Development and utilization of an infectious clone for porcine deltacoronavirus strain USA/IL/2014/026.

We report the generation of a full-length infectious cDNA clone for porcine deltacoronavirus strain USA/IL/2014/026. Similar to the parental strain, the infectious clone virus (icPDCoV) replicated efficiently in cell culture and caused mild clinical symptoms in piglets. To investigate putative viral interferon (IFN) antagonists, we generated two mutant viruses: a nonstructural protein 15 mutant virus that encodes a catalytically-inactive endoribonuclease (icEnUmut), and an accessory gene NS6-deletion virus in which the NS6 gene was replaced with the mNeonGreen sequence (icDelNS6/nG). By infecting PK1 cells with these recombinant PDCoVs, we found that icDelNS6/nG elicited similar levels of type I IFN responses as icPDCoV, however icEnUmut stimulated robust type I IFN responses, demonstrating that the deltacoronavirus endoribonuclease, but not NS6, functions as an IFN antagonist in PK1 cells. Collectively, the construction of a full-length infectious clone and the identification of an IFN-antagonistic endoribonuclease will aid in the development of live-attenuated deltacoronavirus vaccines.

Zika Virus Amplification Using Strand Displacement Isothermal Method and Sequencing Using Nanopore Technology.

Development of novel point of care diagnostic methods in order to help in implementing disease control program and identifying the causative agent of an outbreak is crucial. Classical diagnostic techniques, e.g., real-time polymerase chain reaction (PCR), rely on the presence of the nucleic acid sequence of the target in GenBank. In the case of an emerging new strain of a known or novel pathogen, false-negative results will be recorded by PCR. On the other hand, next-generation sequencing technologies allow rapid whole genome sequencing without previous knowledge of the target. One of these methods is the Oxford Nanopore sequencing technique, which utilizes a portable device named MinION and has a short run time. In this protocol, we describe the development of a novel nanopore sequencing protocol by combining random isothermal amplification technology and nanopore sequencing. The established protocol is rapid (<7 h) and sensitive as less than 4% of the sequenced RNA belonged to the target virus, Zika. Interestingly, we have established an offline BLAST search for the data analysis that facilitates the use of the whole protocol at remote settings without the need of an Internet connection.

Element content and expression of genes of interest in guard cells are connected to spatiotemporal variations in stomatal conductance.

Element content and expression of genes of interest on single cell types, such as stomata, provide valuable insights into their specific physiology, improving our understanding of leaf gas exchange regulation. We investigated how far differences in stomatal conductance (gs ) can be ascribed to changes in guard cells functioning in amphistomateous leaves. gs was measured during the day on both leaf sides, on well-watered and drought-stressed trees (two Populus euramericana Moench and two Populus nigra L. genotypes). In parallel, guard cells were dissected for element content and gene expressions analyses. Both were strongly arranged according to genotype, and drought had the lowest impact overall. Normalizing the data by genotype highlighted a structure on the basis of leaf sides and time of day both for element content and gene expression. Guard cells magnesium, phosphorus, and chlorine were the most abundant on the abaxial side in the morning, where gs was at the highest. In contrast, genes encoding H+ -ATPase and aquaporins were usually more abundant in the afternoon, whereas genes encoding Ca2+ -vacuolar antiporters, K+ channels, and ABA-related genes were in general more abundant on the adaxial side. Our work highlights the unique physiology of each leaf side and their analogous rhythmicity through the day.

Improved library preparation with the new iCLIP2 protocol.

Individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) is a state-of-the-art technology to map the RNA interaction sites of an RNA-binding protein (RBP) across the transcriptome. Here, we present the new iCLIP2 protocol that allows to obtain high-quality iCLIP libraries in a fast and efficient manner. The new protocol comprises separate adapter ligations, two cDNA amplification steps and bead-based size selection. The full procedure can be completed within four days. Our advances significantly increase the complexity of the iCLIP2 libraries, resulting in a more comprehensive representation of RBP binding sites. Overall, the methodological advances in iCLIP2 allow efficient library generation and thereby promote the versatile and flexible application of this important technology.

Descubrimiento de la proteína reguladora de apoptosis 2 como determinante en la muerte de células tumorales.

