Identification Of Shell Matrix Protein Hic-Genes Of Hyriopsis Cumingii And Preliminary Functional Exploration

Hyriopsis cumingii is a kind of unique freshwater mussel of China,accounting for 80%of freshwater pearls.Matrix proteins,as a key component of matrix organic,play a significant role in regulating the polymorph and morphology during the mineralization process of shells and pearls,and research it providing meaningful information for the mechanism of shell formation and biomineralization.In this experiment,we have cloned three matrix protein genes which are involved in the formation of shell from mantle,and preliminary explored its role in the formation of pearls and shells.1.The hichin gene was isolated from the mantle of Hyriopsis cumingii.The full-length of hichin is 645bp containing a 543bp open reading frame,encoding a deduced 20.3kDa protein.Its pI is 4.63.The amino acid sequence covered a chitin binding domain,and the three dimensional structure prediction of hichin had high similarities with invertebrate chitin-binding proteins.The full-length of hic14 is 586bp and has a 456bp open reading frame encoding a deduced 151-amino acids protein,and the pI is 6.32.Analysis of amino acid sequences revealed hic14 exists GS,GSG,and SGS repeats.The cDNA of hic19 is 1127bp containing an open reading frame of 579bp.It encodes a deduced 19.6kDa protein composed of 192 amino acids,and its pI is 9.72.There are basic amino acid in the amino acids sequence.Tissue expression analysis showed that hichin,hic14 and hic19 mainly expressed in the mantle,and located at the dorsal epithelial cells of the mantle pallial by in situ hybridization,indicating the involvement of hichin,hic14 and hic19 in nacreous layer and prismatic layer formation.2.During the process of pearl biominerazation,the type of CaCO₃deposition could be divided into primitive accumulation and regular deposition.qRT-PCR revealed that hichin expressed significantly highly during the accumulation of calcium carbonate,suggesting that hichin may participate in the construction of organic framework during mineralization.The expression level of hic14 and hic19 increased significantly at nucleation period,revealing that hic14 and hic19 may take part in the CaCO₃ nucleation and regulated the growth of aragonite crystals during pearl formation.3.In the RNA interference experiment,the expression levels of the four genes,hichin,hic14,hic19,and hic52,decreased significantly after dsRNA injection.Observation by SEM,we consider that hichin may bind with chitin,participating in the organic framework of shell mineralization.The other three matrix protein genes had no significant effect in prism layer,and only affected the mineralization of the shell nacre.They may be related to the crystal growth and morphology of the nacre.