Search Results (162)

Uncaria rhynchophylla, a medicinal plant extensively used in traditional Chinese medicine, is an important plant source of terpenoid indole alkaloids (TIAs), but the mechanism of TIA biosynthesis at molecular level remains unclear. Geraniol synthase (GES) serves as a crucial enzyme in catalyzing the formation of geraniol from geranyl pyrophosphate (GPP) in various plants, but the functional characterization of the GES gene in U. rhynchophylla has not been investigated. In this study, a GES was identified and characterized through genome mining and bioinformatic analysis. Functional validation was performed via a protein catalysis experiment, transient expression in Nicotiana benthamiana, and methyl jasmonate (MeJA) induction experiments. The full-length UrGES gene was 1761 bp, encoding a protein product of 586 amino acids with an estimated 67.5 kDa molecular weight. Multiple sequence alignments and phylogenetic analysis placed UrGES within the terpene synthase g (TPS-g) subfamily, showing high similarity to known GESs from other plants. Enzymatic assays confirmed that recombinant UrGES catalyzed GPP conversion to a single product of geraniol. The transient expression of UrGES resulted in geraniol accumulation in N. benthamiana, further confirming its function in vivo. UrGES expression was observed in leaves, stems, and roots, where leaves had the highest transcript levels. Moreover, MeJA treatment significantly upregulated UrGES expression, which positively correlated with an increase in alkaloid content. This study functionally characterizes UrGES as a geraniol synthase in U. rhynchophylla, contributing to the current knowledge of the TIA biosynthetic pathway. These findings may offer insights for future metabolic engineering aiming to enhance TIA yields for pharmaceutical and industrial applications. Full article

Plants are frequently exposed to various abiotic stresses, among which low-temperature stress markedly impairs growth and physiological functions. WRKY transcription factors are key regulators in plant responses to abiotic stress. In this study, a novel WRKY transcription factor gene, TrWRKY79, was cloned from white clover. Functional characterization revealed that the full-length TrWRKY79 protein possesses typical features of transcription factors, including transcriptional activation activity located at its C-terminal domain. Heterologous expression of TrWRKY79 in Arabidopsis thaliana significantly enhanced cold tolerance under low-temperature stress. Physiological assays showed that the transgenic lines exhibited higher chlorophyll content and elevated activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) compared to wild-type plants. Furthermore, Protenix was employed to predict the potential target genes of TrWRKY transcription factors, and their expression profiles were analyzed to help elucidate the regulatory network underlying cold tolerance. qRT-PCR analysis confirmed that several cold-responsive genes, such as COR47 and ABI5, were significantly upregulated in the transgenic lines. Collectively, these findings indicate that TrWRKY79 plays a positive regulatory role in enhancing cold tolerance, providing valuable insights into the molecular mechanisms of cold resistance in white clover and offering promising candidate genes for improving stress resilience in forage crops. Full article

Coccidiosis, caused by Eimeria spp., significantly reduces poultry productivity and causes major economic losses. Traditional control methods are limited by drug resistance and high production costs. Recent genomic and bioinformatic advances have enabled the identification of novel antigens, making recombinant subunit vaccines a promising next-generation strategy by eliciting robust cellular and humoral immune responses. This study investigates the E. necatrix gametocyte protein 56 (EnGAM56) as a potential candidate for recombinant subunit vaccines. The full-length E. necatrix gametocyte gam56 gene (Engam56-F) was amplified, expressed in vitro, and characterized via SDS-PAGE and Western blot. Immunofluorescence assays revealed that EnGAM56-F is specifically localized in gametocytes and unsporulated oocysts. Chickens immunized with recombinant proteins (rEnGAM56-F and rEnGAM56-T) were evaluated for immunoprotection against E. necatrix infection through lesion scores, weight gain, oocyst production, anticoccidial index (ACI), and antibody and cytokine levels. The synergistic effects were evaluated by employing various combinations of recombinant proteins, including rEtGAM22, rEtGAM56-T, and rEtGAM59. Results showed that EnGAM56-F encodes a 468-amino acid protein with distinct tyrosine-serine-rich and proline-methionine-rich regions. rEnGAM56-F was specifically recognized by both anti-6 × His tag antibodies and convalescent serum from chickens infected with E. necatrix. Both rEnGAM56-F and rEnGAM56-T provided immune protection, with rEnGAM56-T showing superior efficacy. The combination of rEnGAM (22 + 59 + 56-T) yielded the strongest immune response, followed by rEnGAM (22 + 56-T). These findings highlight the potential of EnGAM56 as a candidate for recombinant subunit anticoccidial vaccines. Full article

