Jasmonic acid, as an important endogenous signaling molecule, determines sex differentiation by inducing programmed death of pistil primordia during maize (Zea mays) flower development. However, its precise regulatory network is still unclear. In this study, the key genes TASSEL SEED 1 (TS1) and SILKLESS 1 (SK1) that regulate sex differentiation by jasmonic acid were used as targets to obtain functional deletion mutants by gene editing, and phenotypic and transcriptome analyses were performed. The results showed that the ts1 mutant showed feminization of male flowers, and the sk1 mutant showed loss of female filaments. Transcriptome analysis showed that ts1 mutation downregulated the expression of genes involved in the jasmonic acid signaling pathway, thereby promoting pistil development. The sk1 mutation might enhance the jasmonic acid signaling pathway and inhibit pistil formation. Integrative analysis based on published single-cell RNA-seq data revealed that 93 key genes were activated in the ts1 mutant during the critical period of floral organformation and sex determination (floret meristem-stage 1 (FM-S1) to FM-S3), which may be involved in the pistil inhibition process. GO enrichment analysis revealed pathways related to floral organ development and identified potential downstream target genes of jasmonic acid regulating sex differentiation. This study provides a theoretical basis for further analysis of the molecular mechanism of jasmonic acid regulating sex differentiation in maize.

Drought stress is a major limiting factor for stable and high wheat (Triticum aestivum) production. Under drought conditions, appropriate deep sowing enables seeds to access deeper soil moisture, thereby improving seedling emergence rate. Coleoptile length determines the maximum sowing depth of wheat; therefore, mining elite genetic resources controlling coleoptile length and developing available molecular markers are of great significance for genetic improvement of deep-sowing tolerance and drought resistance. In this study, a recombinant inbred line (RIL) population derived from 'Gaocheng 8901' and 'Zhoumai 16' was used to evaluate coleoptile length. Genotyping was performed using the wheat 90K SNP array. A total of 4 QTL associated with coleoptile length were detected on chromosomes 1AL, 2BS, 3BS and 7DL through genome-wide linkage analysis, designated as QCL.zaas-1AL, QCL.zaas-2BS, QCL.zaas-3BS and QCL.zaas-7DL, with phenotypic variation explained (PVE) ranging from 7.12% to 7.89%. The favorable alleles of QCL.zaas-1AL, QCL.zaas-2BS and QCL.zaas-3BS were derived from 'Zhoumai 16', whereas those of QCL.zaas-7DL were from 'Gaocheng 8901'. Among these, QCL.zaas-1AL, QCL.zaas-2BS and QCL.zaas-7DL were newly reported loci, while QCL.zaas-3BS was located in close proximity to previously reported QTL. Eleven candidate genes were further identified, involving functional categories such as cytoskeletal formation, auxin homeostasis and polar transport, transcriptional regulation, energy metabolism, membrane lipid composition, and light signal perception. Moreover, 4 kompetitive allele specific PCR (KASP) markers were developed and validated in a natural panel of 130 wheat varieties. Among them, Kasp-CL-1AL and Kasp-CL-2BS exhibited significant allelic effects (P<0.05) in the natural population, serving as candidate tools for marker-assisted selection of coleoptile length. This study provides novel genetic loci and available KASP markers for genetic improvement of coleoptile length and breeding for deep-sowing tolerance and drought resistance in wheat.

Peanut (Arachis hypogaea) is an important oilseed and cash crop that is frequently affected by abiotic stresses such as drought and low temperature during production. To clarify the member composition, structural characteristics, and expression divergence of the lysin motif receptor-like kinase (LYK) gene family in allotetraploid cultivated peanut, genome-wide identification of LYK members was performed using the reference genome of the peanut cultivar 'Fuhuasheng'. Phylogenetic relationships, chromosomal locations, gene structures, conserved motifs, and promoter cis-acting regulatory elements were subsequently analyzed. Publicly available transcriptome data were further used to investigate tissue-specific expression and expression responses to phytohormones, drought, and low temperature, and the low-temperature responses of selected candidate genes were validated by RT-qPCR. The research results showed that a total of 20 LYK genes were identified and classified into 3 subgroups (Ⅰ, Ⅱ and Ⅲ) according to their phylogenetic relationships. These genes were unevenly distributed across the peanut chromosomes, and approximately half were intronless, suggesting their potential for rapid responses. Various light-responsive, hormone-responsive and stress-responsive cis-acting elements were widely distributed in their promoter regions, particularly those associated with abscisic acid and methyl jasmonate responses. Analysis of public transcriptome data showed that several LYK genes exhibited tissue-specific expression patterns. LYK8 was highly expressed in roots and root-related tissues, whereas LYK10 was expressed in roots, root-stem composite tissues, stems, and cotyledons, suggesting their possible involvement in root development or growth regulation in different tissues. Different LYK members exhibited distinct expression responses to phytohormones, drought, and low temperature. In particular, LYK2 showed a marked response to drought, whereas LYK3 was upregulated under multiple phytohormone and low-temperature conditions, indicating possible functional divergence among LYK members in hormone signaling and abiotic stress responses. Overall, the cultivated peanut LYK gene family shows marked divergence in gene structure, regulatory element composition, and expression patterns, and some members may participate in tissue development and abiotic stress responses. This study provides a reference for subsequent functional validation of peanut LYK genes and the identification of candidate genes for stress resistance.

