| Foxp subfamily, which is composed of4members (Foxpl—p4), is defined by its characteristic of a110amino acid long DNA-binding forkhead (FKH) domain. In addition, four members of Foxp subfamily contain a ZnF_C2H2domain and a Leucine zipper-liked domain, which can form homologous or heterologous dimmers to perform functions. In mammals, the latest research shows that Foxp1plays an important role in the immune system by regulating the development of T cells and B cells; Foxp2is the first gene found to be related to speech and language disorder; Foxp3is an essential and sufficient transcription factor in the development and function of the regulatory T cells, and the expression levels of Foxp3is closely associated with the clinical prognosis of anti-tumor immunity, autoimmune diseases, anti-infection immunity and allograft tolerance; Foxp4participates in the histogenesis of lung, gut and nervous system, and has some relevant to T cell mediated immune response. Nowadays, researches on the Foxp subfamily have received considerable attention.Teleost fishes are the most primitive vertebrates with both non-specific and specific immune response, which have a special position in research of immune molecules from the perspective of molecular evolution. However, the study of Foxp in teleosts is scare till now, only a primary study of Foxp from a few teleost fishes such as zebrafish, grass carp was performed, which was mainly about the spatio expression patterns during different developmental stages. On the other hand, more and more studies show that estrogen can affect the immune function by regulating the expression of immune molecules which are involved in the immune response regulation. In teleost, whether estrogen is involved in the expression regulation of Foxp has not yet been reported.Tilapia (Perciformes:Cichlidae) has many advantages, such as feeding miscellaneous, fast growth, so it has a great application value and market potential. However, the immune related knowledge of tilapia is limited. Therefore, the cDNA sequences of Foxp1a/1b/2/4were cloned from tilapia (tilapia referred to Nile tilapia (Oreochromis niloticus) in this study without special instructions) and analyzed by bioinformatics analysis and RT-PCR. On the other hand, the expression patterns and the regulation factors of Foxp1a/1b/2/3/4were intensively investigated. Results in detail are as follows:1. Foxp1a/1bThe full length of the tilapia Foxp la open reading frame was1710bp, which contained15exons and encoded569amino acids. Meanwhile, Foxplb was2040bp, which contained16exons and encoded679amino acids. Protein sequences analysis revealed that tilapia Foxpla/lb shared similarities.with other vertebrate homologs of50-55.4%and65.7-83.8%, respectively. Both of tilapia Foxp1a and Foxp1b contained a FKH domain, a C2H2zinc finger domain and a leucine zipper-like domain. Additionally, Foxplb contained a super-secondary structure of PLNLV that could combine with CtBP1. The synteny and phylogenetic analysis of Foxp revealed that tilapia Foxp1a/1b were orthologous genes of mammal Foxpl, while tilapia Foxp1a/1b might be derived from teleost specific genome duplication. The tissue distributions of Foxpla/lb were detected by qPCR, and the results showed that the expression levels of Foxpla in the immunological tissues were significantly lower than those of Foxplb. The highest expression level of tilapia Foxpla was observed in testis, followed by brain and ovary, while Foxp1b highest in heart, followed by kidney and liver. Stimulation of peripheral blood mononuclear cells (PBMCs) with PHA and PMA led to a significant increase of tilapia Foxp1b at6h, and highest at12h (p<0.05), whilst LPS led to a decrease at12h then increase at24h comparing to the control. The mRNA expression of tilapia Foxplb in the kidney significantly increased at6h (p<0.05) and maintained up to24h (p<0.05) after intraperitoneal injection of2×107live Aeromonas hydrophila. These results suggested that tilapia Foxplb might be involved in lymphocyte activation and immune response. The expression levels of Foxplb mRNA in the kidney, intestine and spleen from6-month-old males were detected by qPCR, the results showed that E2could significantly increase the mRNA expression levels of Foxplb in the kidney and intestine (p<0.05), whilst decrease the mRNA expression level of Foxplb in the spleen (p<0.05). The results suggested that exogenous E2could regulate the expression of Foxplb in teleosts similar to that in mammals.2. Foxp2The full length of the tilapia Foxp2open reading frame was2298bp, which contained16exons and encoded765amino acids. Protein sequences analysis revealed that tilapia Foxp2shared a similarity with other vertebrate homologs of71.5-88.4%, respectively. Tilapia Foxp2contained a FKH domain, a C2H2zinc finger domain and a leucine zipper-like domain. Additionally, Foxp2contained a supersecondary structure of PLNLV that could combine with CtBPl. The synteny and phylogenetic analysis of Foxp revealed that tilapia Foxp2was an orthologous gene of mammal