Molecular characterization and expression patterns of Nanog gene validating its involvement in the embryonic development and maintenance of spermatogonial stem cells of farmed carp, Labeo rohita.

BACKGROUND: The homeobox containing transcription factor Nanog plays crucial roles in embryonic development/proliferation and/or maintenance of spermatogonial stem cells (SSCs) via interacting with transcription factors such as Oct4 and Sox2 in mammals. However, knowledge of its exact mechanistic pathways remains unexploited. Very little is known about teleost Nanog. Information on the Nanog gene of farmed rohu carp (Labeo rohita) is lacking. We cloned and characterized the Nanog gene of rohu carp to understand the expression pattern in early developmental stages and also deduced the genomic organization including promoter elements. RESULTS: Rohu Nanog (LrNanog) cDNA comprised an open reading frame of 1,161 nucleotides bearing a structural homeodomain; whereas, the genomic structure contained four exons and three introns suggesting that it is homologous to mammalian counterparts. Phylogenetically, it was closely related to freshwater counterparts. Protein sequence (386 AA of 42.65 kDa) comparison revealed its low similarity with other vertebrate counterparts except that of the conserved homeodomain. Tissue distribution analysis revealed the existence of LrNanog transcripts only in adult gonads. The heightened abundances in the ovary and proliferating spermatogonia suggested its participations in maternal inheritance and male germ cell development. The potentiating abundances from fertilized egg onwards peaking at blastula stage vis- à-vis decreasing levels from gastrula stage onwards demonstrated its role in embryonic stem cell development. We also provided evidence of its presence in SSCs by western blotting analysis. Further, the promoter region was characterized, predicting a basal core promoter and other consensus elements. CONCLUSION: The molecular characterization of LrNanog and its documented expression profiling at transcript and protein levels are indicative of its functional linkage with embryonic/spermatogonial stem cell maintenance. This is the first report of LrNanog genomic organization including its promoter sequence information with predicted regulatory elements of a large-bodied carp species. This will be useful for elucidating its mechanism expression in future. Nanog could be used as a potential biomarker for proliferating carp SSCs.

In Silico Analysis of nsSNPs of Carp TLR22 Gene Affecting its Binding Ability with Poly I:C.

Immune response mediated by toll-like receptor 22 (TLR22), only found in teleost/amphibians, is triggered by double-stranded RNA binding to its LRR (leucine-rich repeats) ecto-domain. Accumulated evidences suggested that missense mutations in TLR genes affect its function. However, information on mutation linked pathogen recognition for TLR22 was lacking. The present study was commenced for predicting the effect of non-synonymous single-nucleotide polymorphisms (nsSNPs) on the pathogen recognizable LRR domain of TLR22 of farmed carp, Labeo rohita. The sequence-based algorithms (SIFT, PROVEAN and I-Mutant2.0) indicated that three SNPs (out of 27) such as p.L159F (rs76759876) and p.L529P (rs749355507) of LRR, and p.I836M (rs750758397) of intracellular motifs could potentially disrupt protein function. The 3D structure was generated using MODELLER 9.13 and further validated by SAVEs server. The simulated molecular docking of native TLR22 and mutants with poly I:C ligand indicated that mutations positioned at p.L159F and p.L529P of the LRR region affects the binding affinity significantly. This is the first kind of study of predicting nsSNPs of teleost TLR22 with disturbed ligand binding affinity with its extra-cellular LRR domain and thereby likely hindrance in subsequent signal transduction. This study serves as a guide for in vivo evaluation of impact of mutation on immune response mediated by teleost TLR22 gene.

Expressed sequences and polymorphisms in rohu carp (Labeo rohita, Hamilton) revealed by mRNA-seq.

Expressed genes and polymorphisms were identified in lines of rohu Labeo rohita selected for resistance or susceptibility to Aeromonas hydrophila, an important bacterial pathogen causing aeromoniasis. All animals were grown in a common environment and RNA from ten individuals from each line pooled for Illumina mRNA-seq. De novo transcriptome assembly produced 137,629 contigs with 40× average coverage.Forty-four percent of the assembled sequences were annotated with gene names and ontology terms. Of these, 3,419 were assigned biological process terms related to "stress response" and 1,939 "immune system". Twenty-six contigs containing 38 single nucleotide polymorphisms (SNPs) were found to map to the Cyprinus carpio mitochondrial genome and over 26,000 putative SNPs and 1,700 microsatellite loci were detected. Seventeen percent of the 100 transcripts with coverage data most indicative of higher-fold expression(>5.6 fold) in the resistant line pool showed homology to major histocompatibility (MH), heat shock proteins (HSP)30, 70 and 90, glycoproteins or serum lectin genes with putative functions affecting immune response. Forty-one percent of these 100 transcripts showed no or low homology to known genes. Of the SNPs identified, 96 showing the highest allele frequency differences between susceptible and resistant line fish included transcripts with homology to MH class I and galactoside-binding soluble lectin, also with putative functions affecting innate and acquired immune response. A comprehensive sequence resource for L. rohita, including annotated microsatellites and SNPs from a mixture of A. hydrophila-susceptible and -resistant individuals, was created for subsequent experiments aiming to identify genes associated with A. hydrophila resistance.