Powdery mildew is one of the most devastating fungal diseases affecting melon (Cucumis melo) production. The identification of elite resistance sources and the elucidation of their underlying resistance genes are of great practical significance for breeding melon cultivars with improved powdery mildew resistance. This study aimed to elucidate the genetic mechanism underlying powdery mildew resistance and to identify resistance genes in the melon inbred line 19PMR exhibiting immunity to powdery mildew, thereby providing a theoretical foundation and potential gene resources for breeding powdery mildew-resistant varieties. In this study, a segregating population was constructed using 19PMR and the highly susceptible inbred line 19PMS as parents. The powdery mildew disease assay by artificial inoculation with Podosphaera xanthii was conducted for genetic analysis. The candidate gene was preliminary mapped using bulked segregant analysis sequencing (BSA-seq) strategy, and specific molecular markers were developed based on sequence polymorphisms. The results showed that the resistant parent 19PMR and the F₁ plants exhibited immunity to powdery mildew, and the segregation ratio of resistant to susceptible individuals of F₂ population among 292 F₂ plants was 3∶1, indicating that the resistance in 19PMR was controlled by a single dominant gene, tentatively named Pm2R. Subsequently, the Pm2R gene was located within the 0~1.66 Mb interval at the distal end of the short arm of chromosome 2 using the BSA-seq method. This interval contained 2 tandem genes, MELO.jh002936 and MELO3C015354, which encoded nucleotide-binding site and leucine-rich repeat (NBS-LRR) proteins. Further sequence analysis revealed a fragment deletion in the MELO3C015354 gene region of the susceptible parent 19PMS, while multiple sequence variations in MELO.jh002936 gene were identified between the 2 parents. Based on a 10-bp sequence variation in the promoter region of MELO.jh002936, an insertion/deletion (InDel) marker ID1152 was developed, which co-segregated with the resistance phenotype in 292 F₂ individuals. In summary, this study demonstrated that the powdery mildew resistance of melon 19PMR was controlled by a single dominant gene, Pm2R, located on chromosome 2 within the 0~1.66 Mb interval. Furthermore, MELO.jh002936 and MELO3C015354 were identified as potential candidate genes for powdery mildew resistance, and the molecular marker ID1152 is expected to be applicable for marker-assisted selection (MAS) breeding programs for powdery mildew resistance in melon.

MYB transcription factors constitute one of the largest transcription factor families in plants and are extensively involved in various biological processes, including growth and development, secondary metabolism, and stress responses. Opisthopappus longilobus, a wild species closely related to chrysanthemum and endemic to China, has long thrived in arid, nutrient-poor cliff habitats characterized by substantial water fluctuations, exhibiting strong environmental adaptability. In this study, the OlMYB35 gene was cloned from O. longilobus. Sequence analysis revealed that the ORF was 888 bp in length, encoding a protein of 295 amino acids with a predicted molecular mass of approximately 33.12 kD and a theoretical isoelectric point (pI) of 8.59. OlMYB35 belonged to the R2R3-MYB transcription factor family. Secondary structure prediction indicated that the protein was mainly composed of random coils, α-helices, and extended strands. Phylogenetic analysis showed that OlMYB35 was most closely related to homologous proteins from Chrysanthemum species and was evolutionarily conserved. A p35S::OlMYB35-GFP fusion expression vector was constructed, and subcellular localization analysis revealed that the protein was localized in both the nucleus and the cytoplasm. Yeast transcriptional activation assays demonstrated that OlMYB35 possessed transcriptional activation activity. Expression pattern analysis under water stress conditions showed that the OlMYB35 gene was rapidly induced by polyethylene glycol 6000 (PEG6000)-simulated drought stress and significantly upregulated under waterlogging stress, suggesting its potential involvement in regulating the response of O. longilobus to water stress. This study provides a theoretical basis for further functional characterization of the transcription factor OlMYB35 in O. longilobus and for the application of chrysanthemum molecular breeding for stress tolerance.

