Search Results (2,166)

Drought stress poses a significant threat to tree growth, making the development of drought-resistant species essential for ecological restoration. WRKY transcription factors are critical regulators of plant drought responses; however, the role of WRKY22 in the woody species Populus ussuriensis K. remains unclear. In this study, the PuWRKY22 gene was cloned from P. ussuriensis via homologous cloning and was found to be highly expressed in leaves and responsive to abscisic acid (ABA) signaling. Subcellular localization confirmed that PuWRKY22 is a nuclear protein. Using fluorescein enzyme complementation assays, PuWRKY22 was shown to bind specifically to W-box cis-elements, indicating its function as a transcriptional regulator. Under ABA and osmotic (sorbitol) stress, the seed germination rate, root growth, and biomass of tobacco and Populus davidiana × Populus bolleana strains overexpressing PuWRKY22 were significantly increased. Additionally, these overexpressed strains exhibited a reduction in reactive oxygen species (ROS) accumulation and a decrease in membrane lipid peroxidation. Transcriptomic analyses revealed that PuWRKY22 activates expression of the ABA receptor gene Ptr.PYL4 (Potri.006G104100.v4.1), which regulates stomatal closure to minimize water loss. Consistent with this, stomatal observations and photosynthetic measurements demonstrated that PuWRKY22 enhances drought tolerance by protecting photosystem II and preserving chlorophyll content. Collectively, this study elucidates the molecular mechanism by which PuWRKY22 enhances drought resistance in woody plants through ABA signaling, providing a foundation for breeding drought-tolerant forest species. Full article

Phthalic acid esters (PAEs), ubiquitously employed as a plasticizer, have been classified as priority environmental pollutants because of their persistence, bioaccumulation, and endocrine-disrupting properties. As a characterized PAE-degrading strain of marine origin, Mycolicibacterium phocaicum RL-HY01 utilizes di-(2-ethylhexyl) phthalate (DEHP) as its sole carbon and energy source. Genome sequencing and RT-qPCR analysis revealed a previously uncharacterized hydrolase gene (dehpH) in strain RL-HY01, which catalyzes ester bond cleavage in PAEs. Subsequently, recombinant expression of the cloned dehpH gene from strain RL-HY01 was established in Escherichia coli BL21(DE3). The purified recombinant DehpH exhibited optimal activity at 30 °C and pH 8.0. Its activity was enhanced by Co²⁺ and tolerant to most metal ions but strongly inhibited by EDTA, SDS, and PMSF. Organic solvents (Tween-80, Triton X-100, methanol, ethanol, isopropanol, acetone, acetonitrile, ethyl acetate, and n-hexane) showed minimal impact. Substrate specificity assay indicated that DehpH could efficiently degrade the short and long side-chain PAEs but failed to hydrolyze the cyclic side-chain PAE (DCHP). The kinetics parameters for the hydrolysis of DEHP were determined under the optimized conditions, and DehpH had a V_max of 0.047 ± 0.002 μmol/L/min, K_m of 462 ± 50 μmol/L, and k_cat of 3.07 s⁻¹. Computational prediction through structural modeling and docking identified the active site, with mutagenesis studies confirming Ser228, Asp324, and His354 as functionally indispensable residues forming the catalytic triad. The identification and characterization of DehpH provided novel insights into the mechanism of DEHP biodegradation and might promote the application of the target enzyme. Full article

