Research On The Transcription Regulation Of Matrix Protein Genes In Pinctada Fucata

The pearl oyster,Pinctada fucata,is namely its unique ability to produce pearls.The shell and pearl are typical products of biomineralization.Matrix proteins are thought to play key roles in the shell and pearl formation of the pearl oyster,and a primary understanding of the mechanisms associated with shell formation has been obtained through extensive study ofidentification and functional analysis of matrix proteins.However,very little is known about the intracellular regulation of matrix protein genes,and few relevant studies have been reported.This study is from the perspective of promoters of matrix protein genes,in addition with the identification and characterization of transcription factors,to investigate the upstream gene transcriptional regulation mechanism of matrix proteins in pearl oyster.In this study,twotranscription factors,Pf-POU2F1 and Pf-POU3F4,were cloned and characterized.Meanwhile,the promoters of the matrix protein genes Aspeinand Prismalin-14 werecloned.Intransientco-transfection assays,Pf-POU3F4 greatly up-regulated the promoter activities of the Aspein and Prismalin-14 genes in a dose-dependent manner.The structural integrity of Pf-POU3F4 was essential for its activation function.One region of the Aspein gene promoter,-181 to-77 bp,and two binding sites in the Prismalin-14 gene promoter,-359 to-337 and-100 to-73 bp,were required for activation of Pf-POU3F4.Furthermore,in vivo Pf-POU3F4 knockdown led to the decrease of Aspein and Prismalin-14 gene expression.Taken together,these results suggest that the transcription factor Pf-POU3F4 regulates the expression of the matrix protein genes Aspein and Prismalin-14 in pearl oyster.In this study,the promoters of the matrix protein genes Shematrin-2,ACCBP,MSI60 and N19 were also cloned.The basictranscriptional activity of Shematrin-2 gene promoter was the highest.In addition with further study on the function of Shematrin-2,thetranscriptional regulation of Shematrin-2 gene was also studied preliminarily.Three C/EBP transcription factors and three subunits of NF-Y transcription factor were cloned.Intransientco-transfection assays,Pf-C/EBP-Aand Pf-C/EBP-Bgreatlyup-regulatedthepromoter activities of the Shematrin-2 gene in a dose-dependent manner.At the same time,we also made a preliminary study onthe effects of Pf-C/EBP-A and Pf-C/EBP-B on the other sevenmatrix protein genes promoters’ activities.Theresults showed that Pf-C/EBP-A and Pf-C/EBP-B could significantly up-regulatedthepromoter activities of multiple matrix protein genes.Taken together,these results suggest that the transcription factors Pf-C/EBP-A and Pf-C/EBP-B may participate in the transcriptional regulation of multiple matrix protein genes.Our study reveals the transcription factors related to the expression regulation of matrix protein genes,and provide a basis for further research on matrix protein gene transcriptional regulation mechanism.There will be helpful to understand the upstream regulatory mechanism of shell formationat the molecular level.