In recent years, with the rising incidence of typeⅡ diabetes, resistant starch (RS) has gained significant attention as a low-glycemic index food component. Resistant starch offers important physiological benefits, such as reducing postprandial blood glucose levels in diabetic patients and decreasing the risk of intestinal dysfunction and colon cancer. The content of resistant starch in rice (Oryza sativa) is positively correlated with amylose content, and the synthesis of amylose is controlled by the Waxy (Wx) gene. In this study, CRISPR/Cas9 gene editing technology was employed to edit the bases near the 3' splice site AG of the fourth intron of the Wx gene in rice, resulting in 4 different types of mutant lines. By analyzing the expression levels of the Wx gene, the activity of granule-bound starch synthase (GBSS), and the contents of amylose and resistant starch in these mutant lines, it was found that mutations near the 3' splice site AG significantly affected the expression of the Wx gene, thereby regulating the contents of amylose and resistant starch in rice. Correlation analysis revealed a significant positive correlation between Wx gene expression and resistant starch content. This study identified intron mutation genotypes that can effectively regulate the expression level of the Wx gene (either increasing or decreasing it), providing new insights for future improvements in the resistant starch content of rice.

Rice (Oryza sativa) blast is one of the factors affecting the stable yield of rice. The lesion mimic mutant is an ideal material for studying the molecular mechanism of crop disease resistance. The analysis of the disease resistance gene of the mutant is the basis for cultivating stable and disease-resistant rice. In this study, a stable genetic lesion mimic mutant spl52 (spotted leaf 52) was obtained by ethylmethylsulfone (EMS) mutagenesis of japonica rice 'Wuyunjing 21'. The effective panicle number, grain number per panicle, 1000-grain weight and seed setting rate of the mutant plants at maturity stage were significantly lower than those of the wild type. From the early tillering stage, the leaf tip of mutant spl52 appeared disease spots. By 3,3'-Diaminobenzidine (DAB) staining, enzyme activity assay and 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) detection results showed that the mutant spl52 lesion leaves showed reactive oxygen species accumulation, and programmed cell death activation was found by TUNEL assay. Transmission electron microscopy analysis of wild-type and mutant leaves showed that the mutant chloroplasts were smaller oval, and the mitochondria were hollowed out. Genetic analysis showed that the phenotype of the mutant was controlled by a pair of recessive nuclear genes. Six possible candidate intervals were found by Mutmap analysis, and the candidate genes were located between RM3.5-RM4.3 on chromosome 6 by map-based cloning. The candidate genes were analyzed, and no reported lesion-like and rice resistance-related genes were found. The leaves of wild-type and mutant plants were inoculated with rice blast fungus, and the results showed that the mutant plants were more sensitive to rice blast fungus (Magnaporthe oryzae). The results of qRT-PCR showed that the expression levels of OsPR1a, OsPR1b, JIOsPR10, OsWRKY6 and OsRSR1 which positively regulated rice resistance in the mutant were significantly lower than those in the wild type. The results showed that the mutant gene was a new gene that had not been reported and was involved in the regulation of rice blast.

Weeds are a major factor affecting the yield and quality of sugar beet (Beta vulgaris). Acetolactate synthase (ALS) inhibitor herbicides are widely used in agricultural weed management due to their high efficiency, low dosage, and safety. To comprehensively investigate the ALS gene family in sugar beet and explore their potential functions under ALS-inhibitor herbicide stress, this study performed a genome-wide identification of ALS family members using bioinformatics approaches. The physicochemical properties, evolutionary relationships, cis-acting regulatory elements and chromosomal locations of the identified genes were systematically analyzed. Additionally, qRT-PCR combined with morphological traits and photosynthetic parameters was used to examine the transcriptional expression patterns of BvALS genes under herbicide stress. A total of 7 BvALS gene family members and their transcript variants were identified in the sugar beet genome. The predicted BvALS proteins had molecular weights ranging from 51. 305~81.867 kD and theoretical isoelectric points ranging from 5.64 to 9.41. Most members were predicted to be stable proteins localized to the chloroplast. Phylogenetic analysis indicated that the BvALS family members were closely related to ALSs in Arabidopsis thaliana and Spinacia oleracea. A total of 10 conserved Motifs were identified, with most genes containing Motif 7. Notably, BvRB_5g124450, BvRB_2g027380, BvRB_5g103060, and BvRB_8g181940 contained all 3 conserved domains (TPP enzyme N, TPP enzyme M, and TPP enzyme C). These genes were distributed across 5 chromosomes and 2 scaffold regions. Promoter analysis revealed that BvALS genes may participate in various stress response pathways. Under ALS-inhibitor herbicide treatment, all BvALS genes were transcriptionally upregulated, with BvRB_5g103060 showing the most significant induction (3.2 times) higher than the control. These findings suggest that BvRB_5g103060 may play a critical role in the herbicide resistance mechanism of sugar beet, and provide theoretical basis for further studies on the biological function of BvALS genes in response to herbicide stress.