Background/Objectives: The recent COVID-19 pandemic caused by SARS-CoV-2 infection has highlighted the need for protocols for rapid development of efficient screening methods to search for the optimal mRNA vaccine structures against mutable viral agents. The unmatched success of mRNA vaccines by Pfizer and Moderna encoding the spike protein of SARS-CoV-2 confirms the potential of lipid nanoparticles for mRNA delivery for an accelerated development of new vaccines. The efficacy of vaccination and the production cost of mRNA-based vaccines largely depend on the composition of mRNA components, since the synthesis of an immunogenic protein requires precise and efficient translation in vivo. The composition of 5′ and 3′ UTR combinations of mRNA has a strong impact on the translation efficiency. The major objective of this study was to increase the probability of producing the immunogenic protein encoded by vaccine mRNA. For this purpose, we proposed to find a new combination of natural UTRs and, in parallel with that, to design and test the system for in vivo selection of translationally active UTRs. Methods: By using Ribo-Seq analysis, sets of candidate short UTRs were generated. These UTRs were tested both in cell cultures and in mice for effective production of secreted nanoluciferase (NLuc) and the S protein of SARS-CoV-2. A combination of the most effective UTRs was used to generate a prototype of an mRNA vaccine capable of inducing neutralizing antibodies against coronavirus. Results: The usefulness of the selected UTRs for vaccine development was tested by implicating the full-length coding sequence of SARS-CoV-2 S protein to produce the main immunogen. As a result, the system for functional screening of UTRs was created by using the NLuc gene. Conclusions: The proposed approach allows non-invasive quantitative assessment of the translational activity of UTRs in the blood serum of mice. By using the full-length sequence of SARS-CoV-2 S protein as a prototype, we demonstrated that the combination of UTRs selected using our luciferase-based reporter assay induces IgG titers and neutralization rates comparable to those obtained by using UTRs from commercial S-protein-based mRNA vaccines. Full article

Background: In recent years, enzootic nasal tumor virus 2 (ENTV-2) has become prevalent in China, resulting in substantial economic losses for the goat industry. In order to enrich the availability of detection methods for ENTV-2, this study developed an expedited and accurate reverse-transcription quantitative real-time polymerase chain reaction (RT-qPCR) assay to facilitate the detection and quantification of ENTV-2. Methods: Specifically, a pair of primers and a TaqMan probe targeting conserved regions of the pro gene were designed to allow the specific amplification and detection of viral RNA in clinical samples. Moreover, modifying the method for use in a quantitative real-time PCR (qPCR) assay enables the detection of proviral DNA in tumor specimens. Results: Both methods exhibited a detection limit for the ENTV-2 standard plasmid at 10⁰ copies/µL. The detection methods we established exhibited high specificity and sensitivity to ENTV-2, without cross-reactivity with other pathogens causing respiratory diseases or endogenous retroviruses (EBRVs). We performed an ENTV-2 analysis of clinical samples in goats via RT-qPCR using nasal swab samples (n = 558) collected from three geographically distinct flocks in Lingyou County, Baoji City, Shaanxi Province, China, and 58 positive samples were detected for a positivity rate of 10.4%. After euthanasia, the autopsy report showed nasal cavity masses. Histopathological analysis demonstrated an epithelial neoplasm, in compliance with the features of enzootic nasal adenocarcinoma (ENA). Three full-length genomes were sequenced to assess genomic sequence conservation and variation. Multiple-sequence alignment demonstrated the existence of sequence variations among strains. Phylogenetic analysis of the nucleotide sequences revealed that the ENTV-2 SX1~3 isolates were phylogenetically related to the Chinese ENTV-2 isolates, especially the JY strain. Furthermore, recombination analysis suggested that both ENTV-2 SX1 and ENTV-2 SX2 might be recombinant variants. Conclusions: In conclusion, both methods are highly specific for the pro gene of ENTV-2, and the development of this assay has been deemed crucial to the early identification and subsequent control of this viral infection. Our results provide valuable information for further research on the genetic variation and evolution of ENTV-2 in China. Full article