The KNOX transcription factor family is an important class of plant homeodomain proteins that play key regulatory roles in plant growth, development, and stress responses. This study aimed to identify and characterize the KNOX gene family members in Polygala tenuifolia, and to explore their tissue-specific expression patterns as well as their response characteristics to biotic and abiotic stresses. Using bioinformatics approaches, KNOX family genes were screened and identified based on the whole-genome sequence of P. tenuifolia, and their physicochemical properties, gene structures, conserved motifs, and promoter cis-regulatory elements were analyzed. Synteny analyses were performed both intra- and inter specifically. A phylogenetic tree was constructed using MEGA 7.0 software, and the expression profiles of KNOX genes in different tissues (roots, stems, and leaves) as well as under salt and salicylic acid (SA) stresses were examined by qPCR. The results showed that a total of 16 KNOX gene family members were identified in P. tenuifolia, distributed across 12 chromosomes, with 13 pairs of PtKNOX genes exhibiting syntenic relationships. Phylogenetic analysis classified the KNOX genes into 2 major groups: ClassI and ClassⅡ. Tissue-specific expression analysis revealed that different types of KNOX genes exhibited distinct expression patterns across various tissues and organs. qPCR results demonstrated that both salt stress and SA treatment significantly upregulated the expression levels of Pt01c1792.1 and Pt17c1863.1 (P<0.05). This study provides a theoretical basis for further investigations into the biological functions of PtKNOX genes in regulating growth, development, and secondary metabolite biosynthesis in P. tenuifolia.

Membrane transporters, such as aluminium-activated malate transporter (ALMTs) and multidrug and toxic compound extrusion (MATE), are encoded by key genes that confer plants resistance to aluminiumtoxicity. In this study, based on the full-length transcriptome data of Cunninghamia lanceolata, a total of 8 ALMT genes and 44 MATE genes were identified, which were classified into 3 and 4 subfamilies, respectively. Genes within the same subfamily shared similar conserved motifs and domains. Cis-acting element analysis revealed that the promoters of ALMT and MATE genes in C. lanceolata contained abundant hormone-responsive and stress-responsive elements. Further expression analysis indicated that some ALMT and MATE genes of C. lanceolata exhibited certain expression specificity in different tissues and organs. The expression levels of ClALMT3, ClALMT5, ClMATE15 and ClMATE34 were significantly induced by aluminium stress, suggesting their potential involvement in aluminiumtoxicity resistance of C. lanceolata. These results provide theoretical basis for further studies on the functions of ALMT and MATE genes in the aluminiumstress resistance process of C. lanceolata.

The 14-3-3 protein family, constitutes a class of highly conserved regulatory proteins in plants, also known as G?box regulatory factors (GRFs), are extensively involved in various physiological processes such as cellular metabolism, signal transduction, and responses to biotic and abiotic stresses. Tamarix chinensis is a typical recretohalophyte, but the function and regulatory mechanism of its 14-3-3 family genes in response to salt stress remains unclear. To investigate the characteristics of the 14-3-3 gene family in T. chinensis and their roles in salt tolerance, the study cloned, identified, and functionally analyzed 14-3-3 family genes. The results showed that 5 14-3-3 genes, named TcGRF1 to TcGRF5, respectively, were successfully cloned, and all TcGRF proteins contained the typical conserved 14-3-3 domain. qPCR analysis revealed that the expression levels of all 5 TcGRFs genes were up-regulated to varying degrees under 0.2 and 0.4 mol/L NaCl stress, with TcGRF3 showing the most obvious difference. To further clarify the function of TcGRF3 under salt stress, transient transformation technology was used to overexpress and suppress the expression of TcGRF3 in Tamarix chinensis, respectively. Overexpression of TcGRF3 significantly increased chlorophyll content, the activities of superoxide dismutase (SOD) and peroxidase (POD)(P<0.05), and proline accumulation, while decreasing relative electrical conductivity and malondialdehyde (MDA) content (P<0.05). In contrast, TcGRF3-silenced plants showed the opposite trends. Comprehensive analysis indicated that TcGRF3 positively regulated salt tolerance in T. chinensis by enhancing reactive oxygen species (ROS) scavenging capacity, thereby mitigating membrane lipid peroxidation damage. This study provides a theoretical foundation for further elucidating the functional mechanisms of the 14-3-3 protein family in the salt stress response of T. chinensis.

