Isolation, Identification And Functional Analysis Of Matrix Protein Gene Hic09 And Hc-upsalin In Hyriopsis Cumingii

Hyriopsis cumingii,unique freshwater mussel of China,accounts for80 percent of freshwater pearls.And it is well known that the shells and pearls of Hyriopsis cumingii are the products from biological mineralization.The study of their biomineralization mechanism plays a crucial role in improving the quality of pearls.In this study,two matrix protein genes relating to shell formation have been cloned from the mantle,and the function was explored initially.1.The hic09 and hc-upsalin genes were cloned,and characterized from the mantle of freshwater mussel Hyriopsis cumingii.The full length cDNA of hic09 gene is 662bp with a 303bp open reading frame.Hic09encodes a 8.651 KD protein composed of 100 amino acids.Its pI is 6.466.There is no homologous protein in the Genebank,this is a novel protein in the Hyriopsis cumingii.The full length cDNA of hc-upsalin is 553bp with a360bp open reading frame.It encodes a 11.4 KD protein composed of 119amino acids,and the pI of it is 6.93.Hc-upsalin shares high sequence homology with matrix protein upsalin from Unio pictorum.2.Tissue expression analysis indicated that hic09 is specifically expression in the mantle.and in situ hybridization indicated that the signal strong signals occurred in the whole outer epithelial cells of outer fold,and also occurred in the crust groove between out fold and middle fold.This result indicated that hic09 is a multifunctional matrix protein involved in the cuticle,prism layer and nacre layer of the shell.In previous pearl-nucleus-inserting experiment,the results showed that the process of CaCO₃ deposition during pearl formation could be divided into two consecutive stages:the CaCO₃ primitive accumulation and the CaCO₃regular deposition.The quantitative real-time PCR（qRT-PCR）revealed that the expression levels of hic09increased significantly at nucleation period,revealing that hic09 may take part in the CaCO₃ nucleation and regulated the growth of aragonite crystals during pearl formation.In the RNA interference experiment,the expression levels of the hic09 decreased significantly after dsRNA injection.Observation by SEM,we consider that hic09 may participates in the formation of prismatic layer of shell mineralization and affected the mineralization of the shell nacre.They may be related to the crystal growth and morphology of the nacre.3.Hc-upsalin is specifically expressed in muscular tissues mostly in the mantle,with positive signals detected in dorsal epithelial cells of the mantle by in situ hybridization,suggesting its involvement in shell nacreous-layer biomineralization.In previous pearl-nucleus-inserting experiment,the results showed that the expression levels of hc-upsalin increased significantly at nucleation period,revealing that hc-upsalin may take part in the CaCO₃ nucleation and regulated the growth of aragonite crystals during pearl formation.