Modern cultivated maize (Zea mays) exhibits significant differences in glume morphology compared to its wild ancestor, teosinte (Z. mays ssp. parviglumis), with glume traits being subjected to strong artificial selection during domestication. In natural environments, glumes are recognized to effectively prevent pathogen invasion caused by rainy conditions and confer insect resistance.The Eg1 mutant was identified as displaying elongated ear glumes. Phenotypic observations revealed that the Eg1 mutant showed significantly longer ear glumes compared to the wild type, while no obvious differences were observed in tassel glumes. Cytological analysis indicated that glume elongation in Eg1 resulted from an increased cell number rather than cell expansion in ear glumes. Genetic analysis demonstrated that the Eg1 mutant phenotype was controlled by a dominant gene. Using map-based cloning, the EG1 gene was preliminarily mapped to an 80 kb interval on maize chromosome 7, which contained 8 candidate genes. Among these, a Harbinger transposon insertion was identified 567 bp upstream of the promoter of Zm00001eb330490 (ZmERF58), leading to upregulated expression of this gene. ZmERF58 was classified as a member of the AP2/ERF family, functioning as a nuclear-localized transcription factor with autonomous activation activity.To further investigate EG1, transcriptome sequencing analysis was performed on 10 cm ear glumes from both wild-type and Eg1 mutants. A total of 3 264 differentially expressed genes (DEGs) were identified, including 1 266 upregulated and 1 998 downregulated genes. Gene Ontology (GO) enrichment analysis revealed that these DEGs were primarily involved in DNA-dependent transcription regulation, protein phosphorylation, and cell cycle processes. KEGG pathway analysis showed significant enrichment in plant hormone signal transduction, MAPK signaling pathways, and biosynthesis of secondary metabolites. This study aims to provide a theoretical foundation for subsequent functional characterization of the EG1 gene.

BRITTLE1 (BT1) mediates the unidirectional transmembrane transport of ADP-glucose (ADP-Glc), which is critical in starch synthesis. However, systematic studies on the members of the BT1 gene family and their biological functions remain lacking in wheat (Triticum aestivum). In this study, genome-wide identification, gene expression pattern, and elite alleles analysis of the BT1 family genes were conducted in wheat. Phylogenetic analysis, protein structure characterization, prediction of cis-acting elementin promoters, and qRT-PCR were employed to investigate the evolutionary relationships and expression patterns of BT1 family genes in wheat. Additionally, elite alleles of TaBT1 family members were screened. Phylogenetic and structural analysis indicated that the wheat TaBT1 protein shared a close evolutionary relationship with barley (Hordeum vulgare) HvBT1 protein. A total of 9 TaBT1 genes were identified in the wheat genome, all of encoding proteins containing the Mito-carr domain. Moreover, members within the same group exhibited similar conserved protein domains, indicating high evolutionary conservation of the TaBT1 gene family. The promoter regions of the TaBT1 genes contained various plant growth and development related cis-acting elements. TaBT1 genes were predominantly highly expressed in wheat grains, with the highest expression levels detected in the endosperm. Among the TaBT1 gene family members, 8 TaBT1 genes exhibited distinct alleles, with 3 showing alleles significantly associated with grain traits. Elite alleles of TaBT1-1A and TaBT1-1B were showed clear geographical differentiation across major wheat producing regions. This study provides a theoretical foundation for the biological function of the TaBT1 genes in regulating wheat grain development and their potential application in elite allele-based breeding.

