| Freshwater pearls are widely used in modern life as natural jewelry and cosmetic material, and research on its bionic materials is a hotspot of current researches. Determinants of pearl quality include color, size, shape, luster, degree of finish, nucleated pearl nacre thickness, of which color is a mainly indicator, the prices vary greatly according to its qualities. Less is known about the mechanisms of pearl color formation and the genes involved in this process. Given the key role of chitin in organic matrix in nacre and the appearance upon inhibition of chitin synthase, it was speculated that chitin was associated with the pearl size, shape, and luster. However, the molecular mechanism underlying pearl quality remains unclear, but all of which are closely related to the process of pearl formation. This study was intended to identify pearl quality-related genes using the technology of transcriptome analysis based on Roche 454 sequencing with the samples from the purple line mussels and white line mussels selected by our laboratory, and further explore the process of nacre chitin synthesis and the underlying regulatory mechanism, in an attempt to get a preliminary understanding about the molecular mechanism underlying pearl and color formation, and the process of chitin synthesis as well.1. cDNA library construction of the mantle from H. cumingii and analysis of pearl quality-related genes981,302 high-quality EST sequences were obtained from the mantles of H. cumingii secreting the purple and the white nacre using the Roche 454 pyrosequencing technique. Among them, 541,268 ESTs were obtained from c DNA library of the purple line with a mean length of 298 bp, and 440,034 ESTs from c DNA library of the white line with a mean length of 293 bp. These EST sequences were clustered and assembled into 222,158 single sequences with open reading frames. Based on gene annotation GO, KEGG analysis methods and COG analysis, the functions of these sequences were predicted. Thirty-three genes involved in the biomineralization process were identified and classified. In addition to calcium, many other metals were contained in the nacre. According to a previous study, the concentrations of Zn, Mg, Ti, V, Ag and Co in purple pearls were higher than those in white pearls. In this study, six genes involved in metal metabolism were also identified, including multi-copper oxidase, Mg2+and Co2+transporter, ferritin, cadmium matallothionein, matrix metalloproteinase and astacin-like protein. These genes were composed of six genes related to chitin biosynthesis and/or modification, five moderately acidic shell protein genes, two basic shellprotein genes, ten genes involved in calcium metabolism, six genes involved in metal metabolism, two matrix protein genes and two genes binding to or cross-linking the matrix. The six genes associated with chitin included chitin synthase, chitin binding peritrophin-A, chitin deacetylase 5, chitin deacetylase 1, chitinase, and chit3 protein. These gene sequences and molecular markers would facilitate exploring the functions of specific genes involved in the process of pearl formation, and guide genetic breeding by employing molecular markers.2. Analysis of differentially expressed genes associated with nacre color and SSR, SNP screeningTo identify genes involved in pearl color, digital comparative analysis of mRNA expression profiles on mantles secreting purple and white nacre was conducted based on the obtained transcriptome sequence information. The results showed that in a total of 358 differentially expressed genes, 137 genes were highly expressed in the purple library, and 221 genes were highly expressed in the white library(error rate <0.001). Most of these genes were genes with specific functions. Among the 42 genes with functional annotation, 22 genes showed significantly higher expression levels in the purple library, and 20 were highly expressed in the white library. 。. Intrinsic factor gene, which is crucial for the normal absorption of B12, was found to have higher expression levels in the Purple library. Vitamin B12, also called cobalamin, is the only vitamin containing a metal element, and contains the biochemically rare element cobalt. A Mg2+ and Co2+ transporter gene was also found in this study. Whether or not vitamin B12 metabolism was related to nacre color is an interesting question to pursue. In addition to the obvious difference in secreting nacre color between these two samples, the purple nacre was considered to be more exquisite than the white nacre, while the white nacre was secreted faster than the purple nacre. The secretion of the shell matrix was non synchronous in the temporal sequence between the two samples. A greater number of genes involved in modulating crystal polymorph were found to have higher expression levels in the Purple library, while genes involved in basic crystal formation were found to have higher expression levels in the White library. The differentially expressed genes with function annotation were mainly involved in signaling, cell proliferation, differentiation and apoptosis, cell-to-cell and cell-to-matrix