Molecular Cloning,Expression And Functional Analysis Of Chitinase Gene And Octopamine Receptor Gene In Pearl Oyster,Pinctada Fucata

The pearl oyster Pinctada fucata is one of the most popular breeding shellfish for seawater pearl production in the world. In recent years, the pearl quality has declined because of the ocean pollution, disease outbreaks and quality degradation. The formation of the pearl is closely connected with the growth of the shell. Chitin is one of the three important components of shells. And chitinases are regulatory proteins of chitin qualitative. Octopamine receptor(OAR) is common in invertebrates and plays important roles in invertebrate life, including adjusting nerve muscle rhythm contraction and the cAMP level, participating in phosphorylation pathways, etc. Chitinase and octopamine receptor are necessary proteins participate in several biological processes, and are essential for the normal development and function of many forms of organisms, but their functions in pearl oyster, Pinctada fucata are still unknown. From the transcriptome sequencing of Pinctada fucata in our laboratory, we cloned the full lenth of Pinctada fucata chitinase gene(named as Pf-Chi1) and Pinctada fucata octopamine receptor gene(named as PfOAR). Then the two genes were characterized and the potential physiological functions were investigated by qRT-PCR expression, shell damage induced expression and in situ hybridization. These results provided basic information for further research of the growth and development in the pearl oyster, Pinctada fucata. The results are as follows: 1. Cloning and molecular characterization of Pinctada fucata Pf-Chi1 geneBy 5’-RACE and 3’-RACE, we got the full sequence of Pf-Chi1 gene. The full-length cDNA of Pf-Chi1 was 2743bp(GenBank number: KT956975), and it consisted of a 2187 bp ORF(Opening Reading Frame) encoding 728 amino acids, a 47 bp 5’-untranslated region(UTR) and a 509 bp 3’-UTR with a polyadenylation signal(AATAAA). The Pf-Chi1 protein was composed of an N-terminal leading signal peptide and with an estimated molecular mass of 78.41 kDa and a predicted p I of 5.57. Domain analysis were showed that Pf-Chi1 contained functional motifs including 4 conserved domains: a catalytic domain(glycoside hydrolase family 18 domain) followed by 3 chitin-binding domains(CBM14 domain). Homology analysis revealed that Pf-Chi1 shared 22.43-31.4% identity with other Chi1 gene from other species and shared the highest identity(31.4%) with Crassostrea gigas Chi1, the lowest identity(22.43%) with Fenneropenaeus chinensis Chi1. The phylogenetic tree showed that Pf-Chi1 clustered with C.gigas Chi1, which suggests that Pf-Chi1 in this present study belongs to the bivalvia Chi1 family.The tissues expression profile showed that Pf-Chi1 transcripts were expressed in all the seven tissues(muscle, mantle, gill, hepatopancreas, hemocytes, intestines and gonad), with a relatively higher expression in mantle than in other tissues. The developmental stages expression profile showed that Pf-Chi1 transcripts were expressed in all the larval developmental stages(trochophore, D-veliger, umbo veliger, pediveliger and metamorphosis), and the highest level of Pf-Chi1 mRNA was found in trochophore larvae, then, the expression was slightly dropped in D-veliger larvae. Moreover, in umbo veliger, pediveliger and metamorphosis larvaes the expression levels were lowest(P<0.05). The results show that Pf-Chi1 gene may play an important roles in shell formation and growth of the the pearl oyster, Pinctada fucata.In shell damage induced experiment, Pf-Chi1 transcript levels were up-regulated significantly(p<0.05) 24 h after shell damage and expression reduced gradually thereafter, suggesting that the Pf-Chi1 might play a role in shell formation.In situ hybridization showed that Pf-Chi1 was mainly expressed in the mantle edge(ME), especially in the outer epithelial cells of the inner fold(IF) and slight hybridization signals were founded in the inner epithelial cells of the middle fold(MF). No hybridization signals were detected in negative control. The results show that Pf-Chi1 gene may play an important roles in shell formation and recorvery. 2. Cloning and molecular characterization of Pinctada fucata PfOAR geneThe full-length cDNA of PfOAR was 1890bp(GenBank number: KX082720), and it consisted of a 1659 open reading frame(ORF), encoding 552 amino acids. PfOAR has the common features of the vast family of G protein-coupled receptors including 7 hydrophobic transmembrane domains. The PfOAR protein molecular mass was 63.167 kDa and has a predicted pI of 9.19. Homology analysis revealed that PfOAR has a highly identity with other OAR proteins from other species. The phylogenetic tree showed that PfOAR clustered with C.gigas OAR, which suggests that PfOAR in this present study belongs to the bivalvia OAR family.The tissues expression profile showed that PfOAR transcripts were expressed in all the six tissues(mantle, pearl sac, intestines, hepatopancreas, gill and muscle), with a relatively higher expression in mantle than in other tissues. The developmental stages expression profile showed that PfOAR transcripts were expressed in all the larval developmental stages(trochophore, D-veliger, umbo veliger, pediveliger and metamorphosis), and the highest level of PfOAR mRNA was found in metamorphosis larvae(P<0.05). The results show that PfOAR gene may play an important roles in shell formation and growth of the the pearl oyster, Pinctada fucata.In shell damage induced experiment, PfOAR transcript levels were up-regulated significantly(p<0.05) 36 h after shell damage, suggesting that the PfOAR might play a role in shell formation. The results show that PfOAR gene may play an important roles in shell formation and recorvery.