Transcriptome analysis of head kidney in grass carp and discovery of immune-related genes

BMC Veterinary Research (Impact Factor: 1.78). 07/2012; 8(1):108. DOI: 10.1186/1746-6148-8-108
Source: PubMed


Grass carp (Ctenopharyngodon idella) is one of the most economically important freshwater fish, but its production is often affected by diseases that cause serious economic losses. To date, no good breeding varieties have been obtained using the oriented cultivation technique. The ability to identify disease resistance genes in grass carp is important to cultivate disease-resistant varieties of grass carp.

In this study, we constructed a non-normalized cDNA library of head kidney in grass carp, and, after clustering and assembly, we obtained 3,027 high-quality unigenes. Solexa sequencing was used to generate sequence tags from the transcriptomes of the head kidney in grass carp before and after grass carp reovirus (GCRV) infection. After processing, we obtained 22,144 tags that were differentially expressed by more than 2-fold between the uninfected and infected groups. 679 of the differentially expressed tags (3.1%) mapped to 483 of the unigenes (16.0%). The up-regulated and down-regulated unigenes were annotated using gene ontology terms; 16 were annotated as immune-related and 42 were of unknown function having no matches to any of the sequences in the databases that were used in the similarity searches. Semi-quantitative RT-PCR revealed four unknown unigenes that showed significant responses to the viral infection. Based on domain structure predictions, one of these sequences was found to encode a protein that contained two transmembrane domains and, therefore, may be a transmembrane protein. Here, we proposed that this novel unigene may encode a virus receptor or a protein that mediates the immune signalling pathway at the cell surface.

This study enriches the molecular basis data of grass carp and further confirms that, based on fish tissue-specific EST databases, transcriptome analysis is an effective route to discover novel functional genes.

