of goats fed two diets with and without the addition of daidzein. The V6-V8 region of 16S rDNA of bacteria was amplified and the amplicons were then separated based on a linear gradient of denaturants in DGGE, a fingerprinting technique. A clone library was created from complete 16S rDNA. From the library, 16 clones had their V6-V8 regions matched predominant bands on the DGGE gel and their 16S rDNAs were then sequenced and subjected to an online similarity search. Five clones showed their similarities with database sequences over 97%, with one sequence similar to Prevotella sp., the rest were similar to those unidentified rumen bacteria. From the library, eight clones with similarities in the range of 90%~96% and the remaining three clones were less than 90%.