Rice (Oryza sativa) allene oxide cyclase (OsAOC) is an important enzyme in the jasmonic acid (JA) biosynthesis pathway and is involved in development and stress responses. However, the research on the molecular mechanism of OsAOC in rice's disease resistance to rice blast and bacterial blight is rather limited. In this study, the expression levels of OsAOC in wild type 'Nipponbare' (NPB) were induced by the inoculation of Magnaporthe oryzae, methyl jasmonate (MeJA) and salicylic acid (SA) treatments. Then, the OsAOC knockout (OsAOC-KO) and OsAOC overexpression (OsAOC-OE) transgenic rice lines were obtained. Further study showed that OsAOC knockout led to growth defects and even death in rice. This phenotype could be alleviated by spraying MeJA to the seedlings. The plants overexpressing OsAOC showed enhanced resistance to the inoculation of M. oryzae and Xanthomonas oryzae pv. oryzae (Xoo). Under the induction of chitin and bacterial flagellin (flg22), reactive oxygen species (ROS) accumulation in OsAOC-OE was significantly higher than that of NPB (P<0.05). The expression of JA pathway genes (OsAOC and OsJAZ8) and SA pathway genes (OsPR1b and OsPAL1) in OsAOC-OE were up-regulated compared with NPB after inoculation with M. oryzae. Subcellular localization analysis showed that OsAOC protein was localized in chloroplasts. The interactions between OsAOC and OsOPR3 as well as OsbZIP81 were confirmed by yeast two-hybrid, glutathione-S-transferase (GST) pull-down and luciferase complementation assays. This study confirmed the localization and protein interaction of OsAOC in rice. OsAOC was involved in the pattern-triggered immunity (PTI) response of rice and mediated disease resistance response through the JA and SA pathways, positively regulating rice resistance to rice blast and bacterial blight. This study provides genetic resources for the breeding of disease-resistant rice varieties.

Flag leaf width affects rice (Oryza sativa) plant type and photosynthetic efficiency, and is one of the important targets in rice breeding. In the chromosome segment substitution lines constructed by using 'Kolayal' and 'Huazhan' as donor and recurrent parents, it was found that the flag leaves of one certain line were significantly narrower than both parental lines, therefore named after 'narrow Huazhan'. The phenotype of the F₂ segregation population of this line was observed, and the region of the QTL for the flag leaf width was located by map-based cloning and the relative expression of the candidate gene was analyzed. The array analysis of individuals with extreme phenotypes in the F₂ segregation population showed that the QTL for flag leaf width was located on chromosome 6. Through fine mapping and comparative sequencing of critical recombinant individuals, quantitative locus for flag leaf width on chromosome 6 (qFLW6) was finally located in the 1.486 kb interval. As the candidate gene, Os06g0647200 had 2 alternative splicing forms, among which Os06g0647200-2 had a significantly higher expression level in the 'narrow Huazhan' than that in 'Huazhan'. This study identified a novel gene Os06g0647200 involved in regulating flag leaf width in rice, which provides valuable genetic resources for the improvement of flag leaf size and photosynthetic yield in rice.

MicroRNA159 (miR159) is a conserved plant microRNA that regulates anther development and fertility maintenance. Its roles in rice (Oryza sativa) reproductive development are still unclear. In this study, the CRISPR/Cas9 system was employed to generate multi-locus loss-of-function mutants of the OsmiR159 family in rice. These mutants exhibited a distinct phenotype characterized by a failure to transition from vegetative to reproductive growth, resulting in complete absence of flowering and seed set. Integrated transcriptomic and metabolomic analyses revealed that OsmiR159 deficiency disrupts normal rice development by perturbing key biological processes, including hormone signaling, energy metabolism, and cell wall biosynthesis. KEGG pathway enrichment analysis demonstrated that differentially expressed genes and metabolites were significantly enriched in core metabolic pathways, such as starch and sucrose metabolism, phenylpropanoid biosynthesis, and the TCA cycle. Coordinated dysregulation of these pathways led to imbalanced energy supply, aberrant carbon skeleton allocation, and disturbances in secondary metabolism, collectively impairing the developmental phase transition in rice. By integrating multi-omics datasets, we systematically dissected the regulatory interactions underlying the OsmiR159-mediated network in reproductive development, thereby establishing a robust theoretical framework and providing valuable genetic resources for crop improvement.

