Full-length cDNA Sequence Research Articles

Hepatocyte growth factor (HGF) gene: molecular characterisation of complete coding sequence and expression profile in Tarim red deer (Cervus hanglu yarkandensis) antlers

Context The cDNA sequence of hepatocyte growth factor (HGF) gene in Tarim red deer has not been reported yet. Aims This study aims to obtain the full-length cDNA sequence of HGF and analyse its expression in different parts of developing antler tissues. The result provides foundational data for understanding the potential role of HGF in regulating antler growth and development. Methods Rapid amplification of cDNA ends was used to obtain the full-length cDNA sequence of Tarim red deer HGF. The pET28a (+) vector was constructed for prokaryotic expression of the recombinant protein in E. coli BL21 (DE3). The expression of HGF in different antler tissues was detected using real-time quantitative polymerase chain reaction and immunohistochemistry. Key results The full-length cDNA of Tarim red deer HGF was found to consist of a 156 bp 5’untranslated region (UTR), a 112 bp 3’UTR, and a 2193 bp open reading frame encoding a protein of 730 amino acids. The recombinant HGF protein expressed in prokaryotic cells formed inclusion bodies. HGF and its receptor c-Met were expressed in all four different antler tissues, with the highest expression level in velvet skin, followed by bone, cartilage, and the lowest in the mesenchyme. Conclusions The study successfully obtained the full-length cDNA sequence of Tarim red deer HGF and identified the expression profile of HGF and c-Met in different antler tissues. HGF is a candidate gene that may play a role in regulating the growth and development of deer antler. Implications These findings provide fundamental data for further investigations into the role of HGF in antler development. Understanding the function of HGF in antler development could have implications for elucidating the mechanism of antler regeneration.

Allelic variation in the autotetraploid potato: genes involved in starch and steroidal glycoalkaloid metabolism as a case study

BackgroundTuber starch and steroidal glycoalkaloid (SGA)-related traits have been consistently prioritized in potato breeding, while allelic variation pattern of genes that underlie these traits is less explored.ResultsHere, we focused on the genes involved in two important metabolic pathways in the potato: starch metabolism and SGA biosynthesis. We identified 119 genes consisting of 81 involved in starch metabolism and 38 in the biosynthesis of steroidal glycoalkaloids, and discovered 96,166 allelic variants among 2,169 gene haplotypes in six autotetraploid potato genomes. Comparative analyses revealed an uneven distribution of allelic variants among gene haplotypes and that the vast majority of deleterious mutations in these genes are retained in heterozygous state in the autotetraploid potato genomes. Leveraging full-length cDNA sequencing data, we find that approximately 70% of haplotypes of the 119 genes are transcribable. Population genetic analyses identify starch and SGA biosynthetic genes that are potentially conserved or diverged between potato varieties with varying starch or SGA content.ConclusionsThese results deepen the understanding of haplotypic diversity within functionally important genes in autotetraploid genomes and may facilitate functional characterization of genes or haplotypes contributing to traits related to starch and SGA in potato.

The life-history trait trade-offs mediated by reproduction and immunity in the brown planthopper, Nilaparvata lugens Stål

Reproduction and immune defense are costly functions, and they are expected to tradeoff with each other to drive evolution. The brown planthopper (BPH), Nilaparvata lugens Stål (Hemiptera, Delphacidae), is a global superpest that mostly damages rice crops. Yeast-like symbionts (YLS) exist in the abdominal fat body tissue and are tightly associated with the development, growth, and reproduction of BPH. Our previous research demonstrated that mating behavior promotes the release of YLS from the fat body into the hemolymph in the BPH, thereby triggering an immune response. Additionally, the fitness costs related to life-history traits of BPH (such as survival rate) have a strong dependence on the relative abundance of YLS. However, the possible relationship between reproduction and the immune response in BPH has not been identified. In this study, an omics-based approach was used to analyze the transcriptome of fat body tissues in mated and unmated BPH at 72 h post-eclosion, from which two antimicrobial peptide genes, NlDefensin A (NlDfA) and NlDefensin B (NlDfB), were selected since they were highly expressed in mated BPH. Subsequently, the full-length cDNA sequences of the NlDfA and NlDfB genes were cloned and analyzed. qPCR results showed up-regulation of the NlDfA and NlDfB genes in mated BPH when compared to unmated BPH. Spatial-temporal expression analysis indicated that the NlDfA and NlDfB genes were expressed in all tissues and developmental stages, and they were most highly expressed in the fat body at 24 h post-eclosion. Moreover, the symbionts in BPH were significantly inhibited by the in vitro expression of the NlDfA and NlDfB proteins. Furthermore, RNA interference (RNAi)-mediated suppression of NlDfA and NlDfB dramatically increased the relative abundance of YLS in the fat body, while YLS in the hemolymph decreased significantly. These BPHs also displayed some fitness disadvantages in survival, fecundity, hatchability, and possibly the vertical transmission of YLS from hemolymph to egg. Our results indicated that mating could heighten the immunity of BPH by up-regulating the expression of the NlDfA and NlDfB genes, which protect the host from pathogen challenges during reproduction. However, the reduced content of YLS may act as a fitness disadvantage in dictating the life-history traits of BPH. This work has significant theoretical and practical implications for the precise green control technology that involves crucial gene targeting, as well as for the “endosymbionts for pest control” strategy in insects.

