| Tilapia is the dominant species of aquaculture in China with important economic value.But recent years,frequent outbreaks of serious Streptococcus agalactiae disease threat to the healthy development of tilapia industry.Unfortunately,efficient way hasn't been found to prevent this disease.It is necessary to explore the molecular mechanism of disease resistance in tilapia.Innate immune system is the first line of defense system against pathogens and pathogenic products in fish.Pattern recognition receptors(PRRs)are representative of immune innate receptors.They interacts with pathogen-associated molecular patterns(PAMPs),they are the key factors to activate the innate immune response.Ddx41(D-E-A-D(aspartate-glutamate-alanine-aspartate-box)polypeptide 41)is a RNA helicase and encoded by the ddx41 gene,which recognizes the PAMPs produced by the bacteria and viruses as PRR in the host cell and activates the sting-dependent immune response process.Recently,the tripartite motif(TRIM)family of proteins has been found to act on factors which involved in the innate immune signaling pathways and affect innate immune responses recently.In this paper,we cloned ddx41,sting,trim16 and trim25 genes in Oreochromis niloticus,and the gene structure and expression pattern were analyzed to understand their biological function.The eukaryotic expression vectors of these genes were constructed to study the effect of overexpression of these genes on NF-?B activity in 293 T cells.This study provides a theoretical basis for further understanding of the anti-infective immune mechanism of Nile tilapia and exploring new ways of disease prevention and control.1 Isolation and expression analysis of ddx41 and sting genes in Nile tilapiaIn order to evaluate the immune function of ddx41 and sting genes from Nile tilapia,the full-length cDNA sequences of ddx41 and sting genes from Nile tilapia were obtained by RT-PCR and RACE-PCR methods.In addition,the genes structure and secondary structure of deduced protein were also analyzed.Quantitative real-time PCR(qRT-PCR)method was used for analyzing the tissue distribution of ddx41 and sting genes and their expression pattern in the process of embryonic development and response to Streptococcus agalactiae infection.Results showed that the ddx41 gene was 2 230 bp in total length which contained ORF of 1 842 bp encoding 614 amino acid residues.The genome sequence of the ddx41 gene was 8 138 bp and contained 17 exons and 16 introns.The putative protein indicated that Ddx41 had Colied coil domain,DEXDc domain,HELICc domain and ZnF-C2 HC domain.The putative protein of Ddx41 has more than 99.0% identities with Maylandia zebra,Pundamilia nyererei and Neolamprologus brichardi.The putative protein of Ddx41 has 85.6%~97.6% identities with other teleosts and mammals.Tissue distribution analysis revealed that ddx41 gene expressed in all tested tissues with the highest expression level in blood and the lowest expression level in liver.The expression level of ddx41 gene in the blood was 27.51 folds of that in the liver(control group).ddx41 gene was expressed stably in embryonic development of Nile tilapia.Upon stimulation with intraperitoneal injection with S.agalactiae,the expression levels of ddx41 genes were up-regulated in intestine,spleen,gill and kidney.The highest expression levels of ddx41 gene in intestinal,spleen,gill,kidney,were 4.05,5.74,4.12 and 2.27 folds of the 0 h(control group)respectively and the expression level in the blood showed a decreasing trend.Results showed that the sting gene was 2 724-bp in total length which contained ORF of 1 209 bp encoding 403 amino acid residues.The genome sequence of the sting gene was 10 746 bp and contained 6 exons and 5 introns.The putative protein of sting has 28.5%~94.9% identities with other teleosts and mammals.The putative protein of sting exhibited highest identity with Maylandia zebra(95.2%).Tissue distribution analysis revealed that sting genes expressed in all tested tissues with the highest expression level in intestinel and the lowest expression level in heart.sting gene was expressed in the embryonic development of Nile tilapia and the expression level was gradually increased.Upon stimulation with intraperitoneal injection with S.agalactiae,the expression levels of sting genes were up-regulated in intestine,spleen,gill and kidney.The highest expression levels of sting gene in intestinel,spleen,gill,kidney and blood were 4.99,15.19,15.74,5.81,6.33 folds of the 0 h respectively.The results showed that ddx41 and sting genes play an important role in the immunoreaction of Nile tilapia against S.agalactia infection.This study provides a theoretical basis for further understanding of the anti-infective immune mechanism of Nile tilapia and exploring new ways of disease prevention and control.2 Isolation and expression analysis of trim16 and trim25 genes in Nile tilapiaIn order to evaluate the immune function of trim16 and trim25 genes from Nile tilapia,the full-length cDNA sequences of trim16 and trim25 genes from Nile tilapia were obtained by RT-PCR and RACE-PCR methods.In addition,the genes structure and secondary structure of deduced protein were also analyzed.qRT-PCR method was used for analyzing the tissue distribution of trim16 and trim25 genes and their expression pattern in the process of embryonic development and response to Streptococcus agalactiae infection.Results showed that the trim16 gene was 2314-bp total in length which contained an open reading frame(ORF)of 1674-bp encoding 558 amino acid residues.trim25 gene was 2748-bp in total length which contained ORF of 1674-bp encoding 558 amino acid residues.trim16 and trim25 genes were all without introns.The putative protein indicated that the Trim16 and Trim25 have conserved structures of the trim family,such as the RING finger domain and the B-box domain.The putative protein of Trim16 has 29.0%~ 92.6% identities with other teleosts and mammals.The putative protein of Trim25 gene has 20.0%~88.1% identities with other teleosts and mammals.The putative protein of Trim16 and Trim25 genes exhibited highest identity with Maylandia zebra(92.6% and 88.1% respectively).Tissue distribution analysis revealed that trim16 and trim25 genes expressed in all tested tissues with the highest expression level in the blood and the lowest expression level in the liver.The expression levels of trim16 and trim25 genes in the blood were 60.46 and 274.07 folds of that in the liver respectively.Both trim16 and trim25 genes expressed during the embryonic development of Nile tilapia,which suggested that they played an important role in the immune process of Nile tilapia in the early stage of embryonic development.Upon stimulation with intraperitoneal injection with S.agalactiae,the expression level of trim16 and trim25 genes were up-regulated in all test tissues(intestine,spleen,gill,kidney and blood).The highest expression levels of trim16 gene in intestinel,spleen,gill,kidney and blood were 1.30,2.09,1.61,7.81 and 6.10 folds of the 0 h(control group)respectively.The highest expression levels of trim25 gene in the intestine,spleen,gill,kidney and blood were 11.13,1.22,1.26,61.41 and 77.80 folds of the control group respectively.The expression levels of trim16 and trim25 genes were significantly exaltation in the kidney and blood.The results showed that trim16 and trim25 genes play an important role in the immunoreaction of Nile tilapia against S.agalactia infection.This study provides a theoretical basis for further understanding of the anti-infective immune mechanism of Nile tilapia and exploring new ways of disease prevention and control.3 The impact of overexpression of ddx41,sting,trim16 and trim25 genes on NF-?B activity in 293 T cellsTo further study the function of ddx41,sting,trim16 and trim25 genes,the eukaryotic expression vector of these genes was constructed.The pcDNA3.1-ddx41,pcDNA3.1-sting,pc DNA3.1-trim16 and pcDNA3.1-trim25 expression vectors were transfected into 293 T cells with NF-?B firefly reporter plasmid and RL-TK renliia luciferase plasmid.The results showed that ddx41,sting,trim16 and trim25 genes eukaryotic expression vectors could stably express and enhance NF-?B activity in 293 T cells.The results showed that the above genes played an important role by control of NF-?B activity for resistance to S.agalactiae in Nile talipia. |
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