Flower color is one of the most ornamental values of Meconopsis wilsonii, but very limited studies have been reported on its flower color formation. The UDP-glycosyltransferase (UGT) gene family plays a crucial role in plant flower color formation. In this study, the full-length transcriptome data of M. wilsonii was used to identify MwUGTs, focusing on protein physicochemical properties’ subcellular localization, and phylogenetic relationships. In addition, sequence analysis, expression pattern analysis, subcellular localization, and functional validation of MwUGT2 were also performed. A total of 26 MwUGTs were identified in full-length transcriptome and clustered into eight subgroups. Phylogenetic analysis and KEGG database annotation showed that MwUGT2 is associated with anthocyanin synthesis and accumulation. Subsequently, based on the expression of MwUGT2 during flower development and in different tissues, it was preliminarily determined that MwUGT2 plays a role in the flower bud stage. Subcellular localization assays suggested that MwUGT2 is present in the nucleus and cytoplasm. Overexpression in Nicotiana tabacum showed that MwUGT2 significantly increased the content of Cyanidin-3-O-glucoside and resulted in dark pink flowers in transgenic plants. In summary, our findings suggest that MwUGT2 plays a crucial role in the biosynthesis of anthocyanin and will also contribute to understanding the mechanisms of flower color formation in M. wilsonii. Full article

Unique genes refer to genes specific to a particular organism and play crucial roles in the biological functions, evolutionary processes, and adaptations to external environments. However, the roles of unique genes in plant pathogenic fungi remain largely unexplored. In this study, we identified a novel unique gene in the rice blast fungus Magnaporthe oryzae, named MoUNG (M. oryzae unique gene), through T-DNA insertion mutagenesis. The disruption of the MoUNG promoter region in the T-DNA insertion mutant (T30-104) led to an almost loss of MoUNG expression. MoUNG has no functional domains and lacks homologues in other organism. It is highly expressed during the early-infection stage between 16 and 32 h post-inoculation (HPI), in contrast to its expression in mycelia and at the later infection stage of 48 HPI. Notably, attempts to knock out MoUNG were unsuccessful, so we examined the T30-104 mutant and found it showed significantly reduced growth, conidiation, and pathogenicity. Introducing the full-length MoUNG with its promoter into T30-104 restored these phenotypic defects. Additionally, subcellular localization assays revealed that MoUNG exhibits a dot-like distribution within the cytoplasm of mycelium, conidium, appressorium, and invasive hypha. Furthermore, knock-down of MoUNG produced results similar to those observed with the insertion mutation. In conclusion, we identified a novel unique gene MoUNG in M. oryzae and demonstrated its involvement in growth, conidiation, and pathogenicity. Full article

