Cloning And Expression Of Genes In Pinctada Maxima, P. Margaritifera And Spondylus Fragum

The pearl oysters Pinctada maxima and P. margaritifera belong to Mollusca, Bivalvia, Pterioida, Pteriidae, Pinctada. Among them, P. maxima is mainly distributed in tropic and subtropic oceans and seas of China, Australia, and Southeast Asia, etc, while P. margaritifera is mainly distributed in tropic and subtropic oceans and seas of the Pacific and Indian Oceans, as well as in Hainan, Leizhou and Taiwan of China. P. maxima can produce the largest marine pearls, which is famous as"South Sea Pearl"and is the most expensive jewelry. The pearl oyster P. margaritifera can produce the rare black pearls in the world, the only mass production of the black pearls. Spondylus fragum which belongs to Mollusca, Bivalvia, Pterioida, Spondylidae, Spondylus, is mainly distributed in the Philippines, Guangdong and Hainan of China. It lives commonly in shallow waters and intertidal rocks near coral reefs. The mode specimen was collected in the Philippines. This study focused on: (1) Cloning and function of Prismalin-14 gene in P. maxima (pmax Prismalin-14); (2) Cloning and tissue expression of cathepsin D (CathepsinD, CD) in P. margaritifera (pmarCD)pmarCD; and (3) Cloning and tissue expression ofα-amylase in Spondylus fragum(sfAMY). The results are as follows:1. I amplified pmaxPrismalin-14 cDNA sequence in P. maxima according to the EST sequence in GenBank. The cDNA sequence length is 379 bp long, including 24 bp of 5 'untranslated region (UTR), 13 bp of 3' untranslated region (UTR), and 339 bp of open reading frame (ORF), which encodes 113 amino acids of a polypeptide with prediction of molecular weight of 12.7kDa and theoretical isoelectric point of 4.239. The 16 amino acids residues in N-terminal is a signal peptide.2. The recombinant plasmid was constructed by inserting the ORF of pmaxPrismalin-14 into an E.coli vector pET-32a. A recombinant protein of expected size was induced by IPTG and purified by affinity chromatography. Calcium carbonate precipitation inhibition experiments showed that pmaxPrismalin-14 protein can also inhibit the precipitation of calcium carbonate. 3. Tissue-specific expression of pmaxPrismalin-14 in mantle is much higher than the other tissues. Fluorescence quantitative PCR showed that the highest expression of pmaxPrismalin-14 occurred in mantle edge among three different parts of the mantle.4. By RT-PCR and RACE I amplified pmarCD from P. margaritifera. The full length of cDNA is 1840 bp, including 151 bp of 5 'untranslated region (UTR) , 516bp of 3' untranslated region (UTR) , and 1170 bp of open reading frame (ORF) with a AATAAA box and poly A tail. The ORF encodes 390 amino acids with prediction of molecular weight of about 42.18kDa and theoretical isoelectric point of 6.354. The 18 amino acids residues in N-terminal is a signal peptide. Tissue-specific expression of pmarCD in gonad and hepatopancreas is much higher than the other tissues.5. Using RT-PCR and RACE I amplified sfAMY from Spondylus fragum. The full length cDNA is 1717 bp, including 89bp of 5 'untranslated region (UTR) , 98bp of 3' untranslated region (UTR) , and 1527 bp of open reading frame (ORF) with a AATAAA box and polyA tail. The ORF encodes 509 amino acids residues, with prediction of molecular weight of 56.98kDa and theoretical isoelectric point of 7.484. Tissue-specific expression of sfAMY in hepatopancreas is much higher than the other tissues.