Molecular Cloning And Expression Analysis Of IGFBP5、IGF2BP1 And Matrilin-1 Genes In Pearl Oyster, Pinctada Fucata

The pearl oyster Pinctada fucata is one of the most commercially important bivalves. Genetic researches about its growth, mineralization and immunization can assist to know the molecular mechanisms of its regulation to growth traits, and give a base for optimizing the pearl cultivation. This research cloned the full lenth of P. fucata IGFBP5 gene(Pf-IGFBP-5), IGF2BP1 gene(Pf-IGF2BP1) and Matrilin-1 gene, and analysed their expression profile. 1. Cloning and expression of Pinctada fucata Pf-IGFBP-5 geneWe cloned the full lenth of Pf-IGFBP-5 gene sequence and its c DNA. The full lenth of its c DNA is 1319 bp, including a 399 bp ORF(Opening Reading Frame) encoding 132 amino acids. The first 21 amino acids in the protein domains of Pf-IGFBP-5 is a signal peptide. The amino acid sequence of Pf-IGFBP-5 is highly homologous to that of igfbp-5. It contain several cysteine residues, have a conserved IB motif in N-terminal which affect its affinity with JGFs. The full lengh of Pf IGFBP-5 gene sequence is 5884 bp, including 3 exon and 2 intron.Through q RT-PCR tissue expression analysis, we found the m RNA of Pf-IGFBP-5 was expressed in all the tested tissues and had a significant highly expression level in the pearl sac. Also, the Pf-IGFBP-5 was expressed in all developmental period of Pinctada fucata larva and had a much higher expression level in Trochophore. These results suggest that the Pf-IGFBP-5 gene may function during the formation of Pinctada fucata shell and development of its larva.Through hybrization in situ, hybrization signal of Pf-igfbp-5 was detected in the epithelium of exocuticle of mantle. The signals were also detected in epithelium of the whole pearl sac and syndesm. These findings suggest that the Pf-igfbp-5 may work in the development of its larva and the formation of pearl. 2. Cloning and expression of Pinctada fucata Pf-IGF2BP1 genePf-IGF2BP1 gene was cloned from the Pinctada fucata by RACE techniques based on transcriptome sequences. The full lenth of Pf-IGF2BP1 c DNA is 2980 bp, including a 1737 bp ORF encoding 579 amino acids. Same as the other IGF2BP1, Pf-IGF2BP1 contains 2 RRM motif and 4 KH motif. Its homology rate compared to human, musculus, zebra fish, sinonovacula and ostrea gigas thunb is 35.21%, 34.88%, 34.35%, 53% and 53.6% accordingly.q RT-PCR analysis showed that Pf-IGF2BP1 was expressed in all the tested tissues and expressed at the highest level in the pearl sac(P<0.05), which is the main tissue for pearl formation. This result suggest that Pf-IGF2BP1 may be involved in the formation of pearl. Also, the Pf-IGFBP-5 was detected to have expression in all 5 developmental periods of Pinctada fucata larva and had a much higher expression level in pediveliger(P<0.05). The prodissoconchâ…¡ formate during pediveliger, so that the Pf-IGF2BP1 may be involved in regulating the formation of the prodissoconchâ…¡. These results suggest that the Pf-IGFBP-5 gene may function during the formation of Pinctada fucata shell and development of its larva. By hybrization in situ, we can find the Pf-IGF2BP1 have a high expression in the OF(out fold), which located in the margin of the mantle of Pinctada fucata. The prismatic layer developed form the outer epidermal cells in the margin of the mantle, so the Pf-IGF2BP1 probably paticipated in the formation of the prismatic layer. 3. Cloning and expression of Pinctada fucata matrilin-1 geneMatrilin-1 gene was cloned from the Pinctada fucata based on its transcriptome sequences and named as Pfmatrilin-1. The whole length of Pfmatrilin-1 is 2 036 bp, including a 1 194 bp ORF encoding 397 amino acids, in which there were a signal peptide and 2 VWA(von Willebrand factor A) motifs. Phylogeny analysis proved that the matrilins in vertebrates and invertebrates were of a distant relationship. q RT-PCR tissue expression analysis indicated that Pfmatrilin-1 was expressed in all the tested tissues with a significant high expression level in blood cells(P<0.05). The Pf-matrilin1 was also continuously expressed from trochophore to metamorphosis stages, reaching the peak in pediveliger stage(P<0.05). The prodissoconchâ…¡formed during pediveliger stage, so the Pfmatrilin-1 may be involved in regulating the formation of the prodissoconchâ…¡. All of these results proved that the Pfmatrilin-1 plays an important role of biomineralization in P.fucata.