Molecular Cloning Of MMP And TIMP From Stichopus Japonicus And Their Expression Profile During Autolysis

Sea cucumber are extremely sensitive to the external environ ment. Autolysis always induced by change of environment physiochemical factors, which cause degradation of extracellular matrix of sea cucumber body. MMPs (Matrix metalloproteinases, MMPs) are a big family of extracellular proteolytic enzymes, can almost degrade all the extracellular matrix components. MMPs are strictly modulated by TIMPs (Tissue inhibitor of metalloproteinases), endogenous inhibitors of MMPs. Rasearch demonstrated that MMPs were critical autolytic protease in sea cucumber. But there is no report on gene expression of MMP or TIMP during sea cucumber autolysis. So in this presentation, a MMP and TIMP gene was cloned and characterized from Stichopus japonicus. Their tissue distribution and expression during autolysis in the derm (body wall) are investigated, for a better understding of from the point of MMP/TIMP gene expression modulation.Total RNA was isolated from intestine of sea cucumber. MMP and TIMP sequences were obtained by reverse transcription, PCR and RACE. The expression of two genes in six tissues of sea cucumber and during autolysis was investigated by quantitative real-time PCR.First, the cDNA sequence of MMP was of 1691 bp in length, encoding 507 amino acids. MMP is intended to be MMP-1 by analyse structure. The cDNA sequence of TIMP is of 721 bp in length, encoding 192 amino acids. TIMP is intended to be TIMP-1 by analyse structure. Second, research the gene expression of MMP and TIMP. The maximal expression of MMP and TIMP was found in respiratory tree and coelomocyte. The expression of MMP and TIMP increased to a peak level and then decreased during autolysis. At 6h, a maximal expression appeared in tube feet and dorsal epidermis (3.48-fold and 5.41-fold higher than that of Oh), respectively. The expression of MMP in the two tissues was 3.75-fold and 1.53-fold higher than Oh. The increase of MMP expression was stronger than TIMP in tube feet, but the opposite in dorsal epidermis. Third, competent cell of BL21(DE3)T1R with recombinant plasmid of TIMP-pET21b can express recombinant TIMP protein by inducing with IPTG. And the optimization condition were 0.4mM IPTG and 37℃.Results demonstrated a broad distribution of MMP-1 and TIMP-1 in all the selected tissues of sea cucumber. The expression of MMP-1 and TIMP-1 up-regulated in dorsal and tube feet. MMP-1/TIMP-1 ratio balance was broken, which might be a critical reason for the degration of structural protein during autolysis. However, the modulative effect of TIMP on MMP deserved further investigation.