Phosphoribulokinase (PRK) is a key enzyme in the Calvin cycle of photosynthetic reactions and plays a central role in carbon assimilation. Recent studies suggest that PRK may also participate in plant responses to abiotic and biotic stresses, but its molecular mechanisms under stress conditions remain unclear. This study investigated the regulatory mechanism of OsPRK in response to drought and rice blast fungus (Magnaporthe oryzae) stress by generating OsPRK-overexpressing transgenic rice lines and combining metabolomic analysis with phenotypic characterization. Using Agrobacterium-mediated genetic transformation and qPCR, we successfully obtained T₂ generation homozygous transgenic rice plants (Oryza sativa ssp. japonica cv. Nipponbare) carrying a single-copy transgene and exhibiting significantly higher OsPRK expression than wild-type (WT) plants. High-performance liquid chromatography-tandem mass spectrometry approach were employed to analyze metabolites in three-leaf-stage seedlings from transgenic and WT plants, identifying a total of 2 036 metabolites. Orthogonal partial least squares-discriminant analysis screened 136 significantly differentially accumulated metabolites (DAMs), including 93 that were upregulated and 43 that were downregulated. These DAMs were primarily enriched in metabolic pathways potentially related to stress response, such as isoflavonoid biosynthesis, flavonoid biosynthesis, and phenylpropanoid biosynthesis. Phenotypic analysis under stress conditions showed that OsPRK-overexpressing plants exhibited delayed leaf wilting under drought stress and had approximately 17% higher survival rate than WT plants after rehydration. After inoculation with Magnaporthe oryzae, transgenic plants developed a significantly fewer lesions, indicating enhanced resistance. Furthermore, key photosynthetic parameters at the mature stage, including net photosynthetic rate and stomatal conductance, were not significantly affected, demonstrating that OsPRK overexpression enhances stress tolerance without compromising photosynthetic efficiency. Interestingly, it was observed that T₀ generation osprk mutant plants exhibited significantly lower chlorophyll content during the seedling hardening stage compared with WT and overexpressing lines, and eventually died. This study reveals a novel mechanism by which OsPRK regulates stress resistance in rice through metabolic networks remodeling, providing a theoretical foundation for molecular breeding of stress-tolerant crops.

Flavonoids are important bioactive secondary metabolites in plants, playing crucial roles in fruit quality and resistance to pests and diseases. Chalcone synthase (CHS) is the first key rate-limiting enzyme in the flavonoid pathway but has not been comprehensively characterized in rambutan (Nephelium lappaceum). This study identified the CHS family of rambutan and analyzed its structural characteristics. The results showed that 5 members of the CHS family were identified in the genome of rambutan and named NlCHS1~ NlCHS5, all NlCHSs were located in the cytoplasm. Except for NlCHS1 which was an alkaline protein, the others were acidic and hydrophilic proteins. All except NlCHS4 were unstable proteins. Phylogenetic analysis classified the NlCHSs into 2 subfamilies, with members of the same subfamily sharing similar conserved motifs, conserved domains, and gene structures. All members contained the Chal_sti_synt_N and Chal_sti_synt_C domains; however, NlCHS1 and NlCHS3 lack some conserved motifs and domains and contain 2 introns within their gene structures (The remaining members contained only 1 intron). Chromosomal localization analysis revealed that the 5 NlCHS genes were located on Chr5, Chr6, Chr7, Chr9, and Chr12, respectively. The promoter regions of each member contained numerous light-responsive cis-elements; notably, the NlCHS4 promoter region contained a greater number of G-box and MRE elements compared to other members. Expression analysis across different cultivars, fruit tissues, and developmental stages demonstrated that the expression of NlCHS4 was highly correlated with flavonoid content in both the pericarp and aril of rambutan fruit. This suggested that NlCHS4 was a key member involved in flavonoid biosynthesis in rambutan. This study provides a certain foundation for further research on the synthesis regulation of anthocyanins in rambutan.