Milk fat and protein percentages are critical indicators of milk quality in dairy cows (Bos taurus). Acyl-CoA synthetase long chain family member 6 (ACSL6) is a key enzyme involved in fatty acid metabolism and is considered a promising candidate gene influencing milk production traits. This study aimed to investigate single nucleotide polymorphism (SNP) in the ACSL6 gene and their association with milk production traits in Chinese Holstein cattle. A total of 1 152 cows with clear pedigrees and complete production records were genotyped. Ten SNPs within the ACSL6 gene were identified through PCR amplification and Sanger sequencing, and subsequently genotyped using Sequenom mass spectrometry. Associations between SNPs and 5 traits—milk fat percentage, milk protein percentage, milk fat yield, milk protein yield, and 305-day milk yield—were analyzed using a general linear model. The result showed that all SNPs were in Hardy-Weinberg equilibrium (P>0.05) and exhibited moderate polymorphism (0.25<PIC<0.5) in the studied population. Association analysis revealed that the SNP locus g.22501195 C>T significantly influenced milk fat percentage (P<0.05), with the CC and TT genotypes exhibiting higher milk fat percentage. The SNP locus g.22502247 T>C showed significant correlation with milk fat percentage (P<0.05), and individuals with CC genotype demonstrated significantly higher milk fat percentage compared to those with TC and TT genotypes. The SNP locus g.22527204 C>T had significant effects on both milk fat percentage and milk protein percentage (P<0.05), with CC genotype individuals exhibiting elevated levels in both traits. The SNP locus g.22532788 A>T significantly influenced milk protein percentage (P<0.05), and AA genotype individuals demonstrated significantly higher milk protein percentage. These 4 loci formed 2 haplotype blocks (Block1 and Block2): Block1 showed significant association with milk fat percentage (P<0.05), while Block2 exhibited significant association with milk protein yield (P<0.05). The results of this study revealed a potential association between ACSL6 gene polymorphisms and milk production traits in Chinese Holstein cattle, suggesting that this gene could serve as an important candidate gene for screening molecular markers associated with high-yield and high-quality milk. This study provides a theoretical basis for molecular breeding and genetic improvement of high-quality milk traits in dairy cows.

Sterol regulatory element-binding proteins (SREBPs) are key regulators of milk fat synthesis, but the effects of their single nucleotide polymorphisms (SNPs) on lactation traits in dairy sheep (Ovis aries) remain unclear. In this study, 321 lactating Yuansheng Aite dairy sheep were used as experimental subjects. SNPs in the SREBP1 gene were detected by Sanger sequencing and genotyped using the penta-primer amplification refractory mutation system (PARMS). A general linear mixed model (lactation trait=population mean + genotype/allele + parity+age+random effect) was applied to analyze the effects of SNPs on lactation traits. Meanwhile, qRT-PCR was used to detect the expression profile of the SREBP1 gene in 6 tissues, including the mammary gland, of lactating sheep. Four SNPs, namely c.148+4892 G/A, c.148+5262 G/C, c.2439+115 T/C, and c.2923 G/A, were identified in the ovine SREBP1 gene. Among them, c.2923 G/A was a missense mutation, and 3 genotypes were detected at each SNP locus. Correlation analysis revealed that at the c.148+4892 G/A locus, individuals with the GG genotype had a significantly higher daily average milk yield than those with the GA genotype (P<0.05). At the c.148+5262 G/C locus, individuals with the GC genotype showed significantly higher milk fat percentage and dry matter content than those with the GG genotype (P<0.05). At the c.2923 G/A locus, individuals with the GA genotype had significantly higher dry matter content than those with the AA genotype (P<0.05). The qRT-PCR results indicated that the expression level of SREBP1 was the highest in the mammary fat pad, significantly exceeding that in the mammary parenchyma, liver, and other tissues, while its expression was the lowest in perirenal fat (P<0.05). The 3 SNPs screened in this study were shown to affect certain lactation traits in dairy sheep, this study provides a theoretical reference for the molecular breeding of lactation traits in dairy sheep.

