斑马鱼肠道细菌的16S rDNA分子鉴定及PCR-SSCP分析

摘要

Polymerase chain reaction-single-strand conformation polymorphism analysis (PCR- SSCP) of intesti-nal bacteria of zebrafish Danio rerio was carried out by bacterial isolation,purification and 16S rDNA gene molecu-lar identification techniques in order to understand the intestinal bacterial diversity in zebrafish. The results showed that the 12 strains,designated as Zf1-Zf12,were isolated from the intestine in zebrafish. Furthermore,16S rDNA of 7 strains was amplified by PCR and then sequenced from the clones of constructed pMD18-T/16S rDNA. The a-nalysis revealed that the 16S rDNA sequences of Zf1 and Zf11 strains had 99% similarity to Aeromonas veronii,Zf4 and Zf8 99% to Sphingomonas sp.,Zf5 99% to Bacillus subtilis,and Zf7 99% to Aeromonas sp.,with high homolo-gus in Zf10 to an uncultured bacterium clone GI3-M-5-G01(92%). The morphology and 16S rDNA sequence a-nalysis of these bacteria indicated that Zf1,Zf7,and Zf11 were primarily belonged to Aeromonas,Zf4 and Zf8 be-longed to Sphingomonas,Zf5 belonged to Bacillus and Zf10 was identified as a new strain.There were differences in band profiles of 16S rDNA V3 region PCR-SSCP in these bacteria of zebrafish,with the same band pattern in both Zf1 and Zf11,and the same band pattern in both Zf4 and Zf8.The findings can provide a scientific reference for re-vealing the effects of the intestinal microflora structure on fish life activities since SSCP results are consistent with those of the molecular identification of the sequence of 16S rDNA,and provide a scientific reference for the identif-icaiton of intestinal bacteria and effect on the physiology in zebrafish.%为了解斑马鱼Danio rerio肠道细菌多样性,采用细菌分离纯化、16S rDNA基因分子鉴定技术,对斑马鱼肠道细菌进行PCR-SSCP分析.结果表明:从斑马鱼肠道分离纯化出12株细菌,分别命名为Zf1、Zf2、Zf3、Zf4、Zf5、Zf6、Zf7、Zf8、Zf9、Zf10、Zf11和Zf12,其中对7株分离菌构建pMD18-T/16S rDNA的阳性克隆测序显示,Zf1、Zf11菌株与Aeromonas veronii的16S rDNA序列一致性为99%, Zf4、Zf8菌株与Sphingomonas sp.的一致性为99%, Zf5菌株与Bacillus subtilis的一致性为99%, Zf7菌株与Aeromonas sp. M10的一致性为99%, Zf10菌株与uncultured bacterium clone GI3-M-5-G01的一致性为92%; 16S rDNA分子鉴定表明,Zf1、Zf7和Zf11属于气单胞菌属Aeromonas, Zf4、Zf8属于鞘氨醇单胞菌属Sphingomonas, Zf5属于芽孢杆菌属Bacillus, Zf10为一种新菌株;采用PCR-SSCP技术对12株细菌16S rDNA基因V3区进行多态性分析,结果显示,斑马鱼肠道细菌16S rDNA基因V3区存在多态性,其中Zf1与Zf11菌株带型一致,Zf4与Zf8菌株带型一致.本研究结果可为揭示鱼类肠道菌群结构对其生命活动的影响提供科学参考.