应用rpoB和16S rDNA基因的变性梯度凝胶电泳技术对山羊瘤胃细菌多样性的研究

Use of rpoB and 16S rDNA genes to analyze rumen bacterial diversity of goat using PCR and DGGE

采用免培养的rpoB和16S rDNA基因的变性梯度凝胶电泳技术(DGGE)对3种山羊(波尔山羊,内蒙古绒山羊,四川南江黄羊)瘤胃细菌优势菌群结构进行了比较分析。研究结果显示rpoBDGGE图谱中条带数目少于16S rDNA图谱,并且条带分离效果明显,更有利于分析瘤胃细菌群落组成。从两种DGGE图谱中均可以发现3种山羊瘤胃细菌具有一定的相似性,种内个体间相似性明显高于种间相似性,这说明寄主品种是影响瘤胃细菌种群构成的一个重要因素。同时进行了部分优势细菌16S rDNA基因V6-V8区序列的系统发育分析。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4条克隆的序列与基因库最相似菌的相似性大于97%,余下的克隆序列相似性在89%～96%之间,其中13条序列的与之相似性最高的序列均来自于未被鉴定的瘤胃细菌。

DNA extraction from the tureen of three species of goat （boer goat, Nanjiang yellow goat, Inner Mongolia cashmere goat ） was followed by Polymerase Chain Reaction （PCR） amplification of the beta subunit of the RNA polymerase （rpoB） and 16S rDNA genes. PCR products were compared by Denaturing Gradient Gel Electrophoresis （DGGE） to compare the predominant bacterial community structure. The results showed the rpoB DGGE profiles comprised fewer bands than those of 16S rDNA profiles and were easier to analyze. The gene for rpoB is a single copy gene unlike 16S rDNA. So using the rpoB gene offeres a better alternative to the commonly used 16S rRNA gene for microbial community analyses based on DGGE. The bacteria community structure of different goats were similar to each other. The similarities within species were noticeably higher than that between species. Goat species were found to influence the rumen microbe community. Phylogenetic and sequence similarity analyses of the resultant 14 clone sequences inl6S rDNA DGGE libraries revealed that 4 clone show similarity over 97 % with that of database sequences, while the rest present similarity in a range of 89% - 96%, and 13 clone of all were similar to those unidentified rumen bacteria. These results suggest that DGGE followed by clone technique is a practicable protocol to research the complex community of rumen microbe.