The Bactrian camel (Camelus bactrianus) is a typical seasonal breeding animal, under natural conditions, its anestrus period usually coincides with the season of high ambient temperature, during which reproductive function is cyclically suppressed. The purpose of this study was to explore the mechanism of the differentially expressed dual oxidase 2 (DUOX2) gene in testicular heat stress response of the Bactrian camel. The testicular tissues of Bactrian camels in estrus and anestrus were collected. Differentially expressed genes were screened by RNA-seq, and the expression and localization of DUOX2 were verified by qRT-PCR, Western blot, immunohistochemistry (IHC) staining, and ELISA and oxidative stress-related indicators and hormone levels were detected. Testicular Sertoli cells (SCs) were isolated and cultured in vitro, and heat stress model (39 ℃ and 42 ℃) was constructed. The cell model was treated with DUOX2 inhibitor diphenyleneiodonium (DPI). Using cell counting kit-8 (CCK-8) to screen for an appropriate DPI concentration and using flow cytometry to analyze cell apoptosis and cell cycle distribution. A total of 291 differentially expressed mRNA were identified by transcriptome analysis, and the expression of DUOX2 was significantly up-regulated in the anestrus period (P<0.05). KEGG analysis showed that DUOX2 was enriched in oxidative stress and other related pathways. The mRNA transcription and protein level verification showed that the expression level of DUOX2 in testis in anestrus was significantly higher than that in estrus (P<0.05). The hematoxylin-eosin staining (HE) results showed that the seminiferous tubules appeared vacuolization and the spermatogenesis process was stagnant during anestrus. IHC results showed that DUOX2 protein was mainly distributed in the seminiferous epithelium, SCs and interstitial cells. ELISA results showed that the oxidative indexes (H₂O₂ and malondialdehyde (MDA)) were significantly (P<0.05) and extremely significantly (P<0.01) increased, while the levels of antioxidant enzymes (superoxide dismutase (SOD) and catalase (CAT)) and reproductive hormones (testosterone (T) and luteinizing hormone (LH)) were extremely significantly decreased (P<0.01). Cell experiments showed that under heat stress, inhibition of DUOX2 activity could significantly reduce the apoptosis rate of SCs, and the G1 phase block changed to G2/M phase block. The up-regulated expression of DUOX2 in the testis of Bactrian camels during anestrus may aggravate heat stress injury by mediating oxidative stress, and then induce apoptosis and cycle arrest of SCs. Inhibition of DUOX2 activity could alleviate heat stress injury to a certain extent. This study revealed the key role of DUOX2 in heat stress-induced testicular SCs injury in Bactrian camels, and provides a new theoretical basis for elucidating the molecular regulatory mechanism of seasonal reproduction in Bactrian camels.

Exosomes are a type of extracellular vesicle. In the follicular microenvironment, exosomes serve as important mediators of cellular communication and are capable of regulating follicular growth and development through the genes they carry; however, information regarding the genes present in follicular fluid exosomes remains unclear. Therefore, this study aims to elucidate the genetic diversity of follicular fluid exosomes and to analyse the regulatory functions of exosomes in follicular growth and development, ovulation, and atresia, thereby providing a theoretical basis for breeding and reproductive research in domestic pigs (Sus scrofa). This study found that follicular fluid exosomes could promote ovarian proliferation and the secretion of steroid hormones in pigs. Through transcriptomic sequencing, 44 955 SNP/InDel mutations were identified in follicular fluid exosome samples, the majority of which were SNP variants, totalling 43 752. Only 2 430 of these were homozygous SNP and InDel mutations. Analysis of the mutation types in the sample revealed a total of 2.30×10⁴ transitions SNPs and 2.08×10⁴ transversions SNPs, with a ratio of 1.11 between the two, indicating a significant difference. Annotation of mutation locations revealed that SNP and InDel primarily occurred in intergenic and intron regions, followed by exonic regions and the 、3'UTR and 5'UTR, distributed across 1 073 genes. Analysis of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment revealed that genes with mutations in exonic regions were primarily involved in pathways such as cellular oxidative stress, translation, autophagy, and cell cycle regulation. Furthermore, 10 genes associated with the regulation of follicular development were identified, such as β-actin (ACTB), tumour protein p53 (TP53), cathepsin B (CTSB) and lamin B1 (LMNB1). The SNP/InDel data obtained in this study may provide theoretical support for future research into the application of follicular fluid exosomes in swine breeding.