Nuclear factors of activated T cells (NFATs) are pivotal regulatory factors of immune responses, primarily by modulating T cell activity and regulating inflammatory cytokine gene transcription. The grass carp reovirus (GCRV) triggers a serious hemorrhagic condition, posing a significant threat to sustainable grass carp (Ctenopharyngodon idella) aquaculture. However, the precise function of NFAT in the host’s defense against GCRV infection is mostly undefined. This study comprehensively identified and characterized the NFAT genetic family in grass carp, cloned grass carp NFAT1 (CiNFAT1), and investigated its expression and function during GCRV infection. Eight NFAT genes encoding seventeen isoforms have been detected within the grass carp’s genomic sequence, distributed across six different chromosomes. Comparative analysis revealed homology with zebrafish NFATs. CiNFAT1 possesses a 2697 bp open reading frame, encoding 898 amino acids, and contains conserved Rel homology domain (RHD) and NFAT-homology (IPT) domains. Quantitative PCR (qPCR) revealed ubiquitous CiNFAT1 expression in healthy grass carp tissues, with the highest expression in gills and skin and the lowest in liver. Following GCRV challenge in vivo, CiNFAT1 expression in immune tissues (liver, spleen, kidney, gill, intestine) showed dynamic changes over time. In vitro experiments in CIK cells demonstrated that CiNFAT1 expression peaked at 12 h post-GCRV infection. Further functional studies revealed that overexpression of CiNFAT1 significantly reduced GCRV replication at 36 h post-infection. This reduction was accompanied by elevated expression of type I interferon (IFN-I) and interferon regulatory factor 7 (IRF7) at 24 and 36 h, respectively, as well as modulated IL-2, IL-8, and IL-10. Conversely, RNA interference-mediated knockdown of CiNFAT1 enhanced GCRV VP5 and VP7 mRNA levels and suppressed IL-2 and IL-8 expression. These results suggest that CiNFAT1 contributes to anti-GCRV immunity by promoting antiviral and inflammatory cytokine responses, thereby inhibiting viral replication. This study provides a foundational understanding of the NFAT genetic family in grass carp and highlights an important role of CiNFAT1 in mediating the body’s inherent defense mechanism against GCRV infection, offering insights for disease control strategies in aquaculture. Full article

Background: Monoclonal antibodies play an important role in therapeutic and analytical applications. For recombinant expression, the coding sequences of the variable regions of the heavy and light chains are required. In addition, cloning antibody sequences, including constant regions, reduces the impact of hybridoma cell loss and ensures preservation of the naturally occurring full antibody sequence. Method: We combined amplification of IgG antibody variable regions from hybridoma mRNA with an advanced method for full-length cloning of monoclonal antibodies in a simple two-step workflow. Following Sanger sequencing and evaluation of consensus sequences, the best matching variable, diversity, and joining (V-(D-)J) gene segments were identified according to identity scores from IgBLAST reference sequences. Simultaneously, the mouse IgG subclass was determined at the DNA level based on isotype-specific sequence patterns in the C_H1 domain. Knowing the DNA sequence of V-(D-)J recombination responsible for the complementary determining region 3 (CDR 3), variable region-specific primers were designed and used to amplify the corresponding antibody constant regions. Results: To verify the approach, we applied it to the hybridoma clone BAM-CCMV-29-81 and obtained identical full-length antibody sequences as with RNA Illumina sequencing. Further validation at the protein level using an established MALDI-TOF MS-fingerprinting protocol showed that five out of six genetically encoded CDR domains of the monoclonal antibody BAM-CCMV-29-81 could be efficiently correlated. Conclusion: This simple, streamlined method enables the cost-effective determination of the full-length sequence of monoclonal antibodies from hybridoma cell lines, with the added benefit of obtaining the DNA sequence of the antibody ready for recombinant expression. Full article