Heat shock protein 20 (HSP20) is a class of small molecular proteins that play critical roles in responding to temperature and other abiotic stresses. Identification and analysis of AaHSP20 gene family members in Artemisia argyi is helpful to reveal the regulatory mechanism response to temperature. 24 AaHSP20 genes were identified by using bioinformatics, and their physicochemical properties, evolutionary relationships, expression patterns, and so on were analyzed. The results showed that the encoded proteins ranged from 155 to 215 amino acids (aa) in length, the molecular weight was 17.60~24.67 kD, and most were predicted to be located in the cytoplasm. The secondary structures of AaHSP20 proteins were mainly composed of α-helix and random coils. The 24 AaHSP20 genes were unevenly distributed across 10 chromosomes. Some AaHSP20 genes had collinear relationships with homologs in Arabidopsis thaliana and Lactuca sativa. Phylogenetic analysis showed that the 24 AaHSP20 proteins could be divided into 3 classes and 4 subfamilies. All AaHSP20 proteins contained conserved domains and motifs (motif 1 and motif 2), and most genes lacked introns. Protein interaction analysis suggested AaHSP20 might interact with other small heat shock proteins. qRT-PCR analysis demonstrated that 5 candidate AaHSP20 genes exhibited distinct expression patterns in different tissues. AaHSP20-2, AaHSP20-7, AaHSP20-16, and AaHSP20-21 were sensitive to both high and low temperatures, while AaHSP20-20 was only sensitive to high temperature. These findings provide a theoretical foundation for elucidating the functions of AaHSP20 genes and improving resistance breeding of A. argyi.

Terpenoids serve as the fundamental material basis for the medicinal value of Bletilla striata. Geranylgeranyl pyrophosphate synthase (GGPPS) is a key branch point enzyme in the terpenoid metabolic network. However, the characteristics of the BsGGPPS gene remain unclear. In this study, using the wild purple-flowered B. striata as material, the full-length CDS of the BsGGPPS gene was cloned via RT-PCR based on transcriptomic data. Bioinformatics tools were employed to analyze the physicochemical properties, functional domains, and evolutionary relationships of its encoded protein. A fusion expression vector was constructed and transiently transformed into tobacco (Nicotiana tabacum) to determine the subcellular localization of the BsGGPPS protein. Furthermore, its expression levels across different tissues of B. striata were assessed using qRT-PCR. The results showed that the cloned BsGGPPS CDS was 1 083 bp in length, encoding 360 amino acids. Bioinformatics analysis revealed that BsGGPPS was a weakly hydrophobic, unstable protein without a signal peptide or transmembrane domains. It contained conserved aspartate-rich catalytic motifs (FARM and SARM) and belonged to a significant clade within the isoprenyl pyrophosphate synthase family. Subcellular localization indicated that BsGGPPS was primarily distributed in the cytoplasm and plastids. Phylogenetic analysis demonstrated that BsGGPPS formed a monophyletic group with GGPPS from Dendrobium catenatum and Phalaenopsis equestris. qRT-PCR results revealed that the expression level of BsGGPPS in the pseudobulbs of B. striata seedlings was significantly higher than that in the roots, stems, and leaves. These findings suggested that BsGGPPS was likely involved in the terpenoid biosynthesis pathway of B. striata through coordinated action in plastids and the cytoplasm. Its tissue-specific high expression in pseudobulbs implied a putative regulatory role in the storage organs for medicinal terpenoid active compounds. This study not only provides a theoretical foundation for elucidating the regulatory mechanisms of terpenoid metabolism in B. striata but also lays the groundwork for subsequent genetic engineering approaches aimed at directional regulation of the biosynthesis of medicinal terpenoids in B. striata.