Orobanche aegyptiaca is a holoparasitic weed that extracts water, nutrients, and growth regulators from host plants, leading to significant yield and quality losses. Biocontrol microbial metabolites have been shown to enhance plant resistance against parasitic plants, yet the underlying microbial mechanisms remain poorly understood. In this study, we investigated the role of Alternaria alternata JTF001 (J1) microbial metabolites in recruiting beneficial microbes to the tomato rhizosphere and promoting the establishment of a disease-suppressive microbiome. Pot experiments revealed that J1 metabolite application significantly reduced O. aegyptiaca parasitism. High-throughput sequencing of full-length 16S rRNA genes and ITS regions, along with in vitro culture assays, demonstrated an increase in the abundance of plant-beneficial bacteria, particularly Pseudomonas spp. The three candidate beneficial strains (zOTU_388, zOTU_533, and zOTU_2335) showed an increase of 5.7-fold, 5.4-fold, and 4.7-fold, respectively. These results indicate that J1 metabolites induce the recruitment of a disease-suppressive microbiome in tomato seedlings, effectively inhibiting O. aegyptiaca parasitism. Our findings suggest that microbial metabolites represent a promising strategy for managing parasitic plant infestations through microbial community modulation, offering significant implications for sustainable agricultural practices. Full article

The well-being and subsistence of giant pandas, an endangered species with a limited distribution, are currently threatened by a number of viruses, including canine parvovirus (CPV-2), canine distemper virus (CDV), and giant panda rotavirus (GPRV). To allow for timely intervention upon viral infection, it is necessary to execute rapid and accurate diagnosis of potential mixed viral infections. In the present study, we developed and validated a multiplex PCR (mPCR) approach for the detection of CPV-2, CDV, and GPRV infections. The results indicate that the method could selectively amplify the three viruses with high sensitivity and specificity, which are necessary attributes in clinical settings. Utilizing the established method, (sub)clinical giant panda samples were examined, and CPV-2, CDV, and GPRV were found in 19.72% (43 out of 218), 7.34% (16 out of 218), and 6.42% (14 out of 218) of the samples, respectively. Noticeably, mixed infections of two or three viruses were common, and this was generally observed in CDV- or GPRV-positive samples. Meanwhile, mPCR results were further validated with sequencing and the phylogenetic analysis of full-length sequences of viral genes. Taken together, our study provides an approachable assay which enables the quick detection of the three viruses mentioned above, which will benefit clinical diagnosis and laboratory epidemiological-based investigations of the giant panda population. Full article

Vitellogenins (Vtgs) are key yolk precursor proteins in fish, serving as critical indicators of gonadal maturation in females and reliable biomarkers for detecting xeno-oestrogenic pollution, particularly through their expression in juveniles or males. The vtg gene family comprises multiple subtypes that are species-specific, necessitating precise characterisation and quantification for effective use as biomarkers in studies on estrogenic endocrine-disrupting chemicals (EEDCs). In this study, we successfully cloned and characterised the full-length cDNAs of three vtg subtypes (vtgAa, vtgAb, and vtgC) from Scatophagus argus. Differential expression analysis revealed that vtgAb exhibited the highest responsiveness to 17α-ethynylestradiol (EE2) exposure, with a 3-fold increase in vivo at 10.0 μg/g EE2 and a 30-fold increase in vitro at 10⁻⁷ mol/L EE2. The expression patterns were dose- and time-dependent, with peak expression observed 72 h post-exposure. While in vivo assays indicated moderate upregulation, in vitro experiments demonstrated significantly higher expression, attributed to direct hepatocyte interaction with EE2. These findings confirm vtgAb as the most responsive subtype to oestrogen exposure in S. argus and highlight the species’ tolerance to EE2, as compared to more sensitive species like Danio rerio. This study shows the evolutionary conservation of vtg transcripts across teleost species and reinforces the importance of subtype-specific characterisation to advance their application as biomarkers for EEDCs, with significant implications for environmental monitoring and pollution regulation. Full article

