应用16S rRNA基因技术研究仔猪结肠梭菌Ⅳ群变化  

16S rRNA gene-based molecular methods to monitor Clostridium cluster Ⅳ community in the colon of piglets

作　　者：边高瑞[1] 谢飞[1] 苏勇[1] 朱伟云[1] 

机构地区：[1]南京农业大学消化道微生物研究室,南京210095

出　　处：《微生物学报》2010年第10期1373-1379,共7页Acta Microbiologica Sinica

基　　金：国家自然科学基金青年基金项目(30901036);南京农业大学青年科技创新项目~~

摘　　要：【目的】研究从7日龄到35日龄(断奶后两周)仔猪结肠中梭菌Ⅳ群(柔嫩梭菌群,Clostridium Leptumgroup)结构以及数量的变化,探究该菌群与结肠中丁酸浓度的关系。【方法】选取3窝新生仔猪,分别在7、14、21(断奶)、24和35日龄每窝随机屠宰1头,收集结肠内容物,利用气相色谱测定挥发性脂肪酸(Volatilefatty acid,VFA);提取结肠内容物总细菌DNA,利用基于16S rRNA基因的变性梯度凝胶电泳(Denaturinggradient gel electrophoresis,DGGE)和Real-time PCR技术定性、定量分析梭菌Ⅳ群。【结果】对仔猪结肠中梭菌Ⅳ群DGGE图谱进行相似性分析显示,7日龄仔猪样品归于一簇,数值达90%以上,而与其他日龄的样品间相似性较低,断奶前(21日龄)和断奶后3天菌群则没有显著变化。Real-time PCR定量分析显示,仔猪结肠总细菌拷贝数在断奶后3天显著降低,这一趋势与结肠中的总挥发性脂肪酸和丁酸浓度变化相似,而梭菌Ⅳ群拷贝数变化则不显著。克隆和测序分析表明,仔猪结肠梭菌Ⅳ群中的优势菌为Faecalibacteriumprausnitzii,Subdoligranulum variabile以及一些未培养细菌。【结论】7日龄至14日龄仔猪结肠中梭菌Ⅳ群发生改变,但断奶对其影响不大;该菌群和结肠丁酸浓度关系还需进一步研究。[ Objective ] To investigate the change of Clostridium cluster Ⅳ community in the colon of piglets from 7 to 35 days of age, and its correlation with butyrate concentration. [ Methods] Three litters of neonatal piglets were used. One piglet from each litter was sacrificed randomly at the age of 7, 14, 21 （weaning day） , 24 and 35 days, digestive samples in the colon were collected. The concentration of volatile fatty acid （VFA） was determined by gas chromatography. 16S rRNA gene-based denaturing gradient gel electrophoresis （DGGE） and real-time PCR were used for qualitative and quantitative analysis of Clostridium cluster Ⅳ community. [ Results] Similarity analysis of DGGE profile revealed that samples from piglets at the age of 7 days formed a coherent cluster with indices above 90% , no significant changes in Clostridium cluster IV community were found around weaning period. Real-time PCR analysis showed that 16S rRNA gene copies of total bacteria in the colon of piglets decreased significantly 3 days after weaning, this tendency was in accordance with the changes in concentration of total VFA and butyrate in colon, while there was no significant difference in copies of Clostridium cluster Ⅳ group. Sequencing analysis indicated that Clostridium cluster Ⅳ group in the colon of piglets were dominated by Faecalibacterium prausnitzii, Subdoligranulum variabile and other uncultured bacteria. [ Conclusion ] Changes in Clostridium cluster IV community in the colon of piglets were found from the age of 7 days to 14 days, while there was no significant difference during the weaning transition.

关 键 词：16S RRNA基因 变性梯度凝胶电泳(Denaturing gradient gel ELECTROPHORESIS DGGE) 仔猪 梭菌Ⅳ群 

分 类 号：S852.6[农业科学—基础兽医学]