Superovulation represents a critical component of embryo transfer, however, repeated use of the superovulation protocols currently available on the market has the potential to result in a decline in the ovarian reserve of donor yaks (Bos grunniens). The objective of this study was to enhance the economic efficiency of superovulation in donor yak. To this end, the donor yak were randomly divided into 2 groups for a 9-day treatment period. For the initial 7-day period, both groups received the same treatment, after which they were given an ovulation induction hormone injection on day 7. The experimental groups were subjected to different protocols: one group was induced to ovulate with prostaglandin (PG), while the other group was induced to ovulate with gonadotropin-releasing hormone (GnRH). Concurrently, serum concentration of anti-Müllerian hormone (AMH) were measured using an ELISA Kit on day 7, 45 minutes post-injection, and 36 h post-injection. Serum concentration of progesterone (P4) were measured on days 0, 3, 7, and 36 h post-injection on day 7. Furthermore, a protocol (FSH-PG) that was available for commercial use was utilised as the control group. Blood samples were collected 36 h after drug injection on day 7 for combined whole-blood transcriptomics and plasma metabolomics analysis to elucidate the mechanism by which the new experimental protocol restores normal ovarian function. The results demonstrated that the novel protocol restored serum AMH concentrations to physiological levels in yaks and elevated post-ovulatory progesterone concentrations. Integrative transcriptomic and metabolomic analysis revealed enrichment primarily in the cAMP signalling pathway and phosphoinositide 3-kinase-protein kinase B (PI3K-Akt) signalling pathway.This study successfully demonstrated that GnRH-induced ovulation offered advantages over PG-induced ovulation in maintaining ovarian endocrine homeostasis during superovulation. Furthermore, a combined analysis was performed to investigate the molecular mechanisms underlying the maintenance of ovarian endocrine homeostasis. This analysis successfully identified 17 target genes and 2 metabolites that contributed to the maintenance of endocrine homeostasis in the ovaries of donor yaks following superovulation. This study has significant practical value for the conservation and industrial-scale breeding of yaks at high altitudes.

Spring shedding, as a key factor affecting meat sheep production efficiency and animal welfare, requires research into its regulatory mechanisms to develop novel heat-tolerant, easy-to-manage breeds. This study systematically analyzed the structural characteristics of skin hair follicles and the expression patterns of key regulatory proteins—B cell lymphoma-2 (Bcl-2) and glutamate-cysteine ligase catalytic subunit (GCLC) in different phases (rapid growth, early growth, and resting phase) of Dorper sheep (Ovis aries), a breed with a stable genetic shedding phenotype. This study also examined the expression patterns of Bcl-2 and GCLC in skin hair follicles during different growth phases. Hair follicle morphology exhibited distinct cyclical changes across the 3 growth phases. It was found that hair follicle morphology exhibited obvious periodic changes. Compared with the telogen phase, the depth of secondary hair follicles, hair bulb diameter and hair shaft diameter increased significantly in the anagen phase (P<0.05). Data-independent acquisition (DIA) proteomics and Western blot analysis consistently showed that B cell lymphoma-2 (Bcl-2) and glutamate-cysteine ligase catalytic subunit (GCLC) expression peaked during the active growth phase, followed by the telogen phase, and was the lowest during the early growth phase. Immunohistochemistry (IHC) further revealed that Bcl-2 and GCLC were primarily localized in the hair matrix, outer root sheath, and epidermal layer and exhibited distinct cycle-dependent patterns. Bcl-2 showed high expression in the hair matrix and outer root sheath during the active growth phase, whereas GCLC expression also increased during this phase and decreased during the resting phase (P<0.05). In summary, this study characterized the cyclical structural changes in Dorper sheep hair follicles and the expression and spatial distribution patterns of Bcl-2 and GCLC. These findings provide experimental evidence for deciphering the molecular mechanisms underlying seasonal molting and contribute to advancing molecular breeding for developing highly adaptive new sheep breeds.