The Yantai Cankang chicken (Gallus gallus domesticus) exhibits significant phenotypic differentiation within this breed, though whether distinct resource populations have formed remains to be determined. In this study, production phenotypes and blood samples were collected from roosters, mottled hens, yellow partridge hens and black partridge hens, for whole-genome resequencing analysis, whole genome data from 101 individuals were obtained and jointly analyzed with genomic data from 3 other local breeds in Jining city. A comprehensive assessment of different subpopulations of Yantai Cankang chicken was conducted at both mitochondrial and whole-genome levels, focusing on genetic diversity, population structure, genetic relationships, and runs of homozygosity (ROH) distribution characteristics. The results indicated significant genetic differentiation between Yantai Cankang chicken and other local breeds. Despite the presence of multiple feather color groups within the breed, no distinct subpopulations were formed in terms of production phenotypes or genomics, characterized by low genetic differentiation among populations, high genetic diversity, and low inbreeding coefficients. Relatively, roosters exhibited comparatively lower inbreeding coefficients. In conclusion, the Yantai Cankang chicken population demonstrated a favorable conservation status, specialized line breeding and industrial development could be appropriately enhanced. This study provides a scientific foundation for the conservation, selective breeding, and industrial development and utilization of Yantai Cankang chicken genetic resources.

Lactic acid bacteria, as a type of probiotic, is widely used in livestock and poultry breeding, and has been proven to improve the growth performance, intestinal health and immune function of animals. Interferon-γ (IFN-γ) is an important immune regulatory factor with great potential in enhancing the body's immune response and disease resistance. In this study, recombinant Lactococcus lactis expressing IFN-γ (rL. lactis-IFN-γ) was constructed, and its effects on the growth performance, intestinal health and immune response of white-feathered broilers (Gallus gallus domesticus) were evaluated, aiming to provide a theoretical basis for the application of engineered lactic acid bacteria in broiler breeding. To explore the effect of rL. lactis-IFN-γ on the growth performance of white-feathered broilers, twenty 'Avian' white-feathered broilers were selected and randomly divided into 2 groups in this study. The basal diet supplemented with rL. lactis-IFN-γ (rL. lactis-IFN-γ group) and PBS (control group) were fed to the broilers respectively for 35 consecutive days. The effects of rL. lactis-IFN-γ on the production performance of broilers were evaluated by measuring the changes of growth performance indexes including feed conversion ratio, gut microbiome indicators and immune function-related indexes. The results showed that compared with the Control group, the body weight gain of white-feathered broilers in the rL. lactis-IFN-γ group was significantly increased (P<0.05), the feed conversion ratio was significantly decreased (P<0.05), and the breast muscle index was significantly increased at the same time (P<0.05). The total intestinal length and villus height to crypt depth ratio of broilers in the rL. lactis-IFN-γ group were significantly increased (P<0.05), and the microbial abundance in the jejunum of the rL. lactis-IFN-γ group was significantly elevated (P<0.05). In addition, the levels of IgG and IgA in the serum of broilers in the rL. lactis-IFN-γ group were significantly increased (P<0.05). In conclusion, dietary supplementation with rL. lactis-IFN-γ could improve intestinal health, promote nutrient absorption, and enhance the immune function of broilers, thereby improving the production performance of broilers. This study provides a theoretical basis for using recombinant engineered lactic acid bacteria as a new green feed additive in broiler chicken farming, and also offers new ideas for developing strategies to reduce antibiotic use in livestock and poultry farming.