Cloning and Characterization of Chitin Deacetylase from Euphausia superba.

Chitin deacetylase (CDA) can catalyze the deacetylation of chitin to produce chitosan. In this study, we identified and characterized a chitin deacetylase gene from Euphausia superba (EsCDA-9k), and a soluble recombinant protein chitin deacetylase from Euphausia superba of molecular weight 45 kDa was cloned, expressed, and purified. The full-length cDNA sequence of EsCDA-9k was 1068 bp long and encoded 355 amino acid residues that contained the typical domain structure of carbohydrate esterase family 4. The predicted three-dimensional structure of EsCDA-9k showed a 67.32% homology with Penaeus monodon. Recombinant chitin deacetylase had the highest activity at 40 °C and pH 8.0 in Tris-HCl buffer. The enzyme activity was enhanced by metal ions Co2+, Fe3+, Ca2+, and Na+, while it was inhibited by Zn2+, Ba2+, Mg2+, and EDTA. Molecular simulation of EsCDA-9k was conducted based on sequence alignment and homology modeling. The EsCDA-9k F18G mutant showed a 1.6-fold higher activity than the wild-type enzyme. In summary, this is the first report of the cloning and heterologous expression of the chitin deacetylase gene in Euphausia superba. The characterization and function study of EsCDA-9k will serve as an important reference point for future application.

Cloning, Expression, and Functional Analysis of the MYB Transcription Factor SlMYB86-like in Tomato.

MYB transcription factors (TFs) have been shown to play a key role in plant growth and development and are in response to various types of biotic and abiotic stress. Here, we clarified the structure, expression patterns, and function of a MYB TF, SlMYB86-like (Solyc06g071690) in tomato using an inbred tomato line exhibiting high resistance to bacterial wilt (Hm 2-2 (R)) and one susceptible line (BY 1-2 (S)). The full-length cDNA sequence of this gene was 1226 bp, and the open reading frame was 966 bp, which encoded 321 amino acids; its relative molecular weight was 37.05055 kDa; its theoretical isoelectric point was 7.22; it was a hydrophilic nonsecreted protein; and it had no transmembrane structures. The protein also contains a highly conserved MYB DNA-binding domain and was predicted to be localized to the nucleus. Phylogenetic analysis revealed that SlMYB86-like is closely related to SpMYB86-like in Solanum pennellii and clustered with other members of the family Solanaceae. Quantitative real-time PCR (qRT-PCR) analysis revealed that the expression of the SlMYB86-like gene was tissue specific and could be induced by Ralstonia solanacearum, salicylic acid, and jasmonic acid. The results of virus-induced gene silencing (VIGS) revealed that SlMYB86-like silencing decreased the resistance of tomato plants to bacterial wilt, suggesting that it positively regulates the resistance of tomatoes to bacterial wilt. Overall, these findings indicate that SlMYB86-like plays a key role in regulating the resistance of tomatoes to bacterial wilt.