Coenzyme Q10 (CoQ10) is an endogenous liposoluble antioxidant that exerts protective effects on the male reproductive system. To investigate the effects of dietary CoQ10 supplementation on semen quality and spermatogenic function in male rabbits (Oryctolagus cuniculus), 50 adult male Minxinan Black rabbits were used in this study. Different concentrations (0, 30, 60, 120, 240 mg/kg diet) of CoQ10 were added to their daily diet. Parameters assessed included semen quality, hormone levels, testicular histomorphology, antioxidant capacity, and the mRNA expression levels of functional genes (c-KIT, GFRα1, PLZF, Bax, Bcl2, and Caspase-3), as well as the protein expression levels of p-Akt and Akt. The results showed that, compared with the control group, CoQ10 supplementation led to an increasing trend in sperm motility and motion parameters. Specifically in the 120 mg/kg group, ejaculate volume, sperm concentration, total motility, progressive motility, straight-line velocity, and average path velocity significantly increased (P<0.05). The transverse and longitudinal diameters of the left testes were also significantly increased (P<0.05), while the testicular index and the transverse and longitudinal diameters of the right testis showed no significant changes. In terms of histomorphology, the number of seminiferous tubules and the thickness of the seminiferous epithelium were significantly increased (P<0.05), whereas the area of seminiferous tubule tissue, tubule diameter, and spermatogonial density showed no significant changes. With increasing CoQ10 supplementation, FSH and LH levels gradually decreased, while inhibin B levels gradually increased, with significant differences among all groups (P<0.05). Regarding immune parameters, the levels of IgA and IgM were significantly increased in the 240 mg/kg supplementation group (P<0.05), while IgG levels showed no significant difference among groups. In terms of antioxidant capacity, the levels of SOD, GSH-Px, T-AOC, and CAT were significantly increased at 120 and 240 mg/kg CoQ10 supplementation (P<0.05), while MDA content was significantly decreased at 240 mg/kg (P<0.05). Regarding gene expression, Bax expression was significantly reduced (P<0.05), while Bcl2 expression and the Bcl2/Bax ratio were significantly increased (P<0.05). The expression levels of GFRα1 and PLZF showed no significant changes among groups, whereas c-KIT expression was significantly lower in the 240 mg/kg supplementation group compared with the control group (P<0.05). The protein expression level of p-Akt was significantly increased (P<0.05). In conclusion, dietary supplementation with 120 mg/kg CoQ10 enhanced the antioxidant capacity of the testes, improved histomorphology and spermatogenic function, optimized hormone levels, and reduced apoptosis in male Minxinan Black rabbits, thereby improving sperm motility and motion parameters. This study not only provides an effective nutritional intervention strategy to alleviate semen quality issues in local rabbit breeds resulting from long-term lack of selective breeding but also offers theoretical basis and practical reference for the conservation of reproductive capacity and germplasm improvement of other local livestock and poultry genetic resources.

Body shape is an important economical trait for germplasm evaluation and aquaculture in common carp (Cyprinus carpio). Different populations of red common carp have developed distinct morphological differentiation during long-term domestication and selective breeding, yet the underlying genetic basis remains insufficiently understood. Transposable element insertion polymorphism (TIP), an important form of genomic structural variation, may contribute to phenotypic differentiation by affecting the structure and expression of adjacent genes. In this study, 4 populations of red common carp, namely Xingguo red common carp (C. carpio var. xingguonensis), Hebao red common carp (C. carpio var. wuyuanensis), Wanan glass red common carp (C. carpio var. wananensis) and 'whole-red' strain of Oujiang color common carp (C. carpio var. color), were used to investigate potential genetic factors associated with body-shape differences and to evaluate the potential application of TIP in population discrimination and trait association studies, by using multivariate analyses of morphological traits and genome-wide comparisons of TIP. The results based on the 24 morphometric ratio traits showed that Hebao red common carp exhibited significantly higher body depth than the other 3 carps (P<0.05) and consistently formed a stable and independent cluster in both hierarchical clustering and principal component analyses, displaying obvious morphological 'specialization' characteristics. Genome-wide analysis of TIP demonstrated a highly conserved transposable element (TE) family composition among the 4 populations of red common carp, which were dominated by the hAT, Gypsy, and CACTA superfamilies. Meanwhile, the similar chromosomal distribution patterns of TIP were found among these carps. Principal component analysis, phylogenetic reconstruction, and STRUCTURE clustering based on TIP loci consistently revealed stable genetic differentiation among populations in TE insertion patterns, which was further corroborated by analyses of nucleotide diversity (π) and population differentiation (F_ST). Population-specific TIP analysis showed that the highest number of unique TE insertion loci was found in Wanan glass red common carp, but the lowest in Xingguo red common carp. By integrating morphology-associated TIP screening with selective sweep analysis, several candidate genes potentially associated with body-depth variation were detected in Hebao red common carp, including wdr11, rsph14, sox6, and smyd2b. This study provides a reference for genetic association studies on body shape-related traits in common carp and candidate gene verification in the future.

