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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:1282  下载量:466
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.
 
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