Puccinia triticina (Pt), an obligate fungal pathogen, induces leaf rust on wheat and is a major cause of yield loss. This study investigates the function and underlying mechanisms of the Triticum aestivum calcineurin B-like protein 1 (TaCBL1) in the interaction between wheat (Triticum aestivum) and Pt, aiming to lay the groundwork for a deeper understanding of the calcium signaling pathway governing this interaction. Expression patterns of TaCBL1 were analyzed in transcriptome data from wheat-Pt interactions and validated by qRT-PCR. Functional analyses were performed using virus-induced gene silencing (VIGS), RNA interference (RNAi)- and overexpression (OE) lines to assess TaCBL1 function during the wheat-Pt interaction. The effects of TaCBL1 silencing or overexpression on pathogenesis-related genes (PRs) expression were assessed by qRT-PCR, and H₂O₂ accumulation was examined using diaminobenzidine (DAB) staining to gain preliminary insight into the underlying mechanisms. Results showed that following inoculation with Pt, TaCBL1 expression in the incompatible combination was significantly elevated at 6 and 12 h (P<0.05), with expression levels markedly higher than those in the compatible combination (P<0.05). Silencing TaCBL1 by VIGS and RNAi increased the number of haustorial mother cells (HMCs) of the Pt relative to controls, whereas TaCBL1 overexpression significantly suppressed fungal development (P<0.05). These findings indicate that TaCBL1 exerts a positive regulatory role in the wheat-Pt interaction, providing new evidence for the involvement of calcium signaling in the regulation of pathogen invasion, providing the foundation for further systematic analysis of the molecular mechanisms by which TaCBL1 positively regulates wheat resistance, and enriching the notion of disease resistance breeding centered on signal transduction.

Exploring salt tolerant genes in rice (Oryza sativa), analyzing their molecular regulatory mechanisms, and breeding new salt tolerant rice varieties are one of the most economical and effective ways to cope with salt stress. This study used rice cultivar '9311' and the salt-tolerant introgression line IL363 of O. longistaminata, which was created with '9311' as there current parent, as materials. RNA-seq was used to perform whole genome transcriptome analysis on plants treated with salt stress (0.75% NaCl) for 3 and 5 d during the seedling stage. The results showed that, compared with untreated plants, 2 090 and 6 219 differentially expressed genes (DEGs) were identified in '9311' after 3 and 5 d of salt stress, and 169 and 2 535 genes were detected in IL363, respectively. This indicated that IL363 exhibited fewer DEGs during the early stage of salt stress. GO enrichment analysis revealed that the DEGs were primarily enriched in functional categories such as binding, catalytic activity, metabolic processes, and cellular processes. KEGG enrichment analysis showed that DEGs were mainly enriched in MAPK signaling and metabolic pathways, while DEGs in the treatment group were predominantly concentrated in pathways such as plant hormone signal transduction and the biosynthesis of secondary metabolites. Based on whole-genome chip detection results and functional annotations, 19 key candidate genes were selected for structural variation analysis from 53 screened genes potentially related to the differential salt tolerance between the 2 materials. Among these, 15 genes were located within the introgressed chromosomal segments, and 4 genes were important functional genes directly associated with salt stress. The structural variation analysis revealed that out of the 19 genes, 10 genes harbored variations in their exon regions, while 9 genes exhibited variations exclusively in non-coding regions. This study provides a theoretical basis for further unraveling the salt tolerance mechanisms of rice and supplies valuable candidate genes for salt-resistant rice breeding.

