用PCR-DGGE和16S rDNA测序解析吐温对绵羊瘤胃细菌多样性的影响

选取装置永久性瘤胃瘘管的中国美利奴绵羊,采用5×5拉丁方试验设计,在绵羊日粮中分别添加5或10 g/d Tween 20,3或6 g/d Tween 80,以空白为对照,采用PCR-DGGE技术研究不同水平的非离子表面活性剂Tween 20和Tween 80对绵羊瘤胃中细菌多样性的影响;对DGGE指纹图谱中的12个优势DNA条带的16S rDNA进行测序和序列分析,在线寻找其相似的细菌分类。结果显示,5个组之间DGGE指纹图谱的相似性在65.4%～75.4%。与对照组相比,除10 g/d吐温20组外,其他各组的细菌丰度均有不同程度的下降。DNA序列分析表明,DGGE图谱中优势条带的16S rDNA序列中有8个条带序列与GenBank中已知序列的相似性大于97%。受吐温20影响的细菌与Microbacteriumoxydans、Schlegelella aquatica、Brevundi monas sp.、Lachnospir-aceae和Methylobacillus sp.有较高的相似性;而受吐温80影响的细菌与Schlegelella aquatica、Acinetobacter soli和Microbacteriumoxydans有较高的相似性。日粮中添加吐温20和吐温80后,高剂量的吐温20使绵羊瘤胃中的优势菌种类增加,吐温80和低剂量的吐温20使优势菌种类下降。在绵羊瘤胃细菌中,受吐温20和吐温80影响的种类不同。

Analyzing the Effects of Tween on Rumen Bacterial Diversity of Sheep by PCR-DGGE and 16S rDNA Sequencing

Five Chinese Merino male sheep fitted with perpetual rumen fistula were used in a 5×5 Latin square designed experiment to examine the effects of supplementation Tween 80 and Tween 20 on the rumen bacterial diversity.The five treatments were the basal diet（the control） and the basal diet supplemented with 5 g/d or 10 g/d Tween 20,3 g/d or 6 g/d Tween 80.The ruminal bacterial community of sheep was analyzed by polymerase chain reaction-denaturing gradient gel electrophoresis（PCR-DGGE）.16S rDNA of 12 predominant DNA bands of DGGE fingerprint were sequenced and were analyzed by sequence to search the similar bacterial classification.Results showed that the DGGE fingerprint similarity of the five groups was 65.4%-75.4%.The bacterial richness from other groups were decreased in varying degree,compared with the control group,except for the 10 g/d Tween 20 group.DNA sequence analysis showed that similarities between 8 bands in DGGE image and the known sequence in Genbank were over 97%.16S rDNA sequence analysis revealed that bacterial populations which were affected by Tween 20 had higher similarities with Microbacterium oxydans,Schlegelella aquatica,Brevundimonas sp.,Lachnospiraceae and Methylobacillus sp.and bacterial populations which were affected by Tween 80 had higher similarities with Schlegelella aquatica,Acinetobacter soli and Microbacterium oxydans.It is concluded that predominant bacterial were increased by high dosage of Tween 20,and decreased by Tween 80 with low dosage of Tween 20.The bacterial species were impacted by Tween 20 and Tween 80 were different.