| Grouper(Epinephelus spp.)is one of the most popular varieties in the tropics and subtropics due to its delicious taste,rich nutrition and tender meat.In the early stage,the research group carried out the hybrid breeding of(E.fuscoguttatus,♀)×(E.polyphekadion,♂),and successfully completed the breeding of the hybrid grouper.The performance of the hybrid grouper in terms of immune response and disease resistance is not clear.In this study,the following studies were conducted on the differences in immune responses between the hybrid grouper and its female brown spotted grouper:1.Transcriptome sequencing of immune-related tissues of hybrid grouper and brown-spotted grouperAfter hybrid grouper and brown spotted group were injected 24 h with polyI:C,RNA-seq sequencing of hybrid grouper and brown spotted spleen and head kidney was performed using the Illumina/Hiseq-2000 RNA-Seq technique.A total of 62,238 high-quality unigenes were obtained with an average length of 1,150 bp and an N50 of 2,499 bp.The functional annotation results show that at least 27,305(43.94%)of the unigenes are annotated in one database,and 11,162(23.07%)of the unigenes are annotated in all four databases.According to the differential gene screening principle [gene |log2(fold change)| ≥ 1] and false discovery rate(FDR)<0.05,47,920 differentially expressed genes(DEGs)were screened.According to KEGG functional enrichment analysis,differentially expressed genes(DEGs)are enriched in 20 immune-related signaling pathways,such as RIG-I-like receptor signaling pathway,NOD-like receptor signaling pathway and Toll-like receptor signaling pathway.Nine differential genes were randomly selected for qRT-PCR validation.The results showed that the expression trends of these nine differential genes were consistent with the transcriptome results.Based on differentially expressed gene data selected by transcriptome sequencing,PRRs genes(NLRX1,NLRP1,TLR9,NLRP3,NOD2 and TLR5),interleukin IL-1β and IRF3 related to immune response were selected for gene cloning and expression analysis.2.Expression analysis of differentially expressed genes related to immune(1)Invasion of congenital immune resistance pathogens mainly relies on the recognition of pathogens by pattern-recognition receptors(PRRs),which activates immune responses and thus resists the invasion of external pathogens.Peripheral blood lymphocytes(PBL)are produced by lymphoid organs,are cell lines with immune recognition function,and are also important cellular components of the body’s immune response function,PBL is the key to conducting immunology research.First,this study need to collect highpurity and sufficient PBL,In this study,PBL of hybrid grouper were isolated by gradient centrifugation at different Percoll concentrations(15%,20%,25%,30%,35%),and classification and statistics of different cells in blood cells by blood smear.The results showed that the concentration of the leukocytic layer of the hybrid grouper collected by Percoll at 25% concentration was the most obvious,and the number of PBL was the highest(85.56%),the purity was the highest(90%),and the activity was the best(more than 95%).(2)In order to detect the expression of differentially expressed genes related to immune in PBL response to LPS and PolyI:C immunization,PBL(25% concentration)was stimulated by LPS and PolyI:C to detect the temporal expression of differentially expressed genes related to immune.The results showed that after LPS and PolyI:C stimulated PBL1 h,the expression levels of TLR2 and TLR9 in hybrid grouper were not significantly different after LPS stimulation;In brown spotted grouper,only the expression levels of NLRX1 gene and IL-1β gene were not significantly different after PolyI:C stimulation.(3)To understand the role of IRF3 gene in the immune response of hybrid grouper to foreign virus stimulation,in this study,In this study,the IRF3 gene of hybrid grouper was cloned by RACE(rapid-amplification of cDNA ends)and its expression in PBL was analyzed.The full-length cDNA of this gene is 2529 bp,including 5’ non-coding region(5’-UTR)325 bp,3’ non-coding region(3’-UTR)916 bp,open reading frame(ORF)1377 bp,encoding 458 amino acids.The amino acid sequence comprises an N-terminal DNA binding region(DBD)(1-108 aa),a C-terminal interferon related region(IAD)(255-435 aa),and a tryptophan rich region(SRD)three domains.Phylogenetic analysis showed that the hybrid grouper IRF3 and the Japanese sea perch(Lateolabrax maculatus)IRF3 clustered into one,and the genetic relationship was close.Real-time quantitative PCR was used to detect the expression of IRF3 gene in nine tissues including liver,stomach,gill,intestine and spleen of hybrid grouper.The results showed that IRF3 gene was expressed in 9 tissues,and the expression in liver,stomach and intestine was significantly higher than that in other tissues,and the expression of head kidney was the lowest.Additionally,the expression of IRF3 gene in PBL increased gradually after stimulation for 1 h at PolyI:C,and reached the maximum at 4 h(about 9.3 times of the control group),and gradually decreased after 8 h.3.Effects of PolyI:C and LPS stimulation on the activity of three enzymes in PBL of hybrid grouperPBL was stimulated by PolyI:C and LPS,respectively,and the activities of ACP,AKP and CAT were measured at 0h,1h,4h,8h and 12 h.The results showed that the activity of both ACP and AKP hydrolase was significantly higher than that of the control group(0h)after PBL stimulation by PolyI:C,but there was no significant change in CAT activity;After PBL stimulation by LPS,the activities of ACP,AKP and CAT were significantly higher than those of the control group(0h). |
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