Cancer is a multifactorial disease that constitutes a serious public health problem worldwide. Prostate cancer advanced stages are associated with the development of androgen-independent tumors and an apoptosis-resistant phenotype that progresses to metastasis. By studying androgen-independent lymphoid nodule carcinoma of the prostate (LNCaP) cells induced to apoptosis by serum elimination, we identified the activation of a non-selective cationic channel of 23pS conductance that promotes incoming Ca2+ currents, as well as apoptosis final stages. arp2cDNA was isolated and identified to be of the same cell type, and mRNA was expressed in Xenopus laevis oocytes, which was found to be associated with the activation of incoming Ca2+ currents and induction to apoptosis. cDNA, which encodes the ARP2 protein, was overexpressed in LNCaP cells and Chinese hamster ovary cells, which induced apoptosis. Our evidence suggests that protein ARP2 overexpression and transit to the cell membrane allows an increased Ca2+ incoming current that initiates the apoptosis process in epithelial-type cells whose phenotype shows resistance to programmed cell death.

El cáncer es una enfermedad multifactorial que constituye un problema de salud pública mundial. Las etapas avanzadas del cáncer de próstata están asociadas con el desarrollo de tumores independientes de andrógeno y un fenotipo resistente a la apoptosis que progresa a metástasis. Al estudiar células de cáncer de próstata de nódulo linfoide (LNCaP) independientes de andrógeno inducidas a la apoptosis por eliminación de suero, identificamos la activación de un canal catiónico no selectivo de 23pS de conductancia que promueve corrientes entrantes de Ca2+ así como las etapas finales de la apoptosis. El cDNAarp2 fue aislado e identificado del mismo tipo celular y el ARN mensajero fue expresado en ovocitos de Xenopus laevis, asociándolo con la activación de las corrientes entrantes de Ca2+ y la inducción a la apoptosis. El ADN complementario que codifica para la proteína reguladora de apoptosis 2 (ARP2) fue sobreexpresado en células LNCaP y células de ovario de hámster chino, induciendo apoptosis. Nuestra evidencia sugiere que la sobreexpresión y tránsito de la proteína ARP2 a la membrana celular permite una corriente de entrada de Ca2+ aumentada, iniciadora del proceso de apoptosis en células de tipo epitelial cuyo fenotipo muestra resistencia a la muerte celular programada.

Transcriptome profiling of mouse samples using nanopore sequencing of cDNA and RNA molecules.

Our vision of DNA transcription and splicing has changed dramatically with the introduction of short-read sequencing. These high-throughput sequencing technologies promised to unravel the complexity of any transcriptome. Generally gene expression levels are well-captured using these technologies, but there are still remaining caveats due to the limited read length and the fact that RNA molecules had to be reverse transcribed before sequencing. Oxford Nanopore Technologies has recently launched a portable sequencer which offers the possibility of sequencing long reads and most importantly RNA molecules. Here we generated a full mouse transcriptome from brain and liver using the Oxford Nanopore device. As a comparison, we sequenced RNA (RNA-Seq) and cDNA (cDNA-Seq) molecules using both long and short reads technologies and tested the TeloPrime preparation kit, dedicated to the enrichment of full-length transcripts. Using spike-in data, we confirmed that expression levels are efficiently captured by cDNA-Seq using short reads. More importantly, Oxford Nanopore RNA-Seq tends to be more efficient, while cDNA-Seq appears to be more biased. We further show that the cDNA library preparation of the Nanopore protocol induces read truncation for transcripts containing internal runs of T's. This bias is marked for runs of at least 15 T's, but is already detectable for runs of at least 9 T's and therefore concerns more than 20% of expressed transcripts in mouse brain and liver. Finally, we outline that bioinformatics challenges remain ahead for quantifying at the transcript level, especially when reads are not full-length. Accurate quantification of repeat-associated genes such as processed pseudogenes also remains difficult, and we show that current mapping protocols which map reads to the genome largely over-estimate their expression, at the expense of their parent gene.

Discovery of the ARP2 protein as a determining molecule in tumor cell death

Cancer is a multifactorial disease that constitutes a serious public health problem worldwide. Prostate cancer advanced stages are associated with the development of androgen-independent tumors and an apoptosis-resistant phenotype that progresses to metastasis. By studying androgen-independent lymphoid nodule carcinoma of the prostate (LNCaP) cells induced to apoptosis by serum elimination, we identified the activation of a non-selective cationic channel of 23pS conductance that promotes incoming Ca2+ currents, as well as apoptosis final stages. arp2cDNA was isolated and identified to be of the same cell type, and mRNA was expressed in Xenopus laevis oocytes, which was found to be associated with the activation of incoming Ca2+ currents and induction to apoptosis. cDNA, which encodes the ARP2 protein, was overexpressed in LNCaP cells and Chinese hamster ovary cells, which induced apoptosis. Our evidence suggests that protein ARP2 overexpression and transit to the cell membrane allows an increased Ca2+ incoming current that initiates the apoptosis process in epithelial-type cells whose phenotype shows resistance to programmed cell death.