Foxp2. The tissue distribution of Foxp2was detected by qPCR, and the results showed that the highest expression level of tilapia Foxp2was observed in brain, followed by muscle, heart and kidney. The result suggested that tilapia Foxp2might mainly perform functions in central nervous system, as well as the mammals.3. Foxp3(1) The tissue distribution of Foxp3was detected by qPCR, and the results showed that tilapia Foxp3was primarily expressed in immune-related organizations. Among them, the highest mRNA expression level was observed in the spleen, followed by head-kidney, intestine and spleen. Stimulation of PBMCs with PHA led to a significant increase of tilapia Foxp3at6h (p<0.05), LPS led to a significant increase of tilapia Foxp3at24h (p<0.05), whilst PMA led to a significant decrease at24h comparing to the control (p<0.05). The mRNA expression of tilapia Foxp3in the spleen significantly decreased at6h (p<0.05) and maintained down to1w (p<0.05) after intraperitoneal injection of2×107live Aeromonas hydrophila. These results suggested that tilapia Foxp3might be involved in lymphocyte activation and immune response.(2) Among the6-month-old control females, Fadrozole-treated females, control males and E2-treated males, the highest expression levels of tilapia Foxp3in the intestine and kidney were detected in the control females with the highest concentration of E2, while the lowest expression levels of tilapia Foxp3in the control males with the lowest concentrations of E2. After the3-month-old control females were fed with400fj,g the inhibitor of E2synthase (Fadrozole) per g feed for three months, the serum E2concentrations were dramatically down-regulated corresponding with the decreasing expression levels of Foxp3in the intestine and kidney (p<0.05), while the serum E2concentrations were dramatically up-regulated after administrating6-month-old males with100ng E2at48h corresponding with the increasing mRNA expression levels of tilapia Foxp3(p<0.05). These results indicated that the level of estrogen in vivo was positively related with the expression level of Foxp3.(3) Foxp3+colonies were obtained through screening the genome Fosmid library of tilapia. Combination of DNA walking sequencing and PCR amplification, the promoter region of tilapia Foxp3was obtained, which was2266bp. Subsequently, the luciferase reporter gene plasmids containing Foxp3promoter region and its serial deletions were constructed, named pGL3-Foxp3(-2014bp), pGL3-Foxp3(-1280bp) and pGL3-Foxp3(-502bp), respectively. In this study, the complete coding sequences of p65from tilapia were obtained through bioinformatical analysis and RT-PCR, which was1836bp, and the eukaryotic expression vector pcDNA3.1-p65was constructed. Then the luciferase reporter gene plasmids and pcDNA3.1-p65were transfected into HEK293cell line by Lipofectin2000. After48h, the luciferase activities were measured. Our results indicated that pGL3-Foxp3(-1280bp) possessed basal transcriptional activity, whereas pGL3-Foxp3(-2014bp) and pGL3-Foxp3(-502bp) had not any detectable transcriptional activities. What’s more, the trancripition factor p65had almost no effect on the transcriptional activities of Foxp3promoter.4. Foxp4The full length of the tilapia Foxp4open reading frame was2139bp, which contained17exons and encoded712amino acids. Protein sequences analysis revealed that tilapia Foxp4shared a similarity with other vertebrate homologs of66.8-85.8%, respectively. Tilapia Foxp4contained a FKH domain, a C2H2zinc finger domain and a leucine zipper-like domain. The synteny and phylogenetic analysis of Foxp revealed that tilapia Foxp4were an orthologous gene of mammal Foxp4. The tissue distribution of Foxp4was detected by qPCR, and the results showed that the highest expression level of tilapia Foxp4was observed in brain, followed by heart and kidney. Stimulation of PBMCs with PMA led to a significant increase of tilapia Foxp4at6h, and highest at12h (p<0.05), LPS led to a significant increase at24h comparing to the control (p<0.05), whilst PHA had no significant influence. The mRNA expression of tilapia Foxp4in the kidney significantly increased at6h (p<0.05) and maintained up to24h (p<0.05) after intraperitoneal injection of2×107live Aeromonas hydrophila. These results suggested that tilapia Foxp4might be involved in lymphocyte activation and immune response. The expression levels of Foxp4mRNA in the kidney, intestine and spleen from6-month-old males were detected by qPCR, the results showed that E2could significantly increase the mRNA expression levels of Foxp4in the kidney and intestine (p<0.05), while the mRNA expression level of Foxp4in the spleen had not changed (p <0.05). The results suggested that exogenous E2could also regulate the expression of Foxp1b in teleosts.This study will not only provide scientific data for the research on molecular evolution of Foxp members, but also promote further study of the fish molecular immune and lay the foundation for the immune prevention and treatment of fish diseases. |
PDF Full Text Request