Grain shape is a core agronomic trait determining rice (Oryza sativa) yield and quality. As a key component of grain shape, grain thickness directly influences the thousand-grain weight and milling quality of rice. As important post-transcriptional regulators, microRNAs (miRNAs) play key roles in plant growth and development. In this study, taking grain shape regulation as the starting point, loss-of-function mutants of rice miR2871a were successfully generated using CRISPR/Cas9 technology. Systematic phenotypic analysis revealed that the mutants exhibited significantly reduced grain thickness and thousand-grain weight for both paddy and brown rice across multiple environments (P<0.05), accompanied by a significant increase in the chalky grain rate of milled rice (P<0.05). Histological observations demonstrated severe impairment in the development of the aleurone layer in mutant's caryopses, characterized by reduced cell layers, thinner thickness, and delayed developmental progression. Multi-element analysis revealed that defects in aleurone layer structure directly led to a significant decrease in the accumulation of key mineral elements, such as zinc, manganese, and calcium, in brown rice (P<0.05). Transcriptome sequencing (RNA-seq) analysis showed that differentially expressed genes were significantly enriched in thiamine metabolism and other biosynthetic pathways closely related to aleurone layer function. This study reveals the key regulatory role of miR2871a in the development of rice grain thickness and aleurone layer, and provides novel and important genetic resources for the synergistic genetic improvement of rice grain shape and nutritional quality.

The AP2/ERF (APETALA2/ethylene responsive factor) transcription factor family, one of the largest transcription factor families in plants, plays a crucial role in regulating plant growth, development, and abiotic stress responses. In this study, using the potato (Solanum tuberosum) cultivar 'Atlantic' as material, the S. tuberosum ethylene-responsive factor 89 (StERF89) gene (GenBank No. XM_006365280) was cloned, with a CDS of 891 bp. Bioinformatics analysis revealed that this gene lacked introns, and its promoter region contained cis-acting elements associated with drought stress response. Tissue-specificity analysis showed that StERF89 expression levels were highest in potato tubers, followed by leaves. Under natural drought conditions, StERF89 gene expression exhibited an upward trend as drought stress intensified, suggesting potential involvement in drought stress response. Further analysis revealed that the promoter region of S. tuberosum dirigent protein 31 (StDIR31), a lignin synthesis-related gene, contained 2 DRE/CRT elements, and its expression pattern under drought stress was similar to that of StERF89. Using yeast one-hybrid assays, dual luciferase reporter assays, and β-glucuronidase (GUS) staining experiments, it was further confirmed that StERF89 directly bound to the StDIR31 promoter and positively regulated its expression. This study provides crucial evidence for further investigation into the functional regulation of the StERF89 transcription factor.

β-glucosidase (BGLU) is a key enzyme involved in plant hormone regulation and stress response, and functional characterization of BGLU family genes is of great importance for genetic improvement of stress resistance in pepper (Capsicum annuum). Using the inbred line '17CL30' of pepper as the experimental material, this study cloned the pepper β-glucosidase 21 gene (C. annuum β-glucosidase 21, CaBGLU21)(GenBank No. PX753275.1), and performed systematic analysis on its bioinformatics characteristics, expression pattern and cis-acting elements in the promoter region. The results showed that CaBGLU21 encoded a stable alkaline hydrophilic protein containing 406 amino acids, which harbored a conserved Glyco_hydro_1 domain and multiple potential phosphorylation sites. Phylogenetic analysis indicated that CaBGLU21 had the closest genetic relationship with BGLU proteins from Solanaceae species, including Solanum lycopersicum, Nicotiana tabacum and Lycium ferocissimum. Promoter analysis revealed that the promoter sequence of CaBGLU21 contained cis-acting elements associated with light response, hormone response (MeJA, ABA, SA) and adversity stress. qRT-PCR results demonstrated that the expression of CaBGLU21 was significantly induced by various treatments including ABA, JA, IAA, GA₃, H₂O₂, NaCl, high temperature and low temperature, with its expression level reaching the peak at around 12 h after treatment. The results of this study preliminarily showed that CaBGLU21 might play an important role in pepper's response to hormone signals and abiotic stresses. This study provides a reference for in-depth dissection of the gene's function and provided candidate gene resources for pepper breeding for stress tolerance.