Identification and characterization of TOR in Macrobrachium rosenbergii and its role in muscle protein and lipid production

The recent scarcity of fishmeal and other resources means that studies on the intrinsic mechanisms of nutrients in the growth and development of aquatic animals at the molecular level have received widespread attention. The target of rapamycin (TOR) pathway has been reported to receive signals from nutrients and environmental stresses, and regulates cellular anabolism and catabolism to achieve precise regulation of cell growth and physiological activities. In this study, we cloned and characterized the full-length cDNA sequence of the TOR gene of Macrobrachium rosenbergii (MrTOR). MrTOR was expressed in all tissues, with higher expression in heart and muscle tissues. In situ hybridization also indicated that MrTOR was expressed in muscle, mainly around the nucleus. RNA interference decreased the expression levels of MrTOR and downstream protein synthesis-related genes (S6K, eIF4E, and eIF4B) (P < 0.05) and the expression and enzyme activity of the lipid synthesis-related enzyme, fatty acid synthase (FAS), and increased enzyme activity of the lipolysis-related enzyme, lipase (LPS). In addition, amino acid injection significantly increased the transcript levels of MrTOR and downstream related genes (S6K, eIF4E, eIF4B, and FAS), as well as triglyceride and total cholesterol tissue levels and FAS activity. Starvation significantly increased transcript levels and enzyme activities of adenylate-activated protein kinase and LPS and decreased transcript levels and enzyme activities of FAS, as well as transcript levels of MrTOR and its downstream genes (P < 0.05), whereas amino acid injection alleviated the starvation-induced decreases in transcript levels of these genes. These results suggested that arginine and leucine activated the TOR signaling pathway, promoted protein and lipid syntheses, and alleviated the pathway changes induced by starvation.

Regulation of sirt1 and foxO1 in glucose metabolism of Megalobrama amblycephala

Both silent information regulator 2 homolog 1 (sirt1) and forkhead box transcription factor 1 (foxO1) are crucial transcription factors involved in glucolipid metabolism and energy regulation. The presnt study aimed to understand their regulatory roles in glucose metabolism. Molecular cloning and sequencing of sirt1 gene of Megalobrama amblycephala (masirt1) was conducted and cellular localization of both the factors were analysed. Their effects and action patterns in the glucose metabolism of Megalobrama amblycephala (M. amblycephala) were investigated through acute and long-term glucose tolerance assays. The results revealed that the full-length masirt1 cDNA sequence was 2350 bp and closely related to Sinocyclocheilus rhinocerous. Sirt1 and foxO1 were found to be mutually dependent and localized in the nucleus. Acute glucose tolerance tests revealed that the expression levels of both factors in the liver of M. amblycephala showed an initial increase followed by a decrease. Plasma glucose levels in M. amblycephala significantly increased at 2 and 12 h (P < 0.05). In a long-term breeding experiment with high-sugar feeding, the expressions of the sirt1 and foxO1 genes in the kidney and intestine of M. amblycephala exhibited synergistic changes. The 51WS groups had significantly higher levels of sirt1 and foxO1 gene expression in the kidney and intestine compared to the 0WS and 17WS groups (P < 0.05). Overall, masirt1 is evolutionarily highly conserved, and the interaction site of sirt1 and foxO1 is located in the nucleus. In long-term hyperglycemic regulation, sirt1 and foxO1 exhibit synergistic regulatory effects in the kidney and intestine of M. amblycephala. This study provides insights into how sirt1 and foxO1 regulate glucose metabolism in M. amblycephala.

Expression characteristics of the cyp19a1b aromatase gene and its response to 17β-estradiol treatment in largemouth bass (Micropterus salmoides).