Vitamin D₃ (VD₃) has various biological functions. Beyond regulating the absorption and utilization of calcium and phosphorus, it also plays a significant role in the metabolism and overall health of animals. A total of 210 one-day-old male Luhua broilers (Gallus gallus) were randomly assigned to a control (basal diet) and VD₃ group (basal diet supplemented with 3750 IU/kg VD₃), with 7 replicates per group in this study. The 84-day experiment was conducted to investigate the effects of dietary vitamin D₃ supplementation on growth performance, immunity and antioxidant function of Luhua broilers, by determining the relative weight of immune organs, as well as immune factors and antioxidant indexes in serum and adipose tissue. The results showed that dietary VD₃ supplementation significantly increased body weight gain of broilers aged from 1 to 84 d (P<0.05). Supplementary VD₃ significantly increased the serum 25-hydroxyvitamin D₃ (25(OH)D₃) level and upregulated the mRNA expression of vitamin D receptor (VDR), cytochrome P450 family 2 subfamily R member 1 (CYP2R1), cytochrome P450 family 27 subfamily C member 1 (CYP27C1), cytochrome P450 family 24 subfamily A member 1 (CYP24A1), and vitamin D binding protein (VDBP) in the adipose tissue of broilers (P<0.05). Dietary VD₃ significantly decreased serum malondialdehyde (MDA) levels and increased the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) in the serum of broilers at 56 days of age, as well as GSH-Px and CAT activities at 84 days of age; additionally, VD₃ significantly upregulated the mRNA expression of antioxidant-related genes, including nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), NADPH quinone oxidoreductase 1 (NQO1), GSH-Px, and SOD in the adipose tissue of broilers at 56 and 84 days of age (P<0.05). Dietary VD₃ significantly increased the organ indices of thymus and bursa of Fabricius in broilers at 56 and 84 days of age, and serum levels of immunoglobulin A (IgA), IgG, interleukin-2 (IL-2) and interferon-γ (IFN-γ) in broilers at 28, 56 and 84 d; significantly increased levels of IgA, IgG, IL-10 in adipose tissue at all ages, as well as IgM levels at 56 and 84 days of age. It also significantly downregulated the mRNA expression of tumor necrosis factor-α (TNF-α), nuclear factor kappa-B (NF-κB), toll-like receptor-4 (TLR-4) and IL-1β, and upregulated mRNA expression of IL-10 in the adipose tissue (P<0.05). These results indicated that dietary supplementation with 3 750 IU/kg VD₃ could enhance VD₃ metabolism and improve systemic and adipose tissue antioxidant and immune functions, thereby supporting the health and growth of Luhua broilers. The findings of this study provide a basis for a deeper understanding of the nutritional and physiological functions of VD₃ and for optimizing its utilization in broiler production.

Banana Fusarium wilt, caused by Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4), represents a severe threat to global banana (Musa nana) cultivation industry. To develop environmentally sustainable strategies for managing this devastating disease, this study aimed to screen and characterize bacterial biocontrol agents capable of inducing systemic resistance in banana plants. Candidate strains were selected based on their ability to enhance the activity of key defense-related enzymes, including polyphenol oxidase (PPO), peroxidase (POD), and superoxide dismutase (SOD), in banana seedlings. The most promising strain was rigorously identified using integrated taxonomic approaches, encompassing morphological, physiological, and biochemical characterization, as well as phylogenetic analysis of the 16S rRNA gene sequence. Subsequently, its optimal culture conditions were systematically optimized. Antagonistic activity of the selected strain against Foc TR4 was evaluated through dual-culture plate assays and co-cultivation experiments with conidial suspensions to assess inhibition of mycelial growth and spore germination. Its capacity to induce systemic resistance in banana plants was further validated via root drench inoculation under controlled conditions. The results revealed that a single isolate, designated GXMZ-G3, was identified as Bacillus sp. This strain effectively induced systemic resistance in banana plants. In vitro assays demonstrated that GXMZ-G3 exhibited a 62.63% inhibition rate against Foc TR4 mycelial growth and suppressed conidial germination by 85.66%. Microscopic observation revealed severe morphological abnormalities in the inhibited Foc TR4 hyphae, including twisting, knotting, and fragmentation. Following root drench application of the GXMZ-G3 suspension, the activities of key defense-related enzymes (PPO, POD, and SOD) were elevated in banana leaves. Moreover, hydrogen peroxide (H₂O₂) accumulation and callose deposition were markedly enhanced in treated leaves. Jasmonic acid (JA) levels peaked at 0.48 ng/mL at 12 h post-inoculation, representing a 20.00% increase relative to the untreated control. In pot experiments, GXMZ-G3 achieved an 89.48% disease control efficacy against banana Fusarium wilt. In summary, the biocontrol strain GXMZ-G3 exerts a dual mode of action. It directly inhibited Foc TR4 hyphal growth and spore germination, while simultaneously priming systemic resistance in banana plants. These findings highlight GXMZ-G3 as a promising microbial resource for the biological control of banana Fusarium wilt disease.

