The Expansion Of Tlr11 Gene Family In Mudskipper(Boleophthalmus Pectinirostris)

Toll-like recqptors(TLRs)are major molecular pattern recognition receptors,which are essential for triggering a series of innate immune responses against invading pathogens by recognizing their evolutionary conserved molecular patterns.The mudskipper,Boleophthalmus pectinirostris is exceptional among fishes due to its amphibious lifestyle and adaptation to living on mudflats.The whole-genome sequencing of B.pectinirostris has revealed that this species possesses an expansion of Tlrll family[12 Tlr11 family genes(one tlr21,4 tlr22 and 7 tlr23)].In order to investigate the potential functions of Tlr11 family genes in B.pectinirostris,in the present study,we cloned the full-length cDNAs of 12 Tlr11 family genes,developed a high-density genetic linkage map and a chromosome map3 located 11 Tlrll family genes at seven chromosomes,and examined the expression profiles of 12 Tlr11 family genes in thirteen different tissues and the expression profiles of 12 Tlr11 family genes in spleen and kidney after LPS and poly(I:C)challenges in B.pectinirostris.The results were as follows.1.The full-length cDNA sequences of the 12 Tlrll family genes in B.pectinirostris were cloned.The lengths of these 12 tlr cDNAs varied from 2878 to 4904 bp,and the deduced protein sizes were conserved,ranging from 898 to 965 amino acids.The deduced amino acid sequences of 12 Tlr11 family genes possessed a typical TLR domain arrangement.All Tlrll family proteins of B.pectinirostris comprised a signal peptide,several leucine-rich repeats(LRRs),a transmembrane domain and a TIR domain.2.The first high-density genetic map and chromosome map of B.pectinirostris were developed by referring to SNP markers from F1 full-sib family using whole genome resequencing strategy.The 212,126.4 cM genetic linkage map of B.pectinirostris organized 66,129 SNP markers on 23 linkage groups(LGs),representing 23 chromosomes,in agreement with the karyotype.In addition,1209 genome assembly scaffolds were anchored onto the LGs,and these scaffolds comprised 916.23 Mb,accounting for 93.2%B.pectinirostris genome.11 Tlrll family genes of B.pectinirostris were located at seven chromosomes.Tlr21 was located at chr7,tlr22a,tlr22b,tlr22c and tlr22d were located at chr12,chr8,chr7 and chrl7,respectively.Among tlr23 genes,three of them(tlr23c,tlr23d,tlr23f)were present in the same chromosomal region(chrl 1),while tlr23a,tlr23e and tlr23g were found in chr6,chr12 and chr18,respectively.3.The basal expression levels of the 12 Tlrl 1 family genes of B.pectinirostris in brain,heart,spleen,gills,liver,intestine,testis,seminal vesicle,ovary,skin,eye,kidney,blood cells were quantified by Real-time qPCR.These 12 Tlr11 family genes were all highly expressed in immune-related organs,i.e.spleen and kidney,which suggest that 12 Tlrl 1 family genes may be involved in immun-related functions4.The expression levels of the 12 Tlr11 family genes of B.pectinirostris in the spleen and kidney were analyzed following intraperitoneal injections of LPS and poly(I:C).Tlr21 and tlr22b transcripts were significantly up-regulated by LPS,whereas tlr22a,tlr22d,tlr23b,tlr23e,tlr23g were significantly up-regulated by poly(I:C)in the spleen or/and kidney,which implies that the expanded Tlr11 family genes may play roles in protecting the fish from the invasion of gram-negative bacteria and double-stranded RNA viruses.