To investigate the regulatory role of the cyp19a1b aromatase gene in the sexual differentiation of largemouth bass (Micropterus salmoides, LMB), we obtained the full-length cDNA sequence of cyp19a1b using rapid amplification of cDNA ends technique. Tissue expression characteristics and feedback with 17-β-estradiol (E2) were determined using quantitative real-time PCR (qRT-PCR), while gonad development was assessed through histological section observations. The cDNA sequence of LMB cyp19a1b was found to be1950 base pairs (bp) in length, including a 5' untranslated region of 145 bp, a 3' untranslated region of 278 bp, and an open reading frame encoding a protein consisting of 1527 bp that encoded 508 amino acids. The qRT-PCR results indicated that cyp19a1b abundantly expressed in the brain, followed by the gonads, and its expression in the ovaries was significantly higher than that observed in the testes (P < 0.05). After feeding fish with E2 for 30 days, the expression of cyp19a1b in the pseudo-female gonads (XY-F) was significantly higher than that in males (XY-M) (P < 0.05), whereas expression did not differ significantly between XX-F and XY-F fish (P > 0.05). Although the expression of cyp19a1b in XY-F and XX-F fish was not significantly different after 60 days (P>0.05), both exhibited significantly higher levels than that of XY-M fish (P<0.05). Histological sections analysis showed the presence of oogonia in both XY-F and XX-F fish at 30 days, while spermatogonia were observed in XY-M fish. At 60 days, primary oocytes were abundantly observed in both XY-F and XX-F fish, while a few spermatogonia were visible in XY-M fish. At 90 days, the histological sections' results showed that a large number of oocytes were visible in XY-F and XX-F fish. Additionally, the gonads of XY-M fish contained numerous spermatocytes. These results suggest that cyp19a1b plays a pivotal role in the development of ovaries and nervous system development in LMB.

Structural and functional analysis of transforming growth factor beta regulator 1 (TBRG1) in the red swamp crayfish Procambarus clarkii: The initial insight into TBRG1's role in invertebrate immunity

The transforming growth factor beta regulator 1 (TBRG1) is a growth inhibitory protein that acts as a tumor suppressor in human cancers, gaining its name for the transcriptional regulation by TGF-β. While extensive research has been conducted on the tumor-related function of TBRG1 in mammals, its significance in invertebrates remains largely unexplored. In this study, a homolog of TBRG1 was first structurally and functionally analyzed in the red swamp crayfish Procambarus clarkii. The full-length cDNA sequence was 2143 base pairs (bp) with a 1305 bp open reading frame (ORF) encoding a deduced protein of 434 amino acids (aa). The changes of PcTBRG1 transcripts upon immune challenges indicated its involvement in innate immunity. After knocking down PcTBRG1, the decline of bacteria clearance capacity revealed the participation of PcTBRG1 in the immune response. Furthermore, the downregulation of AMPs' expression after the cotreatment of RNAi and bacteria challenge suggested that PcTBRG1 might participate in innate immunity through regulating AMPs’ expression. These results provided initial insight into the immune-related function of TBRG1 in invertebrates.

A Novel Kunitz-Type Serine Protease Inhibitor (HcKuSPI) is Involved in Antibacterial Defense in Innate Immunity and Participates in Shell Formation of Hyriopsis cumingii.

Serine protease inhibitors (SPIs) are abundantly reported for its inhibition against specific proteases involved in the immune responses, but SPI data related to calcareous shells are scarce. Previously, our research group has reported the proteome analysis of non-nucleated pearl powder, and a candidate matrix protein containing two Kunitz domains in the acid soluble fraction caught our attention. In the present study, the full-length cDNA sequence of HcKuSPI was obtained from Hyriopsis cumingii. HcKuSPI was specifically expressed in the mantle, with hybridization signals mainly concentrated to dorsal epithelial cells at the mantle edge and weak signals at the mantle pallium, suggesting HcKuSPI was involved in shell formation. HcKuSPI expression in the mantle was upregulated after Aeromonas hydrophila and Staphylococcus aureus challenge to extrapallial fluids (EPFs). A glutathione S transferase (GST)-HcKuSPI recombinant protein showed strong inhibitory activity against the proteases, trypsin and chymotrypsin. Moreover, HcKuSPI expression in an experimental group was significantly higher when compared with a control group during pellicle growth and crystal deposition in shell regeneration processes, while the organic shell framework of newborn prisms and nacre tablets was completely destroyed after HcKuSPI RNA interference (RNAi). Therefore, HcKuSPI secreted by the mantle may effectively neutralize excess proteases and bacterial proteases in the EPF during bacterial infection and could prevent matrix protein extracellular degradation by suppressing protease proteolytic activity, thereby ensuring a smooth shell biomineralization. In addition, GST-HcKuSPI was also crucial for crystal morphology regulation. These results have important implications for our understanding of the potential roles of SPIs during shell biomineralization.

Molecular characterization and functional analysis of peroxiredoxin 1 (Prx1) from roughskin sculpin (Trachidermus fasciatus).