The ABI3 transcription factor is a key regulator in plant growth and development. Through transcriptome analysis of the resistant soybean cultivar ‘Dongnong L10′ and the susceptible cultivar ‘Heinong 37′ exposed to soybean cyst nematode race 3 (SCN 3) stress, the differentially expressed gene GmABI3VP1 was identified. The GmABI3VP1 gene was then cloned and analyzed through bioinformatics, subcellular localization, and qRT-PCR analysis of resistant and susceptible soybean germplasms, as well as overexpression and gene editing of soybean hairy roots followed by SCN 3 identification analysis. It was found that the protein encoded by GmABI3VP1 is an acidic and hydrophilic protein with transmembrane domains. It has a collinear relationship with Arabidopsis and is widely distributed in plants. Through the analysis of promoter elements, it was shown that this gene contains multiple hormone-responsive promoter elements like ABRE/ABRE3a/ABRE/4a/as-1 and stress-responsive elements such as Myb/MYC/MYc. Transient expression in tobacco indicated that the GmABI3VP1 gene is located in the nucleus. The transcription of GmABI3VP1 responds to the stress of SCN, and its transcriptional level is relatively high in the roots of resistant materials. Genetic transformation mediated by Agrobacterium rhizogenes was used to obtain GmABI3VP1 gene overexpressed and CRISPR-Cas9 gene-edited soybean hairy roots. In comparison to the wild type (WT), the density of nematodes per area was notably lower in hairy roots overexpressing (OX) the gene, whereas the density of SCN per unit area (per cm of lateral root length) significantly increased in gene-edited (KO) soybean hairy roots. Through SCN phenotyping, GmABI3VP1 was identified as a contributor to SCN 3 resistance. This study provides initial insights into the role of the GmABI3VP1 gene in SCN resistance, establishing a robust basis for future research on the mechanisms underlying SCN disease resistance and offering valuable genetic reservoirs for SCN 3 resistance. Full article

Despite significant medical advancements, two major health challenges persist: antibiotic resistance in microbial pathogens and drug resistance in cancer cells. To address these issues, research has increasingly focused on discovering novel natural compounds with dual antimicrobial and anticancer activities. Among such candidates, antimicrobial peptides (AMPs) have attracted attention due to their ability to selectively target microbial and cancer cells while exhibiting minimal toxicity toward normal cells. Although Vietnam possesses rich biodiversity, including a wide range of Anura species, studies on AMPs from these organisms remain limited. In this study, a novel AMP, brevinin-1 E8.13, was identified from the skin secretion of Sylvirana guentheri, a frog species native to Vietnam. The brevinin-1 E8.13 peptide was successfully cloned, sequenced, and chemically synthesized. Functional assays revealed that brevinin-1 E8.13 possesses strong antibacterial activity against Staphylococcus aureus and exerts significant antiproliferative effects on various human cancer cell lines, including A549 (lung), AGS (gastric), Jurkat (leukemia), HCT116 (colorectal), HL60 (leukemia), and HepG2 (liver). The peptide demonstrated moderate to potent cytotoxic activity, with IC₅₀ values ranging from 7.5 to 14.8 μM, depending on the cell type. Notably, brevinin-1 E8.13 exhibited low cytotoxicity toward normal human dermal fibroblast (HDF) cells and even promoted cell proliferation at lower concentrations. Furthermore, Chemically Activated Fluorescent Expression (CAFLUX) bioassay results confirmed that the peptide significantly downregulated Cyp1a1 gene expression in HepG2 cells. Collectively, these findings highlight the therapeutic potential of brevinin-1 E8.13 as a dual-function antimicrobial and anticancer agent derived from the skin secretion of Sylvirana guentheri. Full article

The melon fly Zeugodacus cucurbitae (Coquillett) is a globally invasive pest responsible for substantial economic losses in the fruit and vegetable industries. Insulin-like peptides (ILPs) are evolutionarily conserved neuropeptides that play a crucial role in insect reproduction. In this study, six ZcILPs from the melon fly, designated as ZcILP1–6, were cloned. Phylogenetic analysis demonstrated a strong orthologous link with Dipteran ILPs. Spatiotemporal expression profiling revealed that ZcILP1 and ZcILP3 exhibit preferential enrichment in the adult female fat body, with their expression specifically and significantly upregulated in 5-day-old individuals. Their expression decreased 12, 24, and 48 h post-starvation and increased upon re-feeding. Silencing ZcILP1 and ZcILP3 resulted in reduced ovarian size by 51.42% and 69.17%, respectively. Furthermore, silencing ZcILP1 or ZcILP3 significantly decreased the transcriptional levels of genes downstream of the insulin signaling pathway (ISP), notably the target of rapamycin (ZcTOR) and Forkhead box O (ZcFOXO). Concurrently, the expression of Vitellogenin (ZcVg), a gene associated with reproduction, was significantly downregulated. These findings indicate that ZcILP1 and ZcILP3 regulate ZcVgs expression and ovarian development through ISP, suggesting them as potential targets for green control of Z. cucurbitae. Full article

