The Comparasion Analysis Of Transcriptomes Among Pearl Oyster And Freshwater Mussels And The Studies Of Paritally Functional Genes

Pinctada fucata martensii is one important pearl oyster mainly cultureled for the production of pearl, Hypriosis cumingii and Cristaria plicata is peculiar breeding clams in our country for the production of freshwater pearl. This paper constructed two transcriptome libraries of mantle tissues from freshwater mussel used the Illumina Hiseq platform.Combined with the existing transcriptome database and whole genome database of P.martensii, we obtained the crucial genes involved in mineralization and immunity, and used cDNA end rapid amplification (RACE) and polymerase chain reaction (PCR) technology to clone the some immune-related gene,and real-time fluorescent quantitative PCR technology was used to verify the gene expression in different tissues and the stimulation of LPS to verify the function of immune genes. The main results were as follows:(1)Transcriptome analysis of biomineralization-relatd genes and immunology-related genes in H. cumingii and C. plicata: 47,722 Unigenes were obtained after assembly and redundancy in H. cumingii, and 66,072 Unigenes were obtained in C. plicata utilized the identified mineralization genes in P. martensii. The obtained biomineralization-related genesincluded: VWA-containing protein, chitinase, chitin synthase and tyrosine enzyme and so on. There were 47 unigenes associated with the above functional annotation in P.Martensii; there were 85 unigenes in H. Cumingii and 119 unigenes in C. Plicata(|log2FoldChange (RMO/RMC)| ?5, FDR ? 0.05). The obtained immune-relaed gene contained Histone H3, Histone H4, Histone acetyltransferase (HAT) and Histone deacetylase (HDAC) (|log2FoldChange (RMO/RMC)| ? 2, FDR ? 0.05). The expression level of two sequences of VWA-containing protein, chitin synthase and tyrosinase in three species were verified. The results showed that the quantitative results were consistent with the data of transcriptome.(2)Selection and verification of differentially expressed genes related to biomineralization in the mantle and KEGG pathway of three species: according to the criteria of |log2FoldChange (RMO/RMC)| ? 5, Pvalue ? 0.05 and FDR ? 0.05, we screened the genes with high expression in the mantle central and edge of three species. There were 91 genes with high expression in the mantle edge of P. martensii, there were 286 genes with high expression in the mantle edge of H. cumingii and there were 165 genes with high expression in the mantle edge of C. plicata, and there were 20 genes with high expression in the mantle central zone P. martensii, there were 43 genes with high expression in the mantle central of H. cumingii and there were 84 genes with high expression in the mantle central of C. plicata, we verified the expression of wingless -type MMTV integration site family, member 7 (Wnt) and mucin gene. The results of fluorescence quantitative validation were anastomosis with transcriptome, and could be used for further research. Then though the analysis of KEGG pathway enrichment of differentially expressed genes related to mineralization in the mantle central and edge three species, We obtained and verified Hedgehog signaling pathway (Pvalue ? 0.01), there were 25 unigenes involved in this pathway in P. martensii, as the same in the H. cumingii, and there were 26 unigenes in the C.plicata, the genes expression trend were totally identical with the transcriptome datas.(3)Cloning and functional verification of differentially expressed immunology-related in the mantle of three species: used RACE technology, we have got the cDNA full length of histone H3, Histone H4, Histone acetyltransferase (HAT) and Histone deacetylase(HDAC) from P. martensii (PmH3, PmH4-1, PmH4-2, PmHAT, PmHDAC-1,PmHDAC-2),and the length of them is 627 bp, 556 bp, 599 bp, 2025 bp, 2143 bp, 887 bp, respectively,they respective encode 136,115, 113, 491,479, 479 amino acids; not cloned full genetic sequencein in H. cumingii and C. Plicata. After LPS stimulation, then the relative expression of PmH3, PmH4-2, PmHAT, PmHDAC-1 and PmHDAC-2 had changed significantly. The expression of PmH3, PmH4-2, PmHAT after stimulation were showing significant upward trend after 12 hours (P<0.05) , and PmHDAC-1 and PmHDAC-2 had a significant rise in 6 hours after stimulation (.P<0.05, and the relative expression of PmH4-1 was not significantly changed,these results showed that PmH3, PmH4-2, PmHAT,PmHDAC-1 and PmHDAC-2 are closely related to the immune response of organism.