Peroxiredoxin1(Prx1), also known as natural killer enhancing factor A (NKEF-A), is a crucial antioxidant involving in various cellular activities and immune response against bacterial and viral infection in fish. In the present study, a full-length Prx1 cDNA sequence (TfPrx1) was firstly cloned from roughskin sculpin (Trachidermus fasciatus), which was composed of 1044bp nucleotides encoding a peptide of 199 amino acids with a molecular weight of 22.35kDa and a theoretical pI of 6.42, respectively. The predicted peptide was a typical 2-cys Prx containing two conserved characteristic motifs 43FYPLDFTFVCPTEI56 and 170GEVCPA175 with the two conserved peroxidatic and resolving cysteine residuals forming disulfide bond. Quantitative real-time PCR analysis showed that TfPrx1 was ubiquitously expressed in all tested tissues with the highest expression in the intestine. It could be significantly induced following LPS injection and heavy metal exposure. Recombinant TfPrx1 (rTfPrx1) displayed insulin disulfide reduction and ROS-scavenging activity in a concentration-dependent manner, and further exhibited DNA and cytoprotective effects under oxidative stress. These results suggested that TfPrx1 protein may play an important role in fish immune protection from oxidative damage.

Identification and characterization of β3 integrin in Crassostrea hongkongensis

Crassostrea hongkongensis is a main oyster species farmed in South China Sea offshore area, and commonly suffers from vibriosis. Vibrio parahaemolyticus is one of the principal pathogens that causes the oyster vibriosis. The integrin from C. hongkongensis was studied for its function against V. parahaemolyticus in the current work. The full-length cDNA sequence was 3274 bp with an open reading frame (ORF) of 2397 bp that encoded a polypeptide of 798 amino acids. The polypeptide included typical domains of β integrin (such as INB domain, three EGF-like domains, and highly conserved NPX[Y/F] motif), and phylogenetically clustered with β3 integrins of other invertebrates, thereby, designated as ChIntβ3. The mRNA transcripts of ChIntβ3 were significantly higher in the heart, hemocytes, adductor muscle and hepatopancreas than those in gills and mantle of C. hongkongensis. Following stimulation with V. parahaemolyticus, the mRNA expression level of ChIntβ3 was significantly up-regulated in both hemocytes and hepatopancreas. The recombinant ChIntβ3 had significantly agglutinating activity on Escherichia coli, V. parahaemolyticus, Bacillus amyloliquefaciens, Pichia pastoris and the red blood cells of rabbit. In the one hand, the survival rate (83.33 ± 5.77%) of the oyster after V. parahaemolyticus infection at 30 days was significantly higher in the ChIntβ3 overexpression group than those in the physiological saline control group (26.67 ± 20.82%) (p < 0.01) and the overexpressed GFP group (26.67 ± 5.77%) (p < 0.01); in the other hand, the cumulative survival duration of C. hongkongensis was significantly shorter in the RNAi treatment group (197.6 ± 2.77 h) than that in the control group (247.2 ± 36.24 h) (p < 0.01). The results indicated that ChIntβ3 was involved in the recognition and clearance of V. parahaemolyticus through cell adhesion, and contributed to the further understanding of the function of ChIntβ3 against infection of pathogens.

Characterization and Structural Analyses of Enolase from Shrimp (Litopenaeus vannamei).

Transcriptome analysis had recognized enolase from shrimp Litopenaeus vannamei (L. vannamei), which is termed LvEnolase, as one of the allergens, but its amino acid sequence and protein structure have been lacking. In this study, natural LvEnolase was isolated from L. vannamei and characterized for the first time. The full-length cDNA sequence of LvEnolase was effectively cloned, which encoded 434 amino acid residues. The crystal structure of LvEnolase was successfully determined at a resolution of 2.5 Å by X-ray crystallography (PDB: 8UEL). Notably, it was observed that near the active center, a loop exists in either an open or closed state, and the open loop was associated with the product release phase. Furthermore, enzyme activity assays were conducted to validate the catalytic capabilities of purified LvEnolase. These findings significantly enhance our comprehension of the enolase family and provide valuable support for delving into the functions and characteristics of LvEnolase.

