Cloning And Functional Analysis Of Gene PU3 And PU6 In Pinctada Fucata

Pinctada fucata is one of the most important economic shellfish in China,and its mineralization mechanism has always been a hot spot in the field of biomineralization.In this paper,we analyzed the proteomics and transcriptomics data of Pinctada fucata,and selected two matrix proteins containing fibronectin type III domain,PU3 and PU6.Through the gene cloning and functional identification of these two proteins,the role of PU3 and PU6 in the mineralization process was preliminarily explored.Based on the data analysis of the proteome and transcriptome of Pinctada fucata,we cloned the c DNA sequences of PU3 and PU6 by RACE,and analyzed their sequences using some bioinformatics software.The expression of these two genes in different tissues was explored by q PCR.Shell damage experiment and RNA interference experiment were performed to study the function of PU3 and PU6.The full-length of the PU3 gene is 2361 bp with an open reading frame of 1857 bp.PU3 has 618 amino acid residues(p I=6.53).PU3 contains four fibronectin type III domains.PU3 gene is highly expressed in the mantle.During the process of shell damage repairing,the expression level will increase significantly and continue to maintain a high level.RNAi experiments showed that inhibition of the expression of this gene caused a significant change in the structure of the shell prismatic layer.The full-length of PU6 gene is 2068 bp,with a 1863 bp-long open reading frame,which produces a protein with 620 amino acid residues(p I=8.74).PU6 has a 19-aa-long signal peptide.PU6 contains four fibronectin type III domains.The PU6 gene is specifically expressed in the mantle and presents a positive response to the restoration of the shell damage.RNAi experiments showed that inhibition of the expression of PU6 gene caused a significant change in the structure of the prismatic layer,the boundary became rough,and voids appeared.In summary,this study shows that the matrix proteins PU3 and PU6 will participate in the mineralization process of Pinctada fucata,and mainly play a role in the mineralization process of the shell prismatic layer.Inhibiting their expression will destroy the structure of the prismatic layer.