Genome-wide Identification And Analysis Of Chemokine Receptor Superfamily In Miiuy Croaker

The innate immune system can recognize non self through pattern recognition receptors and provide the first antibacterial host defense line.Therefore,innate immunity plays a very important role in vertebrate resistance to major bacterial diseases.In the innate immune responses,the chemokine receptors act as the main receptors of chemokines which are released at the sites of infection,inflammation and injury.The innate immune system can recognize non-self,danger signals,and pathogen associated molecular patterns and provides a first line of antimicrobial host defense.Therefore,it plays an instructive role and is pretty important in vertebrates.In this study,based on the whole-genome and transcriptome database of miiuy croaker,we used BLAST searches,multiple sequence alignment,phylogenetic analysis,syteny analysis,and molecular evolution analysis to conduct comprehensive identification and analysis of the Chem R family members in miiuy croaker.And expression levels of three members of Chem R family CXCR3.2,CXCR5,and XCR1 were analyzed after being infected by Vibrio harveyi.The major results are as follows:1.We have identified 27 Chem Rs in miiuy croaker: 13 CCR genes,11 CXCR genes,and 3 XCR genes based on whole-genome and transcriptome database,and no member of CX3 CR subfamily was identified in miiuy croaker.2.Gene structure and location analysis showed that the structure of the miiuy croaker Chem R gene family is quite simple.Most genes contain 1 to 2 exons,three genes(CCR7,CCR11,and CCR4Lc)have 3 exons,and CXCR4 a is relatively special,which has 4 exons.Twenty-seven Chem Rs were unevenly distributed into 12 chromosomes of miiuy croaker.Chromosome 7 had the largest number of genes(6;22.2%),besides,Chromosome 16,11,17,and 22 contained 5,4,3,and 2 gene family members respectively.However,the other chromosomes shown here had only one gene.3.Amino acid sequence structure analysis showed that all of miiuy croaker Chem R proteins contain the seven membranes structures,including a cell outer,three extracellular loops,and three cell rings,and a carbon end.There is a highly conserved "“DRY”" motif in the second inner ring of CCR and CXCR subfamily of miiuy croaker,while the XCR subfamily don’t have the motif.Moreover,in the TM7,we have also discovered a NPxx Y motif(in motif 1)which acts as an integral part in G-protein activation.MEME analysis showed that all Chem R proteins have similar motif compositions.Motif 1,2,3,4,5,6,and 7 are present in each protein,motif 8 is only missing in CCR6 b and CCR10,and motif 9 is absent in CCR4 Lc,CCR6a,and CCR10.4.Phylogenetic and synteny analysis showed that these genes experienced tandem and segmental duplication event in several species,and tandem duplication might be the main expansion way in miiuy croaker.gene duplication analysis showed that 3 gene clusters were detected as tandem repeat genes,accounting for 29.6%(8 of 27),which is also the main reason for the large quantity of Chem Rs on the chromosome 7 and 16.In addition,only one pair of Chem Rs(CXCR4a and CCRL1b)were regarded as segmentally duplicated genes(Fig.4),and these genes with replication may play an important role in the evolution of chemr gene family.5.The major Chem Rs of each orthologous group in vertebrates were selected for molecular evolution analysis,the results of which indicated that compared with vertebrates,Chem Rs of teleost fishes may have a relatively high evolutionary dynamic.In addition,a total of 21 positively selected codons were detected in vertebrate Chem Rs under Model 8,these sites were mainly located in the extracellular domains which were the ligand binding and pathogen interaction regions.6.RNA-Seq analysis and q RT-PCR verification demonstrated that CXCR3.2,CXCR5,and XCR1 genes were up-regulated significantly upon the Vibrio harveyi infection in immune tissue of miiuy croaker.