| China is a freshwater pearl producing country, accounted for more than 95% all the world total output of fresh water pearl. But the scarcity of large high-quality pearls and low yield, it is difficult to meet the needs of domestic and international pearl market. Freshwater resources are rich in China, over the years the production in the mantle of pearl farming. But because the mantle pallisl thin film, pearl space is limited, is not easy to cultivate large pearls. However, the visceral mass of a larger space, can be inserted into the larger diameter of nucleus, shorten the cycle, to cultivate bigger pearls, increase economic benefits. But because the freshwater shellfish is an open circulatory system, visceral mass of pearl spot near the stomach, kidney, gonad and other organs, the pearl not as luster of the mantle produced pearl. Our research group has long aimed at how to cultivate high quality large pearl in visceral mass and carried out the research of different levels, to explore the aspects of physiological and biochemical condition, the visceral mass of pearl nucleus inserting, immune response and calcium metabolism related gene function.Hyriopsis cumingii as China unique mainly freshwater pearl mussel, is widely cultured pearls. The main component of pearl is Ca CO3, so to strengthen the study has important implications for biological process and functional genes of Hyriopsis cumingii calcium metabolism. In recent years, with the development of molecular biological technology, provides an experimental platform for the research of functional genes of different species. But at present the gene data mollusk is a lack of the research of marine shellfish is relatively rich, but because of the difference of water environment greatly, marine shellfish and freshwater shellfish gene are quite different, long drag on freshwater mollusks functional gene. With the rapid development of the promotion of new generation sequencing technology, combined with the digital gene expression profiling transcriptome sequencing technology can provide high throughput analysis of data for the no reference genome species.This research take the mantle of the front、middle and rear、visceral mass、gonad and hemolymph 6 pearl related organizations of Hyriopsis cumingii, total RNA was extracted. Through the enrichment of m RNA by reverse transcription into c DNA and sequencing of c DNA library construction joints, the detection of qualified by Illumina Hi Seq 2000 sequencing. The clean reads obtained after filtering by raw reads, assembled into 257457 Unigenes, with 223345 genes out of annotated 7 database, through the GO and KEGG Pathway function significant enrichment analysis were screened out of 156 calcium metabolism related genes homology rate is high.A total of 200 size differences smaller at the age of 2 Hyriopsis cumingii, 20 rats were randomly selected as the control group, the remaining 180 of experimental group were inserted diameter 3mm nucleus and a mantle pieces to mantle and visceral mass, conventional breeding in the same waters. Total RNA of 5 the mussel of the control group and after nucleus inserting 5d, 20 d, 50 d, 90 d the mantle and visceral mass pearl cultured was extracted and the detection of qualified for DGE sequencing. 10 the DGE library clean reads 7M, and transcriptome sequence annotation mapping rate in more than 90%, high quality to meet the requirements of the follow-up data analysis of biological information. By means of differential gene expression analysis, GO and KEGG function significant enrichment analysis, screened 58 differentially expressed significantly from the transcriptome of 156 calcium metabolism related genes. The 58 calcium metabolism related genes were different expression of pearl period, summarizes the expression change trend of among the formation of pearl.EF-hand protein is a kind protein of related calcium metabolism function, the study found 7 EF-hand genes through the analysis of transcriptome and DGE. Firstly, the analysis of amino acid sequence alignment and phylogenetic tree of these 7 genes, to find out the mutual similarity relation. Selected the containing EF-hand calcium binding domain 1(EF-hand calcium-binding domain-containing protein 1, EFCB1) gene c DNA sequence was cloned and its nucleotide sequence and amino acid sequence by bioinformatics analysis. In different period of pearl 7 genes of EF-hand family and EFCB1 gene in various tissues by expression analysis. The results showed that EF-hand gene in the mantle and visceral tissues and in pearl formation patterns had significant difference. More deeper and more research to influence the deposition of nacre by regulating the expression of key genes.Provides genetic data rich information of the transcriptome data platform constructed and 10 DGE Library of Hyriopsis cumingii in this study, laid the foundation for further research of functional genes and molecular mechanism of the formation of pearl, the more molecular research and researcher effort will eventually in the visceral mass of Hyriopsis cumingii breeding high-quality pearls, accelerate the sustainable development of Chinese pearl industry. |
PDF Full Text Request