Animal bioreactors represent an advanced biotechnology platform that uses specific organs of transgenic animals (such as mammary gland, blood, bladder, etc.) as biosynthesis “factories” to produce high value-added bioactive substances. With the continuous growth in demand for recombinant proteins in the biopharmaceutical industry, traditional microbial expression systems and cell culture technologies have gradually encountered development bottlenecks due to limitations in post-translational modification or excessively high production costs. In contrast, animal bioreactors possess the advantages of low production cost, high expression yield, and the ability to achieve complex post-translational modifications, thus becoming an important approach to break through the above technical bottlenecks. From an integrated perspective of cutting-edge technologies including multi-gene co-expression, CRISPR/Cas9 precise gene editing, non-invasive product harvesting, and integrated ethical and safety evaluation, this review systematically reviews the technical principles and application progress of major animal bioreactors such as mammary gland, silkworm (Bombyx mori) silk gland, avian eggs, and bladder; Compares and analyzes the selection strategies of different expression tags. In response to current challenges including low transgenic efficiency, controversies over animal ethics, and imperfect safety evaluation systems, this review proposes improvement strategies based on optimized CRISPR/Cas9 gene editing and non-invasive product collection methods, and prospects future development directions of these technology in the regulation of multi-gene co-expression and large-scale production of biopharmaceuticals, which provides theoretical references for fundamental research, system optimization and industrial development of animal bioreactors for recombinant medicinal proteins.

Gene chips are high-throughput tools that detect specific DNA sequences by leveraging DNA double-strand complementary pairing or DNA-RNA complementary hybridization, enabling efficient genotyping and supporting individual genetic assessment and selective breeding. Currently, mainstream commercial breeding chips primarily utilize SNPs—distributed evenly across the genome—and are widely applied in areas such as the genetic analysis of economically important traits in pigs (Sus scrofa domesticus), genomic selection, marker-assisted breeding, breed identification, and germplasm conservation. These chips have facilitated significant progress in breeding for meat quality, growth, reproduction, and resilience (including disease and stress resistance), driving the transition of pig breeding from traditional phenotypic selection toward precision, large-scale, and high-efficiency operations. Meanwhile, the introduction of novel genetic markers, such as structural variations (SVs), helps address the limitations of conventional SNP chips regarding complex genomic regions and large-scale variation detection, offering new technical avenues for deciphering complex traits. This article reviews the technical principles, classification, and domestic development process of gene chips, systematically elaborating on their application effecacy and key advancements in improving pork quality, enhancing growth performance, optimizing reproductive efficiency, and developing disease-resistant and stress-tolerant pig breeds. It analyzes current application bottlenecks and envisions the prospects of gene chips, through multi-technology integration, empowering the independent and controllable development of the pig breeding industry and promoting its high-quality development. The aim is to provide theoretical reference and practical guidance for the innovative research and industrial application of gene chips in the field of pig breeding in China.

Cancer is a major disease that seriously threatens the health of humans and animals, and its treatment strategies are still under continuous exploration. Anti-tumor nanovaccines represent an emerging immunotherapy following monoclonal antibodies, immune checkpoint inhibitors, and adoptive cell therapy. This approach utilizes nanomaterials as carriers to assemble functional components such as antigens and adjuvants into nano-scale vaccine formulations. By mimicking pathogen characteristics, these vaccines effectively activate the host immune system's surveillance and clearance functions against tumor cells. This article systematically reviews recent advances in anti-tumor nanovaccines from four aspects: nanovaccine materials, tumor antigens, adjuvants, and combination therapies for enhanced efficacy. Based on this foundation, it particularly summarizes current research and applications of nanovaccines for tumors in livestock and poultry, and aims to provide theoretical references for the prevention and control of animal tumors and tumor immunotherapy in agricultural animals.

The commercialization of genetically modified (GM) maize (Zea mays) in China has accelerated significantly, creating an urgent need for rapid field detection technologies as conventional nucleic acid extraction and detection methods are often too complex and time-consuming for efficient screening. To develop an accurate and efficient on-site detection technology to enhance regulatory oversight, this study systematically evaluated 5 cell lysis methods for their compatibility with 3 detection platforms: Conventional PCR, quantitative real-time PCR (qPCR), and recombinase polymerase amplification (RPA), while also investigating the effects of high-temperature incubation on DNA extraction efficiency. Results demonstrated that the alkaline lysis method showed the best cross-platform compatibility, with rapid extraction reagent 1 performing optimally. Although high-temperature treatment significantly increased nucleic acid yield, it simultaneously caused the release of chlorophyll and other fluorescent substances that severely interfered with detection accuracy. Based on these findings, an integrated portable detection device combining quartz sand mechanical lysis, membrane filtration, and isothermal amplification technologies was developed. This system achieved complete sample-to-result detection within 25 minutes, representing a 6-fold efficiency improvement over conventional methods. Field validation confirmed 100% detection specificity for both DBN9936 GM maize event and Ruifeng125 GM maize event, with effective discrimination from non-GM crops and other transgenic varieties. In conclusion, this study successfully identified rapid extraction reagent 1 as a versatile extraction method compatible with multiple detection platforms, and innovatively developed a 'sample-to-result' on-site detection system that provides a rapid (<30 minutes), efficient solution for GM crop regulation.

