Molecular Cloning,Molecular Evolution And Expression Analysis Of 5 TLR Genes In Great Blue-Spotted Mudskipper (Boleophthalmus Pectinirostris)

Boleophthalmus pectinirostris is a wide-temperature and wide-salt fish,the salinity of the living environment often changes,and it also needs to face the invasion of various pathogens.Studies showed that Vibrio anguillarum is a pathogen that can cause serious loss of aquaculture.Innate immunity is the first sensor and barrier against pathogen invasion and infection,and TLR is an important innate immune member.In present study,5 TLR genes were analyzed by bioinformatics,and in order to explore the immune patterns of 5TLR genes stimulated by bacteria in different salinities,V.anguillarum was used to stimulate B.pectinirostris that lived in different salinities.In addition,in order to explore the molecular evolution of the 5 TLR genes,molecular evolution analysis was performed by using the constructed phylogenetic tree.The main results are as follows:(1)The structure of B.pectinirostris TLR3 gene was conserved.TLR3 gene was expressed in 9 healthy tissues(intestine,gill,eye,kidney,liver,brain,muscle,spleen and skin),but the expression of liver,spleen and skin were high.In different salinities,TLR3 gene responded differently to V.anguillarum invasion.In normal circumstances,the spleen responds faster when it is resistant to V.anguillarum invasion,but it was opposite in high salinity.Neither site model nor branch-site model detected an evolutionary locus in TLR3 gene.(2)The structure of B.pectinirostris TLR5 gene was conserved.TLR5 gene was expressed in 9 healthy tissues,but the expression of liver and spleen were high.In different salinities,TLR5 was responded differently to V.anguillarum invasion.In normal circumstances,when the pathogen invades,the spleen reacts faster,but it was opposite in high salinity.In normal salinity,the reaction of TLR5 in liver and spleen was faster than that in high salt,and the spleen was more intense than the liver,while the high salt group is opposite.Two evolutionary sites(101H,453N)were detected in site model,but no evolutionary sites were detected in branch-site model.(3)The structure of B.pectinirostris TLR7,TLR8 and TLR9 genes were conserved.The expression of TLR7,TLR8 and TLR9 in liver,spleen and gill were relatively high in 9healthy tissues.In addition,the expression of TLR8 in the intestine,muscle and skin were relatively high,and the expression of TLR9 in muscle and skin were relatively high.In different salinities,TLR7,TLR8 and TLR9 were responded differently to V.anguillarum invasion.In normal salinity,the reaction of TLR7 in liver and spleen was faster than that in high salt.The response of TLR7 in spleen was higher than that in liver,while the high salt group was opposite.In contrast to TLR7,high salinity stress caused TLR8 to react morestrongly than the normal group.When Vibrio anguillarum invades,the response rate of TLR9 in liver was faster than in spleen after V.anguillarum invasion.In addition,salinity stress caused the response of TLR9 to the invasion of V.anguillarum become sluggish.Molecular evolution analysis were performed by using TLR7,TLR8 and TLR9 phylogenetic trees,site model showed that two(649Q,674E)and one evolutionary sites(410S)were detected in TLR7 and TLR9,respectively,and no evolutionary sites was detected in TLR8.The branch-site model showed that an evolutionary site was detected in each of the ancestors of B.pectinirostris in TLR7(485S)and TLR9(373R).In a word,the 5 TLR genes of B.pectinirostris were conserved.The results of tissue differences showed that 5 TLR genes were expressed in 9 healthy tissues(Intestinal,Gill,Eye,Kidney,Liver,Brain,Muscle,Spleen and Skin)and were highly expressed in liver and spleen.In addition,after B.pectinirostris in the different salinity were infected by V.anguillarum,the trend of expression of 5 TLR genes in liver and spleen indicates that TLR gene plays an important role in dealing with pathogen invasion,and to a certain extent,salinity stress affects the reaction of 5 TLR genes.According to the results of the site model,TLR3 and TLR8 were highly conserved,while TLR5,TLR7 and TLR9 experienced adaptive evolution during long-term evolution.Finally,the branch-site model results showed that B.pectinirostris TLR7 and TLR9 have undergone adaptive evolution from the aquatic environment to the amphibious environment.