Molecular Cloning And Expression Analysis Of TLR21,TLR22 And Their Related Genes From Golden Pompano(Trachinotus Ovatus)

Toll-like receptors(TLRs),as an important pattern recognition receptor family(pattern-recognition receptors,PRRs),are responsible for recognizing pathogen associated molecular patterns(PAMPs)and ultimately initiate the innate immunity to eliminate the invasion of pathogenic microorganisms.Besides their functions in initiating innate immunity,TLRs can modulate adaptive immune response,known as a bridge to linking native and adaptive immunity.TLR21 and TLR22 are two important members of TLRs family,mainly identified in birds,amphibians or fish,whilst TRAF3 and IRAKI are key molecules in TLR signaling pathway,involved in TLR signaling cascase.Trachinotus ovatus,an economically important marine fish species,is widely cultured in South China.With the development of a large scale fanning,a variety of infectious diseases outbreak frequently and resulted in great economic loss in Trachinotus ovatus industry.Due to the crucial roles that TLR21,TLR22,TRAF3 and IRAKI play in immunity,the full-length of cDNA sequences of TLR21,TLR22,TRAF3 and IRAK1 gene from Trachinotus ovatus(named as TrTLR21,TrTLR22,TrTRAF3 and TrIRAK1)were cloned by RACE-PCR and RT-PCR in this study.Additionally,the sequence characteristics and protein structures of TrTLR21,TrTLR22,TrTRAF3 and TrIRAK1 were analyzed by using bioinformatics.Furthermore,the distribution of TrTLR21?TrTLR22?TrTRAF3 and TrIRAK1 in tissues from healthy Trachinotus ovatus and the expression change in tissues,incuding gill,spleen,head kidney,and liver after the stimulation by different immune stimulants(Vibrio alginilyticus,LPS and PolyI:C were analyzed by real-time quantitative PCR.The main research results are as follows:1.The full-length cDNA of TrTLR21,TrTLR22,TrTRAF3 and TrIRAKI are 3184 bp,3454 bp,2564 bp and 2818 bp,respectively.The ORF of TrTLR21 and TrTLR22 are 2931 bp and 2880 bp in length,encoding a putative protein of 976 and 959 amino acids,respectively.Both predicted proteins(TrTLR21 and TrTLR22)consist of an extracellular domain with a signal peptide and LRR motifs,a transmembrane domain and a TIR domain,both have typical characteristics of TLRs.However,the number of LRRs in TrTLR21 and TrTLR22 is different.TrTLR21 contains 4 LRR-TYP motifs and 14 LRR motifs,while TrTLR22 contained 3 LRR-TYP motifs,13 LRR motifs and LRR-CT motifs.Homology alignment showed that TrTLR21 and TrTLR22 shared 53.1%-81.8%and 41.3%-82%identities with that of other fishes,respectively.Phylogenetic tree analysis revealed that the putative amino acid sequence of TrTLR21 and TrTLR22 was clustered with their counterparts from other teleosts,respectively,and with closest relationship with yellowtail(Seriola Lalandi).The ORF of TrIRAK1 and TrTRAF3 are 2247 bp and 1788 bp,respectively,encoding a putative protein of 748 and 595 amino acids.TrIRAKI protein contains a death domain and a Serine/Threonine protein kinases catalytic domain.TrTRAF3 protein contains RING domain,zinc fingers,coiled-coil regiion and MATH domain.Homology alignment showed that TrIRAKI and TrTRAF3 shared 43%-72.1%and 74.7%-93.9%identities with that of other fishes,respectively.Phylogenetic tree analysis showed that TrIRAKI was closest to red snapper(Lutjanus sanguineus),TrTRAF3 was closest to greater amberjack(Seriola dumerili).These results showed that TLR21,TLR22,TRAF3 and IRAK1 were highly conserved in evolution.2.TrTLR21,TrTLR22,TrTRAF3 and TrIRAK1 were constitutively expressed in the all examined tissues from healthy Trachinotus ovatus.However,their expression levels were significantly different in different tissues.TrTLR21 was observed highest expression of in spleen,followed in head kidney,skin,heart,intestine and gill,with lowest expression in muscle.Similarly,TrTLR22 expression level was highest in head kidney,followed in spleen,gill and intestine,with lowest level in heart.As for TrIRAK1 and TrTRAF3,TrIRAK1 was observed highest expression in gill,followed in skin,brain,spleen,liver and head kidney,with lowest expression in muscle.Whilst TrTRAF3 expression level was highest in spleen,followed in liver and head kidney,with lowest level in muscle.3.The mRNA expression levels of TrTLR21,TrTLR22,TrTRAF3 and TrIRAKI were up-regulated in spleen,head,kidney and other immune organs after stimulation by vibrio alginolyticus,LPS and PolyI:C.However,the expression change folds of genes mentioned above were various in different time points and in different tissues.The expression levels of TrTLR21,TrTLR22 and TrIRAKI were highest in the spleen,reaching the peak at 24 h,6 h and 24 h after LPS stimulation,respectively.While TrTRAF3 highest in the gill,reaching the peak at 24 h.The expression levels of TrTLR21,TrTLR22 and TrTRAF3 were highest in spleen,reached the peak at 24 h,6 h and 12 h post the infection with vibrio alginolyticus,respectively.While TrIRAKI was highest in liver and reached the peak at 12 h.The highest expression levels of TrTLR21,TrTLR22 and TrIRAK1 were observed in liver,reaching the peak at 24 h,6 h and 6 h post PolyI:C stimulation,respectively,while TrTRAF3 in spleen,reaching the peak at 24 h.These results indicate that TrTLR21,TrTLR22,TrTRAF3 and TrIRAK1 are involved in anti-bacterial and anti-viral signaling pathways of Trachinotus ovatus.