The MADS-box transcription factor family is widely involved in fruit ripening, cell wall remodeling, and texture regulation in plants, playing a central role in maintaining and softening fruit flesh. However, the molecular mechanisms of this family in regulating flesh hardness in watermelon (Citrullus lanatus) remain unclear. To investigate the regulatory function of MADS-box transcription factors in the formation of fruit flesh firmness in watermelon, fruit flesh tissues of the hard-flesh line '51' and the soft-flesh line '1338' were used as experimental materials. Through transcriptome analysis and qRT-PCR validation, a candidate gene associated with fruit flesh firmness, designated ClMADS37, was screened. The coding sequence of this gene was cloned, and its structural and expression characteristics were analyzed using bioinformatics methods. The results showed that the open reading frame (ORF) of ClMADS37 was 1 176 bp in length, encoding 391 amino acids. The protein was predicted to be an acidic, stable, hydrophilic protein localized in the nucleus. Expression analysis revealed that ClMADS37 expression levels in the flesh were significantly positively correlated with fruit firmness (r=0.7558). Furthermore, its expression was highest in stems and lowest in leaves. Promoter region analysis identified multiple light-responsive and hormone-responsive elements. In summary, ClMADS37 may positively regulate watermelon flesh hardness, and this study provides a theoretical basis and genetic resource for improving fruit texture and molecular breeding in watermelon.

Class Ⅲ peroxidases (PRXs) play essential roles in plant stress responses and immune regulation. To investigate the expression patterns and functional roles of PRXs in tobacco (Nicotiana tobacum) resistance against bacterial wilt induced by diallyl disulfide (DADS), this study conducted a systematic identification and analysis of the tobacco PRX gene family based on previous transcriptome data (NCBI accession number: PRJNA1470963). A total of 19 NtPRX genes were identified, unevenly distributed on 12 chromosomes. All encoded proteins contained typical secretory peroxidase domains, and their promoter regions were enriched with hormone-responsive, light-responsive, and stress-related cis-acting elements. Phylogenetic, conserved motif, and collinearity analyses indicated that the NtPRX gene family was evolutionarily conserved yet exhibited functional diversification. Furthermore, combining transcriptome data from roots and leaves of tobacco pretreated with DADS and subsequently inoculated with Ralstonia solanacearum, and validating with qRT-PCR. The result showed that DADS pretreatment could induce high-level expression of NtPRX4, NtPRX9, NtPRX11, NtPRX13, and NtPRX19 in both roots and leaves, and significantly enhanced post-inoculation leaf peroxidase (POD) activity. In conclusion, DADS may enhance tobacco resistance to bacterial wilt by modulating the temporal expression of key NtPRX genes and activating antioxidant and defense responses. This study provides a molecular basis for understanding DADS-induced plant disease resistance.

The FLOWERING LOCUS T (FT) gene family plays a critical role in regulating floral bud differentiation and is highly conserved across diverse plant species. This study conducted a genome-wide identification and systematic analysis of the FT gene family in tobacco (Nicotiana tabacum) using bioinformatics approaches, including gene structure characterization, conserved motif prediction, chromosomal localization, cis-regulatory element annotation, and expression pattern. A total of 11 FT family members (NtFT) were identified in tobacco, distributed across 3 chromosomes with preferential localization in low gene-density regions. All members harbored the conserved motif 1 and motif 4, and 73% of the members (8/11) exhibited a conserved structure consisting of 4 exons and 3 introns. Promoter cis-element analysis demonstrated that hormone-responsive elements (25.9%) and light-responsive elements (44.7%) were the most abundant. Tissue-specific expression profiling indicated that most NtFT genes were highly expressed in leaves, with only NtFT-09 showing specific expression in buds and flowers. qRT-PCR analysis of gene expression levels at different developmental stages revealed that NtFT-01, NtFT-02, NtFT-05, and NtFT-06 were significantly upregulated during the central flower opening stage (P<0.05), and their expression patterns were closely correlated with the enrichment of photoperiod- and hormone-responsive elements in the promoter regions. In contrast, NtFT-09 exhibited stage-specific expression patterns, suggesting its potential role in early floral bud differentiation. This study identifies NtFT-01, NtFT-02, NtFT-05, and NtFT-06 as core candidates driving reproductive growth and NtFT-09 as a potential regulator of floral initiation. These findings provide key genetic resources for further elucidation of the molecular regulatory mechanisms underlying flowering time in tobacco and provides a reference for future targeted editing of NtFT gene expression to develop early-flowering-resistant tobacco germplasm.