African swine fever (ASF) is a highly pathogenic and hemorrhagic swine infectious disease caused by the African swine fever virus (ASFV). It encodes over 150 proteins, among which the CD2v protein plays multiple roles throughout the infection process. Single B-cell antibody technology is a cutting-edge method for preparing monoclonal antibodies (mAbs), which has the advantages of rapid, efficient, and high yield in antibody production, while possessing natural conformations. In this study, by cloning and expressing antibody genes in vitro, 14 murine-derived mAbs were prepared using recombinant CD2v proteins as immunogenic sources, which brings sufficient enrichment and selectivity for the development of antibodies based on the single B-cell antibody technique. All 14 mAbs demonstrated reactivity with CD2v protein by indirect ELISA, whereas 8 mAbs successfully detected CD2v in ASFV-infected PAM cells by IFA, indicating the tested mAbs can effectively recognize and bind to ASFV CD2v. Finally, a blocking ELISA method for detecting CD2v antibodies using CD2v mAb C89 was established, which holds significant potential for broad application in the serological diagnosis of ASFV with determination of the CD2v-blocking ELISA specificity, sensitivity, reproducibility, and compliance rate. It could be used for the rapid clinical detection of ASFV CD2v protein to provide a powerful tool for the monitoring of epidemics. Full article

Tea polyphenol oxidase (CsPPO) is a crucial enzyme involved in the production of tea and tea products. However, the recombinant expression of CsPPO in microorganisms is often hindered by challenges such as inclusion body formation and extremely low enzyme activity. In this study, the CsPPO gene (1800 bp) from Camellia sinensis cv. Yihongzao was cloned and 14.5% of its codons were optimized for Pichia pastoris expression. Compared to pre-optimization, codon optimization significantly enhanced CsPPO production in P. pastoris KM71, yielding a 42.89-fold increase in enzyme activity (1286.67 U/mL). The optimal temperature and pH for recombinant CsPPO were determined to be 40 °C and 5.5, respectively. This study demonstrates that codon optimization effectively improves the expression of plant-derived enzymes such as CsPPO in eukaryotic expression systems. Future research should explore the long-term stability of recombinant CsPPO and its potential applications in tea fermentation processes and functional food development. Full article

TasA gene, encoding a functional amyloid protein critical for biofilm formation and antimicrobial activity, was cloned from the endophytic strain Bacillus amyloliquefaciens BS-3, isolated from rubber tree roots. This study identified the shortest functional TasA variant (483 bp, 160 aa) reported to date, featuring unique amino acid substitutions in conserved domains. Bioinformatics analysis predicted a signal peptide (1–27 aa) and transmembrane domain (7–29 aa), which were truncated to optimize heterologous expression. Two prokaryotic vectors (pET28a and pCZN1) were constructed, with pCZN1-TasA expressed solubly in Escherichia coli Arctic Express at 15 °C, while pET28a-TasA formed inclusion bodies at 37 °C. Purified recombinant TasA exhibited potent antifungal activity, achieving 98.6% ± 1.09 inhibition against Colletotrichum acutatum, 64.77% ± 1.34 against Alternaria heveae. Notably, TasA completely suppressed spore germination in C. acutatum and Oidium heveae Steinmannat 60 μg/mL. Structural analysis via AlphaFold3 revealed that truncation enhanced protein stability. These findings highlight BS-3-derived TasA as a promising biocontrol agent, providing molecular insights for developing protein-based biopesticides against rubber tree pathogens. Full article

