Identification of TgTLR3 and Its Immune Response to Hypoxia and Sulfide in Tegillarca granosa
In order to investigate the biological characteristics and immunoregulatory effects of the Toll-like receptor 3(TLR3)on hypoxia and sulfide exposure in the blood clam Tegillarca granosa,we cloned the core sequence of TgTLR3 and examined mRNA the expression level of TgTLR3 in haemolymph,gill,hepatopan-creas,mantle,adductor muscle,foot and haemolymph.The mRNA level of TgTLR3 in haemolymph was also examined when exposed to hypoxia and sulfide using quantitative real-time fluorescence PCR(qRT-PCR).The results showed that the TgTLR3 has an open reading frame(ORF)of 2 634 bp which encodes 877 amino acids.Among them,the position of 49-593 represents extra cellular leucine-rich repeats(LRRs),at position of 658-680 and 20-865 represent the transmembrane domain and the interleukin receptor 1 structural domain(TIR),respectively.The molecular weight(Mw)and putative isoelectric point(pI)of TgTLR3 were 99.4 kD and 7.76,respectively.The amino acid homology of T.granosa comparison showed that the TgTLR3 sequence was most similar to that of Anadara sativa,with the identity and similarity of 80.05％.The phylogenetic tree analysis showed that TgTLR3 was clustered with A.sativa and was the closest relative to A.sativa.TgTLR3 was ex-pressed in all tissues and it was most expressed in the haemolymph.After hypoxia exposure,TgTLR3 in the haemolymph was significantly up-regulated at 48-96 h.After the combined hypoxia and sulfide exposure,TgTLR3 was significantly lower than the hypoxia group at 48 h but it was still significantly higher than the control group at 96 h.The findings suggest that TgTLR3 might play an important role in the immune response to hypoxia and sulfide in T.granosa.This study provides a basis data for investigating for the function of TLR3 and the immune mechanism in response to hypoxia and sulfide stresses in mollusc.
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