A novel short-type peptidoglycan recognition protein with unique polysaccharide recognition specificity in sea cucumber, Apostichopus japonicus

Pattern recognition receptors (PRRs) are the first line of immune defense in invertebrates against pathogen infection; they recognize pathogens and transmit signals to downstream immune pathways. Among these, peptidoglycan recognition proteins (PGRPs) are an important family in invertebrates that generally comprise of complicated isoforms. A comprehensive understanding of PGRPs in evolutionarily and economically important marine invertebrates, such as the sea cucumber, Apostichopus japonicus, is crucial. Previous studies have identified two PGRPs in sea cucumber, AjPGRP-S and AjPGRP-S1, and another novel short-type PGRP, AjPGRP-S3, was additionally identified here. The full-length cDNA sequence of AjPGRP-S3 was obtained here by PCR-RACE, followed by which showed its gene expression analyses by in situ hybridization that showed it to be relatively highly expressed in coelomocytes and tube feet. Based on an analysis of the recombinant protein, rAjPGRP-S3, a board-spectrum pathogen recognition ability was noted that covered diverse Gram-negative and -positive bacteria, and fungi. Moreover, according to the results of yeast two-hybridization, it was suggested that rAJPGRP-S3 interacted with multiple immune-related factors, including proteins involved in the complement system, extracellular matrix, vesicle trafficking, and antioxidant system. These findings prove the important functions of AjPGRP-S3 in the transduction of pathogen signals to downstream immune effectors and help explore the functional differences in the AjPGRP isoforms.

Phosphate deficiency responsive TaSPX3 is involved in the regulation of shoot phosphorus in Arabidopsis plants

SPX (SYG/PHO81/XPR1) domain genes have been reported to play vital roles in the Phosphorus (Pi) signaling network in Arabidopsis thaliana and rice. However, the functions of SPX proteins in wheat remain largely unknown. In this study, the full-length cDNA sequence of the TaSPX3 gene was cloned from the common wheat variety Zhengmai9023. The expression of TaSPX3 was up-regulated in eight different genotypes of wheat under low phosphorus (LP) stress, indicating that TaSPX3 responds to Pi limitation in multiple wheat genotypes. The transcription level of TaSPX3 was also detected in the absence of seven different elements, showing certain specificity for Pi deficiency in wheat. Over expressing TaSPX3 in Arabidopsis can alleviate Pi deficiency symptoms at the seedling stage and promote the growth of plant, and advance the flowering period at the adult stage. The expression of 7 genes associated with the Pi starvation signal pathways was analyzed using qRT-PCR. The results showed that TaSPX3, along with AtSPX1, AtRNS1, AtIPS1, AtPAP2, AtPAP17 and AtAT4, were all induced by Pi deficiency. This study reveals that the TaSPX3 gene in wheat is involved in the response to phosphorus stress and may affect shoot phosphorus levels through AT4 or PAPs-related pathways. Overall, our study provides new insights into the regulation of plant response under LP conditions and the molecular mechanism underlying the role of the wheat SPX gene in coping with LP stress.

Kidney cell culture of Rana dybowskii and study molecules of TRIF in Rana dybowskii cells under the stress of Aeromonas hydrophila

As a model organism, the cell culture method of Rana dybowskii (Rd) has not received widespread attention. We obtained kidney cells from Rana dybowskii by tissue block culture and enzyme digestion, and optimized the culture method by changing different culture conditions. We can more conveniently study the operation of the Rana dybowskii immune system through cell lines. One of the most important adaptor proteins in the Toll-like receptors (TLRs) signaling pathway, TIR-domain-containing adaptor inducing interferon-β (TRIF), plays a role in the apoptosis pathway and participates in the ubiquitin-mediated protein hydrolysis pathway. This rationalizes the significance of TRIF in the immune cascade signaling pathway. To obtain and analyze the full-length sequence of cDNA, sequential structures of amino acids, as well as Rana dybowskii TRIF expression profiles, the present work carried out multiple-sequence alignment, rapid amplification of cDNA ends (RACE), the establishment of a phylogenetic tree, search for the conserved domain, and qRT-PCR. Results revealed 1733-bp full-length cDNA of RdTRIF with an open reading frame (ORF) of 1281 bp, which encoded one protein containing 426 amino acid residues. The amino acid sequence of RdTRIF confirmed the presence of one Toll/interleukin-1 receptor (TIR) domain. As a kind of hydrophilic protein, the pI and molecular weight of RdTRIF were 5.67 and 24.76 kDa, respectively. We predicted 51 candidate phosphorylation sites, including 32 serine residues, 16 threonine residues along with 3 tyrosine residues. The phylogenetic tree and multiple-sequence alignment substantiated the highest homology of RdTRIF protein to Xenopus tropicalis homologs. According to the qRT-PCR results, the expression of RdTRIF was noted under the stress of Ah in the kidney.

