Molecular Cloning And Expression Analysis Of Toll-like Receptors Signal Pathway Elements In Small Abalone Haliotis Diversicolor

Small abalone is one of the most important mollusc species for commercial production inseveral southern provinces of China. But since1990s, small abalone aquaculture in China hasbeen experiencing a continual large-scale mortality, which not only caused a great economic lossbut also threatened the existence development of the culture industry. Enhancing the diseaseresistance of abalone is recognized an important way to controlling diseases. The cloning andexpression of the genes involved immune defense are now considered to be a basic solution inthe disease control because of their potential use in the development of therapeutic agents, studyof immune defense mechanism and genetic improvement to increase the resistance to disease.TLR signal pathway plays an important role in immune process. In our studies, six TLRsignal pathway elements, Gram-negative bacteria-binding proteins (saGNBP1, saGNBP2),Interleukin-1receptor-associated kinases4(saIRAK-4), Interleukin-1receptor-associatedkinases1binding protein1(saIRAK1BP1), TNF receptor-associated factor6(saTRAF6) andTumor necrosis factor-alpha (saTNF-α) were cloned by EST analysis and SMART RACEtechniques. Real time PCR was used to analyze their expression patterns in different tissues,different developmental stages and the expression patterns after Vibrio Parahaemolyticusinfection. Results are indicated as follows:1) The full-length of saGNBP1cDNA sequence was1430bp, encoding a protein of222aa. saGNBP2cDNA sequence was1289bp, encoding aprotein of396aa. Both saGNBP1and saGNBP2had a Glycoside hydrolase family16domain.Amino acid sequence analysis revealed both of the domains were conserved. Phylogenetic treeanalysis showed that saGNBP1and saGNBP2were different, the result may indicate that GNBPsplay different roles in abalone.2) The full-length of saIRAK-4cDNA sequence was2062bp,with a1548bp open reading frame encoding a protein of516aa. Amino acid sequence analysisrevealed saIRAK-4shares conserved signature motifs with other IRAK-4proteins, including thedeath domain (DD), serine/threonine/tyrosine protein kinase domain (STYKc). Quantitativereal-time PCR was employed to investigate the tissue distribution of saIRAK-4mRNA, and itsexpression in abalone under bacteria challenge and larvae at different developmental stages. ThesaIRAK-4mRNA could be detected in all examined tissues, with the highest expression level ingills, and was up-regulated in hemocytes and gills after bacteria injection. saIRAK-4was alsoconstitutively expressed at all examined developmental stages. Additionally, in situ hybridization analysis showed that the distribution patterns of saIRAK-4mRNA were identical in gills andhybridization signals were strong in the epithelial cells.3) The full-length of saIRAK1BP1cDNA sequence was1047bp, encoding a protein of249aa. Amino acid sequence analysisrevealed saIRAK1BP1shares a conserved SIMPL domain. Quantitative real-time PCR wasemployed to investigate the tissue distribution of saIRAK1BP1mRNA, and its expression inabalone under bacteria challenge and larvae at different developmental stages. The saIRAK1BP1mRNA could be detected in all examined tissues, with the highest expression level in hemocytes,and was up-regulated in gills and kidneys after bacteria injection. saIRAK1BP1wasconstitutively expressed at all examined developmental stages. Additionally, His-saIRAK1BP1recombination protein was induced by IPTG with the prokaryotic expression method.4) Thefull-length of saTRAF6cDNA sequence was795bp, encoding a protein of170aa. Amino acidsequence analysis revealed saTRAF6shares a conserved RING domain. Quantitative real-timePCR was employed to investigate the tissue distribution of saTRAF6mRNA, and its expressionin abalone under bacteria challenge. The saTRAF6mRNA could be detected in all examinedtissues, with the highest expression level in kidneys and hepatopancreas, and was up-regulated ingills and kidneys after bacteria injection.5) The full-length of saTNF-α cDNA sequence was954bp, encoding a protein of250aa. Amino acid sequence analysis revealed saTNF-α shares aconserved TNF domain. Quantitative real-time PCR was employed to investigate the tissuedistribution of saTNF-α mRNA, and its expression in abalone under bacteria challenge andlarvae at different developmental stages. The saTNF-α mRNA could be detected in all examinedtissues, with the highest expression level in kidneys, and was up-regulated in kidneys, however,down-regulated in hemocytes after bacteria injection. Additionally, saTNF-α was constitutivelyexpressed at all examined developmental stages.