Moleculer Cloning And Preliminary Functional Studies Of BMP3 And BMP10 Gene From Pearl Oyster, Pinctada Fucata

The pearl oyster Pinctada fucata is the most important shellfish which can produce seawater pearls. It has a series of excellent economic traits. But recent years the pearl oyster has suffered from some challenges such as degeneration of genetic characterization and low in ability of pearls production, which are serious constraints of the healthy development of pearl industry. It appears particular urgent that the pearl industry should be promoted though breeding fine varieties. Enhancing selection of good traits, especially the traits relation to growth and pearl production through Molecular marker-assisted selection technology is a feasible approach. So that the basic science study, which includes development regulation, shell formation and so on about the pearl oyster should be enhanced.Bone morphogenetic proteins(BMPs) are a series of multiple functional cytokins,which include many members. These proteins mostly have the competence of promotion osteogenesis, acting in embryonic development, histogenesis, reproductive manipulation and so on. But there are not many researches about the BMPs in molluscs species until now.The study cloned two BMP family genes, BMP3 and BMP10, based on the transcriptome data, and conducted preliminary functional studies. Specific results are as follows:1.Moleculer cloning and preliminary functional studies of BMP3 gene from pearl oyster, Pinctada fucata. In this study, The c DNA sequence of BMP3 gene from the Pinctada fucata was obtained through RACE technology based on the sequence fragment with the annotation of BMP3 homologous gene from the transcriptom data.The full length c DNA is 2710 bp, including an open reading frame(ORF) of 1470 bp, 5’untranslated region(UTR) 350 bp, 3’ UTR 890 bp and 3’ typical polyadenylation signal(AATAAA). The gene was named as Pf BMP3(Gene Bank accession number:KT956999).The Pf BMP3 gene encodes 489 amino acids and has a predicted molecular weight of 56.64 k Da, the theoretical isoelectric point(PI) of 9.80. The prediction of transmembrance structure showed that Pf BMP3 had no transmembrane structure and was located extracellular. The analysis of the deduced amino acids sequence showed Pf BMP3 contained a signal peptide(1-21 aa), a pro-domain(22-361 aa) and a mature peptide(362-489 aa). The mature peptide was produced by cleaving off the pro-domain in the putative maturation site Arg-X-X-Arg. The mature protein consisted of 128 amino acids with estimated molecular weight of 14.59 k Da and PI of 8.90. The mature peptide includes a TGF-β domain(387-489 aa) with 7 conservtive cysteine residues.The q RT-PCR assay of different developmental stages proved that the Pf BMP3 expressed at all the five stages analyzed(trochophore, D-veliger larvae, umbo veliger larvae, eyespot larvae and metamorphosis), and highly expressed at trochophore and metamorphosis significantly. The result indicateed that the Pf BMP3 might involve in development regulation during trochophore and metamorphosis of P. fucata. The q RT-PCR assay proved the Pf BMP3 expressed in all the eight tissues analyzed(hepatopancreas, adductor muscle, hemocyte, gonad, intestine, pearl sac, mantle and gill), and the highest relative expression level appeared in gill and mantle. In situ hybridization of the mantle showed that the Pf BMP3 high expressed in the inner epidermal of the inner fold, the outer epidermal of the middle fold and the outer epidermal of mantle. These result indicated that the Pf BMP3 might play an role in shell formation of P. fucata.In shell notching experiment, the Pf BMP3 gene showed high expression significantly in gill at 36 hours after treatment, and 24 hours in mantle. The result indicated Pf BMP3 gene played an role in shell repair after damaged.The expression level of Pf BMP3 in gill and mantle were analysed after the pearl oysters were cultured in sea water with different concentration of calcium ion. The result showed that the expression level of Pf BMP3 in low calcium group(adding EDTA group) was higher than in high calcium group(adding Ca Cl2 group). The change tendency was similar to calmodulin(Ca M) gene which had been demonstrated participation of calcium ion metabolism during shell formation. The result indicated Pf BMP3 gene might play an role in the regulation of calcium ion metabolism during shell formation.RNA interference(RNAi) was performed aimed at Pf BMP3 gene and then theexpression level of Pf BMP3 and Pf Ca M were analysed. The result showed that the expression level of Pf Ca M appeared down-regulated after RNAi, which indicated Pf BMP3 gene might participate in calcium ion metabolism though regulation of Pf Ca M.2. Moleculer cloning and preliminary functional studies of BMP10 gene from pearl oyster, P. fucata. In this study, two sequence fragments with the annotation of BMP10 homologous gene from the transcriptom data were complemented and spliced. Then the c DNA sequence of BMP10 gene from the P. fucata was obtained through RACE technology. The full length c DNA is 1752 bp, including an open ORF of 1344 bp, 5’UTR 293 bp and 3’ UTR 115 bp. The gene was named as Pf BMP10(Gene Bank accession number: KU529148).The Pf BMP10 gene encodes 447 amino acids and has a predicted molecular weight of 51.92 k Da, the theoretical isoelectric point(PI) of 6.14. The prediction of transmembrance structure showed that Pf BMP10 has no transmembrane structure and was located extracellular. The analysis of the deduced amino acids sequence showed Pf BMP10 contains a signal peptide(1-23 aa), a pro-domain(24-337 aa) and a mature peptide(338-447 aa). The mature peptide is produced by cleaving off the pro-domain in the putative maturation site Arg-X-X-Arg. The mature peptide included a TGF-βdomain(344-447 aa) with 7 conservtive cysteine residues.The q RT-PCR assay of different developmental stages proved that the Pf BMP10 expressed at all the five stages analyzed(trochophore, D-veliger larvae, umbo veliger larvae, eyespot larvae and metamorphosis), and highly expressed at trochophore and metamorphosis significantly. The result indicateed that the Pf BMP10 might involve in development regulation during trochophore and metamorphosis of P. fucata. The q RT-PCR assay proved the Pf BMP10 expressed in all the eight tissues analyzed(hepatopancreas, adductor muscle, hemocyte, gonad, intestine, pearl sac, mantle and gill), and the highest relative expression level appeard in gill and Mantle. The result indicated that the Pf BMP10 maight play an role in sheell formation of P. fucata.In shell notching experiment, the Pf BMP10 gene showed high expression significantly in gill and mantle at 24 hours after treatment. The result indicated Pf BMP10 gene played an role in shell repair after damaged.The study provides some bases for the functional study of BMP family genes and the mechanism study of shell formation.