| Pinctada fucata martensii is the major marine pearl oyster cultured to produce artificial pearl. The core part of marine pearl culture is the necleus-inserting operation. The operation is to insert pearl necleus and mantle pieces from the donor pearl oyster into the visceral mass of the recipent for nucleation. The issues and mantle pieces form pearl sac together. The pearl sac cells secrete nacre which deposited on the necleus forming the pearl. The mantle pieces inserted could cause immune rejection of the pearl oyster for nucleating, which may cause the discharge of necleus and even the death of pearl oyster. Therefore, researchers and culturists have taken various measures in an attempt to ease the immune rejection after necleus-inserting opration for a long time. But so far these problems have not been solved, for which the fundamental reason is that the stressed immune response key factors in the process of necleus-inserting opration are unknown.In this study, haemolymph transcriptome libraries from different stages of P. fucata martensiiwere constructed, sequenced and assembled by RNA-seq approachand analyzed. The differential expression of functional genes and key signal transduction pathways were analyzed and screened in order to identify the immune-related genes. Furthermore, the immune-related proteins were screened and identified by comparing the proteins in hemocytes using isobaric tags for relative and absolute quantitative(iTRAQ) technology and liquid chromatogram and tandem mass spectrum(LC-MS-MS). The results are as follows:Analysis from the seven different haemolymph transcriptome libraries were collected and combined into a transcriptome text set. The combined transcriptome text set generated 81,390 unigenes, of which there are 36,604 unigenes with various length can be annotated. Bioinformatic analysis with NR, Swiss-Prot, COG and GO databases indicated,31,689, 25,253,12,065 and 12,720 transcripts were annotated. In addition, the biological function of 1,636 transcripts were unkown in the COG function classfication. About 21,811 transcripts may participate in 302 metabolic or signalling pathways. When compared the transcriptome library at the 0th day with other time point haemolymph libraries, there were 3,345,6,845,4,636,4,167 and 6,679 genes expressed differentially at 5th,10th,15th, 20th,30th and 60th day, respectively. Interestingly, the number of differencially expressed genes increased significantly at the 10th day.46 immune-related genes showed significant difference in expression at different time after nucleus-insertion.These genes includethe lysozyme, scavenger receptor, glutathione peroxidase, superoxide dismutase, mannose receptors, letin, Toll-like receptor and etc.. The pathways of cell adhesion molecules and primary immunodeficiencies were significantly riched (P<0.05) in the differentially expressed genes of comparsion between any two periods.By combining iTRAQ technology with LC-MS/MS, comparative analysis ofhemocyte proteomesat 0th,10th,20th and 30th dayafter nucleus-insertion were performed for differentially expressed proteins. A total of 277,226 secondary mass spectrograms were collected through LC-MS/MS. There were 43,421 peptides and 2,702 proteins identified. The differentially expressed proteins were enriched according to GO function and KEGG pathway analysis. Using the hemocyte proteome beforenecleus-insertion(designated as 0th day) as the internal control,97,103 and 101 proteins expressed differentially significantly were found at 10th,20th and 30th day, respectively. As compared to day 0, there were 39 up-regulated proteins and 60 down-regulated proteins in day 10. As compared to day 10, 21 up-regulated proteins and 8 down-regulated proteins were identified in day 20. Similarly,12 proteins were up-regulated and 9 proteins were down-regulated from day 20 to day 30. The differentially expressed proteins include Protocadherin, Plasminogen, caspase, Interferon-induced protein, Interleukin-17 receptor, tumor necrosis factor, TNF receptor-associated factor, Heat shock protein 70, Tripartite motif-containing protein, TNF-alpha induced protein, Neurogenic locus notch-like protein, skin mucus antibacterial L-amino acid oxidase,E3 ubiquitin-protein ligase, Integrin, death-associated protein kinase, peptidoglycan recognition protein and so on.In this study, the differentially expressed immune-related genes and proteins were studied using transcriptome and proteome technology. The results enriched the information of cDNA and protein databases, and provided further reference to investigate the immune mechanism during necleus-insertion in P. fucata martensii. Meanwhile, the study could provide reference materials for invertebrates non-specific immune study. |
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