Molecular Cloning And Functional Identification Of Cathepsin D And Matrix Metalloproteinase 2 Gene Of Tilapia(oreochromis Niloticus)

Cathepsin D, as a lysosomal aspartyl proteinase, that can degrade intracellular and endocytosed proteins, and was reported to be expressed constitutively in many eukaryotic cells. Matrix metalloproteinase 2(MMP2), Zinc-binding endopeptidase, that it can degrade the proteins of extracellular matrix. They play an important role in fish pathological processes. Tilapia(Oreochromis niloticus), an important cultured freshwater fish in our country, used in this experiment. The research studied the cloning, functional prediction, and the expression of cat hepsin D and MMP2 by RAC E-PCR, molecular cloning and Real Time PCR from tilapia. The results of research are as follows:(1)In this current study, the full-length cathepsin D cDNA(1642bp) of tilapia was cloned by 5′-and 3′-RACE, and it contained a 57 bp 5′UTR, a 394 bp 3′UTR and a 1191 bp CDS encoding 396 amino acids. The cathepsin D protein includes signal peptide(1-18aa), leading peptide(19-46aa) and mature peptide(47-396aa). The tilapia pre-procathepsin D isoelectric point is 6.1 and molecular weight is 43.2 KDa were predicted by ProtParam tool. The isoelectric point of the mature cathepsin D protein is 5.52, and the molecular weight is 38 KDa. The tilapia mature cathepsin D protein was an stable protein for its instability index(II) is 34.24. And t he mature protein was classified as an hydrophilic protein for its Aliphatic index is79.09 and its Grand average of hydropathicity is-0.114. Alignment analysis revealed that the cathepsin D gene is well conserved in many species, and tilapia cathepsin D amino acid sequence showed the highest identity of 89% to Lates calcarifer. Unlike many teleosts, the tilapia cathepsin D contained three N-glycosylation sites at amino acid residues 131, 249 and 314. Like zebrafish, the tilapia cathepsin D genomic DNA contains 9 exons and 8 introns.(2)In present research the MMP2 gene of 3033 bp was cloned by PCR, and it contained a 95 bp 5 ′UTR, a 967 bp 3′UTR and a 1971 bp CDS encoding 656 amino acids. The MMP2 protein includes signal peptide(1-29aa), leading peptide(43-97aa) and mature peptide(98-656aa). The mature MMP2 protein consists of three domains: a catalytic domain, a hinge region and a C-terminal region. The catalytic domain contains three repeats of fibronectin-typeⅡ domain, and the C-terminal region contains four repeats of hemopexin- like domain. The tilapia pre-proMMP2 isoelectric point is 5.01 and molecular weight is 74.33 KDa were predicted by ProtParam tool. The isoelectric point of the mature MMP2 protein is 4.91, and the molecular weight is 70.99 KDa. The mature MMP2 protein was an stable protein for its instability index(II) is 29.16. And the mature protein was classified as an hydrophilic protein for its Aliphatic index is 57.11 and its Grand average of hydropathicity is-0.529. Alignment analysis shows that the MMP2 gene is well conserved in many vertebrates, tilapia MMP2 amino acid sequence showed the highest identity of 89% to Takifugu rubripes.(3)Real-time quantitative RT-PCR showed that tilapia cathepsin D mRNA was ubiquitously expressed in twelve tissues(intestines, liver, brain, spleen, gill, kidney, heart, gonad, muscle, fin, blood, eye), highly expressed in the kidney and spleen, follows in the intestine brain and blood, low expressed in heart, fin, eye, muscle, gonad and liver. The expression levels of cathepsin D in k idney and spleen were 13 and 7 times higher than those in heart, respectively. The cathepsin D expression of spleen, intestine, liver and kidney were increased significantly after challenged with Aeromonas hydrophila, reached the peak at 5h, 24 h, 48 h and 48 h and decreased to the normal level at the end.The MMP2 mRNA was expressed in all eleven tissues(brain, eye, fin, gill, heart, intestines, kidney, liver, muscle, spleen, testis), highly expressed in the testis, heart and eye, follows in the muscle and fin, low expressed in liver. The expression level of MMP2 in testis was 14.27 times higher than that in liver. The expression level of MMP2 in heart and eye were 8.12 and 7.04 times higher than those in liver. The expression level of MMP2 in spleen, kidney and liver were increased significantly after challenged with Aeromonas hydrophila, reached the peak at 5h, 24 h, 48 h and decreased to the normal level.The expression of cathepsin D and MMP2 increased first and decreased afterwards after challenged with pathogen indicated that tilapia may against the pathogen in this time. Conclusion: It is suggested that cathepsin D and MMP2 gene may plays an important role in the innate immune response of tilapia.