Expression Pattern Analysis Of Immune-Related Genes And Molecular Cloning,Characterization Of Aquaporin 3 From Lamprey

Lampreys,the surviving representative of jawless vertebrates,have been a focal point in the research for the evolutionary origin of adaptive immunity.TLR/MyD88 signaling pathway plays a central role in the immune system by regulating the interaction of MyD88 with several genes(such as IRAK1,TRAF6,IKK?,TRAF3 and others).In this work,we have searched EST sequences of the four genes(IRAK1,TRAF6,IKK? and TRAF3)from a cDNA library generated from leukocyte cells,and analyzed the expression pattern of the important members of the TLR/MyD88 signaling pathway in the lamprey(Lampetra japonica).Furthermore,upon in vivo stimulation of lampreys with lipopolysaccharide(LPS),the mRNA levels of the three genes(IRAK1,TRAF6 and IKK?)were incresed in gills,and the mRNA levels of the three genes(IRAK1,TRAF6 and TRAF3)were up-regulated in heart.Those results provide some clues for the subsequent cloning to get the full length genes and exploring their functions in lamprey.In this report,to determine the cellular gene expression changes in lamprey Lampetra japonica leukocytes in response to LPS treatment,Affymetrix GeneChip? Human Genome U133 Plus 2.0 Array were used to screen transcriptome differences.Expression levels of 155 genes were significantly altered by at least two-fold upon treatment with LPS compared with controls.92 of the 155 genes were up-regulated and 63 genes were down-regulated,which involved in signal transduction,cell growth and differentiation,cell metabolism,immune defense,apoptosis,proliferation,and many other aspects.The results obtained in this report provide new valuable clues,which are helpful to shed more light in the immune response of the oldest jawless vertebrates.Aquaporin 3 gene,Aqp3,which was up-regulated by the experimental results of gene chip of lamprey leukocytes in response to LPS treatment,was explored in depth.AQP3 belongs to aquaglyceroporin subfamily which contributes to transporting water,urea and glycerol and promoting cell proliferation and migration.In this study,the complete cDNA sequence of Aqp3(designated LjAqp3)was cloned from Arctic lamprey Lampetra japonica(GenBank accession number KR054618).The open reading frame of LjAqp3 was 900 bp,encoding 299 amino acids,with a predicted six transmembrane domains and a putative signal anchor peptide.Two highly conserved NPA(asparagine-proline-alanine)motifs and the ar/R(aromatic/arginine)constriction are contained in LjAQP3.Additionally,LjAQP3 shares high sequence homology with AQP3 of other species.Real-time quantitative PCR revealed constitutive expression of LjAqp3 in multiple tissues and organs.Furthermore,after lipopolysaccharide(LPS)stimulation the expression levels in gills and intestine incresed visibly.This study provides evidence for aquaporin 3 playing a vital role in the regulation of the body fluid homestasis and immune defense in jawless vertebrates.