Anthocyanidin synthase (ANS) catalyzes the conversion of colorless proanthocyanidins into colored anthocyanidins. This study used 4 crape myrtle (Lagerstroemia indica) cultivars with white, pink, purple, and red petals to investigate the characteristics of the LiANS gene. Through multiple sequence alignment, phylogenetic tree construction, homology modeling, detection of the activity of prokaryotically expressed fusion protein LiANS-His₆, qPCR, and promoter cloning, the characteristics of LiANS in different cultivars were analyzed. The results showed that the ORF of the LiANS gene was 1 068 bp, encoding 355 amino acids. According to the differences among LiANS, it was found that there were 4 LiANS allelic variants in 4 cultivars. Homology modeling and enzyme activity assays of the 3 purified LiANS-His₆ variants showed that these variations had no effect on enzyme activity. qPCR analysis revealed that the LiANS gene was expressed constitutively in leaves from upper, middle, and lower part of shoots, and the flower at 5 developmental stages, but the expression patterns were different. The overall anthocyanin content peaked at the S3 stage of petal development. Among the samples, the red-flowered cultivar 'Dynamite' maintained relatively high expression levels during the full bloom and senescence stages (S3 and S4). The expression levels of the LiANS gene were not always positively correlated among the 4 varieties. Promoter sequence analysis indicated that 3 allelic variants of the promoter existed among the 4 cultivars, featuring different G-box elements and MYB binding sites. Notably, the white-flowered cultivar 'Pure white' exhibited a 195 bp deletion in the proANS region. This study provides genetic resources for molecular breeding of crape myrtle cultivars with different flower colors and lays a foundation for further elucidation of its regulatory mechanisms.

Branched-chain amino acid aminotransferase 1 (BCAT1) is a key enzyme in the catabolism of branched chain amino acids (BCAAs). This study aimed to obtain the CDS sequence of the BCAT1 gene and preliminarily investigate the effect of this gene on milk protein synthesis in mammary epithelial cells of dairy goats (Capra hircus). Mammary epithelial cells of dairy goats were used as materials, the CDS region of the BCAT1 gene was cloned, and bioinformatics analysis of the CDS sequence was performed using online software. The pcDNA3.1-BCAT1 overexpression vector was constructed, and the effect of the BCAT1 gene on milk protein synthesis in mammary epithelial cells was explored. The results showed that the full-length CDS region of the BCAT1 gene was 1 161 bp, encoding 386 amino acids. The molecular weight of the BCAT1 protein was 43 173.42 D, and the theoretical pI value was 5.05. No transmembrane domain or signal peptide was detected, and it was identified as a negatively charged, hydrophobic and stable protein. Protein-protein interaction analysis showed that the BCAT1 protein interacted with branched chain keto acid dehydrogenase E1 subunit alpha (BCKDHA), BCKDHB and other proteins. Tissue expression profile analysis indicated that the expression level of the BCAT1 gene was relatively high in mammary gland tissue during the dry period. After transfection of the BCAT1 gene recombinant overexpression vector into mammary epithelial cells, the expression levels of milk protein-related genes, including beta-lactoglobulin (BLG) (P<0.01), casein alpha s1 (CSN1S1) (P<0.05), casein beta (CSN2) (P<0.05), and casein kappa (CSN3) (P<0.05), were significantly increased. It was indicated that the BCAT1 gene played a regulatory role in milk protein synthesis in goat mammary epithelial cells. This study provides a theoretical foundation for further research on the regulatory mechanism of the BCAT1 gene in goat milk protein synthesis.

