A novel tissue inhibitor of metalloproteinase 3 in blood clam Tegillarca granose:molecular cloning and immune-related research of Cd

On the basis of cDNA library of Tegillarca granosa, Tg-TIMP-3 cDNA was cloned by SMART RACE(Rapid Amplification of cDNA Ends)techniques and then the bioinformatics, expression and immune defense analysis of Tg-TIMP-3 were carried out.The full length cDNA of Tg-TIMP-3 gene was 804 bp, containing a complete ORF(Open Reading Frame)of 570 bp encoding 189 amino acids.The deduced amino acid sequence of Tg-TIMP-3 did not share high similarity with others, only shared 35.8% similarity with TIMP-3 from Crassostrea gigas, but the two functional domains of TIMPs were relatively conservative.The results of tissue-specific expression by real time PCR showed that the mRNA transcript level of Tg-TIMP-3 in gill was higher than those in other tissues including haemocytes, adductor muscle, foot, hepatopancreas and mantle.After being challenged by different concentration of Cd, the level of relative expression in gill of Tg-TIMP-3 mRNA was first slightly reduced and then significantly increased the highest.Those challenged by the concentrations of 25 μg/L and 250 μg/L of Cd reached the highest at 6 h(P<0.01), 500 μg/L reached the highest at 9 h, but finally inhibited to different degrees after 48 h.The results indicated that Tg-TIMP-3 played a vital role in the immune defense responses to Cd.However, the temporal sequence and sensitivity of mRNA expression were different in different challenge groups.