Construction and biological characterization of an Agrobacterium-mediated infectious cDNA of squash mosaic virus.

Squash mosaic virus (SqMV), a member of the species Squash mosaic virus in the genus Comovirus (family Comoviridae), is an important seed-borne virus that causes serious economic losses in cucurbit crops. Here, we constructed infectious cDNA clones of SqMV genomic RNAs (RNA1 and RNA2) under the control of the cauliflower mosaic virus (CaMV) 35S promoter by Gibson assembly. The infectious cDNA clones of SqMV could infect zucchini squash (Cucurbita pepo) plants systemically by agrobacterium-mediated inoculation. The virus progeny from the infectious clones showed no difference from the wild type in terms of pathogenicity and symptom induction. It could be mechanically transmitted to zucchini squash (Cucurbita pepo), pumpkin (Cucurbita moschata), cucumber (Cucumis sativus), and muskmelon (Cucumis melo) but not watermelon (Citrullus lanatus) or Nicotiana benthamiana. This is the first report of construction of a SqMV infection clone and will facilitate the investigation of viral pathogenesis and host interactions.

A novel 86-bp indel of the motilin receptor gene is significantly associated with growth and carcass traits in Gushi-Anka F2 reciprocal cross chickens.

1. A previous whole-genome association analysis has identified the motilin receptor gene (MLNR), which regulates gastrointestinal motility and gastric emptying, as a candidate gene related to chicken growth.2. MLNR mRNA was expressed in all tissues tested, and the expression level in digestive tissues was greater than in other tissues. Expression levels in the pancreas, duodenum and glandular stomach at day old and one, two and three weeks of age indicated a possible correlation with the digestive system. This suggested that the MLNR gene plays a central role in gastrointestinal tract function and affects the growth and development of chickens. Moreover, there was a significant difference in expression in the glandular stomach tissue between Ross 308 and Gushi chickens at six weeks of age.3. Re-sequencing revealed an 86-bp insertion/deletion polymorphism in the downstream region of the MLNR gene. The mutation locus was genotyped in 2,261 individuals from nine different chicken breeds. MLNR expression levels in the glandular stomach of chickens with DD genotypes were greater than those in chickens with the ID and II genotypes. The DD genotype was the most dominant genotype in commercial broiler's (Ross 308 and Arbor Acres broilers), and the D allele frequency in these breeds exceeded 91%. The deletion mutation tended towards fixation in commercial broilers.4. Association with growth and carcass traits analysed in a Gushi-Anka F2 intercrossed population, showed that the DD genotype was significantly associated with the greatest growth and carcass trait values, whereas values associated with the II genotype were the lowest in the F2 reciprocal cross chickens.5. The results suggest that the mutation is strongly associated with growth related traits and it is likely to be useful for marker-assisted selection of chickens.

Infectious cDNA clones of two strains of Mayaro virus for studies on viral pathogenesis and vaccine development.

Mayaro virus (MAYV; family Togaviridae, genus Alphavirus) is an emerging global threat that can cause severe clinical manifestations similar to Zika, dengue, and chikungunya viruses. Currently, there is a lack of molecular tools to enable a better understanding of the transmission and pathogenesis of MAYV. Here, we detail the development and characterization of infectious clones of two strains of MAYV that produce infectious virus and replicate in mammalian and mosquito cells similarly to wild-type virus. Additionally, clone-derived viruses produced identical infection rates and phenotypes in CD-1 mice compared to the parental strains. This infectious clone system will provide a resource to the research community to analyze MAYV genetic determinants of virulence, determine vector competence, and develop vaccines.

Isolation and characterization of abundantly-expressed cDNAs from the Harderian gland of the garter snake (Thamnophis sirtalis: Colubridae).

