您的位置：首页 > 论文页面
小鼠肠道菌群胞外DNA的提取及指纹图谱分析
发表时间：2016-08-15 浏览量：1710 下载量：671
| 全部作者： | 李 亚，王盼盼，随子房，乐国伟，孙 进 |
| 作者单位： | 江南大学食品学院，食品营养与功能因子研究中心；江南大学食品科学与技术国家重点实验室 |
| 摘 要： | 肠道菌群胞外DNA（extracellular DNA，eDNA）是维持黏膜免疫稳态的重要分子，其主要来源目前尚不清楚。对肠道切片进行TOTO-1染色后用激光共聚焦扫描显微镜观察黏液层原位eDNA分布；并对其提取方法进行优化，制备正常（CT）和高脂饲料处理（high fat diet，HFD）C57BL/6J小鼠肠道eDNA，进行末端限制性片段多态性（terminal restriction fragment length polymorphism，T-RFLP）指纹图谱分析。结果表明，肠道切片黏液层存在丰富的eDNA. 用含0.5 mmol•L-1二硫苏糖醇（DTT）的PBS缓冲液可充分回收黏液，两步酚提可高效制备黏液eDNA，用引物334F/939R进行16S rRNA基因PCR扩增，结合内切酶AluⅠ、DdeⅠ消化，为eDNA进行T-RFLP分析的优化参数。主成分分析（principal component analysis，PCA）结果显示eDNA和细胞内DNA构成存在显著差异。 |
| 关 键 词： | 食品营养学；小鼠；肠道菌群；细胞外DNA；T-RFLP |
| Title： | Preparation for mouse gut microbiota extracellular DNA and fingerprint analysis |
| Author： | LI Ya, WANG Panpan, SUI Zifang, LE Guowei, SUN Jin |
| Organization： | Food Nutrition and Functional Factors Research Center, School of Food Science and Technology, Jiangnan University; State Key Laboratory of Food Science and Technology, Jiangnan University |
| Abstract： | Bacterial extracellular DNA (eDNA) plays important role in keeping gut immune homeostasis. Its composition is not known till now. Mucus layer eDNA was observed in situ with a laser scanning confocal fluorescence microscope after TOTO-1 staining on intestinal sections. Method for eDNA preparation was further optimized. And eDNA from control (CT) and high fat diet (HFD) treated C56BL/6J mice was prepared and its diversities were analyzed by (terminal restriction fragment length polymorphism, T-RFLP). The results revealed that eDNA was enriched in mucus layer. Mucus could be prepared using PBS containing 0.5 mmol•L-1 dithiothreitol (DTT). Then eDNA was obtained using two step phenol extraction. For eDNA specific T-RFLP, 16S rRNA gene should be amplified using 334F/939R primers, the PCR products should be treated with restriction endonuclease AluI and DdeI. Principal component analysis (PCA) of T-RFLP results revealed that eDNA was significantly different from intracellular DNA. |
| Key words： | food nutriology; mice; gut microbiota; extracellular DNA; T-RFLP |
| 发表期数： | 2016年8月第15期 |
| 引用格式： | 李 亚，王盼盼，随子房，等. 小鼠肠道菌群胞外DNA的提取及指纹图谱分析[J]. 中国科技论文在线精品论文，2016，9（15）：1593-1602. |
2
评论数 0请您登录
暂无评论
下载全文
多维论文