Cloning, Evolution And Expression Analyses Of Toll-like Receptor Genes From Sea Cucumber (Apostichopus Japonicus)

Apostichopus japonicus belongs to Echinidermata, Holothuroidea, Apostichopus, which isan economically important aquaculture species. Theoratically, the studies on the immune genesand their mechnisms in A. japonicus will lay the foundation for diseases prevention.Toll-like receptors (TLRs), a family of type I integral membrane glycoproteins, are the mostancient pattern recognition receptors (PRRs) in innate immunity. The full-length cDNAs of twoA. japonicus TLRs (AjTLRs) were obtained by RACE method. They were named as AjTLR3and AjToll according to the BLASTX results that these two sequences were similar to TLR3sand Tolls from other animals, respectively. The full-length cDNA sequences of AjTLR3andAjToll are3484bp and4211bp, with an open reading frame (ORF) of2679bp and2853bp,encoding892and950amino acids, respectively. SMART analysis show that both AjTLR3andAjToll are composed of a leucine-rich repeat (LRR) domain, a transmembrane (TM) domain andan intracellular Toll/interleukin-1receptor (TIR) domain indicating they are conserved duringvertebrate evolution process. Phylogenetic analysis revealed that AjTLR3and AjToll wereclustered with the vertebrate-like TLRs (V-TLRs) and the protostome-like TLRs (P-TLRs)respectively confirming the coexistence of two different types of TLRs.To investigate the expression profiles of AjTLR3and AjToll, quantitative real-time PCR(qRT-PCR) was used to analyze their transcript levels in five tissues from the healthy and theimmune challenged sea cucumbers. These two genes were widely expressed in all five testedtissues (body wall, coelomocytes, tube feet, intestine and respiratory tree), showing highlyexpressed in respiratory tree but at relatively low levels in other four tissues. When challengedwith peptidoglycan (PGN), lipopolysaccharides (LPS), Zymosan A andpolyinosinic-polycytidylic acid (PolyI:C) which represent the synthetic analogs of the mainpathogens: Cram-positive bacteria, Gram-negative bacteria, fungi and double-stranded RNA(dsRNA) viruses, AjTLR3and AjToll showed different expression patterns. In PGN group, bothAjTLR3and AjToll were strongly up-regulated in coelomocytes. After LPS and PolyI:Cchallenges, AjTLRs expression levels were significantly up-regulated in tube feet. Whenchallenged with Zymosan A, these two genes were highly expressed in respiratory tree. Aboveresults suggested that AjTLRs are widely involved in immune responses.