WRKY transcription factors are a class of transcription factors that are unique and conserved in plants, playing important regulatory roles in plant growth, development, and secondary metabolism. To explore the WRKY genes involved in the regulation of flavonoid metabolism in safflower (Carthamus tinctorius), the WRKY family genes of safflower, were identified and analyzed at the genome level in this study. Utilizing the genome and transcriptome data of safflower, this study identified 87 CtWRKY genes, distributing across 12 chromosomes. Subcellular localization prediction showed that the vast majority (85) of CtWRKY family members were located in the nucleus. Evolutionary analysis showed that CtWRKY genes could be categorized into 3 groups (GroupⅠ/Ⅱ/Ⅲ) and 5 subgroups (GroupⅡa/b/c/d/e) in GroupⅡ, with the highest number of WRKYs (52.87%). The analysis of protein motif showed that all CtWRKY proteins contained a highly conserved motif WRKYGQK. Collinearity analysis indicated that there were 39 pairs of segmental duplication genes in safflower CtWRKY gene family. RNA-seq analysis showed that there were obvious differences in the expression of CtWRKY gene family members in different tissues. Furthermore, the transcriptional profiles of 9 CtWRKY genes highly expressed in full bloom petals were investigated using qRT-PCR analysis. Results showed that a total of 9 CtWRKY genes were differentially expressed in response to abscisic acid (ABA) and methyl jasmonate (MeJA) treatment. This study provides a basis for further elucidating the function of WRKY transcription factors in the biosynthesis of flavonoids in safflower.

The methyl jasmonate (MeJA)-induced TwWRKY1 (GenBank No. GAVZ01022264.1) gene apparently regulated the biosynthesis of wilforgine in Tripterygium wilfordii. To elucidate the regulation mechanism and key target gene, the T. wilfordii sesquiterpene cyclase (Twses-CS) was selected for further exploring, the Twses-CS was screened as the key enzyme involved in the sesquiterpene biosynthesis pathway, the cis-acting element was analyzed in the Twses-CS promoter, and the expression of the TwWRKY1 and alkaloid content changes were detected. The results showed that the WRKY transcription factor might regulate the biosynthesis of sesquiterpene-pyridine alkaloids through regulating Twses-CS gene in T. wilfordii. Yeast one hybrid (Y1H) assays revealed that TwWRKY1 binded to the promoter of Twses-CS. Subcellular localization analysis indicated that the TwWRKY1 was located in the nucleus, consistent with its role as a typical transcription factor. Electrophoretic mobility shift assays (EMSA) further confirmed that TwWRKY1 formed a DNA-protein complex with a 30 bp probe containing 2 W-box sequences, indicating its binding ability to the W-box element. Overexpression of TwWRKY1 in hairy roots resulted in a significant increase in wilforgine and wilforine content about 1.87 and 1.90 times compared with the control, the content of wilforgine and wilforine was 249.53 and 638.25 μg/g DW, respectively. These results suggested that TwWRKY1 likely regulated the content of sesquiterpene-pyridine alkaloids in T. wilfordii by binding to the W-box element in the promoter of Twses-CS. This study provides basis for further exploration of alkaloid biosynthesis pathway and the transcription regulatory mechanism for sesquiterpene alkaloids, and provides a theoretical basis for using metabolic engineering and synthetic biology ways to solve the alkaloid shortage.