Cloning, computational analysis and expression profiling of steroid 5 alpha-reductase 1 (SRD5A1) gene during reproductive phases and ovatide stimulation in endangered catfish, Clarias magur

The cloning and characterization of the complete coding sequence of the Clarias magur SRD5A1 (CmSRD5A1) gene, which encodes an enzyme responsible for regulating steroid levels by converting testosterone into 5α-dihydrotestosterone (DHT), have been successfully achieved. DHT plays a vital role in enabling the complete expression of testosterone's actions in neuroendocrine tissues. The ORF of the full-length cDNA sequence of SRD5A1 was 795 bp, translating into 265 amino acids, with a total length of 836 bp including UTRs. Like other vertebrates, the signal peptide analysis revealed that SRD5A1 is a non-secretory protein, and hydropathy profiles indicated that it is hydrophobic in nature. The 3D structure of CmSRD5A1 sequence generated above was predicted using highly accurate AlphaFold 2 in Google Colab online platform. CmSRD5A1 contains seven transmembrane helices connected by six loops, with the N-termini located on the periplasmic side and C-termini on the cytosolic side. Structural superimposition with known bacterial and human SRD5As showed very high structural similarity. The electrostatic potential calculation and surface analysis of CmSRD5A1 revealed the presence of a large cavity with two openings one highly electropositive towards the cytosolic side and another relatively neutral towards the transmembrane region. The structural comparison revealed that the electropositive side of the cavity should bind to NADPH and the steroid hormone in the hydrophobic environment. Polar residues binding to NADPH are highly conserved and the same as known strictures. The conserved residues involved in hydrogen bonding with the ketone group at C-3 in the steroids hence fevering Δ4 double-bond reduction are identified as E66 and Y101. Our findings showed that SRD5A1 expression was lower during the spawning phase than the preparatory phase in female fish, while the administration of Ovatide (a GnRH analogue) resulted in up-regulation of expression after 6 h of injection in the ovary. In males, the lowest expression was observed during the preparatory phase and peaked at 16 h post- Ovatide injection in the testis. The expression of SRD5A1 in the brain of female fish was slightly higher during the Ovatide stimulation phase than the spawning phase. This study represents the first report on the cloning and characterization of the full-length cDNA of SRD5A1 in Indian catfish.

Characterization and expression analysis of seven lipid metabolism-related genes in yellow catfish Pelteobagrus fulvidraco fed high fat and bile acid diet

SREBPs, such as SREBP1 and SREBP2, were the key transcriptional factors regulating lipid metabolism. The processing of SREBPs involved many genes, such as scap, s1p, s2p, cideb. Here, we deciphered the full-length cDNA sequences of scap, srebp1, srebp2, s1p, s2p, cideb and cidec from yellow catfish Pelteobagrus fulvidraco. Their full-length cDNA sequences ranged from 1587 to 3884 bp, and their ORF length from 1191 to 2979 bp, encoding 396–992 amino acids. Some conservative domains were predicted, including the multiple transmembrane domains in SCAP, the bHLH-ZIP domain in SREBP1 and SREBP2, the ApoB binding region, ER targeting region and LD targeting region in CIDEb, the LD targeting region in the CIDEc, the conserved catalytic site and processing site in S1P, and the transmembrane helix domain in S2P. Their mRNA expression could be observed in the heart, spleen, liver, kidney, brain, muscle, intestine and adipose, but varied with tissues. The changes of their mRNA expression in responses to high-fat (HFD) and bile acid (BA) diets were also investigated in the brain, heart, intestine, kidney and spleen tissues. In the brain, HFD significantly increased the mRNA expression of seven genes (scap, srebp1, srebp2, s1p, s2p, cideb and cidec), and the BA attenuated the increase of scap, srebp1, srebp2, s1p, s2p, cideb and cidec mRNA expression induced by HFD. In the heart, HFD significantly increased the mRNA abundances of six genes (srebp1, srebp2, scap, s2p, cideb and cidec), and BA attenuated the increase of their mRNA abundances induced by HFD. In the intestine, HFD increased the cideb, s1p and s2p mRNA abundances, and BA attenuated the HFD-induced increment of their mRNA abundances. In the kidney, HFD significantly increased the scap, cidec and s1p mRNA expression, and BA diet attenuated the increment of their mRNA expression. In the spleen, HFD treatment increased the scap, srebp2, s1p and s2p mRNA expression, and BA diet attenuated HFD-induced increment of their mRNA expression. Taken together, our study elucidated the characterization, expression profiles and transcriptional response of seven lipid metabolic genes, which would serve as the good basis for the further exploration into their function and regulatory mechanism in fish.

