PCR-SSCP analysis and 16S rDNA identification of intestinal bacteria in zebrafish
HU Xiu-cai1， Lü Ai-jun1， SUN Jing-feng1， SHI Hong-yue1，YEONG Yiksung1， PEI Chao2， ZHANG Chao2， LI Li2
1.Tianjin Key Lab of Aqua-Ecology and Aquaculture， College of Fisheries， Tianjin Agricultural University， Tianjin 300384， China； 2.College of Fisheries， Henan Normal University， Xinxiang 453007， China
Abstract: Polymerase chain reaction-single-strand conformation polymorphism analysis(PCR-SSCP)of intestinal bacteria of zebrafish Danio rerio was carried out by bacterial isolation，purification and 16S rDNA gene molecular 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 analysis 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 homologus in Zf10 to an uncultured bacterium clone GI3-M-5-G01(92%).The morphology and 16S rDNA sequence analysis of these bacteria indicated that Zf1， Zf7， and Zf11 were primarily belonged to Aeromonas， Zf4 and Zf8 belonged 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 revealing 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 identificaiton of intestinal bacteria and effect on the physiology in zebrafish.
2018, Vol. 33 