interactions and extracellular matrix. In addition, 9,474 SSR markers were identified from these ESTs as well as 27,303 SNP locus markers. Among them, 13,087 conversion sites, 9,944transversion sites, and 4,272 insersion and deletion sites were identified. This study provides valuable data for the study of the formation and evolution mechanism about colored shell, pearl or shell nacre. Besides, the molecular markers discovered in this study may provide a basis for genetic linkage analysis and quantitative trait locus(QTL) analysis of H. cumingii in the future.3. Sequence analysis, homology comparison and tissue distribution of seven genes in chitin synthesis pathwayBased on the early transcriptome sequencing results and online BLAST, the partial gene sequences of trehalase, hexokinase, glucose-6-phosphate isomerase, glutamine:fructose-6-phosphate aminotransferase, glucosamine-6-phosphate N-acetyltransferase, phosphoacetylglucosamine mutase, and UDP-N-acetylglucosamine pyrophosphorylase were identified from the triangle sail mussel. Using performing sequence analysis, secondary and tertiary structure simulation of translated proteins, sequence homology comparison, phylogenetic analysis, spatial and temporal expression pattern analyses, we demonstrated that these sequences shared high similarities with their respective cunterparts from insects, suggesting that a chitin synthesis pathway similar to that in insects may exist in H. cumingii. q RT-PCR results showed that trehalase gene was mainly expressed in the mantle. The mantle is known as the main organ to generate shells and pearls, this gene might be functionally similar to the trehalase gene in insects, participating in regulating chitin biosynthesis in shells and pearls. In addition to trehalase gene and UDP-N-acetylglucosamine pyrophosphorylase gene with specific expression profiles, the other genes were constitutively expressed in all detected tissues. This suggests that these genes might also participate in other biological anabolic processes other than chitin biosynthesis.4. Sequence analysis and expression patterns of chitin synthase gene(HcCHS1) in H. cumingiiIn the present study, we assembled two HcCHS1 fragments obtained from transcriptome sequencing and 5′RACE, and obtained a c DNA sequence of 6,906 bp coding a polypeptide of 2,300 amino acids. The structure prediction of this deduced amino-acid sequence showed that this protein contained a My Sc domain, a characteristic structure of Chitin synthase domain, and 12 transmembrane domains. BLAST online analysis showed that the deduced amino-acid sequence of Hc CHS1 shared the highest identity(67%, for both) with the CHS1 of Pinctada fucat(Gen Bank accession no.: BAF73720) and Atrina rigida(Gen Bank accession no.: AAY86556). Phylogenetic analysis indicated that HcCHS1 was more similar to the CHS1 of marinemolluscs. The 3D model results showed that the spatial structure was highly conserved in various species. Although there were only 12 transmembrane domains, fewer than 16 domains from the previously reported in Atrina rigida, and 16-18 domains in insects, the basic conformation of the protein molecules remained unchanged, especially the structure of the catalytic center remained unchanged, thus guaranteeing the basic function of the molecule, with the protein still being able to perform the function of chitin synthase. Homologous comparisons revealed that Hc CHS1 shared the highest similarity with chitin synthase A from Mytilus gallopovincialis and Atrina rigida. Tissue distribution also showed that the gene mainly expressed in the mantle, and less in other tissues such as the hemocyte and intestine. q RT-PCR in this study showed that Hc CHS1 was up-regulated in the mantle at 12 hours after shell breaking, and reached the highest level(two fold the normal) at 24 h post shell damage, suggesting that this gene was involved in shell regeneration. Knowing that the mantle is the counterpart of the cuticle in insects due to its key role in shell and pearl formation in molluscs, we speculated that Hc CHS1 might be similar to cuticle chitin synthase A in insects, and participate in the formation of shells and pearls in the triangle sail mussel.In conclusion, this study was intended to screen genes associated with biological mineralization that are involved in pearl forming in H. cumingii. Our digital expression analysis identified a large number of differentially expressed genes from the Purple-library and the White-library. At the same time, eight key genes in the chitin synthesis pathway were also discovered and identified. These genes, especially the chitin synthase gene, may participate in the regulation of shell and pearl formation. In addition, a set of SSRs and SNPs between the two samples were identified from the ESTs, which provide markers for genetic linkage and QTL analysis in genetic breeding. These EST sequences may also provide valuable information to further understand the molecular mechanisms involved in the formation, color determination and evolution of the pearl formation in H. cumingii, thus accelerating the breeding process. |
PDF Full Text Request