As one of the high-workload functional dogs, police dogs (Canis lupus familiaris) typically perform demanding tasks during routine training and missions, often accompanied by issues such as decreased endurance and reproductive disorders. Therefore, this study employed response surface methodology to optimize the extraction of bovine bone peptide (BBP), subsequently investigated its effects on preventing fatigue-induced reproductive damage in fatigued Beagle dogs. Beagles were randomly divided into 4 groups: Normal (normal feeding, no training), Model (exhaustion training), BBP-L (exhaustion training + 5 g/kg BBP), and BBP-H (exhaustion training + 10 g/kg BBP). Beagles underwent 4 weeks of BBP supplementation combined with downhill treadmill training at a 20° incline and 30 m/min speed, for 30 minutes daily, 5 days per week. After 4 weeks, a 30-minute non-weight-bearing exhaustive swimming test was conducted. At the conclusion of the experiment, blood samples were collected from the dogs, and testes were removed to assess sperm quality within the epididymides. Serum levels of blood glucose, blood urea nitrogen (BUN), lactic acid (LA), and lactate dehydrogenase (LDH) were measured in the Beagles. Measure pyruvate kinase (PK), succinate dehydrogenase (SDH), malondialdehyde (MDA), and superoxide dismutase (SOD) levels in testicular homogenates; Detect glycogen content in liver and muscle tissues; Finally, perform hematoxylin and eosin (HE) staining and immunohistochemical staining on testicular tissues from experimental dogs to analyze the effects of BBP on testicular histology and zonula occludens-1 (ZO-1) expression. The highest extraction rate of BBP was achieved under the following conditions: Boiling for 5 h, pH 10 during enzymatic hydrolysis, hydrolysis temperature of 55 ℃, and hydrolysis time of 6 h, yielding a rate of (35.12±1.25)%. Sperm quality and oxidative stress indicators revealed that after exhaustion training, the total sperm count, sperm density, sperm motility, and SOD levels in the trained dogs were significantly reduced (P<0.01), while MDA levels were extremely significantly elevated (P<0.001). These indicators were reversed after BBP supplementation. Serum biochemical indicators revealed that after exhaustive training, blood glucose, liver glycogen, muscle glycogen, SDH, and PK levels significantly decreased (P<0.01), while BUN, LA, and LDH levels significantly increased (P<0.01). After supplementation with BBP, blood glucose, liver glycogen, muscle glycogen, SDH, and PK levels significantly increased (P<0.01), while BUN, LA, and LDH levels significantly decreased (P<0.01). Histopathological examination revealed enlarged intercellular spaces and loose tissue structure in testicular supporting cells, accompanied by seminiferous tubule abnormalities. ZO-1 expression was significantly downregulated (P<0.01), with marked improvement after BBP supplementation (P<0.01). This study possesses important application value for alleviating problems such as endurance decline and reproductive dysfunction in police dogs during duty operations and high-intensity training.