As a protein feed with wide sources in China, cottonseed meal provides an effective solution to alleviate the shortage of protein feed in the animal husbandry industry. In sheep farming, replacing soybean meal with a certain proportion of cottonseed meal is conducive to improving economic benefits. The aim of this study was to analyze the effects of cottonseed meal replacing 50% and 100% soybean meal on the growth performance and digestive physiological indices of fattening lambs (Ovis aries), in order to provide a technical solution for soybean meal-free diets in fattening lambs. Healthy lambs at the age of 2 months with similar body weights were selected and fed with diets in which 0% (groupⅠ, control group), 50% (groupⅡ) and 100% (groupⅢ) cottonseed meal instead of soybean meal, with a pre-feeding period of 10 d and a regular feeding period of 60 d. During the feeding period, daily feed intake was recorded, and fasting body weights were measured on the morning of 0 and 60 d to calculate average daily gain and feed-to-gain ratio. On the morning of 60 d, blood samples were collected via venipuncture for analyzing serum immune indices and serum nutritional metabolism indices. Fecal samples were collected to determine the digestibility of dry matter, crude protein, crude fat, neutral detergent fiber, and acid detergent fiber. Rumen fluid was collected using an oral rumen cannula for analyzing rumen fermentation parameters and the rumen microbial community. The results showed that the average daily weight gain and feed weight ratio was no significant difference (P>0.05), the diets with 50% and 100% of soybean meal replaced by cottonseed meal had no significant effect on the growth performance.There was no notable divergence among groups in terms of the levels of serum nutritional metabolism indices and immune indices such as immunoglobulin A, immunoglobulin G, immunoglobulin M, tumor necrosis factor-α, interleukin-6, interleukin-4, interleukin-10, heat shock protein-70, creatinine, urea nitrogen, alanine aminotransferase, aspartate aminotransferase, serum protein (P>0.05). There was no notable divergence among groups in terms of the levels of major nutrients such as dry matter digestibility, crude protein digestibility, crude fat digestibility, neutral detergent fiber and acid detergent fiber digestibility (P>0.05). Among groups, the levels of rumen fermentation parameters such as rumen pH, ammonia nitrogen, bacterial protein and volatile fatty acid content did not display significant variances (P>0.05). At the phylum level of rumen microorganisms, Firmicutes and Bacteroidetes were the dominant phyla in all groups, with no significant differences (P>0.05) in the relative abundance of the top 10 phyla among groups. At the genus level, Prevotella was the dominant genus in the rumen microorganisms of all groups, and there were no significant differences (P>0.05) in the relative abundance of the top 10 genera among groups. The results of economic benefit analysis indicated that the income of lambs fed with 50% cottonseed meal and 100% cottonseed meal had an additional income of 7.08 yuan and 8.63 yuan per lamb respectively compared with the group fed with 100% soybean meal. In summary, replacing soybean meal with 50% and 100% cottonseed meal had no significant effect on the growth and major digestive physiological indices of lambs, and the economic benefits were significant. It is feasible to completely replace soybean meal with cottonseed meal in the fattening diet of lambs. This study provides the technical guide for cottonseed meal usage in meat sheep and goats.

Fish are typical ectothermic animals. Under high temperatures, fish are susceptible to endoplasmic reticulum (ER) stress, which activates the eukaryotic translation initiation factor 2α kinase 3 (EIF2AK3) / C/EBP-homologous protein (CHOP) signaling pathway, thereby inducing apoptosis and tissue damage. To investigate the response characteristics of eif2ak3 and chop in small yellow croaker (Larimichthys polyactis) under high-temperature stress, this study used small yellow croaker as the research subject and cloned the CDS of eif2ak3 and chop. The CDS of eif2ak3 was cloned, comprising 3 321 bp and encoding 1 106 amino acids, with conserved domains STKc_EIF2AK3_PERK and Luminal_EIF2AK3. Additionally, the CDS of chop was obtained, spanning 939 bp and encoding 263 amino acids, featuring a single BRLZ conserved domain. Phylogenetic analysis indicated that eif2ak3 and chop in the small yellow croaker exhibit the closest evolutionary relationship with their orthologs in the large yellow croaker (L. crocea). qPCR analysis revealed that eif2ak3 and chop were ubiquitously expressed across various tissues of the small yellow croaker, albeit with significant tissue-specific variation. Specifically, eif2ak3 expression was highest in the liver, whereas chop expression was highest in the heart. Under heat stress at (32±0.5) ℃, the liver expression profiles of eif2ak3 and chop were synchronized, peaking immediately upon reaching 32 ℃ and declining progressively thereafter. This suggested that eif2ak3 and chop playing a critical regulatory role during the initial phase of the heat stress response. This study reveals the expression characteristics of eif2ak3 and chop in response to heat stress, providing important data for further elucidating the molecular mechanisms of fish response to heat stress.

