Designing prokaryotic gene expression regulatory elements: From genomic mining to artificial intelligence-driven generation.

Expression and functional characterization of recombinant soluble CD40 Ligand in prokaryotic systems.

This study integrated transcriptome and hormone profiling of Colletotrichum camelliae-infected tea leaves to identify the methyl esterase (MES) gene family. Genome-wide analysis identified 17 MES genes, with CsMES4 as a crucial candidate localized in the nucleus and cell membrane. Through prokaryotic expression, purification, in vitro enzymatic assays, and transient silencing experiments in tea plants, we demonstrated that CsMES4 hydrolyze methyl salicylate (MeSA), methyl indole-3-acetate (MeIAA), and methyl jasmonate (MeJA) to produce salicylic acid, indole-3-acetic acid, and jasmonic acid, respectively. Transient silencing of CsMES4 reduced these hormones and enlarged lesions, confirming its role in disease resistance. Key catalytic residues predicted by docking were validated by mutagenesis: the F60L mutation significantly decreased CsMES4's catalytic activity toward all three substrates, while the S127Y mutation enhanced its catalytic efficiency. This study highlights the pivotal role of CsMES4 in phytohormone metabolism and provides insights into its multifunctional catalytic mechanism in tea plants.

Structure-guided design of a minimal epitope tag and nanobody affinity system.

Develop subunit vaccines against Nocardia seriolae in hybrid snakehead (Channa maculata ♀ × Channa argus ♂) using two resuscitation-promoting factors.

Revolutionizing recombinant protein production in prokaryotic platforms - Methodologies and advances.

Multi-route induction of Salmonella-based vaccine delivery elicits protective immunity against FV3-like ranavirus in an ornamental fish model.

PIMR, a novel Kunitz-type protease inhibitor from the Macrovipera razii, with dual anticoagulant and anticancer activities.

Design and development of a novel recombinant multi-epitope antigen: A promising candidate for melioidosis disease diagnosis.

Soil salinization severely threatens crop productivity and agricultural sustainability. Mulberry (Morus notabilis) is an economically important woody crop, but the molecular mechanisms of its salt tolerance remain unclear. The conserved Salt Overly Sensitive 3 (SOS3) gene regulates ion homeostasis under salt stress, making it a key target for exploring mulberry's salt adaptation. We cloned MnSOS3 from M. notabilis, constructed a prokaryotic expression vector (pCold-TF-MnSOS3) for transformation into Escherichia coli BL21, and generated MnSOS3-overexpressing transgenic tobacco (Nicotiana benthamiana) via Agrobacterium-mediated transformation. Salt tolerance was evaluated in E. coli (0.6 M NaCl) and tobacco (300 mM NaCl), with Na⁺/K⁺ content quantification in tobacco leaves (250 mM NaCl, 24 h) and verification of MnSOS3-MnSOS2 interaction via yeast two-hybrid assay. Recombinant E. coli expressing MnSOS3 showed enhanced growth (maximum OD₆₀₀ = 0.338) vs. near-zero growth in the control. Transgenic tobacco line MnSOS3-2 (highest expression) exhibited 33.3% survival under 300 mM NaCl, 2.5-fold higher than wild-type (WT, 13.3%). Transgenic leaves accumulated 54.8% less Na⁺ (0.95 ± 0.02 vs. 2.10 ± 0.07 mmol/g FW) and 45.8% more K⁺ (1.75 ± 0.02 vs. 1.20 ± 0.02 mmol/g FW) than WT, leading to a 67.8% lower Na⁺/K⁺ ratio (0.55 ± 0.01 vs. 1.71 ± 0.08). The yeast two-hybrid assay confirmed MnSOS3-MnSOS2 interaction. These findings show MnSOS3 boosts salt tolerance via Na⁺/K⁺ homeostasis and SOS pathway activation, a key resource for salt-tolerant mulberry breeding.

Sugarcane smut caused by Sporisorium scitamineum is one of the major diseases of sugarcane. Antimicrobial peptides are eco-friendly agents that can effectively control fungal diseases. The antimicrobial peptide AG-AFP is derived from Aspergillus giganteus and has the advantages of high stability and low biosafety risk. We tested the inhibitory effect of AG-AFP on Sporisorium scitamineum aiming to provide biocontrol resources for diseases in sugarcane. AG-AFP was obtained through prokaryotic expression. We examined the inhibition of AG-AFP on the cell growth of Sporisorium scitamineum and its effects on the integrity and fluidity of the cell membrane to verify the effectiveness of this antimicrobial peptide in the prevention and control of Sporisorium scitamineum. AG-AFP obtained by prokaryotic expression had a good inhibitory effect on Sporisorium scitamineum, with a minimum inhibitory concentration of 23.5 μg/mL. Moreover, it enhanced the cell membrane fluidity, destroyed the cell membrane integrity, and inhibited the dikaryotic hyphae of Sporisorium scitamineum. AG-AFP demonstrates a significant antifungal effect and potential application value for controlling diseases in sugarcane.