The barbel chub (Squaliobarbus curriculus) exhibits remarkable resistance to grass carp reovirus (GCRV), a devastating pathogen in aquaculture. To reveal the molecular basis of this resistance, we investigated complement factor D (DF)—a rate-limiting serine protease governing alternative complement pathway activation. Molecular cloning revealed that the barbel chub DF (ScDF) gene encodes a 1251-bp cDNA sequence translating into a 250-amino acid protein. Crucially, bioinformatic characterization identified a unique N-glycosylation site at Asn¹³⁹ in ScDF, representing a structural divergence absent in grass carp (Ctenopharyngodon idella) DF (CiDF). While retaining a conserved Tryp_SPc domain harboring the catalytic triad (His⁶¹, Asp¹⁰⁹, and Ser²⁰⁴) and substrate-binding residues (Asp¹⁹⁸, Ser²¹⁹, and Gly²²¹), sequence and phylogenetic analyses confirmed ScDF’s evolutionary conservation, displaying 94.4% amino acid identity with CiDF and clustering within the Cyprinidae. Expression profiling revealed constitutive ScDF dominance in the liver, and secondary prominence was observed in the heart. Upon GCRV challenge in S. curriculus kidney (SCK) cells, ScDF transcription surged to a 438-fold increase versus uninfected controls at 6 h post-infection (hpi; p < 0.001)—significantly preceding the 168-hpi response peak documented for CiDF in grass carp. Functional validation showed that ScDF overexpression suppressed key viral capsid genes (VP2, VP5, and VP7) and upregulated the interferon regulator IRF9. Moreover, recombinant ScDF protein incubation induced interferon pathway genes and complement C3 expression. Collectively, ScDF’s rapid early induction (peaking at 6 hpi) and multi-pathway coordination may contribute to barbel chub’s GCRV resistance. These findings may provide molecular insights into the barbel chub’s high GCRV resistance compared to grass carp and novel perspectives for anti-GCRV breeding strategies in fish. Full article

Molecular chaperones, especially heat shock proteins (HSPs) have vital functions in cells’ responses to stress. Here, we cloned and sequenced the complete complementary DNA encoding HSP90 (MjHSP90) from the shrimp Marsupenaeus japonicus. The MjHSP90 cDNA comprised 3162 bp, including a 2172 bp coding region encoding a 724 amino acid-protein (predicted molecular mass = 83.12 kDa). Homology and phylogenetic analyses showed that MjHSP90 was highly conserved and most homologous to Litopenaeus vannamei HSP90. MjHSP90 is expressed in all tested tissues, with high expression in gill tissue and the hepatopancreas. Cold stress significantly upregulated MjHSP90 expression in the gill and hepatopancreas (p < 0.05). Following RNA interference knockdown of MjHSP90, the cold stress-related death rate of the shrimp increased significantly, accompanied by significantly upregulated expression of apoptosis-related genes Mjcaspase-3 and Mjbcl-2 (p < 0.05) and an increase in the number of apoptotic cells. The results indicated that MjHSP90 might play a pivotal role in the shrimp’s immune response to cold stress. Full article

The innate immune response is an important defense against invading pathogens. Stimulator of interferon gene (STING) plays an important role in the cyclic GMP-AMP synthase (cGAS)-mediated activation of type I IFN responses. However, some viruses have evolved the ability to inhibit the function of STING and evade the host antiviral defenses. Understanding both the mechanism of action and the viruses targets of STING effector is important because of their importance to evade the host antiviral defenses. In this study, the STING (IpSTING) of Ictalurus punctatus was first identified and characterized. Subsequently, the yeast two-hybrid system (Y2HS) was used to screen for proteins from channel catfish virus (CCV, Ictalurid herpesvirus 1) that interact with IpSTING. The ORFs of the CCV were cloned into the pGBKT7 vector and expressed in the AH109 yeast strain. The bait protein expression was validated by autoactivation, and toxicity investigation compared with control (AH109 yeast strain transformed with empty pGBKT7 and pGADT7 vector). Two positive candidate proteins, ORF41 and ORF65, were identified through Y2HS screening as interacting with IpSTING. Their interactions were further validated using co-immunoprecipitation (Co-IP). This represented the first identification of interactions between IpSTING and the CCV proteins ORF41 and ORF65. The data advanced our understanding of the functions of ORF41 and ORF65 and suggested that they might contribute to the evasion of host antiviral defenses. However, the interaction mechanism between IpSTING, and CCV proteins ORF41 and ORF65 still needs to be further explored. Full article