Hyperlipidemia is a group of blood lipid disorders caused by dyslipidemia, which seriously endangers human health. Sterols possess multiple important physiological activities, such as lipid-lowering, anticancer, and antioxidant effects. To enhance the sterol yield from the liquid fermentation of Pleurotus tuber-regium, this study optimized the culture medium formulation using single-factor experiments and response surface methodology. The lipid-lowering activity of the sterol extract was evaluated through bile salt binding capacity assays and a high-lipid human hepatoblastoma G2 (HepG2) cell model. The results showed that the optimal medium consisted of fructose 52.51 g/L, corn protein 3.14 g/L, and MgSO₄ 0.14 g/L. Under this formulation, the sterol yield reached 362.25 mg/L, representing a 105.70% increase compared to that before optimization. The 2.0 mg/mL sterol extract exhibited significantly higher binding rates to sodium cholate, sodium taurocholate, sodium glycocholate, and mixed bile salts compared to the 1.0 mg/mL extract (P<0.05), corresponding to 86.21%, 86.05%, 89.76%, and 87.56% of the positive control cholestyramine, respectively. Treatment with 5.0 μg/mL extract reduced total cholesterol, triglycerides, and low-density lipoprotein cholesterol levels in HepG2 high-lipid cells by 26.95%, 71.42%, and 38.40%, respectively. The expression of lipid decomposition and transport genes CPT1, PPARα, and ABCG5 was upregulated by 437.30%, 235.85%, and 71.79%, respectively. In contrast, the expression of lipid synthesis and absorption genes SREBP2, NPC1L1, and ACAT2 was downregulated by 36.32%, 33.10%, and 17.53%, respectively. The expression of the rate-limiting enzymes in cholesterol synthesis, HMGCS1 and HMGCR, was downregulated by 47.31% and 20.80%, respectively. In summary, the sterol extract from P. tuber-regium not only demonstrated strong bile salt binding capacity, effectively interfering with cholesterol absorption, but also significantly modulated the expression of lipid metabolism-related genes through multiple targets, exhibiting considerable lipid-lowering activity. The findings provide a scientific basis for the development of lipid-lowering products derived from P. tuber-regium.

Camellia oleifera oil deodorizer distillate (CODD) is rich in high-value lipid components such as phytosterols, vitamin E, and unsaturated fatty acids, yet its direct utilization as a feed additive is hampered by poor dispersibility and low bioavailability. To develop green feed additives as alternatives to dietary antibiotics and achieve high-value utilization of CODD. In this study, Paecilomyces cicadae was inoculated into a solid-state fermentation system with soybean meal and wheat bran as carriers to ferment CODD. Non-targeted metabolomics combined with multivariate statistical analysis, KEGG pathway enrichment, and FELLA(functional enrichment analysis and visualization using sparse network-based activity diffusion) network analysis was employed to systematically elucidate the differential characteristics and regulatory mechanisms of lipid metabolism before and after fermentation. The results showed that the metabolic profiles of the fermentation and control groups exhibited significant separation. The expression patterns of differential lipid metabolites could be categorized into 4 functional groups: Comprehensive up-regulation of flavonoids (e.g., chrysin increased by over 4-fold), bidirectional regulation of steroids (anti-inflammatory pregnanes up-regulated while inert sterones down-regulated), uniform down-regulation of pro-inflammatory prostaglandins, and significant up-regulation of terpenoids. Mechanistic studies revealed that cutin/suberine/wax biosynthesis, steroid hormone biosynthesis, and arachidonic acid metabolism constitute 3 core regulatory pathways. The FELLA (functional enrichment analysis and visualization using sparse network-based activity diffusion) network further elucidated a cross-pathway synergistic regulatory mechanism with diacylglycerol and phosphatidylethanolamine as hub nodes and lipolytic enzymes together with acyltransferases as key enzyme targets. In conclusion, P. cicadae fermentation achieves pre-conversion of inert plant sterols present in CODD into highly active C21 steroids and directional restructuring of hesperetin-type precursor flavonoids into anti-inflammatory flavonoids such as chrysin, thereby enhancing the anti-inflammatory and antioxidant functional potential of the fermented products. This study provides a theoretical basis for the resource utilization of CODD and the development of green feed additives.