Identification and molecular characterization of Rab7 form Yellow River Carp Cyprinus carpio

Rab7 is a small GTPase that regulates vesicular traffic from early to late endosomal stages of the endocytic pathway. In this study, full-length cDNA sequences of Rab7 were cloned from Yellow River Carp Cyprinus carpio, which were designated as CcRab7, and its function during pathogen infection was also investigated. The results showed that the full-length cDNA of CcRab7 was composed of 1511 bp, and open reading frame is 618 bp encoding 205 amino acids with 23.10 kDa. The deduced amino acid sequence of CcRab7 shared high similarity to that of Pimephales promelas (99.02%). In this paper, real-time fluorescence quantitative PCR (qRT-PCR) was used to detect the expression levels in 11 tissues and organs of healthy Yellow River Carp, and the tissue distribution of CcRab7 as well as its defense mechanism against Spring viremia of carp virus (SVCV) and Aeromonas hydrophila in Yellow River Carp C. carpio was investigated. The results revealed that the mRNA transcripts of CcRab7 were widely detected in various tissues of C. carpio, with the highest expression levels in head kidney and blood, indicating that CcRab7 was involved in the innate immune process. Yellow River Carp was immunized with A. hydrophila and SVCV respectively, and the expression changes of CcRab7 were detected in gill, spleen, liver and skin of Yellow River Carp. The mRNA transcripts of CcRab7 in the main organs (gill, and liver, skin and spleen) were significantly affected by SVCV and A. hydrophila infection. The results showed that the CcRab7 expression levels was obviously up-regulated at different time points. The eukaryotic recombinant plasmids of CcRab7 and pEGFP-N3 transfected into Grass carp ovary cell lines (GCO) cells for subcellular localization. The results provide a basis for further investigation into the immunological role of CcRab7 in Yellow River Carp.

Identification of transcriptionally active transposons in Barley

BackgroundThe genomes of many major crops including barley (Hordeum vulgare) consist of numerous transposons. Despite their important roles in crop genome evolution and morphological variations, most of these elements are silent or truncated and unable to be mobile in host genomes. Thus far, only a very limited number of active transposons were identified in plants.ResultsWe analyzed the barley full-length cDNA (FLcDNA) sequences and detected 71 unique FLcDNAs exhibiting significant sequence similarity to the extant transposase proteins. These FLcDNAs were then used to search against the genome of a malting barley cultivar ‘Morex’, seven new intact transposons were identified. Sequence alignments indicated that six intact transposons contained the entire FLcDNAs whereas another one served as 3’ untranslated region (3’ UTR) of a barley gene. Our reverse transcription-PCR (RT-PCR) experiment further confirmed the expression of these six transposons and revealed their differential expression. We conducted genome-wide transposon comparisons and detected polymorphisms of three transposon families between the genomes of ‘Morex’ and other three genotypes including the wild barley (Hordeum spontaneum, B1K-04-12) and two cultivated barley varieties, ‘Golden Promise’ and ‘Lasa Goumang’. Lastly, we screened the transcripts of all annotated barley genes and found that some transposons may serve as the coding regions (CDSs) or UTRs of barley genes.ConclusionWe identified six newly expressed transposons in the barley genome and revealed the recent mobility of three transposon families. Our efforts provide a valuable resource for understanding the effects of transposons on barley genome evolution and for developing novel molecular tools for barley genetic improvement and other research.