As an important economic crop, the yield and quality of Camellia sinensis susceptible to environmental stresses, SHR subfamily transcription factors play key roles in plant development and stress response, but their functions in C. sinensis are still poorly studied. In order to analyse the regulatory mechanisms of SHR subfamily transcription factors in C. sinensis growth and development and abiotic stress response, this study used bioinformatics to analyse the physicochemical properties, phylogenetic relationships, protein structures, promoter cis-acting elements and chromosomal positioning of CsSHR subfamily members. Tissue-specific expression of the genes and their expression patterns under shade, drought, salt stress, methyl jasmonate (MeJA) and exogenous hormone (gibberellin A3 (GA3); 6-benzylaminopurine (6-BA)) treatments were further analysed by combining transcriptome data with qRT-PCR. The results showed that a total of 8 CsSHR genes were identified, and the encoded proteins were all unstable hydrophilic nuclear localised proteins, distributed on 6 chromosomes and with promoter regions enriched in light-responsive, hormone- and stress-related regulatory elements. Phylogenetic analyses showed that the subfamily of CsSHR was closely related to the SHR subfamily of Populus, and the gene structure showed that the small number of introns. Expression analysis showed that the members of the CsSHR gene family showed a tissue-specific expression pattern, in which CsSHR-1/4/7/8 was highly expressed in roots, while CsSHR-2/3/5 was predominantly expressed in young leaves. In response to abiotic stress, CsSHR-2 and CsSHR-5 were significantly up-regulated under drought and salt stress. Hormone treatment experiments showed that GA3 treatment induced the up-regulation of CsSHR-1~CsSHR-4 in the early stage, but inhibited the expression of CsSHR-5~CsSHR-8. However, 6-BA treatment led to a biphasic expression pattern in which most members were first inhibited and then up-regulated. The study showed that members of the CsSHR subfamily play important roles in the growth and environmental adaptation of C. sinensis through the regulation of tissue development and stress response. This study provides a new theoretical perspective for the study of the molecular breeding of C. sinensis stress tolerance.

Walnut (Juglans regia) is frequently subjected to extreme weather conditions such as winter frost and severe cold waves, which severely hinder the healthy development of the walnut industry. F-box genes, as critical components of the E3 ubiquitin ligase complex, have been demonstrated to participate in plant stress responses; however, their role in walnut cold resistance remains unclear. To explore the molecular cold resistance mechanism of walnuts, this study selected one-year-old branches of cold-resistant resources Juglans hopeiensis 'Mahetao' and cold-sensitive 'LL29' as experimental materials. Physiological indices and transcriptome analyses of the phloem were conducted following low-temperature stress treatment. The results revealed that 'Mahetao' exhibited higher soluble sugar and soluble protein contents under low-temperature stress compared with 'LL29'. Furthermore, a substantial number of genes were upregulated in one-year-old branches of 'Mahetao' under cold stress. KEGG pathway enrichment analysis indicated that the differentially expressed genes in 'Mahetao' under low-temperature stress were primarily associated with starch and sucrose metabolism, plant hormone signal transduction pathway, etc. Based on these findings, 24 candidate genes related to cold resistance were further screened, including 10 genes involved in starch and sucrose metabolism pathways and 14 transcription factors. Functional analysis of 4 F-box transcription factors revealed their classification into the FBL (F-box and Leucine-Rich Repeat Protein), FBP (F-box and phloem protein 2), and FBU (F-box with unknown functional domain) subfamilies. The promoter regions of the 4 F-box genes contained 8 distinct types of stress-responsive cis-acting elements, with the JrFBU1 gene harboring a low-temperature-responsive LTR element. qPCR analysis demonstrated that the expression levels of JrFBU1 and JrFBP were significantly upregulated under low-temperature stress and consistently higher in 'Mahetao' than in 'LL29', suggesting their potential critical roles in walnut cold resistance. This study provides theoretical and practical insights for advancing cold tolerance improvement and cultivation techniques in walnut.

