Cloning And Functional Studies On Genes Related To TGFβ Signal Pathways Of Pinctada Fucata

Dozens of matrix proteins have been isolated or cloned from the pearl oyster Pinctada fucata. They are involved in a series of processes in shell and pearl formation such as nucleation, growth, orientation and morphology of calcium carbonate crystallites. Therefore, study on the regulation effects of signal pathways on the oyster matrix proteins expressions is bound to open a breakthrough for the investigation of molecular mechanisms on molluscan biomineralisation. In this study, several genes related to the oyster TGFβsignaling pathways were cloned and characterized. The regulation effect of Pf-Smad3 on the mRNA expression level of the oyster matrix protein KRMP was demonstrated. On the other hand, a promoter region of the oyster matrix protein KRMP was identified and analyzed. The results provide much important information to identify a more specific and detailed TGFβsignaling pathway that would take part in regulating matrix proteins transcriptional expressions.The full-length cDNAs of ALR1 and Smad3, two transforming growth factors superfamily members of the oyster Pinctada fucata (short for Pf-ALR1 and Pf-Smad3, in respective) were cloned by polymerase chain reaction (PCR) and rapid amplification of cDNA ends methods (RACE) and were sequenced. The amino acids sequences of their conserved motifs are highly homologous to those of the vertebrates. Semi-quantitative Reverse Transcription-PCR results show that Pf-ALR1 and Pf-Smad3 mRNAs are ubiquitously expressed in all the tissues of the adult oyster but their mRNA levels have significant changes in different periods of embryonic development, shell notching repair and pearl sac formation processes. In situ hybridization results show Pf-ALR1 and Pf-Smad3 mRNAs are expressed mainly at the outer epithelial cells of the middle fold and the inner epithelial cells of the outer fold in the mantle tissue. The results of the drug treatment assays with primary cultured oyster mantle tissue cells indicate there are correlations among Pf-Smad3 mRNA expressions and the mRNA expressions of several matrix proteins and some members of other signal pathways such as engrailed and NF-κB in the oyster. In vivo knockdown of Pf-Smad3 by RNA interference in the oyster led to significant decrease of mRNA expressions of the matrix protein KRMP. The above results suggest that TGFβsignal pathways may play important roles in many psychological processes, including biomineralisation of shell and pearl formation, in a conservative way in the oyster.Using simple specific primers for PCR, a fragment of the 5'-end untranslated region of the oyster matrix protein KRMP was obtained. It contains a long fragment (about 200 bp) that is also shared by the oyster matrix proteins Pearlin and Prisilkin-39. The core promoter region and two Sox binding sites of KRMP were also predicted by bioinformatic softwares. Then the promoter region (-487 bp - -1 bp) of KRMP was ligated into the luciferase reporter vector pGL2-Basic and transfected into mouse cell line MC3T3-E1. The results of the dual luciferase assays show that the high activity of the promoter of KRMP and the mRNA expression level of the mouse Sox9 gene can be increased by overexpression of Pf-Smad3 in mouse cells. Therefore we propose that there are Smad3-dependent signal pathways that can influence the oyster matrix proteins transcripts levels by adjusting the mRNA expression level of the Sox homolog protein in the oyster.All our observations provide foundation to elutriate the signal pathways network on oyster matrix proteins transcriptional expressions and theoretical basis for selecting proper drugs to stimulate the nacre secretion of the oyster mantle in order to enhance the quality of the pearls products.