To explore the molecular targets for regulating glucose metabolism in carnivorous fish, the turbot (Scophthalmus maximus) was selected as the research object to study. Farnesoid X receptor (FXR; NR1H4), as a ligand-activated transcription factor, plays an important role in glucose metabolism in mammals. However, the mechanisms controlling glucose metabolism mediated by FXR in fish are not understood. It was first found that the protein levels of FXR and its target gene, small heterodimer partner (SHP), were significantly decreased in the high-glucose group (50 mM, HG) compared with those in the normal glucose group (15 mM, CON) in primary hepatocytes of turbot. By further exploring the function of FXR in turbot, the full length of FXR in turbot was cloned, and its nuclear localization function was characterized by subcellular localization. The results revealed that the FXR had the highest expression in the liver, and its capability to activate SHP expression through heterodimer formation with retinoid X receptor (RXR) was proved, which proved RXR could bind to 15 binding sites of FXR by forming hydrogen bonds. Activation of FXR in both the CON and HG groups significantly increased the expression of glucokinase (gk) and pyruvate kinase (pk), while it decreased the expression of cytosolic phosphoenolpyruvate carboxykinase (cpepck), mitochondrial phosphoenolpyruvate carboxykinase (mpepck), glucose-6-phosphatase 1 (g6pase1) and glucose-6-phosphatase 2 (g6pase2), and caused no significant different in glycogen synthetase (gs). ELISA experiments further demonstrated that under the condition of high glucose with activated FXR, it could significantly decrease the activity of PEPCK and G6PASE in hepatocytes. In a dual-luciferase reporter assay, the FXR could significantly inhibit the activity of G6PASE2 and cPEPCK promoters by binding to the binding site ‘ATGACCT’. Knockdown of SHP after activation of FXR reduced the inhibitory effect on gluconeogenesis. In summary, FXR can bind to the mpepck and g6pase2 promoters to inhibit their expression, thereby directly inhibiting the gluconeogenesis pathway. FXR can also indirectly inhibit the gluconeogenesis pathway by activating shp. These findings suggest the possibility of FXR as a molecular target to regulate glucose homeostasis in turbot. Full article

Kallmann syndrome is a rare disorder characterized by hypogonadotropic hypogonadism and an impaired sense of smell (anosmia or hyposmia) caused by congenital defects in the development of the gonadotropin-releasing hormone (GnRH) and olfactory neurons. Mutations in several genes have been associated with Kallmann syndrome. However, genetic testing of this disorder often reveals variants of uncertain significance (VUS) that remain uninterpreted without experimental validation. The aim of this study was to analyze the functional consequences of a heterozygous missense VUS in the CHD7 gene (c.4354G>T, p.Val1452Leu), in a patient with Kallmann syndrome with reversal of hypogonadism. The variant, located in the first nucleotide of exon 19, was analyzed using minigene assays to determine its effect on ribonucleic acid (RNA) splicing. These showed that the variant generates two different transcripts: a full-length transcript with the missense change (p.Val1452Leu), and an abnormally spliced transcript lacking exon 19. The latter results in an in-frame deletion (p.Val1452_Lys1511del) that disrupts the helicase C-terminal domain of the CHD7 protein. The variant was reclassified as likely pathogenic. These findings demonstrate that missense variants can exert more extensive effects beyond simple amino acid substitutions and underscore the critical role of functional analyses in VUS reclassification and genetic diagnosis. Full article

