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Identification and Characterization of Lipopolysaccharide Induced TNFα Factor from Blunt Snout Bream, Megalobrama amblycephala.

ABSTRACT: Lipopolysaccharide induced TNFα factor (LITAF) is an important transcription factor responsible for regulation of tumor necrosis factor α. In this study, a novel litaf gene (designated as Malitaf) was identified and characterized from blunt snout bream, Megalobrama amblycephala. The full-length cDNA of Malitaf was of 956 bp, encoding a polypeptide of 161 amino acids with high similarity to other known LITAFs. A phylogenetic tree also showed that Malitaf significantly clustered with those of other teleost, indicating that Malitaf was a new member of fish LITAF family. The putative maLITAF protein possessed a highly conserved LITAF domain with two CXXC motifs. The mRNA transcripts of Malitaf were detected in all examined tissues of healthy M. amblycephala, including kidney, head kidney, muscle, liver, spleen, gill, and heart, and with the highest expression in immune organs: spleen and head kidney. The expression level of Malitaf in spleen was rapidly up-regulated and peaked (1.29-fold, p < 0.05) at 2 h after lipopolysaccharide (LPS) stimulation. Followed the stimulation of Malitaf, Matnfα transcriptional level was also transiently induced to a high level (51.74-fold, p < 0.001) at 4 h after LPS stimulation. Taken together, we have identified a putative fish LITAF ortholog, which was a constitutive and inducible immune response gene involved in M. amblycephala innate immunity during the course of a pathogenic infection.

SUBMITTER: Lv Y

PROVIDER: S-EPMC5343772 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND: Blunt snout bream (Megalobrama amblycephala) is an economically important fish species in the Chinese freshwater polyculture system for its delicacy and high economic value. MicroRNAs (miRNAs) play important roles in regulation of almost all biological processes in eukaryotes. Although previous studies have identified thousands of miRNAs from many species, little information is known for miRNAs of M. amblycephala. To investigate functions of miRNAs associated with growth of M. amblycephala, we adopted the Solexa sequencing technology to sequence two small RNA libraries prepared from four growth related tissues (brain, pituitary, liver and muscle) of M. amblycephala using individuals with relatively high and low growth rates. RESULTS: In this study, we have identified 347 conserved miRNAs (belonging to 123 families) and 22 novel miRNAs in M. amblycephala. Moreover, we observed sequence variants and seed edits of the miRNAs. Of the 5,166 single nucleotide substitutions observed in two libraries, the most abundant were G-to-U (15.9%), followed by U-to-C (12.1%), G-to-A (11.2%), and A to G (11.2%). Subsequently, we compared the expression patterns of miRNAs in the two libraries (big-size group with high growth rate versus small-size group with low growth rate). Results indicated that 27 miRNAs displayed significant differential expressions between the two libraries (p < 0.05). Of these, 16 were significantly up-regulated and 11 were significantly down-regulated in the big-size group compared to the small-size group. Furthermore, stem-loop RT-PCR was applied to validate and profile the expression of the differentially expressed miRNAs in ten tissues, and the result revealed that the conserved miRNAs expressed at higher levels than the novel miRNAs, especially in brain, liver and muscle. Also, targets prediction of differentially expressed miRNAs and KEGG pathway analysis suggested that differentially expressed miRNAs are involved in growth and metabolism, signal transduction, cell cycle, neural development and functions. CONCLUSIONS: The present study provides the first large-scale characterization of miRNAs in M. amblycephala and miRNA profile related to different growth performances. The discovery of miRNA resource from this study is expected to contribute to a better understanding of the miRNAs roles playing in regulating the growth biological processes and the study of miRNA function and phenotype-associated miRNA identification in fish.

Project description:BackgroundBlunt snout bream (Megalobrama amblycephala) is an herbivorous freshwater fish species native to China and has been recognized as a main aquaculture species in the Chinese freshwater polyculture system with high economic value. Right now, only limited EST resources were available for M. amblycephala. Recent advances in large-scale RNA sequencing provide a fast, cost-effective, and reliable approach to generate large expression datasets for functional genomic analysis, which is especially suitable for non-model species with un-sequenced genomes.Methodology and principal findingsUsing 454 pyrosequencing, a total of 1,409,706 high quality reads (total length 577 Mbp) were generated from the normalized cDNA of pooled M. amblycephala individuals. These sequences were assembled into 26,802 contigs and 73,675 singletons. After BLAST searches against the NCBI non-redundant (NR) and UniProt databases with an arbitrary expectation value of E(-10), over 40,000 unigenes were functionally annotated and classified using the FunCat functional annotation scheme. A comparative genomics approach revealed a substantial proportion of genes expressed in M. amblycephala tanscriptome to be shared across the genomes of zebrafish, medaka, tetraodon, fugu, stickleback, human, mouse, and chicken, and identified a substantial number of potentially novel M. amblycephala genes. A total number of 4,952 SSRs were found and 116 polymorphic loci have been characterized. A significant number of SNPs (25,697) and indels (23,287) were identified based on specific filter criteria in the M. amblycephala.ConclusionsThis study is the first comprehensive transcriptome analysis for a fish species belonging to the genus Megalobrama. These large EST resources are expected to be valuable for the development of molecular markers, construction of gene-based linkage map, and large-scale expression analysis of M. amblycephala, as well as comparative genome analysis for the genus Megalobrama fish species. The identified SSR and SNP markers will greatly benefit its breeding program and whole genome association studies.

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Identification and Characterization of Lipopolysaccharide Induced TNFα Factor from Blunt Snout Bream, Megalobrama amblycephala.

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