Colorectal cancer is one of the malignant tumors that severely threaten human health. Safe and effective therapeutic vaccines are urgently required to induce tumor-specific anti-tumor immunity. Wilms tumor antigen 1 (WT1) is highly expressed in various solid tumors but restricted in normal tissues, making it an ideal tumor-associated antigen. In this study, a LADS-WT1 vaccine was constructed using Listeria-based live attenuated double substitution (LADS) as a vector (LADS-WT1). Furthermore, a mouse (Mus musculus) colon cancer cell line (CT26-WT1) stably expressing WT1 was established to evaluate the specific therapeutic efficacy of LADS-WT1. In vitro growth analysis showed that the proliferation kinetics of the recombinant strain LADS-WT1 in brain heart infusion (BHI) broth were basically consistent with those of the parental LADS strain, indicating that the insertion of the heterologous antigen did not significantly impair its growth capacity. In addition, hemolysis assays demonstrated that the hemolytic activity of LADS-WT1 in liquid culture was significantly reduced, suggesting that the function of listeriolysin O (LLO), a virulence-related protein, was inhibited and its safety was further improved. The LADS-WT1 vaccine was administered via tail vein injection to CT26 and CT26-WT1 tumor-bearing BALB/c mice, respectively, and changes in tumor growth and immune responses were dynamically monitored. The results showed that following continuous LADS-WT1 vaccine treatment for 20 d, CT26-WT1 tumor-bearing mice exhibited a slower tumor growth rate and significantly smaller tumor volumes compared with CT26 tumor-bearing mice, indicating that the LADS-WT1 vaccine could inhibit the growth of WT1-expressing colorectal cancer cells. Flow cytometry analysis revealed an increased proportion of CD4⁺ T cells and CD8⁺ T cells in the spleens of CT26-WT1 tumor-bearing mice immunized with the LADS-WT1 vaccine, indicating that the vaccine effectively activated systemic cellular immune responses. In conclusion, the LADS-mediated delivery of the WT1 vaccine exerts anti-colon cancer effects through cellular immune responses, which provides a therapeutic strategy for other WT1-positive malignancies.

Porcine deltacoronavirus (PDCoV) is an emerging enteric coronavirus that causes acute watery diarrhea, vomiting, and dehydration in neonatal piglets (Sus scrofa). PDCoV infection leads to villus atrophy, crypt hyperplasia, and disruption of the intestinal barrier, resulting in high mortality and substantial economic losses to the swine industry. Occludin, a key component of tight junctions, plays critical roles in maintaining epithelial barrier integrity and mediating signal transduction. Using swine testicular (ST) cells as a model, the effect of PDCoV infection on Occludin expression was assessed by Western blot and qRT-PCR analyses in this study. Results demonstrated that both Occludin mRNA and protein levels decreased significantly over time post-infection (P<0.01), suggesting a correlation between PDCoV infection and Occludin downregulation. To further clarify the functional role of Occludin in viral replication, Occludin overexpressed or knocked down via plasmid overexpression and short hairpin RNA (shRNA) interference, respectively. The results showed that Occludin overexpression significantly promoted PDCoV infection (P<0.0001), while Occludin knockdown significantly inhibited PDCoV infection (P<0.0001), indicating that Occludin was a critical host factor involved in PDCoV replication. Furthermore, to explore the entry pathways through which Occludin promotes viral infection, cells were treated with the clathrin inhibitor Pitstop 2 and the macropinocytosis inhibitor 5-(N-ethyl-N-isopropyl) amiloride (EIPA). Both inhibitors markedly attenuated the Occludin overexpression-mediated enhancement of PDCoV infection. Collectively, The findings elucidated that Occludin positively regulated PDCoV entry via clathrin-mediated endocytosis and macropinocytosis, providing a theoretical basis for in-depth understanding of PDCoV pathogenesis and for developing novel host factor-targeted control strategies.