Actin-related protein 2/3 complex subunit 3 (ARPC3) and ARPC4, as important signaling molecules regulating actin cytoskeleton, plays an important role in maintaining the integrity of epithelial barrier function during the development of inflammation. In order to explore the mechanism of ARPC3/4 regulating barrier injury and oxidative stress in bovine (Bos taurus) mammary alveolar cells-large T antigen (MAC-T), cell counting kit-8 (CCK-8) was used to screen the optimal concentration of lipopolysaccharide (LPS) and ARPC3/ARPC4 inhibitor (CK666) in this study. qPCR and Western blot were used to detect the expression of nuclear factor-κB (NF-κB) signaling pathway, antioxidant and tight junction related proteins and genes. The results showed that LPS significantly increased the content of reactive oxygen species (ROS) and up-regulated the expression of myeloid differentiation factor 88 (MyD88), Toll-like receptor 4 (TLR4), nuclear factor-κB inhibitor kinase β (IKKβ) and NF-κB. The expression of catalase (CAT), superoxide dismutase 1 (SOD1), glutathione peroxidase 1 (GPx1), Claudin1, Occludin and zonula occludens-1 (ZO-1) were down-regulated. After inhibiting ARPC3/ARPC4 by CK666 treatment, the expression of heat shock protein 70 (HSP70) was further up-regulated compared with LPS group, and the up-regulation of NF-κB, MyD88, TLR4 and IKKβ induced by LPS was reversed. The expression of CAT, SOD1, GPx1, Claudin1, ZO-1 and Occludin was down-regulated, and the content of ROS was reduced. The results showed that in the inflammatory model of bovine mammary epithelial cells, ARPC3/ARPC4 impaired the epithelial cell barrier by destroying the tight junction, thereby activating the NF-κB signaling pathway and inhibiting the expression of antioxidant genes to promote inflammation and oxidative stress, indicating its potential as a therapeutic target for mastitis. This study provides new ideas and theoretical basis for the prevention and treatment of dairy cow mastitis.

The classical Wnt/β-catenin signaling pathway is highly conserved and widely present in a variety of organisms and cells, and is involved in regulating the growth and development of follicular granulosa cells. To investigate the effects of Wnt/β-catenin signaling pathway on the secretion of estradiol (E2) and progesterone (P4) in yak (Bos grunniens) follicular granulocyte cells. Cultured yak follicle granulocytes were treated with exogenous Wnt/β-catenin signaling pathway agonist SKL2001. The cell proliferation was detected by Cell Counting Kit-8 (CCK-8), and the contents of estradiol and progesterone in granulocyte supernatant were detected by ELISA kit. qRT-PCR and Western Blot were used to detect the expression levels of genes related to cell proliferation, Wnt/β-catenin signaling pathway and steroid hormone synthesis. The results showed that exogenous SKL2001 could up-regulate the expression of beta-catenin (beta-catenin, β-catenin) and axin-related protein 2 (Axin2), the key factors of Wnt/β-catenin signaling pathway, but had no effect on the expression of glycogen synthase kinase 3β (GSK3β ) in yak follicular granulocyte in the range of 2.5~10.0 µmol/mL. SKL2001 significantly decreased the expression of proliferation-related genes proliferating cell nuclear antigen (PCNA), cyclin D2 (CCND2), cell division cycle 42 (CDC42) in granulosa cells and decreased the proliferative activity of granulosa cells. The addition of SKL2001 could decrease the content of estradiol and increase the content of progesterone in culture medium. It inhibited the expression of key genes of estradiol synthesis cytochrome P450 family 19 subfamily A member 1 (CYP19A1) and cytochrome P450 17A1 (CYP17A1) in granulosus cells, and promoted the expression of key genes of progesterone synthesis cytochrome P450 family 11 subfamily A member 1 (CYP11A1), hydroxy-Δ⁵-steroid dehydrogenase-3β-androsten-Δ⁵-diol dehydrogenase type 1 (HSD3B1) and steroidogenic acute regulatory protein (StAR). These results suggested that SKL2001, after activating Wnt/β-catenin signaling pathway, inhibited the proliferation of follicular granulosa cells and promoted the transformation of follicular granulosa cells from estrogen-secreting type to progesterone secreting type by regulating the content of key enzymes of steroid hormone synthesis, which might be a potential mechanism of follicular atresia and luteinization in yaks. This study accumulates basic data for further understanding the function of follicular granulosa cells and the regulation mechanism of follicular development in yaks.

