PCR-SSCP analysis and 16S rDNA identification of intestinal bacteria in zebrafish
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.
zebrafishintestinal bacteriumisolation and identification16S rDNAPCR-SSCP
NETL
NSTL
万方数据
维普