The Harderian gland (HG) is an orbital structure whose proteinaceous secretions pass through the nasolacrimal duct to the vomeronasal organ (VNO). Though these three structures occur in many tetrapod vertebrates, the garter snake (Thamnophis sirtalis) is one of the few vertebrates in which the passage of the proteinaceous secretions have been experimentally shown. Secreted proteins from the HG may function as transporters for chemical signals to the VNO epithelium. To investigate the proteins being produced by the HG of the garter snake, cDNA libraries were constructed from HG mRNA, and several individual cDNAs were analyzed by sequencing, RT-qPCR, and PCR on genomic DNA. Two of the three cDNAs that were characterized are abundantly expressed only in the Harderian gland and contain putative signal sequences for secretion, which makes them candidates for transporter proteins secreted from the HG. One is a member of the large lipocalin family of proteins, based on its similarity to other members of that protein family. Many lipocalins are binding/carrier proteins for a variety of ligands. The other is a family of proteins, with five members identified so far, all of unknown structure and function and present in the garter snake genome but not in other squamate genomes.

Discovery of the ARP2 protein as a determining molecule in tumor cell death.

Cancer is a multifactorial disease that constitutes a serious public health problem worldwide. Prostate cancer advanced stages are associated with the development of androgen-independent tumors and an apoptosis-resistant phenotype that progresses to metastasis. By studying androgen-independent lymphoid nodule carcinoma of the prostate (LNCaP) cells induced to apoptosis by serum elimination, we identified the activation of a non-selective cationic channel of 23pS conductance that promotes incoming Ca2+ currents, as well as apoptosis final stages. arp2cDNA was isolated and identified to be of the same cell type, and mRNA was expressed in Xenopus laevis oocytes, which was found to be associated with the activation of incoming Ca2+ currents and induction to apoptosis. cDNA, which encodes the ARP2 protein, was overexpressed in LNCaP cells and Chinese hamster ovary cells, which induced apoptosis. Our evidence suggests that protein ARP2 overexpression and transit to the cell membrane allows an increased Ca2+ incoming current that initiates the apoptosis process in epithelial-type cells whose phenotype shows resistance to programmed cell death.

Thioredoxin o-mediated reduction of mitochondrial alternative oxidase in the thermogenic skunk cabbage Symplocarpus renifolius.

Thermogenesis in plants involves significant increases in their cyanide-resistant mitochondrial alternative oxidase (AOX) capacity. Because AOX is a non-proton-motive ubiquinol oxidase, the dramatic drop in free energy between ubiquinol and oxygen is dissipated as heat. In the thermogenic skunk cabbage (Symplocarpus renifolius), SrAOX is specifically expressed in the florets. Although SrAOX harbours conserved cysteine residues, the details of the mechanisms underlying its redox regulation are poorly understood. In our present study, the two mitochondrial thioredoxin o cDNAs SrTrxo1 and SrTrxo2, were isolated from the thermogenic florets of S. renifolius. The deduced amino acid sequences of the protein products revealed that SrTrxo2 specifically lacks the region corresponding to the α3-helix in SrTrxo1. Expression analysis of thermogenic and non-thermogenic S. renifolius tissues indicated that the SrTrxo1 and SrAOX transcripts are predominantly expressed together in thermogenic florets, whereas SrTrxo2 transcripts are almost undetectable in any tissue. Finally, functional in vitro analysis of recombinant SrTrxo1 and mitochondrial membrane fractions of thermogenic florets indicated its reducing activity on SrAOX proteins. Taken together, these results indicate that SrTrxo1 is likely to play a role in the redox regulation of SrAOX in S. renifolius thermogenic florets.

Analysis of differentially expressed genes and the promoters in bovine endometrium throughout estrus cycle and early pregnancy.

Endometrial gene expression is primarily regulated by the ovarian steroids and pregnancy recognition factors. This study was aimed to characterize differential expression genes (DEGs) in bovine endometrium together with the analysis of their promoter region. Bovine uteri at follicular stage (FS), luteal stage (LS), and implantation stage (IS) at Day 18 of pregnancy were collected. Total RNA extracted and prepared cDNA were then subjected to high-throughput sequencing. For promoter analysis, 1 kb upstream promoter region of each DEG was analyzed. The numbers of highly expressed DEGs were 496 and 597 at FS and LS, respectively. When compared the gene expression of IS with LS, 383 and 346 DEGs showed higher and lower expression at IS, respectively. It was also observed that 20-30 transcription factors (TFs) were included in each DEGs. In addition, promoter analyses estimated 150-160 TFs for each stage. DLX4 and interferon regulatory factor 4 (IRF4) at FS, and IRF5, IRF9, STAT1, and STAT2 at IS were in common to DEGs and estimated TFs, respectively. This study highlighted potential molecular mechanisms controlling endometrial function during estrus cycle and IS, which will further guide to better understand the endometrial functions in future studies.