Potato anthracnose caused by Colletotrichum coccodes represents a significant phytopathological constraint in both potato (Solanum tuberosum) cultivation and postharvest storage. In this study, C. coccodes was used as the pathogen, and 2 endophytic bacteria (QB-2-7 and BM-7 ) with good bacteriostatic effect on C. coccodes preserved in the early stage of the laboratory were used to explore the control effect of its synthetic microbial community on potato anthracnose through in vivo and pot control experiments.Through comprehensive morphological characterization, physiological profiling, and molecular phylogenetic analysis, the isolates were taxonomically identified as Bacillus atrophaeus (QB-2-7) and B. safensis (BM-7), respectively. Biosafety assessment confirmed the non-hemolytic nature of both strains. Optimal consortium performance was achieved at a QB-2-7∶BM-7 ratio of 7∶3, demonstrating excellent microbial compatibility. Comparative analysis of different fermentation fractions revealed that the crude fermentation broth exhibited maximal antifungal activity (64.13% inhibition). In vivo evaluation of tuber protection efficacy demonstrated superior disease suppression in preventive applications (78.5% control efficiency) compared to both curative treatments and conventional azoxystrobin applications. The synthetic microbial community significantly enhanced host defense responses, as evidenced by elevated superoxide dismutase (SOD) and peroxidase (POD) activities, coupled with reduced polyphenol oxidase (PPO) activity and malondialdehyde (MDA) accumulation. Additional characterization revealed robust environmental tolerance of the fermentation products, including resistance to photodegradation, UV irradiation, and thermal stress, along with pronounced biofilm formation capacity. At the same time, the potato pot experiments showed that the synthetic microbial community had a significant prevention and control effect on potato anthracnose. These findings collectively suggest that the QB-2-7 and BM-7 synthetic microbial community consortium holds substantial promise as a biocontrol agent against potato anthracnose, providing both theoretical foundations and practical methodologies for developing microecological preparations targeting this economically important disease.

The banana (Musa acuminata) anthracnose disease caused by Colletotrichum musae is one of the most common post harvest diseases of bananas. To screen biocontrol bacterial strains with significant efficacy against banana anthracnose, in this study, a strain, Y-2-3, was isolated from soil samples collected from an abandoned salt field in Beihai, Guangxi, China, showing an inhibition rate of 75.62% against C. musae, which mycelia treated with Y-2-3 became swollen, deformed, and intertwined. Functional characterization revealed that Y-2-3 possesses growth-promoting properties, including nitrogen fixation, ammonia (NH₃) production, and phosphate solubilization, as well as the ability to produce cellulase and amylase. 16S rRNA and gyrase subunit B (gyrB) gene sequencing identified Y-2-3 as Bacillus velezensis. Dual culture assays confirmed that Y-2-3 exhibited broad-spectrum antifungal activity, with inhibition rates exceeding 50% against multiple pathogenic fungi. Moreover, volatile compounds produced by Y-2-3 exhibited a strong inhibitory effect on C. musae. Postharvest preservation experiments demonstrated that banana disease lesions were controlled within 40% under volatile treatment with Y-2-3. In conclusion, strain Y-2-3 exhibits both growth-promoting and biocontrol properties, showing great potential for application in banana anthracnose management and sustainable agriculture. This study provides theoretical basis and high-quality bacterial resources for the biological control of banana anthracnose.