Angiotensin-converting enzyme 2 (ACE2) serves as a negative regulator of the renin-angiotensin system (RAS) and also acts as a key functional receptor for multiple coronaviruses, including Severe acute respiratory syndrome coronavirus (SARS-CoV), facilitating viral entry into host cells. To explore the relationship between chicken (Gallus gallus domesticus) ACE2 protein and avian coronaviruses including Infectious bronchitis virus (IBV), this study amplified the complete coding sequence of the chicken ACE2 gene by RT-PCR and cloned it into the pcDNA3.1(+) eukaryotic expression vector via homologous recombination. After verification by colony PCR, restriction enzyme digestion, and nucleotide sequencing, the recombinant plasmid was transfected into Chinese hamster ovary (CHO) cells using liposome-mediated transfection. Stable cell lines constitutively expressing chicken ACE2 were then established through geneticin (G418) selection combined with monoclonal expansion. The expression of ACE2 in CHO cells was assessed by RT-PCR, Western blot, and immunofluorescence staining, while the enzymatic activity of the recombinant ACE2 protein was quantitatively measured using a double-antibody sandwich assay. The results demonstrated that the recombinant eukaryotic plasmid pcDNA3.1(+)-ACE2 was successfully constructed in vitro, yielding a single restriction fragment of approximately 7 872 bp, and sequencing data matched the predicted sequence perfectly. After efficient transfection into CHO cells, RT-PCR confirmed abundant transcription of the target gene, immunofluorescence revealed uniform distribution of the recombinant protein on the cell membrane with high transfection efficiency, and Western blot further confirmed robust expression of the recombinant protein with an apparent molecular weight of about 105 kD. Following G418 selection and monoclonal culture, 4 stable cell lines were obtained, designated pcDNA3.1(+)-ACE2-A/B/C/D. The double-antibody sandwich assay showed that the ACE2 enzymatic activity reached 601.77 U/L, indicating high catalytic activity. In summary, this study successfully established a eukaryotic expression system for chicken ACE2 and generated stable cell lines producing the recombinant protein, thereby providing a solid foundation for further investigations into the biological functions of chicken ACE2, particularly its involvement with coronaviruses.

African swine fever (ASF) is a highly lethal infectious disease caused by African swine fever virus (ASFV), inflicting devastating losses on the global pig industry. Currently, no safe and effective commercial vaccines are available, and elucidating the functions of viral key proteins serves as the core prerequisite for vaccine development and antiviral strategy design. Traditional research on ASFV protein functions heavily relies on specific antibodies, which is limited by long preparation cycles and cumbersome procedures. C129R is a structural protein encoded by the central region of ASFV, while its precise localization in viral particles, dynamic distribution and host interaction mechanisms remain unclear. To investigate the functions of C129R during viral infection and pathogenesis, in this study, a recombinant ASFV strain with a C-terminal 3×Flag-Strep double tag fused to the structural protein C129R was constructed. Using homologous recombination technology, the gene sequences encoding the 3×Flag-Strep double tag and GFP selection marker were inserted into the C-terminus of the C129R gene of ASFV CN/GS/2018 strain, and the recombinant virus ASFV-C129R-3×Flag-Strep was obtained through clonal screening and purification. Western blot analysis confirmed that the recombinant virus correctly expressed the C129R fusion protein carrying the double tag. qPCR revealed that tag insertion had no significant effect on the transcription levels of C129R and its upstream and downstream genes M448R and C84L. One-step growth curve, hemadsorption assay and transmission electron microscopy showed no significant differences in in vitro replication kinetics, morphological structure and viral assembly between the recombinant virus and the parental strain.This study successfully constructed a double-tagged recombinant ASFV targeting C129R with stable biological characteristics, providing a technical reference for further analyzing the precise localization and structural features of C129R in viral particles, as well as its interaction mechanism with host proteins.