Cloning And Functional Analysis Of Three Shell Matrix Protein Genes From Hyriopsis Cumingii

Hyriopsis cumingii,unique freshwater mussel of China,accounts for 80 percent of freshwater pearls yield.And it is well known that the shells and pearls of H.cumingii are the products from biological mineralization,the process of depositing calcium carbonate crystals under the control of various matrix proteins.The identification and functional exploration of matrix proteins can help to understand the biomineralization mechanism of shells and pearls.In this study,three matrix protein genes,silkmaxin,hic6 and hic7,were isolated and identified from H.cumingii.Structural and functional characters of three genes were analyzed.Silkmaxin,hic7 and hic6 genes were cloned from the mantle of H.cumingii,and the cDNA sequence of silkmaxin gene was obtained through experiments,with a total length of 1151 bp,and an ORF of 885 bp,encoding a total of 294 amino acids and contains a signal peptide consisting of 15 amino acids.After removal of the signal peptide,the theoretical molecular mass of this gene was 28.39 kda and the isoelectric point was 9.52,indicating that the protein has high similarities with an silk protein.The cDNA of hic7 encodes a total of 79 amino acids and contains a signal peptide consisting of 19 amino acids.After removal of the signal peptide,the theoretical molecular mass of this gene was 6.8 kDa and the isoelectric point was 6.12.Hic6 has a cDNA length of449 bp,in which ORF is 219 bp and encodes a total of 73 amino acids,includinga signal peptide consisting of 20 amino acids.After removal of the signal peptide,the theoretical molecular mass of this gene was 6.43 kDa and the isoelectric point was 4.28.Tissue expression analysis indicated that silkmaxin,hic6 and hic7 are specifically expression in the edge of mantle,and in situ hybridization indicated that hic6 and silkmaxin genes are expressed in the dorsal epithelial cells of the mantle pallial,while hic7 was of a strong signal in epithelial cells of the entire mantle.It is speculated that silkmaxin and hic6 might be involved in the mineralization of the nacreous layer,whilehic7 might account for nacreous layer and prismatic layer formation.In the RNA interference experiment,the expression levels of the three genes,silkmaxin,hic6 and hic7 decreased significantly after dsRNA injection.By RT-PCR analysis,quantity of the expression of the experimental group had undertook significantly lower.The SEM images showed that matrix protein genes silkmaxin and hic6 had no significant effect in prism layer,and only affected the mineralization of the shell nacre.These two genes may be related to the crystal growth and morphology of the nacre.But the SEM images showed that the expression of hic7 gene was significantly changed in the experimental group compared with the control group.The experimental results showed that hic7 obtained by cloning was related to the formation of pearl and prismatic layers.The obtained high-throughput genetic data results were compared with the sequences encoded by transcript target genes,and the positions of differentiated exons in the genome were compared.Primers were designed based on the cDNA sequence of hic6 in H.cumingii.Genomic sequence fragments were amplified according to cDNA fragments of hic6 genes.Five SNPs were identified from hic6 gene after sequencing the PCR products and blasting.Regarding these SNPs,the genotype and gene frequency were further analyed from 144 individuals of H.cumingii.The result showed the polymorphic information(PIC)of these SNPs were low and medium genetic diversity.Associations were analyzed between SNPs and shell nacre colour related traits with Chi-square test.The results showed that there were 5 SNPs on hic6 gene were significantly associated with shell nacre colour(L,a and dE).Haplotype analysis revealed that the frequence of one major predominant haplotype(T1)in the white stock was significantly higher than that in the purple stock.And the other four major predominant haplotypes(T2,T3,T4 and T5)showed significantly higher frequency in the purple stock.The results suggested that the polymorphic loci in hic6 could be potential genetic markers for future molecular selection of different nacre in H.cumingii.