Background: Anoectochilus roxburghii (Wall.) Lindl. (A. roxburghii) was widely used in traditional Chinese medicine and also as a health food in China. Gibberellins (GAs) are plant hormones that regulate various aspects of growth and development in A. roxburghii. Ent-kaurene synthase (KS) plays a crucial role in the biosynthesis of GAs in plants. However, there is limited functional analysis of KS in GA biosynthesis and its effect on salt tolerance, especially in A. roxburghii. Methods: The ArKS genes were cloned from A. roxburghii, and its salt tolerance characteristics were verified by prokaryotic expression. Under salt stress, analyze the regulation of KS gene on GA and active ingredient content by qRT-PCR and HPLC-MS/MS, and explore the mechanism of exogenous GAs promoting active ingredient enrichment by regulating the expression level of the KS under salt stress. Results: The ArKS protein was highly homologous to KSs with other plant species; subcellular localization of KS protein was lacking kytic vacuole. The transformants displayed a significant increase in salt tolerance under the stress conditions of 300 mM NaCl. And the expression of ArKS genes and the GAs accumulation was downregulated under the salt stress; among them, the contents of GA3, GA7, GA8, GA24, and GA34 showed a significant decrease. It was further found that there was an increase (1.36 times) in MDA content and a decrease (0.84 times) in relative chlorophyll content under the salt conditions from A. roxburghii. However, the content of active constituents was elevated from A. roxburghii under the NaCl stress, including polysaccharides, total flavonoids, and free amino acids, which increased by 1.14, 1.23, and 1.44 times, respectively. Interestingly, the ArKS gene expression and the chlorophyll content was increased, MDA content showed a decrease from 2.02 μmoL·g⁻¹ to 1.74 μmoL·g⁻¹ after exogenous addition of GAs, and the elevation of active constituents of polysaccharides, total flavonoids, and free amino acids were increased by 1.02, 1.09, and 1.05 times, implying that GAs depletion mitigated the damage caused by adversity to A. roxburghii. Conclusions: The ArKS gene cloned from A. roxburghii improved the salt tolerance of plants under salt stress by regulating GA content. Also, GAs not only alleviate salt tolerance but also play a key role in the synthesis of active components in A. roxburghii. The functions of KS genes and GAs were identified to provide ideas for improving the salt tolerance and quality of ingredients in artificial cultivation from A. roxburghii. Full article

Barley leaf stripe, caused by Pyrenophora graminea (Pg), significantly reduces yields across various regions globally. Understanding the resistance mechanisms of barley to Pg is crucial for advancing disease resistance breeding efforts. In this study, two barley genotypes—highly susceptible Alexis and immune Ganpi2—were inoculated with the highly pathogenic Pg isolate QWC for 7, 14, and 18 days. The number of differentially expressed genes (DEGs) in Alexis was 1350, 1898, and 2055 at 7, 14, and 18 days, respectively, while Ganpi2 exhibited 1195, 1682, and 2225 DEGs at the same time points. Gene expression pattern analysis revealed that Alexis responded more slowly to Pg infection compared to Ganpi2. A comparative analysis identified 457 DEGs associated with Ganpi2’s immunity to Pg. Functional enrichment of these DEGs highlighted the involvement of genes related to plant-pathogen interactions and kinase activity in Pg immunity. Additionally, 20 resistance genes and 24 transcription factor genes were predicted from the 457 DEGs. Twelve candidate genes were selected for qRT-PCR verification, and the results showed that the transcriptomic data was reliable. We conducted cloning of the candidate Pg resistance gene HvLRR_8-1 by the barley cultivar Ganpi2, and the sequence analysis confirmed that the HvLRR_8-1 gene contains seven leucine-rich repeat (LRR) domains and an S_TKc domain. Subcellular localization in tobacco indicates that the HvLRR_8-1 is localized on the cell membrane. Through the functional analysis using virus-induced gene silencing, it was demonstrated that HvLRR_8-1 plays a critical role in regulating barley resistance to Pg. This study represents the first comparative transcriptome analysis of barley varieties with differing responses to Pg infection, providing that HvLRR_8-1 represents a promising candidate gene for improving durable resistance against Pg in cultivated barley. Full article