【作者】 李素娟； 张伟武； 苏长城； 虞慧芳； 王欣； 王靖；

【Author】 Li Sujuan, Zhang Weiwu, Su Changcheng, Yu Huifang, Wang Xin, Wang Jing (Zhejiang Academy of agriculture sciences, HangZhou,310021; HangZhou Academy of agriculture sciences,HangZhou,310021; Hangzhou Aopal Biotech. Co. Lid; Biochemical Engineering, China university of petroleum,BeiJing, 102249)

【机构】 浙江省农业科学院； 杭州市农业科学院； 杭州华扬奥博生物技术有限公司； 中国石油大学生物化工；

【摘要】 雏鸡肠道微生物菌群的组成变化与肉鸡的营养和免疫状况密切相关。PCR-DGGE结合16S rDNA序列分析的方法是在DNA水平上进行微生物生态研究的有力手段。0日龄雏鸡分为试验和对照两组,试验组在第一次饮水中添加106微生态制剂华扬雏宝。2、3、4日龄从雏鸡肠道内容物中提取基因组总DNA,扩增山16S rDNA V6-V8区片段的PCR产物经变性梯度凝胶电泳分离。从DGGE 图谱上清楚显示出了雏鸡肠道微生物菌群随雏鸡不同发育时期的变化。欧式聚类距离分析显示肠道菌群结构直接受到饲用微生态制剂的影响。同时,对3个不同DGGE条带切胶回收DNA并克隆测序得到的23个序列,BLAST分析检索并选定部分亲缘关系最近的菌种。发现其中10个序列和 Genebank中已发表序列的相似性超过98％。其余序列的相似性超过92％。乳杆菌属、肠杆菌属和瘤胃球菌属出现的频率比较高。但大部分序列为不可培养菌或未鉴定细菌。在对照组(无菌水)肠道中鉴定出志贺氏菌的序列而微生态制剂组肠道中发现丁酸产生菌。这些结果为详细了解雏鸡肠道微生物群落组成和饲料微生态添加剂对雏鸡肠道微生物群落结构的作用影响提供了有用信息,对家禽养殖业中防止家禽肠道疾病、提高肉料比有重要实用价值。文章还讨论了使用PCR-DGGE方法进行微生物生态研究的优越性和局限性。更多还原

【Abstract】 Compositional variation of intestinal microflora is closely correlated with nutrition absorption and disease resistance in broilers. The method combining PCR-DGGE(denaturing gradient gel electrophoresis) with 16SrDNA sequence analyses has been proved as a powerful approach for micro-ecological studies at DNA level. Bacterial genomic DNA was extracted from the cecum samples of two groups of broilers with and without direct feed microbial supplements. PCR products of the V6-V8 region of 16S rDNA amplified were separated by DGGE. Results from the band pattern analysis clearly demonstrated the compositional change accomplished with different developmental stage of broilers and the impacts of direct feed microbial additives. DNA sequencing was then carried out on 23 clones obtained from cloning of DNA isolated from 3 different DGGE bands. 10 out of 23 sequences demonstrated 98% similarity to the published sequences in Genebank, indicating they are belong to the same species. 19 out of 23 sequences have 95% similarity to the published sequences, indicating they are belong to the same genus. Bacterial species represented by these sequences were mostly uncultured and unidentified bacteria although Lactobacillus, Ruminococcus and Enterobacter appeared more popular. It is interested to see that there were one pathogenic bacteria identified from control group and one butyrate producing bacteria species identified from probiotic group. The results provided useful information in better understanding the composition and variation of intestinal microflora and the influence of direct feed microbial additives on the change of microflora composition. The advantage and limitation of applying PCR-DGGE method in micro-ecological studies were also discussed.更多还原