| Summary: | 碩士 === 國立臺灣海洋大學 === 水產養殖學系 === 104 === In this study, we would like to understand the molecular mechanism of immune regulation in Oreochromis niloticus against Streptococcus iniae. Firstly, we used transcriptomes and RNAseq to analyze expression changes of whole transcriptome of Nile tilapia NT1 strain before and after infection by nonlethal dose (1 x 10^4 CFU/g BW) of S. iniae and revealed differential regulation of three distinct HAMP genes, encoding HAMP1 (87 a.a.), HAMP2 (90 a.a), and HAMP3 (83 a.a.) precursor proteins. Mature hepatic antimicrobial peptide (HAMP/Hepcidin) is a ~20 a.a. AMP and an iron regulatory hormone, and its structure composed of 6 to 8 cysteines to form disulfide bonds are conserved in evolution. Moreover, we used quantitative RT-PCR to analyze relative expression of three HAMP genes in 14 tissues including brain, liver, muscle, eye, heart, skin, gill, head kidney, kidney, spleen, stomach, intestines, fin, and gonad of Nile tilapia in male and female with no infection. Three Nile tilapia HAMP genes were all abundantly expressed in the liver. HAMP2 also expressed in spleen and head kidney whereas HAMP1 and HAMP3 little expressed in these two tissues. The LD50 of S. iniae for Nile tilapia NT1 strain was estimated to be around 1.5 x 10^5 CFU/g BW. To study their differential expression profiles of three HAMP genes in Nile tilapia after infection by S. iniae in dose of LD50, total RNAs from liver, spleen, head kidney and gill were isolated at time interval of 0, 3, 6, 9, 12, 18 hours post-infection (hpi) and analyzed by quantitative RT-PCR. HAMP1 was strongly activated in liver and spleen of Nile tilapia at 6 hpi and reached to maximum at 12 hpi in liver, spleen, head kidney and gill. HAMP3 was early activated at 3 hpi in liver and reached to highest level at 12 hpi in liver, spleen, head kidney and gill of Nile tilapia. HAMP2 was highly activated at 3 hpi and 6 hpi in liver, spleen and head kidney but reached to peak at 12 hpi in gill. As upstream stimulator of the HAMP gene in mammals, Nile tilapia IL-6 was early activated at 3 hpi and highest expressed at 6 hpi in spleen to induce expression of HAMP genes via activation of STAT3. Overall, HAMP1 gene had highest expression level in response to S. iniae infection than HAMP2 and HAMP3 genes not only in liver but also in spleen, head kidney and gill of Nile tilapia. HAMP3 activation was stronger by S. iniae in liver, head kidney and gill than HAMP2 whereas HAMP2 activation was stronger in spleen than HAMP3. Due to their liver-abundant and pathogen-inducible expression, 3.8 kb HAMP1 promoter and two distinct HAMP3 genes, HAMP3a and unpredicted HAMP3b, 2.6 kb promoters of Nile tilapia were obtained by genomic walking and PCR amplification. Putative binding sites of liver-enriched and immune-related transcription factors including HNF1α, HNF3β, HNF4α, C/EBPα, NFκB, IRFs and STAT3 were predicted. Especially, duplicated HAMP3 genes, HAMP3a and HAMP3b encoding identical HAMP3 protein composed of three disulfide bonds in 19-a.a. HAMP3 mature peptide but under the control of different promoter regulation due to sequence variations and deletions in promoters were firstly identified in tilapia. In conclusion, Nile tilapia HAMP genes including HAMP1, HAMP2 and HAMP3, were differentially activated in the liver, spleen, head kidney and gill in response to S. iniae infection to contribute for defense against S. iniae. Furthermore, in vivo tissue-specific and Streptococcus-inducible promoter activities of Nile tilapia HAMP genes in the liver, spleen, head kidney and gill will be investigated in transgenic zebrafish and tilapia.
|
|---|