N⁶-methyladenosine (m⁶A), as the most prevalent post-transcriptional modification in eukaryotic mRNA, is widely involved in the regulation of gene expression. Methyltransferase-like 3 (METTL3), as the core catalytic enzyme for m⁶A modification, regulates gene expression by mediating m⁶A methylation, thereby significantly influencing key physiological processes in livestock and poultry, such as growth and development, fat metabolism, immune response, and disease resistance. This review focuses on the molecular structure and function of the METTL3 gene, as well as the latest advances in its regulatory roles in muscle development, fat metabolism, embryonic development, immune function, and disease resistance in livestock and poultry. This review provides theoretical support and potential molecular intervention targets for genetic improvement of livestock and poultry, providing a reference for continuously enhancing production efficiency.

Phalaenopsis spp. suffers from complex genetic backgrounds and difficult in vitro regeneration, compounded by the low efficiency of traditional stable genetic transformation, which hampers research on gene function and molecular breeding. Virus vector-mediated transient transformation technology offers an effective approach to address these issues. In this study, Phalaenopsis 'Zishuijing' was used to compare the transient genetic transformation efficiencies mediated by 3 viral vectors: Cymbidium mosaic virus (CymMV), Tobacco rattle virus (TRV), and Cabbage leaf curl virus (CaLCuV). Recombinant vectors carrying the phytoene desaturase (PDS) gene were introduced into leaves via Agrobacterium-mediated injection. The silencing efficiency of the 3 vectors was evaluated based on the photobleaching phenotype induced by PDS silencing and analysis of gene expression levels. The results indicated that significant differences in performance among the 3 vectors, with pCymMV demonstrating the highest transformation efficiency. Color fading was observed on inoculated leaves 4 weeks post-inoculation, and albinism emerged in newly developed leaves at 7 weeks. Moreover, the expression level of PePDS showed a nearly linear decreasing trend. In conclusion, pCymMV was the optimal vector for mediating transient transformation in Phalaenopsis. This study provides an efficient technical foundation for gene function analysis and molecular breeding in Phalaenopsis.

Peste des petits ruminants (PPR) counts as an acute virulent animal disease that spreads efficiently through direct contact, where goats (Capra hircus) and sheep (Ovis aries) are the primary susceptible small ruminant species. This study aimed to prepare polyclonal antibodies against the nucleocapsid (N) protein of the Peste des petits ruminants virus (PPRV) to provide biological materials for research on PPRV pathogenesis and disease control. Based on the N gene sequences of virulent and attenuated PPRV strains, the dominant antigenic epitopes were screened and analyzed using the online bioinformatics tools SEMA and IEDB. The dominant antigenic regions of the N gene from both strains were amplified separately by overlap PCR and then linked in tandem. The tandem gene encoding the dominant antigenic epitopes of the N protein was cloned into the pET-28a vector via BamHⅠand EcoRⅠrestriction sites to construct the prokaryotic expression plasmid, pET28a-PPRV-2N. After sequencing verification, the recombinant plasmid was transformed into Escherichia coli Rosetta competent cells for induced expression and optimization of the conditions. BALB/c mice (Mus musculus) were immunized using emulsified mixtures of purified target protein and adjuvant. The harvested polyclonal antibodies were subjected to reactivity identification assays. The size of the tandem 2N gene fragment amplified by PCR was approximately 1 607 bp, which was consistent with the expected result. The recombinant expression strain successfully expressed the target protein upon induction with IPTG, and the protein was mainly present in the form of inclusion bodies with an apparent molecular weight of approximately 60 kD. Optimal expression was achieved under the conditions of 30 ℃, 1 mmol/L IPTG, and 14 h induction. After denaturation, renaturation, and purification, the target protein was used to immunize BALB/c mice. Following the 3 immunizations, blood was collected, and the sera were separated to obtain polyclonal antibodies. The titer of the polyclonal antibodies against the PPRV N protein reached 1:10 000, as determined by Western blot. ELISA showed that the purified 2N protein exhibited good reactivity with positive serum. This study successfully obtained the expression product, 2N recombinant protein. The prepared mouse polyclonal antibody exhibited good immunoreactivity against both the N protein and the virus, providing theoretical and technical support for further exploration of the pathogenic mechanism of PPRV.