| Tilapia is one of the most important fresh-water aquaculture species in China. However, the development of tilapia cultivation has been severely threatened due to the fish disease in recent years. Streptococcus agalactiae infection is considered to be the most important pathogen. Unfortunately, there are not the effective prevention and treatment measures against the disease. Research of the immune-related genes and their functional analysis can reveal the immune regulation mechanism in Nile tilapia. This can provide the conditions for further screening of disease-related markers and breeding of disease-resistance strain of Nile tilapia. The pathogen identification of pattern recognition receptors is a critical step to trigger innate immune response. Toll-like receptors(TLRs), the key pattern recognition receptors, have important roles in defense against infectious pathogens. In the present study, the full lengths of TLRs(TLR2、TLR3、TLR5、TLR13、TLR21、TLR22 and TLR23) c DNA sequences were obtained from Nile tilapia(Oreochromis niloticus) by RT-PCR and rapid amplification of c DNA ends(RACE) approaches. And according to the sequences of TLRs c DNA, specific primers were designed and Quantitative real-time PCR was used to detect the expression levels in various tissues and organs, and the development after fertilization, and bacteria treated tilapia. Recombinants pc DNA3.1-TLR2, pc DNA3.1-TLR21, pc DNA3.1-TLR22 and pc DNA3.1-TLR23 were successfully constructed. The impact on expression profiles of TLR signaling pathway-related genes and NF-κB activation after overexpression the TLRs recombinants were detected. Methods of cloning and sequencing were used to understand the relationship between polymorphism of the TLR22 and TLR23 genes and the disease resistance of nile tilapia. This study has identified potential candidate genes for further investigation into the breeding of disease-resistant strain of Nile tilapia. The main results are as follows in this dissertation.1. TLRs c DNA cloning and sequences polymorphism analysis in Nile tilapiaThe full lengths of TLRs(On TLR2, On TLR3, On TLR5, On TLR13, On TLR21, On TLR22 and On TLR23) c DNA were obtained from Nile tilapia(Oreochromis niloticus) by homology cloning, RT-PCR and rapid amplification of c DNA ends(RACE) approaches. And then the sequences of the full-length TLRs gene(On TLR3, On TLR21, On TLR22 and On TLR23) were able to obtain. The On TLR2 c DNA was 3677 bp in length, and its ORF was 2670 bp, encoded 889 amino acids. The full length of On TLR3 gene 5662 bp, four exons and three introns were identified in On TLR3, its ORF was 2739 bp, encoded 912 amino acids. The On TLR5 c DNA was 3500 bp in length, and its ORF was 2664 bp, encoded 887 amino acids. The On TLR13 c DNA was 3905 bp in length, and its ORF was 2835 bp, encoded 944 amino acids. The full length of On TLR21 gene was 3196 bp(Gen Bank No. KJ010824), its ORF was 2940 bp, encoded 979 amino acids. The full length of On TLR22 gene was 3440 bp(Gen Bank No.KJ010825), three exons and two introns were identified in On TLR22, its ORF was 2808 bp, encoded 935 amino acids. The full length of On TLR23 gene 3789 bp, three exons and two introns were identified in On TLR23, its ORF was 2817 bp, encoded 938 amino acids.2. Tissue expression of Nile tilapia TLRs geneTo determine the expression of TLRs genes in untreated tilapia and the development after fertilization, and bacteria treated tilapia primer pairs were designed based on the TLRs c DNA sequences of Nile tilapia. Quantitative real-time RT-PCR demonstrated that TLRs genes were ubiquitously expressed in ten tissues of the Nile tilapia, but the expression level was different among different tissues. The high levels of On TLR2 transcripts were detected in brain and stomach, and low expression in liver. The high levels of On TLR3 transcripts were detected in spleen, and low expression in liver. The high levels of On TLR5 transcripts were detected in blood, and low expression in kidney. The high levels of On TLR13 transcripts were detected in spleen, and low expression in liver. The high levels of On TLR21 transcripts were detected in gill, and low expression in liver. The high levels of On TLR2 transcripts were detected in spleen, and low expression in liver. The high levels of On TLR23 transcripts were detected in blood, and low expression in stomach.The expression pattern of TLR during embryonic development showed that the expression levels of On TLRs at 5- 6.5 days post fertilization(dpf) were significantly higher than that of other 8 time points(2.5 dpf, 3.5 dpf 4.5 dpf, 5 dpf, 6 dpf, 6.5 dpf, 7.5 dpf and 8.5 dpf).Besides, On TLR2, On TLR3, On TLR5, On TLR13 and TLR21 transcriptional levels were significantly up-regulated in test tissues after intraperitoneal injection with Streptococcus agalactiae. While On TLR22 and On TLR23 transcriptional levels were significantly down-regulated in test tissues.3. Overexpression the TLRs in Nile tilapia, the expression profiles of TLR signaling pathway-related genes after Streptococcus agalactiae infectionHealty Nile tilapia was administered intramuscularly with pc DNA3.1/CT-GFP-TOPO?-TLRs eukaryotic expression vector at 20 ug. At 48 h post-intramuscularly, the fish were administered intramuscularly with PGN at 20 ug. At 24 h post-intramuscularly, the fish were challenged intraperitoneally 100ul(103CFU) of S. agalactiae. Then the expression profiles of TLR signaling pathway-related genes were detected. The results were found that the expression of Myd88 in intestin and liver after overexpression TLRs was significantly up-regulation, down-regulation were tested in brain. Significant up-regulation of TRAF3 after overexpression TLRs and Myd88 was observed in the intesin. At the same time, the expression of TRAF6 and TRF3 in intestine was also up-regulation at most time point after overexpression TLRs and Myd88.4. the impact on NF-κB activation after overexpression the TLRs in 293 T cellsRecombinant pc DNA3.1-TLR2, pc DNA3.1-TLR21, pc DNA3.1-TLR22, pc DNA3.1-TLR23 and pc DNA3.1-Myd88 were successfully expressed in 293 T cells. The signal adapter molecules of TLR2, TLR21, TLR22 and TLR23 in Nile tilapia were Myd88. The pc DNA3.1-TLR2, pc DNA3.1-TLR21, pc DNA3.1-TLR22, pc DNA3.1-TLR23 and pc DNA3.1-Myd88 expression vectors were all successfully transfected into 293 T cells with NF-κB firefly reporter plasmid and RL-TK renliia luciferase plasmid. The results showed that the overexpression of pc DNA3.1-TLR2, pc DNA3.1-TLR21, pc DNA3.1-TLR22 and pc DNA3.1-TLR23 could fail to activate NF-κB, but pc DNA3.1-TLRs and pc DNA3.1-Myd88 were all overexpression together in 293 T cells, they could increased NF-κB activation.5. TLRs polymorphism and its relationship with Streptococcus susceptibility in Nile tilapiaIn order to understand the relationship between polymorphism of the TLRs genes and the disease resistance of Nile tilapia, the On TLR22 and On TLR23 gene fragment was amplified from 20 susceptible nile tilapia individuals and 20 resistant nile tilapia individuals in 20 families. 25 polymorphic loci and nine insert/delete mutation were founded in On TLR22. 16 polymorphic loci and three insert/delete mutation might be associated with the resistance of Nile tilapia to S. susceptibility. On TLR23 sequence analysis showed that 20 mutations site. 14 of them locate in the coding sequence region. No disease-related genotypes were founded in On TLR22 and On TLR23 genes. These variabile sites were only obtained in susceptible and resistant groups, and we were going to intended to verify its performance in famlies in order to further analyze the potential applications. This study laid the foundation for disease resistance breeding of Nile tilapia. |
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