Background/Objectives: The non-canonical GC-5′ splice sites (5′ss) are the most common exception (~1%) to the classical GT/AG splicing rule. They constitute weak 5′ss and can be regulated by splicing factors, so they are especially sensitive to genetic variations inducing the misrecognition of their respective exons. We aimed to investigate the GC-5′ss of the breast/ovarian cancer susceptibility genes, ATM (exon 50), BRIP1 (exon 1), and PALB2 (exon 12), and their dysregulation induced by DNA variants. Methods: Splicing assays of the minigenes, mgATM_49-52, mgBRIP1_1-2, and mgPALB2_5-12, were conducted to study the regulation of the indicated GC-5′ss. Results: A functional map of the splicing regulatory elements (SRE) formed by overlapping exonic microdeletions revealed three essential intervals, ATM c.7335_7344del, PALB2 c.3229_3258del, and c.3293_3322del, which are likely targets for spliceogenic SRE-variants. We then selected 14 ATM and 9 PALB2 variants (Hexplorer score < −40) located at these intervals that were assayed in MCF-7 cells. Nine ATM and three PALB2 variants affected splicing, impairing the recognition of exons 50 and 12, respectively. Therefore, these variants likely disrupt the active SREs involved in the inclusion of both exons in the mature mRNA. DeepCLIP predictions suggested the participation of several splicing factors in exon recognition, including SRSF1, SRSF2, and SRSF7, involved in the recognition of other GC sites. The ATM spliceogenic variants c.7336G>T (p.(Glu2446Ter)) and c.7340T>A (p.(Leu2447Ter)) produced significant amounts of full-length transcripts (55–59%), which include premature termination stop codons, so they would inactivate ATM through both splicing disruption and protein truncation mechanisms. Conclusions: ATM exon 50 and PALB2 exon 12 require specific sequences for efficient recognition by the splicing machinery. The mapping of SRE-rich intervals in minigenes is a valuable approach for the identification of spliceogenic variants that outperforms any prediction software. Indeed, 12 spliceogenic SRE-variants were identified in the critical intervals. Full article

Background: Benign prostatic hyperplasia (BPH) is a growing issue due to an ageing population. Our study investigated the possible associations between BPH and ageing hallmarks, including the telomere length (TL) and mitochondrial genome copy number (mtDNA CN), along with genetic variations in the TP53 gene and mtDNA. Methods: Prostate tissue samples were obtained from 32 patients with BPH, together with 30 blood samples. As a healthy control group, age-matching blood DNA samples were used. For the comparison of mtDNA sequence data, 50 DNA samples of the general Latvian population were used. The full mtDNA genome was analyzed by using Next-Generation Sequencing (NGS), the TP53 gene by Sanger sequencing, and the mtDNA copy number (mtDNA CN) and telomere length (TL) byqPCR assay. Results: The results showed that in BPH patients, telomeres in the prostate tissue were significantly longer than in blood cells, while the TL in blood cells of the healthy controls was the shortest. Also, the mtDNA amount in the prostate tissue of BPH patients was significantly greater in comparison with blood cells, and controls had the smallest mtDNA CN. We did not find any mutations in the TP53 gene that could be linked to BPH; however, in mtDNA, we found several unique mutations and heteroplasmic changes, as well as genetic changes that have been previously associated with prostate cancer. Conclusions: In conclusion, prolonged telomeres and changes in the mtDNA amount might be involved in the molecular mechanisms of BPH. Some of the heteroplasmic or homoplasmic mtDNA variants might also contribute to the development of BPH. Additional studies are needed to substantiate these findings. Full article

Porcine epidemic diarrhea (PED) is an acute, highly contagious, and infectious disease caused by porcine epidemic diarrhea virus (PEDV). PEDV can affect pigs of all ages, with 50~100% mortality in neonatal piglets and substantial economic losses in the swine industry. In the present study, 347 fecal and intestinal samples were collected from seven regions in China during 2020–2022. A comprehensive molecular investigation of the spike (S) gene of PEDV strains was carried out, which included phylogenetic analysis of the obtained PEDV sequences. Epidemiological surveillance data indicate that the GIIc subgroup strains are widely distributed among pigs. A PEDV strain was successfully isolated from positive small intestine samples and identified through RT-PCR detection using specific N gene primers of PEDV, indirect immunofluorescence assay (IFA), TEM analysis, genome sequencing, and full-length S gene analysis, named PEDV/SC/2022. RDP and SimPlot analysis showed that the isolate originated from the recombination of PEDV/AH2012 and PEDV/AJ1102. In conclusion, our findings contribute to the current understanding of PEDV epidemiology and provide valuable information for the control of PED outbreaks in China. Full article