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    • "Grass carp (Ctenopharyngodon idella), family Cyprinidae, is an herbivorous freshwater fish widely distributed in the major river systems of China (Chen et al., 2012). The species is also used for fish farming in China where it has an annual production of over 1 million tons; the fish is characterized as delicious and nutritious, and it grows rapidly and has a low cost of breeding (Ye et al., 2012). "
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    ABSTRACT: Here, we characterized the structure and function of the coagulation factor II (FII) gene in grass carp and determined its role in coagulation mechanisms. The FII gene EST was obtained using a constructed splenic transcriptome database; the full-length FII gene sequence was obtained by 3' and 5' RACE. The open reading frame (ORF) of FII was cloned and the full-length gene was found to be 1718 bp, with an ORF of 1572 bp; the gene contained a 25 bp 5'-untranslated region (UTR) and 108 bp 3'-UTR. The ORF encoded 524 amino acids, including 74 alkaline amino acids (arginine and lysine) and 69 acidic amino acids (aspartic acid and glutamic acid). The theoretical pI was 6.22. The calculated instability index (II) was 39.81, indicating that FII was a stable protein; the half-life period was predicted to be approximately 30 h. Amino acid sequence comparisons indicated that grass carp FII showed most similarity (71%) to FII of Takifugu rubripes, followed by Oplegnathus fasciatus (48% similarity) and Larimichthys crocea (47% similarity). A real-time reverse transcription PCR analysis showed that under normal circumstances, FII was most highly expressed in the liver, followed by the gill, spleen, thymus, and head-kidney (P < 0.001). After injection of the grass carp reovirus 873 (GCRV873), the pattern of FII expression was significantly altered (P < 0.001); gene expression was high after injection, suggesting a response involving the initiation of the coagulation system and defense of the body in combination with the platelet and complement system.
    Preview · Article · Nov 2015 · Genetics and molecular research: GMR
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    • "GCRV, identified as the fiercest slayer for grass carp, provokes hemorrhage in about 85% of fingerling and yearling populations [28]. But to date, no excellent breeding varieties have been obtained by the oriented cultivation technique [29]. Together with the particularity immune systems of fish and the important role of innate immune response as the first line of Table 4 Result of the T-test of the expression of CiTLR8 mRNA in the secondary challenge experiment. "
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    ABSTRACT: Toll-like receptor 8 (TLR8), a prototypical intracellular member of TLR family, is generally linked closely to antiviral innate immune through recognizing viral nucleic acid. In this study, 5'-flanking region of Ctenopharyngodon idella TLR8 (CiTLR8), 671 bp in length, was amplified and eight SNPs containing one SNP in the intron, three SNPs in the coding region (CDS) and four SNPs in the 3'-untranslated region (UTR) were identified and characterized. Of which 4062 A/T was significantly associated with the susceptibility/resistance to GCRV both in genotype and allele (P < 0.05), while 4168 C/T was extremely significantly associated with that (P < 0.01) according to the case (susceptibility)-control (resistance) analysis. Following the verification experiment, further analyses of mRNA expression, linkage disequilibrium (LD), haplotype and microRNA (miRNA) target site indicated that 4062 A/T and 4168 C/T in 3'-UTR might affect the miRNA regulation, while the exertion of antiviral effects of 4062 A/T might rely on its interaction with other SNPs. Additionally, the high-density of SNPs in 3'-UTR might reflect the specific biological functions of 3'-UTR. And also, the mutation of 747 A/G in intron changing the potential transcriptional factor-binding sites (TFBS) nearby might affect the expression of CiTLR8 transcriptionally or post-transcriptionally. Moreover, as predicted, the A/G transition of the only non-synonymous SNP (3846 A/G) in CDS causing threonine/alanine variation, could shorten the length of the α-helix and ultimately affect the integrity of the Toll-IL-1 receptor (TIR) domain. The functional mechanism of 3846 A/G might also involve a threonine phosphorylation signaling. This study may broaden the knowledge of TLR polymorphisms, lay the foundation for further functional research of CiTLR8 and provide potential markers as well as theoretical basis for resistance molecular breeding of grass carp against GCRV. Copyright © 2014. Published by Elsevier Ltd.
    Full-text · Article · Dec 2014 · Fish & Shellfish Immunology
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    • "Although immune genes and pathways in fish tissues such as gill, liver, spleen, head kidney and larvae of turbot [50] and head kidney of grass carp [51] have been previously characterized using RNA-seq, the immune factors in the mud loach skin remains unknown. As shown in Figure 6, immune system was the most highly represented KO sub-category from the mud loach skin transcriptome, suggesting a large number of genes expressed in the skin are associated with immunity. "
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    ABSTRACT: Fish skin serves as the first line of defense against a wide variety of chemical, physical and biological stressors. Secretion of mucus is among the most prominent characteristics of fish skin and numerous innate immune factors have been identified in the epidermal mucus. However, molecular mechanisms underlying the mucus secretion and immune activities of fish skin remain largely unclear due to the lack of genomic and transcriptomic data for most economically important fish species. In this study, we characterized the skin transcriptome of mud loach using Illumia paired-end sequencing. A total of 40364 unigenes were assembled from 86.6 million (3.07 gigabases) filtered reads. The mean length, N50 size and maximum length of assembled transcripts were 387, 611 and 8670 bp, respectively. A total of 17336 (43.76%) unigenes were annotated by blast searches against the NCBI non-redundant protein database. Gene ontology mapping assigned a total of 108513 GO terms to 15369 (38.08%) unigenes. KEGG orthology mapping annotated 9337 (23.23%) unigenes. Among the identified KO categories, immune system is the largest category that contains various components of multiple immune pathways such as chemokine signaling, leukocyte transendothelial migration and T cell receptor signaling, suggesting the complexity of immune mechanisms in fish skin. As for mucin biosynthesis, 37 unigenes were mapped to 7 enzymes of the mucin type O-glycan biosynthesis pathway and 8 members of the polypeptide N-acetylgalactosaminyltransferase family were identified. Additionally, 38 unigenes were mapped to 23 factors of the SNARE interactions in vesicular transport pathway, indicating that the activity of this pathway is required for the processes of epidermal mucus storage and release. Moreover, 1754 simple sequence repeats (SSRs) were detected in 1564 unigenes and dinucleotide repeats represented the most abundant type. These findings have laid the foundation for further understanding the secretary processes and immune functions of loach skin mucus.
    Full-text · Article · Feb 2013 · PLoS ONE
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