Induced pluripotent stem cells (iPSCs) possess both self-renewal capability and multidirectional differentiation potential. Compared with embryonic stem cells (ESCs), iPSCs are associated with fewer ethical concerns and exhibit broad application prospects in medical and agricultural fields. However, it is limited in its differentiation efficacy and clinical translation by the persistence of residual exogenous reprogramming genes. Although integration-free technologies for generating iPSCs in rodents and humans (Homo sapiens) have become increasingly mature, obtaining integration-free iPSCs lines with stable pluripotency traits in livestock species remains challenging. This review focused on the core strategies for producing integration-free livestock iPSCs (such as integration-free vector delivery, novel reprogramming factor combinations and culture systems). Key regulatory mechanisms underpinning successful cases in large animals like pigs (Sus scrofa domesticus) were specifically analyzed. It further evaluated their translational potential and challenges from the dual perspectives of sustainable agricultural development and biomedical applications, thereby providing insights for advancing research and industrial applications of genome-safe livestock iPSCs.

N6-methyladenosine (m⁶A), the predominantly internal epigenetic modification in eukaryotic RNA, plays an important role in a variety of physiological and pathological processes. With the rapid development of high-throughput sequencing and single-base resolution detection technologies, an increasing number of m⁶A modification-related proteins and their target genes have been successfully identified in aquatic animals, and the corresponding regulatory functions and molecular mechanisms have gradually been clarified. This review systematically summarized the functions and mechanisms of m⁶A modification in aquatic animals such as reptiles, amphibians, fish, crustaceans, mollusks, and echinoderms, which aim to reveal the functional conservation and species specificity of this modification during evolution, and to provide potential molecular targets and biomarkers for the growth and development, stress-resistant breeding, and disease control of aquatic animals.

Leuconostoc citreum is a type of heterofermentative lactic acid bacteria that have garnered attention due to their ability to produce a variety of functional substances. The bacteria themselves also possess excellent fermentation and flocculation capabilities. This paper reviewed the current research progress of L. citreum, both domestically and internationally. The key functional substances synthesized, including exopolysaccharides, enzymes, mannitol, and bacteriocins, as well as the applications of the bacteria themselves as the starter culture, microbial flocculant, and probiotic in the fields of food processing and agricultural production were summarized. This study systematically analyzed the current challenges and developmental bottlenecks hindering the industrial application of L. citreum and explores prospective research directions, aiming to provide a theoretical basis for its further development and industrial utilization.

Brassica napus, an economically vital oilseed crop in China, exhibits significant commercial and ornamental value. Phytochrome A (phyA), a core photoreceptor responsible for far-red light perception, plays crucial roles in regulating plant growth and abiotic stress responses. In this study, BnphyA gene family members in B. napus were systematically identified through bioinformatics screening and their expression patterns across tissues and under diverse abiotic stress conditions were analyzed. Fifteen BnphyA members were identified and categorized into 3 subfamilies. Phylogenetic analysis showed that Subfamily Ⅱmembers, including BnaA06G0057900ZS and BnaA09G0661700ZS, exhibited the closest evolutionary relationship with Arabidopsis thaliana AtphyA (AT1G09570). Structural analysis revealed conserved PHY-GAF-PAS_2-HATPase domains across all members. Cis-regulatory element screening identified light-responsive, developmental, and stress-associated motifs in BnphyA promoters. Chromosomal mapping indicated that homologs were predominantly localized near chromosomal termini. Tissue-specific expression profiling revealed upregulated BnphyA expression in roots and seeds, with predominant root-specific responses to cold, heat, drought, and osmotic stresses. Subcellular localization assays indicated cytoplasmic distribution of inactive BnphyA proteins. Heterologous complementation of A. thaliana phyA-211 mutants failed to restore wild-type phenotypes. CRISPR/Cas9-mediated editing generated multiple mutant lines in '19YB437' cultivars, including base substitutions (TGC→CGC) and deletions at the BnaA06G0057900ZS target site. This study provides a theoretical foundation and essential genetic resources for further investigation of the BnphyA gene family's functions.