The insulin-like growth factor 2 receptor (IGF2R) plays a crucial role in physiological processes such as regulating animal growth and development and promoting protein synthesis. To explore the correlation between IGF2R gene polymorphism and growth traits in yak (Bos grunniens). In this study, with 336 individuals from Plateau yak, Huanhu and Xueduo yak in Qinghai province, the SNPs sites were screened by DNA mixed pool sequencing, detected the SNP sites of yak IGF2R gene by direct sequencing, and analyzed the association with growth traits. The results showed that 2 SNPs were screened on the yak IGF2R gene, namely g.96261388T>C (intron 20) and g.96292456A>G (exon 43). The g.96261388T>C locus detected only TT and TC genotypes in 3 yak populations. Three genotypes of AA, AG and GG were detected at g.96292456A>G sites. Analysis of genetic polymorphisms showed that g.96261388T>C and g.96292456A>G met the Hardy-Weinberg equilibrium adaptability test in 3 yak populations (P>0.05). The results of association analysis showed that chest circumference and tube circumference of Xueduo yak in TC genotype at g.96261388T>C were significantly better than TT genotype; In the g.96292456A>G locus of Plateau yaks, the body length and cannon circumference of individuals with the AA genotype were significantly better than those with the GG genotype (P<0.05); In Huanhu yaks, the body height, body length, chest circumference and cannon circumference of individuals with the GG genotype were significantly better than those with the AG genotype (P<0.05). Therefore, the IGF2R genes g.96261388T>C and g.96292456A>G sites were significantly associated with partial yak growth traits, which could be used as molecular markers for yak growth trait breeding. This study provides theoretical basis for yak breeding work.

Early pregnancy is a critical factor influencing sheep (Ovis aries) reproduction. Accurate identification of non-pregnant sheep can effectively reduce the empty period and enhance reproductive efficiency. Exosomes, which are extracellular vesicles with diameters ranging from 30 to 150 nm, are secreted by cells and play a crucial role in nearly all aspects of mammalian pregnancy. In this study, JunKen mutton sheep ewes aged 2 to 3 years were selected as research subjects. Plasma samples were collected from 3 ewes at non-pregnant (0 d) and early pregnant stages (14, 30 d). Plasma exosomes were isolated to construct miRNA libraries from these JunKen mutton sheep at different early pregnancy time points. Significantly differentially expressed miRNAs were identified in the plasma exosomes of JunKen mutton sheep at various early pregnancy stages. Through library analysis and database alignment, a total of 248 differentially expressed miRNAs were detected, including 79 known and 169 novel miRNAs. Comparative analysis revealed 40 differentially expressed miRNAs between 14 and 0 d (30 up-regulated, 10 down-regulated), 72 between 30 d and 0 d (43 up-regulated, 29 down-regulated), and 35 between 30 d and 14 d (9 up-regulated, 26 down-regulated). Target gene annotation and functional enrichment of differentially expressed exosomal miRNAs were performed by aligning with the GO and KEGG databases using BLAST software. Enriched biological processes were found to be associated with early pregnancy. Notably, oar-miR-27a exhibited high expression levels and significant differences between non-pregnant 0 d and pregnant 14 d and 30 d, suggesting its potential role in early pregnancy and embryonic development in sheep. To validate the sequencing results, 5 differentially expressed miRNAs were randomly selected for expression detection using qRT-PCR, and the results were consistent with the sequencing data. This study demonstrated that plasma exosomal miRNAs might participate in the regulation of early pregnancy in JunKen mutton sheep. These findings provide new insights into the regulatory processes of early pregnancy in sheep and offer a theoretical basis for exploring the molecular mechanisms by which differentially expressed exosomes influence early pregnancy in this species.

E-twenty-six proto-oncogene 1 (ETS1) gene plays an important role in autoimmune diseases (such as systemic lupus erythematosus, rheumatoid arthritis, etc.), and its deletion leads to abnormal differentiation of immune cells. The ETS1 gene has been extensively studied in the immune systems of mice (Mus musculus)and humans (Homo sapiens), but it has not been fully studied in large model animals like pigs (Sus scrofa). To investigate the role of the ETS1 gene in large model animals, this study constructed cloned pigs with an ETS1 gene knockout using gene editing technology. Firstly, the homology of the ETS1 gene in humans and pigs was analyzed through bioinformatics. Then, an ETS1 knockout cell line was constructed using CRISPR/Cas9 technology. The knockout cell line was subsequently used as the donor cell for constructing a lupus-like pig model with an ETS1 gene defect via somatic cell nuclear transfer technology. The results showed that the ETS1 amino acid sequence in pigs had high homology with humans. Fourteen monoclonal cell lines with gene deletions were obtained, including 7 with monoallelic deletions and 7 with biallelic deletions. One ETS1 monoallelic deletion pig and three wild-type pigs were successfully cloned using somatic cell nuclear transfer. This model provides a pivotal tool for elucidating the function of the ETS1 gene in the immune systems of large-animal models such as pigs, and lays the groundwork for developing large-animal models of autoimmune diseases.

