| Toll-like receptors(TLR)are a class of pattern recognition receptors(PRRs)that can recognize pathogen-associated molecular patterns(PAMPs)and play important roles in the innate immune system in vertebrates.In this study,we identified a teleost fish-specific tlr22 gene from yellow catfish(Pelteobagrus fulvidraco),cloned its tlr22 gene,and analyzed its molecular and evolutionary biological characteristics.The expression pattern of tlr22 gene in different tissues and developmental stages of yellow catfish was detected by real-time PCR.To detect the expression pattern of tlr22 gene after injection of Aeromonas hydrophila and Poly(I:C).To detect the expression pattern of tlr22 gene after LPS and Poly(I:C)infection with macrophages of yellow catfish.The promoters of the immune-related tlr22 gene in yellow catfish were analyzed,and the binding sites of important transcription factors on the promoters of their genes were predicted.The transcription level regulation of transcription factors(Myb,Oct-1,Sox5)in various processes and tissues was studied by real-time PCR and other methods.The primary findings are shown as follows:(1)The open reading frame(ORF)of the tlr22 was 2892 bp in length,encoding a protein of 963 amino acids.Multiple protein sequences alignment,secondary and three-dimensional structure analyses revealed that TLR22 is highly conserved among different fish species.Phylogenetic analysis showed that the phylogenetic topology was divided into six families of TLR1,TLR3,TLR4,TLR5,TLR7 and TLR11,and TLR22 subfamily was clustered into TLR11 family.(2)Meanwhile,synteny and gene structure comparisons revealed functional and evolutionary conservation of the tlr22 gene in teleosts.Further-more,tlr22 gene was shown to be widely expressed in detected tissues except barbel and eye,with highest expression level in liver.Early development results showed that tlr22 gene was expressed at all stages,including unfertilized eggs,and its expression was highest at 0 h,suggesting the maternal expression of tlr22 gene.(3)The transcription of tlr22 was significantly increased in spleen,kidney,liver and gill tissues at different timepoints after Poly(I:C)infection,suggesting tlr22 plays critical roles in defensing virus invasion.Similarly,the transcription of tlr22 was also dramatically up-regulated in spleen,kidney and gill tissues with different patterns after A hydrophila infection,indicating that tlr22 is also involved in resisting bacteria invasion.Further verification showed that after LPS and Poly(I:C)stimulated macrophages of yellow catfish,the expression of tlr22 decreased significantly at different time points.These results imply that the tlr22 gene may play a role in protecting fish from invasion by gram-negative bacteria and doublestranded RNA viruses.(4)Promoter predictive analysis showed that the promoter of tlr22 contained binding sites of transcription factors including Myb,Oct-1,and Sox5,and one Cp G island site was also observed,implying these transcription factors may have potential regulatory roles on tlr22 transcription.Furthermore,the expression level of myb,oct-1 and sox5 in various treatment groups and tissues was also detected,and the results showed that the change trend of myb was consistent with the expression pattern of tlr22,revealing its potential positive regulatory effect.Differently,the expression patterns of oct-1 and sox5 genes were totally opposited to those of tlr22,revealing their potential negative regulatory effects.In summary,our results are helpful to systematically investigate the immune response mechanisms and defense mechanisms of yellow catfish in response to different pathogens at the molecular and cellular levels.Our findings provide a solid foundation for studying the immune function of teleost tlr22 gene and provides useful information for the prevention and treatment in yellow catfish.It also provides a theoretical basis for improving fish immunity and improving aquaculture disease prevention technology. |
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