Cloning Of Amphioxus Leucocephalus COMMD Gene Family And Preliminary Analysis Of Immune Function

COMMD protein is a kind of protein family that has been found in recent years and is widely presented in multicellular organisms.The COMMD protein family includes ten members(COMMD 1?10).The analysis of COMMD protein sequence showed that COMMD protein contains about 200 amino acid residues that play the role of interface when protein interacts with each other.The carboxyl end of all COMMD protein members has a highly conservative COMM domain composed of 70-85 amino acids.The COMMD protein is a pleiotropic factor that participates in various biological processes,such as the regulation of copper and sodium transport,NF-?B activity and cell cycle progression.NF-?B is activated in the immune response,which can be up-regulated to help body resist the invasion of external pathogens,and be down-regulated to prevent excessive immunity from causing damage to the body.Recent studies have found that COMMD can inhibit the excessive activity of NF-?B in nucleus,and then participate in the immune regulation,avoid the damage to the body.Amphioxus is a classical species evolved from invertebrates to vertebrates which can be used as a model for vertebrate ancestors in structure and development,and it's an important mode in the study of the origin of zoology and evolutionary biology because of its' special status.In order to futher understand the function and evolution of COMMD family gene,we select amphioxus COMMD family genes as the research object,and the function and evolution of the family members were analyzed by gene cloning,quantitative RT-PCR and bioinformatics analysis.The results are as follows.(1)We cloned full-length sequence of COMMD family genes(AmphiCOMMDs)from Branchiostoma belcheri through the PCR and RACE-PCR.The bioinformatics analysis on the AmphiCOMMDs sequence showed that the AmphiCOMMD protein contains about 200 amino acid residues,the carboxyl end of COMMD protein has a conservative COMM domain composed of 70-85 amino acids,while the members were significantly different in the amino terminal.The genomic structure and phase analysis of the COMMD family showed that COMMD3 and COMMD8 were conservative in chordates,while COMMD1,2,4,7,9 were only conservative among vertebrates.In addition,COMMD5 and COMMD6 are only conservative in amphioxus and zebrafish.In general,the genetic structure and phase changes of the COMMD family are diverse.Compared with sea squirt which belong to urochordata,the gene structure of the COMMD family is closer to vertebrates.(2)In order to understand the origin and evolution of COMMD family,the phylogenetic tree was constructed by bayesian method using the sequeneces of AmphiCOMMDs and the representative species in different evolutionary status.Phylogenetic analysis indicated that these ten members of AmphiCOMMDs family are clustered into the COMMD1?10 subfamily cluster,respectively.Among the subclusters,the AmphiCOMMDs gene is between invertebrates and vertebrates.The AmphiCOMMDs gene of amphioxus is closely related to the vertebrates when compared with sea squirt,which was consistent with the genetic structure and phase analysis,and the drosophila melanogaster were located at the bottom of each subcluster.The analysis of conserved motif showed that all ten COMMD subfamilies contain conserved motif 1 and motif 2.However,motif 7 is only included in COMMD3,4,5,7,10,and motif 11 is only in COMMD4,5,7,10.The remaining motifs appear only to present in a specific subfamily,may be related to the differentiation of protein functions.The selective pressure analysis of the COMMD family genes showed that the COMMD family genes underwent very strong purifying selection in the evolutionary history,the positive selective sites might result in functional diversity of COMMD family genes.Our findings provide important clues to better understanding the function and evolution of the COMMD family gene.(3)To study the antibacterial function of AmphiCOMMDs gene,we used the quantitative RT-PCR method to detect the expression of AmphiCOMMDs gene in in different tissues(muscles,gills,intestine,heaptic cecum and notochord)and different time points after LPS injection.The results showed that the expression level of AmphiCOMMDs was similar except for AmphiCOMMD3 and AmphiCOMMD5,AmphiCOMMD3 has a high expression level in the heaptic cecum,intestinal and notochord,and the expression level of AmphiCOMMD5 was relatively high in the intestine.The AmphiCOMMDs expression level has varying degrees of fluctuation after LPS stimulation,it is indicated that the members of COMMD in amphixous may participate in the regulation of NF-?B pathway in response to external stimulation.