The susceptibility of tilapia (Oreochromis spp.) to streptococcicosis is an important industry problem that hindering the healthy development of tilapia culture. However, studies on the resistance mechanisms of tilapia to streptococcal diseases and their related molecular markers is still relatively limited. In this study, molecular markers were identified associated with tilapia resistance to S. agalactiae based on resistant and susceptible phenotypes. Through artificial challenge with S. agalactiae, individuals were selected from a Nile tilapia (Oreochromis niloticus) population that exhibited resistance or susceptibility to S. agalactiae based on their survival or death following the challenge. Whole-genome resequencing were then performed on these resistant and susceptible individuals and conducted a genome-wide association analysis (GWAS) based on the resistant and susceptible phenotypes. The results showed that in this population, a total of 9 major-effect molecular markers associated with tilapia resistance to S. agalactiae were identified (P<2.50×10^-6), as well as 787 minor-effect molecular markers (2.50×10^-6<P<1.00×10^-4). These major-effect molecular markers were primarily located on tilapia linkage groups 13 (LG13), LG17, and LG22 and annotated to genes associated with the tumor immune microenvironment and immune cell infiltration (such as WD repeat domain 27 (WDR27), thrombospondins 2 (THBS2) and collagen type XIX α-1 chain (COL19A1)) as well as genes related to innate immunity (such as F10 α chain (class Ⅰ histocompatibility antigen, F10 α chain-like)). Meanwhile, GO and KEGG analyses indicated that the genes annotated by minor-effect markers were enriched in immune and inflammatory pathways, including the urokinase-type plasminogen activator signaling pathway, Ras signaling pathway, cyanine amino acid metabolism, arachidonic acid metabolism, and taurine metabolism. In addition, validation based on kompetitive allele specific PCR (KASP) revealed that the GG genotype at locus LG13_20881657 and the TT genotype at locus LG13_20548718 were advantageous genotypes associated with tilapia resistance to S. agalactiae. In summary, this study has preliminary revealed the genes and molecular markers associated with tilapia resistance to streptococcosis, and provides a scientific basis for the molecular marker-assisted breeding of tilapia resistant to streptococcosis.

The ubiquitin-proteasome system (UPS) is a critical protein degradation pathway in fungal cells, playing a vital role in regulating protein homeostasis and maintaining cellular functions. Previous studies have shown that during the early stages (0~72 h) of Alternaria solani infection in potato (Solanum tuberosum), the expression of the AsPR45 gene, which encoded the P45 subunit of the 26S proteasome in A. solani, was significantly upregulated. To further explore the function of the AsPR45 gene, this study first optimized a genetic transformation method for A. solani mediated by magnetic nanoparticles (MNPs). Using this transformation assay, a premature termination construct of AsPR45 was developed based on the pTOR-mRFP vector backbone and introduced into the wild-type strain TA-0410. Functional validation was carried out on 2 positive mutant strains. The transformation efficiency was the highest when the mass ratio of MNPs to the pTOR-mRFP vector was 1∶4, with binding at 37 ℃ for 30 min, followed by co-incubation with spores at room temperature for 1.5 h, achieving a transformation rate of (36.77±2.91)%. Biological function assays revealed that premature termination mutants of AsPR45 exhibited no significant difference in hyphal growth rate compared with the wild-type strain. However, the mutants produced less melanin in hyphae, had a significantly reduced conidiation rate, and showed markedly decreased pathogenicity on the leaves of potato cultivar 'Desiree'. Additionally, the EC₅₀ values for difenoconazole resistance differed significantly between the wild-type strain and the mutants. These results indicated that AsPR45 was a key gene in the ubiquitination pathway in A. solani, playing an important role in fungal morphogenesis, potato infection, and stress resistance. This study not only provides a technical foundation for functional gene research in A. solani but also contributes to the identification of potential fungicide targets, accelerating the development of strategies for controlling potato early blight disease.