Molecular characterization of a hepcidin homologue in starry flounder (Platichthys stellatus) and its synergistic interaction with antibiotics.

Hepcidins are small cysteine-rich antimicrobial peptides that play an important role in host immunity against pathogenic organisms. Most fish hepcidins exert bactericidal activities against a wide range of pathogens. In this study, we identified a cDNA sequence encoding a hepcidin homologue (PsHepcidin) in the starry flounder Platichthys stellatus. The predicted amino acid sequence of PsHepcidin comprises a signal peptide and a prodomain, which are followed by the mature peptide. Sequence analysis revealed that PsHepcidin belongs to the fish HAMP2 cluster and that it is closely related to mudskipper hepcidin-2. Expression of PsHepcidin mRNA was detected in all examined immune-related tissues, with the highest transcript levels being found in the liver. In response to lipopolysaccharide treatment, PsHepcidin was significantly up-regulated in the liver, kidney, and spleen in a time-dependent manner. Chemically synthesized mature peptides of PsHepcidin were found to exhibit broad antimicrobial activity in vitro. We also investigated the combined effect of PsHepcidin and conventional antibiotics and found that these combinations showed synergistic effects against most of the examined bacterial strains. Collectively, the results of this study indicate that PsHepcidin exhibits potent antibacterial activity both independently and when used in combination with conventional antibiotics.

Molecular identification of single hormone-encoding proglucagon cDNA isoforms from squamates and their abundant expression.

Among ectothermic reptiles, the order Squamata has adapted most successfully to the terrestrial environment. However, the physiological background of this success remains unknown. Since the regulation of energy metabolism provides an important insight into terrestrial adaption by ectothermic animals, we focused on proglucagon-derived peptides (PGDPs). In the process of cloning proglucagon mRNA in geckos, we identified several novel proglucagon (PG) cDNA isoforms. They were tissue-specifically and strongly expressed in the pancreas and small intestine of the geckos, suggesting their biological relevance. Therefore, in order to clarify whether these novel cDNA isoforms are phylogenetically conserved, we performed the additional molecular characterization of proglucagon cDNAs from several representative species of the Squamata and Testudine clade and examined the expression of proglucagon mRNAs in the small intestine and pancreas. In the present study, a total of 7 proglucagon cDNA isoforms were identified and divided into two groups (Classes A and B) based on the 3'-UTR sequence of each isoform. The longest isoform of each group (named PG-A1 and PG-B1, respectively) had the same molecular characteristics as those previously reported from chickens and reptiles, namely, PG-A and PG-B. Other 5 isoforms were novel-type cDNAs, and were the products of exon skipping (named PG-A2, PG-A2s, PG-B2, PG-B2s, and PG-B3). Some of these isoforms coded for only one peptide hormone (GLP-1 or GLP-2). This is the first identification of single hormone-encoding proglucagon cDNAs in vertebrates. Moreover, an expression analysis of these isoforms revealed that single hormone-encoding proglucagon mRNAs were predominantly expressed with tissue and lineage specificities in the reptile clade. Collectively, the present results suggest an independent regulatory system for GLP-1 and GLP-2 secretion and indicate the plasticity of proglucagon genes in expressing different isoforms in different tissues in Squamata. These results also provide insights into the plastic energy metabolic system of Squamata in accordance with various habitats in the terrestrial environment, supporting their successful prosperity.

Molecular cloning, identification of GSTs family in sunflower and their regulatory roles in biotic and abiotic stress.

Glutathione-S-transferase (GST) genes exist widely in plants and play major role in metabolic detoxification of exogenous chemical substances and oxidative stress. In this study, 14 sunflower GST genes (HaGSTs) were identified based on the sunflower transcriptome database that we had constructed. Full-length cDNA of 14 HaGTSs were isolated from total RNA by reverse transcription PCR (RT-PCR). Sunflower was received biotic stress (Sclerotinia sclerotiorum) and abiotic stress (NaCl, low-temperature, drought and wound). GST activity was measured by using the universal substrate. The results showed that most of the HaGSTs were up-regulated after NaCl and PEG6000-induced stresses, while a few HaGSTs were up-regulated after S. sclerotiorum, hypothermia and wound-induced stressed, and there was correlation between the changes of GST activity and the expression of HaGSTs, indicating that HaGSTs may play regulatory role in the biotic and abiotic stress responses. 14 HaGSTs from sunflower were identified, and the expression of HaGSTs were tissue-specific and played regulatory roles in both stress and abiotic stress.