Growth differentiation factor 11 (GDF11), as a member of the transforming growth factor β (TGF-β) superfamily, primarily participates in cell growth, differentiation, and tissue regeneration, while also playing a crucial role in embryonic development and the maintenance of adult tissues. To elucidate the role of GDF11 in liver diseases and hepatic aging and to address the deficiency of animal models in liver-related GDF11 research, this study employed the Cre recombinase-loxP site system (Cre-loxP) conditional gene knockout strategy to construct a liver-specific growth differentiation factor 11 (GDF11) knockout mouse (Mus musculus) model (C57BL/6J). This model was developed to investigate the regulatory mechanisms of GDF11 in liver aging and overall senescence. Initially, GDF11 gene-edited mice harboring loxP sites GDF11 (flox/+) were crossbred with wild-type C57BL/6J mice to generate a substantial population of GDF11 (flox/+) heterozygous mice. These were subsequently crossed with Albumin-Cre recombinase (Alb-Cre) transgenic mice, which expressed Cre recombinase specifically in the liver, to produce GDF11 (flox/+) Alb-cre (+/-) double transgenic mice. Through intercrossing, a liver-specific GDF11 knockout mouse model was established. Three mice each of GDF11 (flox/flox) Alb-cre (+/-), GDF11 (flox/+) Alb-cre (+/-), and GDF11 (flox/flox) Alb-cre (-/-) genotypes, aged 3~4 weeks, were selected. The efficiency and specificity of gene knockout in the target organ were assessed, and combined RNA and protein level analyses confirmed the successful construction of the liver-specific GDF11 knockout mouse model. GDF11 expression remained unaffected in the heart, spleen, lungs, and kidneys. Quantitative analysis revealed significant differences in GDF11 expression among the groups, and immunohistochemistry and Western blot results indicated a reduction in GDF11 protein levels in liver tissues. The established mouse model provides a robust platform for exploring the physiological and pathological roles of GDF11 in liver aging and systemic senescence.

Alveolar echinococcosis (AE) is a severe zoonotic parasitic disease caused by the larval stage (alveolar hydatid) of Echinococcus multilocularis, which parasitizes the liver of humans and animals. This study aimed to prepare the E. multilocularis Em-AGO2 (Argonaute 2) recombinant protein using prokaryotic expression technology, establish an indirect enzyme-linked immunosorbent assay (ELISA), and preliminarily evaluate its potential for early diagnosis of AE. The N-terminal 1~738 bp fragment of the Em-AGO2 gene was selected to construct the truncated recombinant expression vector pET-28a-Em-AGO2 (1~738 bp). This vector was transformed into an Escherichia coli expression system, and high-level expression was achieved through induction. The Em-AGO2 truncated recombinant protein was then purified by affinity chromatography to obtain a high-purity product. Western blot analysis confirmed that serum samples from E. multilocularis-infected mice specifically recognized the truncated Em-AGO2 recombinant protein, indicating its strong antigenicity. The purified truncated Em-AGO2 recombinant protein was used as the coating antigen. Through stepwise parameter adjustment reaction conditions were optimized: 1 μg/mL of truncated Em-AGO2 recombinant protein was coated onto microplates using 0.05 mol/L (pH 9.6) carbonate coating buffer (CBS) at 4 ℃ overnight; Plates were blocked with 5% skimmed milk at 37 ℃ for 1 h; Test sera (1∶200 diluted in blocking buffer) were added (100 μL/well) and incubated at 37 ℃ for 1 h; Horseradish peroxidase (HRP)-conjugated secondary antibody (1∶10000 diluted in blocking buffer) was added (100 μL/well) and incubated at 37 ℃ for 1 h; after chromogenic substrate development in the dark for 10 min, 50 μL of stop solution was added, and OD₄₅₀ were measured. ROC-curve analysis revealed that the indirect ELISA based on truncated recombinant Em-AGO2 exhibited 100% diagnostic sensitivity and 100% specificity (AUC=1.00) for Echinococcus multilocularis infection in mice. With no cross-reactivity observed against positive serum samples from sheep infected with Echinococcus granulosus, Taenia multiceps, Fasciola hepatica, Toxoplasma gondii, Haemonchus contortus, or rabbits infected with Echinococcus granulosus cysticercus. When applied to detect early-stage E. multilocularis-infected mouse serum samples, the method showed that the following performance: At 7 d post-infection (dpi), the positive detection rates using crude parasite antigen, Em18 recombinant protein, and truncated Em-AGO2 recombinant protein were 18.75%, 75%, and 71.88%, respectively; at 10 dpi, these rates increased to 34.38%, 90.63% and 84.38%; at 15 dpi, they reached 81.25%,100%, and 100%; At 20 dpi, all 3 assays maintained 100% positivity. In summary, this study successfully developed an indirect ELISA detection method for AE based on the truncated Em-AGO2 recombinant protein. This method demonstrates significant potential in the early diagnosis of animal alveolar echinococcosis and holds important application prospects.