Sarco/endoplasmic reticulum Ca²?-ATPase (Atp2a) is a key transmembrane protein regulating muscle contraction and calcium homeostasis. In this study, 6 members of the Atp2a gene family (Afatp2a1-Afatp2a6) were identified in the genome of Acrossocheilus fasciatus, distributed across 6 chromosomes and divided into 3 subfamilies. Bioinformatics analysis revealed that all gene family members contain characteristic domains of P-type ATPases and 10 conserved motifs, with exon numbers ranging from 18 to 22. The proteins were acidic, weakly hydrophobic, and highly thermostable. Subcellular localization prediction indicated that Afatp2a1, Afatp2a4 and Afatp2a6 were localized to the endoplasmic reticulum, while Afatp2a2, Afatp2a3 and Afatp2a5 were localized to the cell membrane. Phylogenetic and collinearity analysis revealed high evolutionary conservation of this family in teleost fish. Tissue expression profiling analysis showed significant specificity: Afatp2a6 was widely and highly expressed in non-muscle tissues, Afatp2a1 was highly expressed in the brain, Afatp2a2 was enriched in the skin, Afatp2a3 was highly expressed in the gills, Afatp2a4 was highly expressed in the heart and skin, and Afatp2a5 was preferentially expressed in muscle and skin. Flow velocity stress experiments showed with increasing flow velocity, the relative expression level of Afatp2a2 decreased, whereas that of Afatp2a5 showed an increasing trend. Specifically, Afatp2a2 expression was significantly lower in the 2 and 4 BL/s groups than in the 0 BL/s group (P<0.0001), while Afatp2a5 expression was significantly higher in the 4 BL/s group than in the 0 BL/s group (P<0.001), presenting a synergistic "down-up" response pattern. These findings suggested that under long-term acclimation, A. fasciatus might maintain calcium homeostasis through functional differentiation of Atp2a gene family members and post-transcriptional regulation, rather than relying solely on transcriptional level changes. This study provides a theoretical reference for elucidating the roles of Atp2a gene family members in the response of A. fasciatus to flow velocity stress.

Coxiella burnetii, the sole species of the genus Coxiella, is a microorganism with characteristics intermediate between viruses and bacteria. It can infect a wide range of hosts via multiple transmission routes and is the pathogen responsible for worldwide zoonotic Q fever (query fever). This study aimed to express recombinant outer membrane protein 34 (Omp34) from C. burnetii using a prokaryotic expression system, followed by purification to prepare rabbit polyclonal antibodies, which provided biological materials for establishing C. burnetii-specific serological detection methods and developing recombinant vaccines. The Omp34 gene was amplified from clinically positive samples using PCR. Bioinformatics analysis predicted the physicochemical properties and structural characteristics of the Omp34 protein. The recombinant plasmid pET-32a-Omp34 was constructed and transformed into Escherichia coli BL21 (DE3) competent cells. Following induction with isopropyl β-D-1-thiogalactopyranoside (IPTG), Omp34 protein was purified using Ni-IDA agarose column gravity purification and identified via SDS-PAGE and Western blot. Polyclonal antibodies were generated by immunizing healthy rabbits (Oryctolagus cuniculus) with Omp34 protein, and antibody titers were measured using indirect ELISA. A 918 bp fragment of the Omp34 gene from C. burnetii was obtained. Bioinformatics analysis revealed that this gene encoded 285 amino acids, with a protein molecular weight of approximately 32 kD, a theoretical isoelectric point of 5.67, an instability index of 48.96, an aliphatic index of 78.81, and a hydrophilicity of -0.869. No transmembrane domains or signal peptides were detected. The secondary structure of the Omp34 protein comprised α-helices (57.54%), extended chains (0.70%), and disordered coils (41.75%). Additionally, the protein contained 55 phosphorylation sites: 24 serine (Ser) phosphorylation sites, 23 threonine (Thr) phosphorylation sites, and 8 tyrosine (Tyr) phosphorylation sites. The molecular weight of the expressed recombinant protein matched the predicted 51.31 kD. After purification, the polyclonal antibody prepared from immunized rabbits (Oryctolagus cuniculus) reached a titer of 1∶12 800. This study provides a reference for exploring the biological functions of Omp34 and supplies critical materials for developing serological diagnostic methods for C. burnetii.