Molecular characterization and expression analysis of strbp in Chinese tongue sole (Cynoglossus semilaevis).

Spermatid perinuclear RNA-binding protein (Strbp) is a kind of double-stranded (ds) RNA specific binding protein that plays important roles in mammalian spermatogenesis. In this study, we have isolated and characterized the strbp gene of Chinese tongue sole, Cynoglossus semilaevis, termed as CS-strbp. The CS-strbp genomic sequence contained fifteen exons and fourteen introns. Its cDNA was 2655 bp in length, encoding a 666-amino-acid protein with two conserved ds binding motifs. Using quantitative PCR, we found that CS-strbp mRNA exhibited sex-biased and tissue-specific distribution, predominantly expressed in the fertile male testis, though the expression levels varied throughout different developmental stages. Comparison of methylation profile in different sexual genotypes demonstrated the low methylation level of CS-strbp promoter in male and pseudo-male, which is consistent with the high expression levels in those genotypes. In situ hybridization revealed that CS-strbp mRNA mainly localized in male germ cells, especially in spermatids and spermatozoa. Given these findings, we postulate that CS-strbp might function in spermatogenesis of Chinese tongue sole.

Expressed exome capture sequencing: A method for cost-effective exome sequencing for all organisms.

Exome capture is an effective tool for surveying the genome for loci under selection. However, traditional methods require annotated genomic resources. Here, we present a method for creating cDNA probes from expressed mRNA, which are then used to enrich and capture genomic DNA for exon regions. This approach, called "EecSeq," eliminates the need for costly probe design and synthesis. We tested EecSeq in the eastern oyster, Crassostrea virginica, using a controlled exposure experiment. Four adult oysters were heat shocked at 36°C for 1 hr along with four control oysters kept at 14°C. Stranded mRNA libraries were prepared for two individuals from each treatment and pooled. Half of the combined library was used for probe synthesis, and half was sequenced to evaluate capture efficiency. Genomic DNA was extracted from all individuals, enriched via captured probes, and sequenced directly. We found that EecSeq had an average capture sensitivity of 86.8% across all known exons and had over 99.4% sensitivity for exons with detectable levels of expression in the mRNA library. For all mapped reads, over 47.9% mapped to exons and 37.0% mapped to expressed targets, which is similar to previously published exon capture studies. EecSeq displayed relatively even coverage within exons (i.e., minor "edge effects") and even coverage across exon GC content. We discovered 5,951 SNPs with a minimum average coverage of 80×, with 3,508 SNPs appearing in exonic regions. We show that EecSeq provides comparable, if not superior, specificity and capture efficiency compared to costly, traditional methods.

Gonadotropin receptors of Labeo rohita: Cloning and characterization of full-length cDNAs and their expression analysis during annual reproductive cycle.

Follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh), secreted from pituitary, stimulate gonadal function by binding to their cognate receptors FSH receptor (FSHR), and LH/choriogonadotropin receptor (LHCGR). Rohu (Labeo rohita) is a commercially important seasonal breeder freshwater fish species, but till date, the regulation of expression of gonadotropins and their receptors gene during different phases of annual reproductive cycle has not been investigated. We envisaged the critical role of these molecules during seasonal gonadal development in this carp species. We cloned full- length cDNAs of fshra and lhcgrba from rohu testis using RACE (Rapid amplification of cDNA ends) and analyzed their expression along with fsh and lh by quantitative real time PCR (qRT-PCR) assay at various gonadal developmental stages of the annual reproductive cycle. Full-length rohu fshra and lhcgrba cDNA encodes 670 and 716 amino acids respectively, and in adult fish, they were widely expressed in brain, pituitary, gonad, liver, kidney, head kidney, heart, muscle, gill, fin, eye and intestine. In male, both fsh and fshra transcripts showed high level of expression during spermatogenesis, however, in female, expression level was found to be higher in the fully grown oocyte stages. The expression of rohu lh and lhcgrba mRNA increased with increment of gonadosomatic index and showed highest level during spermiation stage in male and fully matured oocyte stage in female. These results together may suggest the involvement of fshra and lhcgrba in regulating function of seasonal gonadal development in rohu.