The porcine Senecavirus A (SVA) is the sole member of the Senecavirus genus in the family of small RNA viruses. It has been demonstrated that the virus can cause vesicular diseases in pigs (Sus scrofa) and lead to the death of newborn piglets. This poses a threat to the development of the global pig farming industry. The objective of this study was to establish a novel method based on reverse transcription-recombinase polymerase amplification (RT-RAA) and CRISPR/Cas12a techniques for the visual detection of SVA. The methodology involved the design of 3 pairs of RT-RAA primers and guide RNA targeting sequence (crRNAs) according to the conserved sequence of the SVA genome. Furthermore, a single-strand DNA probe (ssDNA) was designed. The optimal reaction concentration ratio of crRNA and LbCas12a was then optimized by orthogonal experiment. Concurrently, the specificity, sensitivity and repeatability of the RT-RAA-CRISPR/Cas12a method were evaluated. Finally, 110 clinical samples with known background were examined. The results showed that the optimal reaction concentrations for crRNA and LbCas12a were 100 and 200 nmol/L, respectively. Utilizing standard RNA (SD-RNA) as the template, the sensitivity of the RT-RAA-CRISPR/Cas12a method was determined to be 15.1 copies/μL. The established RT-RAA-CRISPR/Cas12a method demonstrated no cross-reactivity with Classical swine fever virus (CSFV), Porcine epidemic diarrhea virus (PEDV), Porcine circovirus type 2 (PCV2), Porcine reproductive and respiratory syndrome virus (PRRSV) and Foot-and-mouth disease virus (FMDV). The repeatability test demonstrated that the coefficient of variation within batches was less than 5%, and the coefficient of variation between batches was less than 10%, indicating that the method exhibited excellent repeatability. The clinical sample results were consistent with those of qPCR, exhibiting a 100% coincidence rate. In summary, the study successfully established a visual RT-RAA-CRISPR/Cas12a detection method with strong specificity, high sensitivity and good repeatability, which can be used for early, effective surveillance and epidemiological investigation of SVA.

Carp edema virus (CEV) is a DNA virus popular in recent years, which seriously endangers the healthy development of koi carp (Cyprinus carpiohaematopterus) and carp (Cyprinus carpio) culture. In this study, specific primers were designed and the full genome sequence of CEV P4a was amplified. The pET-32a-P4a recombinant plasmid was constructed using prokaryotic expression system. The recombinant protein P4a was obtained after induction by isopropyl β-D-thiogalactopyranoside (IPTG). After SDS-PAGE identification, purification and renaturation, the His-tagged fusion protein was obtained, and immunized New Zealand white rabbits (Oryctolagus cuniculus) were prepared to prepare polyclonal antibodies. The concentration of the antibody was detected by bicinchoninic acid assay (BCA) method and identified by Western blot. The construction of the pET-32a-P4a prokaryotic expression vector was successfully verified through experimental validation. The NCBI Blast analysis showed that the homology of the P4a vector sequence and the theoretical sequence of P4a protein was 99%. The concentration of the antibody was 2.05 mg/mL by BCA method. SDS-PAGE and Western blot showed that the antibody has good immunogenicity. In summary, the polyclonal antibody against CEV P4a protein was successfully prepared in this experiment, which provides data support for the immunological detection and vaccine development of CEV.