Classical swine fever (CSF) is an acute, febrile and contact infectious disease of swine caused by Classical swine fever virus (CSFV), which belongs to the genus Pestivirus within the family Flaviviridae. The E2 protein is an envelope glycoprotein on the surface of CSFV and also the major protective antigen that induces the production of neutralizing antibodies in the host. Based on previous research, to reduce the downstream processing cost of rice (Oryza sativa)-derived E2 vaccine and improve vaccine stability, this study optimized the purification conditions of rice-derived E2 protein and evaluated the stability of the vaccine under different conditions. According to the physicochemical properties of E2 protein and rice endosperm proteins, the purification conditions of E2 protein were optimized by screening anion and cation chromatography resins. Purified E2 protein (10 μg) was then used to prepare vaccines under different formulations and storage conditions to immunize 5~7 week old female BALB/c mice (Mus musculus). Antibody blocking values in mice were detected using an IDEXX antibody blocking kit, antibody neutralizing titers were determined by microneutralization assay, and the levels of IL-2, IFN-γ, IL-4 and IL-10 cytokines were measured by ELISA. The results showed that the purity of E2 protein reached approximately 80% after purification with two cation-exchange resins, High-S and Sulfate-650F. After immunization, the antibody blocking values of all groups were ≥40% at 28 days post-immunization except for the PBS control group. At 35 days post-immunization, the antibody blocking values of Group 6 (buffer: PB; adjuvant: 50V; temperature: 4 ℃; formaldehyde concentration: 0.1%), Group 10 (buffer: Tris; adjuvant: 201VG; temperature: 4 ℃; formaldehyde concentration: 0.05%), Group 12 (buffer: Tris; adjuvant: 50V; temperature: 4 ℃; formaldehyde concentration: 0.05%) and the inactivated vaccine group were ≥80%. The neutralizing antibody titer of Group 6 reached 2?, while those of Group 12 and the inactivated vaccine group were 2⁷. The levels of IL-2, IL-4 and IL-10 in Group 6, Group 12 and the inactivated vaccine group peaked at 35 days post-immunization. The content of IFN-γ remained relatively high in Groups 4, 6, 7, 8, 9, 10, 12 and the inactivated vaccine group. This study optimized the purification method of E2 protein and screened the stability conditions of the vaccine, providing basic data and references for subsequent research and development of rice-derived E2 vaccine.

Accurate diagnosis of Feline calicivirus (FCV) infection relies on specific antibody tools, and single-chain variable fragments (scFvs) show important application potential in clinical practice. In this study, the FCV VP1-CDE protein expressed and purified through prokaryotic expression was used as the immunogen to immunize chickens (Gallus gallus domesticus). A phage-displayed scFv library was constructed and positive clones were obtained by solid-phase panning. Eukaryotic expression vectors were constructed and expressed in human embryonic kidney 293F cells, followed by purification. The binding activities were verified by Western blot, indirect immunofluorescence assay (IFA), and bio-layer interferometry (BLI). The results showed that soluble VP1-CDE protein was successful obtained. The titer of chicken serum was higher than 1∶64 000 after 5 immunizations. A phage-display library with a capacity of 5.25×10⁷ CFU/mL and good diversity was constructed. Thirty-five recombinant phage cloned with strong reactivity were obtained after panning, and 5 unique scFv sequences were acquired. Western blot analysis demonstrated that 5 clones (X1A2, X2B2, X2C1, X2E1 and X2G5) could specifically bind to the VP1-CDE protein. IFA results indicated that 3 of them (X2B2, X2E1 and X2G5) exhibited good binding activity to intact viral particles, which was further confirmed by BLI. In conclusion, this study successfully constructed a phage-displayed chicken scFv library against FCV VP1-CDE protein and obtained 3 scFv clones with high binding activity, providing valuable materials for basic research and diagnostic development of FCV.

