Cloning And Functional Study Of Shell Matrix Protein Genes Related To Prismatic Layer Formation In Pinctada Fucata Martensii

Shell matrix proteins?SMPs?are the important participants and function executors of shell formation.They play vital roles in the regulation of organic framework formation and mineral deposition.SMPs of prismatic layer were obtained by analyzing the full spectrum of SMPs from Pinctada fucata martensii in our previous research.We cloned the full-length sequences of five genes from prismatic layer matrix protein.Then the functional studies were carried out by q RT-PCR,in situ hybridization?ISH?,RNAi and prokaryotic expression techniques.The results are as follows:1.Beta-hexosaminidase/Beta-N-acetylhexosaminidase?HEX?belong to Glycoside hydrolase family 20?GH20?,and it is the key enzyme in chitin metabolism.Chitin participates in shell formation as the main component of organic framework,and its metabolism plays an important role during shell formation process.Two HEX genes of P.f.martensii were cloned and named as Pm HEX and Pm HEXL.The full-length of sequences Pm HEX was 3 813 bp,encoding 1 141 amino acids.The full-length sequences of Pm HEXL was 2 760 bp,encoding 774 amino acids.Each of Pm HEX and Pm HEXL contained two typical Glyco_hydro₂0 domain and a Glyco_hydro₂0b domain,as well as a CHB-HEX domain.QTR-PCR analysis exhibited that both Pm HEX and Pm HEXL were significantly high expression in mantle edge?ME?and mantle pallial?MP??P<0.05?.And ISH detection showed that Pm HEX and Pm HEXL were mainly located in the outer epidermal cells of the outer fold of ME and MP.After the RNAi of Pm HEX,the expression of Pm HEX significantly down-regulated in the ME and MP,and the microstructure of both prismatic layer and nacreous layer crystallized irregularly.These studies indicated that Pm HEX and Pm HEXL could regulate the formation of prismatic layer and nacreous layer of P.f.martensii.2.Chitin binding protein?CBP?plays an important role in the process of specific binding with chitin and calcification regulation.We cloned the full-length sequences of Pm CBP was 6 365 bp,encoding 2 044 amino acids.Pm CBP contained twenty typical Chitin-binding domains type 2?Cht BD2 domains,CBDs?,and a signal peptide sequence.QRT-PCR analysis exhibited that Pm CBP was most abundant in the MP?P<0.05?.And ISH detection showed that Pm CBP mainly distributed in the outer epidermal cells of MP.After down-regulating Pm CBP expression using RNAi technology in MP?P<0.05?,and the microstructure of nacreous layer showed a disordered growth.In addition,association analysis showed that expression level of Pm CBP gene was significantly correlated with shell weight and shell length.These results indicated that Pm CBP might regulate the formation of nacreous layers of P.f.martensii.3.We cloned the full-length sequences of Pm EGFCP?PU12?was 1 394 bp,encoding 363 amino acids.Pm EGFCP has two conservative epidermal growth factor-like domain?EGF?,a zona pellucida domain?ZP?and a signal peptide sequence.QRT-PCR analysis exhibited that Pm EGFCP was significantly highly expressed in ME and MP?P<0.05?.ISH detection showed that Pm EGFCP mainly distributed in the inner and the outer epidermal cells of the outer fold of ME,the inner epidermal cells of the middle fold of ME and the outer epidermal cells of MP.And after down-regulating Pm EGFCP expression using RNAi technology in ME?P<0.05?,and the shell prismatic layer showed a disordered growth.These results showed that Pm EGFCP could affect the formation of prismatic layer of P.f.martensii.In addition,the Pm EGFCP was inserted into the expression vector p ET32 a,and successfully expressed the recombinant proteins Pm EGFCP.Western blotting results showed that the recombinant protein could integrate with His-Tag monoclonal antibody,in accordance with the predicted molecular weight.Finally,3mg of the recombinant protein Pm EGFCP were obtained by refolding,concentration and freeze-drying.4.The full-length sequences of SMP gene Pm-PNU7 was 1 434 bp,which without functional annotation,encoding 363 amino acids.It contained a special repeating sequence “KGG” and a aspartic acid-rich region “DDDDDDHDD”.QRT-PCR analysis exhibited that Pm-PNU7 was significantly highly expressed in ME and MP?P<0.05?.ISH detection showed that Pm-PNU7 mainly distributed in the inner and the outer epidermal cells of the outer fold of ME,the inner epidermal cells of the middle fold of ME,and the outer epidermal cells of MP.After down-regulating Pm-PNU7 expression using RNAi technology in ME and MP?P<0.05?,the microstructure of both prismatic layers and nacreous layers appear distortion.These results showed that Pm-PNU7 could affect the formation of prismatic layer and nacreous layers of P.f.martensii.