Cloning And Expression Analysis Of Calmodulin-Like Protein?Calreticulin And Carbonic Anhydrase In Galaxea Astreatas

In recent years,due to the environmental deterioration and the increasing human activities,the marine ecosystems are damaged seriously.As the lower vertebrates in the sea,coral reefs are facing serious crises.In order to understand the mechanism of coral calcification and protect the coral reef ecosystem,researchers began studying on coral calcification related genes.In this study,we used Galaxea astreatas as the experimental object,the calmodulin-like protein,calreticulin and carbonic anhydrase gene were cloned and their prokaryotic expression were studied.At the same time,the expression level of calmodulin-like protein gene was analyzed under temperature stress,and also under the calcium channel blockers verapamil.The results are as follows:1.The full length of calmodulin-like protein(Ca LP)c DNA is 1290 bp,with a 109 bp 5' UTR,683 bp of 3' UTR and an ORF of 498 bp,coding 165 amino acid residues.Its theoretical molecular mass is 18.89 k Da and theoretical isoelectric point is 4.22.Ca LP gene prokaryotic expression system was constructed with p ET-32 a vector and BL21(DE3)host strain,it can induced a specific fusion protein and detected in supernatant after disrupted by sonication.It was found that its expression was increased at first and then decreased under both 20°C and 30°C over time.In addition,after verapami treatment,we noted that the down regulation of Ca LP gene.2.The full length of calreticulin(CRT)c DNA is 1792 bp,with a 77 bp 5' UTR,380 bp of 3' UTR,and a 1335 bp ORF,coding 444 amino acid residues with 51.85 k Da and p I of 4.43.Its prokaryotic expression system was constructed as the description above,which can be induced the protein in the predicted molecular mass.The purified recombinant protein was able to promote both the Gram-positive bacterium Micrococcus luteus and Gram-negative bacterium Escherichia coli agglutination,which indicated that Galaxea astreatas CRT possesses bacterial agglutinative function.3.The full length of carbonic anhydrase(CA)c DNA is 1120 bp,with a 148 bp 5' UTR,a 3' UTR of 228 bp and an ORF of 744 bp,coding 247 amino acid residues with 27.54 k Da and p I of 5.88.Its prokaryotic expression system was constructed as the description above,which can be induced the protein in the predicted molecular mass,and detected in the insoluble fraction of E.coli cell lysates.It was found that 0.2 mmol/L IPTG was the best inducing concentration and 37? was the best inducing temperature.