This study aimed to identify key genes regulating the bulb development of Fritillaria thunbergii by utilizing transcriptome data from the mature stage of 'Zhebei No.1' and 'Zhebei No.3' cultivars, which exhibited differences in propagation coefficient and single bulb weight. Furthermore, functional validation was performed to elucidate the mechanisms underlying their effects. Key candidate genes were functionally validated via bioinformatics analysis, prokaryotic expression, tobacco overexpression, and subcellular localization techniques, and their upstream regulatory sequences were analyzed. Relevant physicochemical data were integrated to investigate the impacts of these key genes on bulb development. The differentially expressed gene FtGGPS encoded a GGPS1 family member involved in gibberellin (GA), abscisic acid (ABA), and phylloquinone (VK1) biosynthesis, as well as chloroplast formation. Its upstream regulatory sequence contained GA-responsive elements such as the GARE-motif and transcription factor binding sites such as ERF, enabling GA signal-responsive regulation of FtGGPS expression. Correlation analysis indicated that under low FtGGPS expression levels, GA and ABA synthesis remained at low levels. Conversely, under high FtGGPS expression levels, rapid GA accumulation triggered feedback inhibition of FtGGPS expression. This inhibitory effect was alleviated with GA metabolism, which thereby maintained high-level synthesis of GA and ABA within the bulb. FtGGPS modulates the bulb development of F. thunbergii by regulating GA and ABA levels through distinct expression patterns. Elucidating the function of FtGGPS will help refine the hormonal regulatory network of bulb development in F. thunbergii and provide a theoretical basis for molecular breeding.

IntroductionAn atypical porcine pestivirus (APPV) causes congenital tremor (CT) type A-II in piglets and has caused considerable financial losses to the pig industry worldwide. This study aimed to develop an effective vaccine candidate against APPV.MethodsWe constructed a prokaryotic expression system for the APPV E2 protein in Escherichia coli. The immunogenicity of the APPV E2 subunit vaccine, formulated with three different adjuvants (ISA 201VG, IMS 1313VG, and Gel 02), was evaluated in piglets. Assessment was based on E2-specific antibody levels measured by an in-house enzyme-linked immunosorbent assay (ELISA) and protection against an APPV challenge. Successful expression of the E2 protein was confirmed by western blot analysis.ResultsThe E2 protein was successfully expressed, and an E2-ELISA detection method was established. Vaccinated piglets mounted virus-specific IgG responses. The E2 protein emulsified with ISA 201VG and Gel 02 adjuvants induced significantly higher antibody levels than the formulation with IMS 1313VG adjuvant. The vaccine with ISA 201VG adjuvant yielded the highest antibody levels and provided protection against APPV infection. Histopathological examination confirmed normal organ morphology in vaccinated pigs, whereas the control group displayed significant pathological alterations.DiscussionThe results demonstrate that the Escherichia coli-expressed E2 protein elicits an immune response in pigs. The E2 subunit vaccine is a potential candidate for preventing APPV infection, and ISA 201VG and Gel 02 adjuvants are suitable candidate adjuvants for vaccine preparation. This study provides a foundation for further APPV vaccine research.

Rice bakanae disease, a devastating disease caused by Fusarium fujikuroi infection, is primarily managed through chemical agents. Fluazinam, an oxidative phosphorylation uncoupler, is registered for controlling rice bakanae disease, yet its precise mechanism of action remains unclear. Our previous studies found that deletion mutants of ATP synthase subunits FfATPh, FfATP5, and FfATPb in Fusarium fujikuroi showed reduced sensitivity to fluazinam, therefore we aimed to investigate whether fluazinam is capable of binding with FfATPh, FfATP5, and FfATPb. In this study, the ATP synthase subunits FfATPh, FfATP5, and FfATPb were obtained via prokaryotic expression and nickel-column affinity purification. Microscale thermophoresis (MST) results showed that fluazinam binds with His9-MBP-TEV-ATPh and the Kd was 25.34 ± 11.71 nM. In contrast, its Kd values for His9-MBP-TEV-ATP5 and His9-MBP-TEV-ATPb were 1.49 ± 1.32 μM and 1.04 ± 0.75 μM, respectively, both similar to that of the His9-MBP-TEV control (2.57 ± 2.02 μM). These findings indicate that fluazinam exhibits specific affinity with FfATPh. Molecular docking analysis showed that fluazinam can form hydrogen bonds with Asp42 and Phe43 of FfATPh. Further affinity measurements of purified FfATPh point-mutant proteins showed that the binding affinity of fluazinam with His9-MBP-TEV-ATPh-D42A, D42H, F43A, and F43S was reduced to varying degrees. Mutations at residue Asp42 led to a more significant decrease in affinity, with Kd values of 639 ± 431 nM for D42A and 1.61 ± 1.36 μM for D42H. Based on these data, we speculate that fluazinam binds with FfATPh, with Asp42 serving as key binding sites. These findings provide a new molecular perspective on the mode of action of fluazinam and lay the foundation for its precise structural optimization. © 2025 Society of Chemical Industry.

Functional analysis of three glucoside hydrolase family 1 genes revealed their positive regulatory roles in the release of bonded-state aroma from strawberry fruits.

β-defensin 2 enhances antiviral immunity via autophagy regulation during Micropterus salmoides rhabdovirus (MSRV) infection and promotes survival in largemouth bass.

Identification and characterization of two novel linear B cell epitopes on the envelope protein of Japanese encephalitis virus.

Two interleukin-1β types reinforce innate immune response modulation in Japanese medaka, Oryzias latipes.

A recombinant adeno-associated virus expressing Eimeria stiedai HSP70 proteins exhibited a promising efficacy against rabbit coccidiosis.

Expression and purification of C14orf119 and generation of its polyclonal antibody.