Cloning,Expression And Functional Analysis Of Mmplg And Mmctl From Meretrix Meretrix

Meretrix meretrix is an important aquaculture species in our country. But recent years, a lot of "bottleneck" problems which severely restricted the development of the aquaculture appeared and caused great economic losses. Fibrinolytic enzyme and cathepsin L play important roles in growth and development of animals. Understanding the growth process and regulation mechanism of clam are meanful for artificial breeding and the development of aquaculture in theory and reality.In this paper, we used Meretrix meretrix as the research object, the content is divided into five sections:Part1:With the rapid-amplificationof cDNA ends (RACE) technique, two gene cDNA sequences of plasminogen (MmPlg) and cathepsin L (MmCtl) were cloned from meretrix meretrix, and their nucleotide and protein sequences were analyzed with bioinformatic methods;The results are as follows:(1) The full-length cDNA of MmPlg is921bp, containing a819bp open reading frame and encoding a protein of272amino acid residues. The first16amino acid of the protein make up the signal peptide. The predicted molecular weight of MmPlg is28.7kD with the isoelectric point (pi) of6.42, and it contain typical serine protease active sites (His77, Asp126, Ser220). Sequence alignment analysis showed that the MmPlg amino acid sequense was most similar to Urechis unicinctus and Chlamys farreri’s fibrinolytic enzyme.(2) The full-length cDNA of MmCtl is1304bp, containing a1005bp open reading frame and encoding a protein of343amino acid residues. The first16amino acid of the protein make up the signal peptide. The predicted molecular weight of MmCtl is37.5kD with the isoelectric point (pI) of5.27. The Conserved Domain Search analysis showed that the MmCtl is a member of Peptidase CIA subfamily, and it contains specific active catalytic sites and substrate binding sites of papain.Part2:The-actin gene as a internal control, using real-time fluorescence quantitative PCR (qRT-PCR) to detect MmPlg and MmCtl genes’mRNA expression in different development periods (trochophore, D-veliger, pediveliger and postlarvae) of M. meretrix; Using real-time fluorescence quantitative PCR (qRT-PCR) technique we detected MmPlg and MmCtl genes’ mRNA expression level in different tissues (muscle, mantle, gill, enteron and hepatopancreas) of adult clam.The results are as follows:(1)The tissues quantitative results showed that MmPlg were expressed in various tissues, expression level in enteron was significantly higher than the one in the other four, and also was approximately two times of the one in gill; The development quantitative experiment results show that, from trochophore to D-veliger period, relative expression of MmPlg did not change significantly, but the expression of MmPlg raised greatly in pediveliger period. In postlarvae, the MmPlg expression showed a slight cut, but still far higher than the previous two periods. We analysed that the MmPlg might be involved in the digestion of nutrients in M. meretrix;(2) The tissues quantitative results showed that MmCtl were also expressed in various tissues and the highest expression level were found in hepatopancreas; The development quantitative experiment results show that, along with the development of Meretrix meretrix, relative expression of MmCtl showed ascendant trend obviously. From trochophore to D-veliger period, the expression of MmCtl had increased slightly. But the expression of MmCtl in pediveliger is20times as much as in trochophore. In postlarvae, the MmCtl expression is highest. We analysed that the MmCtl may involved in the development of clam larvae through the cell apoptosis mechanisms.Part3:The prokaryotic expression vector of MmPlg was constructed, and then transform the vector to Escherichia coli (DE3) in which we induced it expression, then the fusion protein DsbA-MmPlg was purified for making antibody in New Zealand rabbit.The results are as follows:DsbA-MmPlg were induced and expressed as inclusion body. The molecular weight of the recombined protein is approximately52.7kD. The fusion protein purified for the preparation of antiserum. The Western Blot result shows that the antiserum has good specificity.Part4:Constructed the MmCtl prokaryotic expression vector, and then a fusion protein DsbA-MmCtl was purified to making antibody.The results are as follows:The molecular weight of DsbA-MmCtl is60.5kD, and expressed as inclusion body. The fusion protein purified for the preparation of antiserum. The Western Blot result shows that the antiserum has good specificity.