Increasing yield and reducing labor input are the main directions of cotton (Gossypium hirsutum) production. Plant architecture is a key factor affecting cotton mechanical harvesting and yield, and breeding cotton varieties with ideal plant architecture has been an important breeding goal. Cotton plant architecture is jointly controlled by genotype and environmental conditions, belongs to a quantitative trait with a complex genetic background and lacks major QTL for application in plant architecture breeding of cotton and so it is difficult to improve plant architecture by conventional breeding methods. This review summarized the latest research advances in the laws of inheritance, the analysis of regulatory mechanisms, and QTL mapping and gene discovery of cotton plant architecture traits. This work clarified the molecular mechanism of the formation of cotton plant architecture traits and provides references for the molecular breeding of cotton plant architecture.

Soybean (Glycine max) is an important grain-oil-forage crop as well as a major source of plant protein. Its varietal improvement is of great significance for ensuring food and edible oil security, enhancing agricultural production resilience, and addressing climate change. With the rapid development of genomics, phenomics, and intelligent algorithms, the knowledge structure and technological pathways of global soybean breeding research are continuously evolving. To systematically reveal the research progress, hotspot evolution, and collaboration characteristics in global soybean breeding, this study used 1 831 English-language publications indexed in the Web of Science Core Collection (WoSCC) from 2016 to 2025 as data sources and applied CiteSpace to conduct a visual bibliometric analysis of collaboration networks, keyword co-occurrence, keyword clustering, and burst terms. The results showed that global soybean breeding research maintained a relatively high level of activity during 2016~2025. The national collaboration network was characterized by the leading roles of China and the United States, with participation from multiple countries, while deeper cross-regional collaboration at the institutional and author levels still needs to be strengthened. Research hotspots were mainly concentrated on QTL mapping, genome-wide association studies, candidate gene mining, gene expression, genetic diversity, yield and quality traits, agronomic traits, stress resistance, and genomic selection. Frontier topics have gradually shifted from genetic linkage maps, molecular markers, and QTL mapping to candidate gene analysis, gene function validation, and machine-learning-assisted breeding. These findings provide references for future research topic selection, international collaboration, and optimization of soybean breeding technical strategies.

The Tibet Autonomous Region boasts abundant and unique animal genetic resources, serving as a critical national reserve for germplasm resources. Restricted by plateau geography and transportation conditions, animal tissue samples collected in the wild cannot be promptly transported to laboratories for processing, which severely impedes the establishment of primary fibroblast cell lines. To improve the efficiency of somatic cell line establishment from endangered livestock breeds in remote plateau regions such as Tibet, a vacuum low-temperature preservation method capable of stably maintaining ear marginal tissue at 0~4 ℃ during transport, as well as a corresponding cell line establishment system, was developed in this study. The feasibility of this approach was first validated in the indigenous Bama pig (Sus scrofa domestica). Ear marginal tissues were collected, subjected to vacuum treatment, and preserved at 4 ℃ for 0, 7, and 14 d prior to cell line initiation, and the success rate was 98%. Subsequently, this vacuum low-temperature preservation method was applied to ear marginal tissues of the Tibet-endangered Apeijiaza cattle (Bos taurus) following long-distance transportation and off-site cell line establishment. Compared with the conventional PBS-immersion method, the vacuum low-temperature preservation protocol yielded a greater number of viable cells at initiation, with cell viability consistently exceeding 85%. The success rate of primary fibroblast establishment from Apeijiaza cattle ear tissues was significantly increased by 37.5% relative to the control group (P<0.05). Furthermore, supplementation of the culture medium with 10 ng/mL basic fibroblast growth factor (bFGF) effectively retarded the senescence progression of Apeijiaza cattle somatic cells during serial passaging in vitro. Under bFGF-supplemented conditions, cells maintained high proliferative activity up to passage 20. There was no obvious apoptosis and senescence morphology, accompanied by significant downregulation of senescence-associated genes, including tumor protein p53 (TP53), insulin-like growth factor-binding protein 2 (IGFBP2), and thrombospondin-1 (THBS1). Collectively, the findings demonstrated that the combined strategy of vacuum low-temperature preservation and bFGF supplementation in culture medium effectively overcame the long-standing bottleneck associated with prolonged sample preservation of livestock genetic resources in remote areas, thereby providing a robust and reliable technological platform for the off-site establishment of somatic cell genetic resources from endangered species.