The highly pathogenic hydropericardium hepatitis syndrome (HHS) caused by fowl Adenovirus serotype 4 (FAdV-4) poses a serious threat to the poultry industry in China. There is an urgent need for an efficient vaccine for its prevention and control. As a key protective antigen of the virus, the truncated expression strategy and immune mechanism of the fiber2 protein remain unclear, which restricts the industrial application of subunit vaccines.To explore the immunological characteristics and application potential of different structural domains of the fiber2 protein of FAdV-4 and develop a safe, efficient vaccine more suitable for the poultry industry, this study analyzed and determined the regions of FAdV-4 fiber2 with good antigenicity and hydrophilicity using bioinformatics software. The full-length gene of fiber2, the gene sequences of fiber2 from 184 to 1 440 bp, and from 841 to 1 440 bp were selected to construct recombinant plasmids pET30a-FF, pET30a-FS, and pET30a-FK, respectively. Using the Escherichia coli expression system, 3 recombinant proteins of FAdV-4 fiber2, namely the full-length (FF), truncated (FS), and knob (FK) proteins, were obtained. These were prepared into subunit vaccines and inoculated into specific-pathogen-free (SPF) chickens (Gallus gallus domesticus) to evaluate their immunogenicity. The results showed that all 3 proteins were mainly expressed in a soluble form. When SPF chickens were immunized at a dose of 30 μg per bird, 100% protection was achieved after challenge. Among them, the FS protein could induce a faster and stronger specific antibody response than the inactivated whole-virus vaccine, and achieve complete protection against virus challenge at a low level of neutralizing antibodies (NAb).The minimum immunization dose test showed that when the FS recombinant protein was inoculated into chickens at a dose of 10 μg per bird, 100% protection against the virulent strain of FAdV-4 was achieved. In summary, this study innovatively established a highly efficient soluble expression system for FAdV-4 fiber2. The developed FS protein antigen had the characteristics of a rapid immune response (14 d) and low-dose protection at 10 μg per bird. This study provides basic data for further research on the immunological characteristics of the fiber2 protein and lays a theoretical foundation and technical reserve for the industrial development of new FAdV vaccines.

As a special microbial community that forms a stable symbiotic relationship with the host, tobacco (Nicotiana tabacum) endophytes are valuable in biocontrol, bioprophylaxis and host ecological adaptation regulation. This review summarized the species diversity of tobacco endophytes and their functional research progress, and found that Bacillus, Fusarium and Streptomyces, as the dominant flora, could promote tobacco growth and development through biological nitrogen fixation, organic phosphorus solubilisation, phytohormone synthesis and systemic resistance induction. In addition, this paper also discussed the analysis of the functional genes of tobacco endophytes and the challenges they faced in the process of production and application, and provides a theoretical basis for the selection and breeding of speciality high-quality tobacco in China.

Plant endophytes represent a class of important microbial resources ubiquitously colonizing plant tissues and intracellular spaces, capable of secreting a variety of enzymes and novel secondary metabolites, which exhibit significant application potential across various fields, including agriculture, medicine, food, and horticulture. This article summarized the domestic and international research advancements regarding the contributions of endophytes to the growth and development of traditional Chinese medicinal materials, the accumulation of bioactive components, the mitigation of allelopathic effects, and the enhancement of stress resistance. It also explored the mechanisms by which endophytes improved to the yield and quality formation of traditional Chinese medicinal materials, aiming to provide a theoretical support for the application of endophytes in the cultivation of traditional Chinese medicinal materials and ultimately promote the protection of resources and the green and sustainable development of the industry.

Peste des petits ruminants virus (PPRV) infection induces diverse pathological damage in infected hosts and poses a serious threat to ruminant health. Transcriptome sequencing analysis of host cells following PPRV infection, conducted by our research group in the early stage, revealed a significant alteration in the expression level of the growth arrest and DNA damage-inducible 45A (GADD45A) gene. To investigate the potential role of GADD45A in PPRV replication, a recombinant lentiviral vector encoding the GADD45A gene was constructed, and a stable Vero cell line constitutively expressing GADD45A was established. qPCR, Western blot, and serial passage experiments confirmed sustained GADD45A expression at both mRNA and protein levels and demonstrated the stability of the cell line over multiple passages. The result showed that the established cell line maintained good viability and stable GADD45A expression throughout continuous passaging. The Vero cell line stably expressing GADD45A protein significantly enhanced the replication of PPRV. This study provides certain of biological tools and technical supports for further elucidating the molecular mechanisms underlying PPRV pathogenesis.

Senecavirus A (SVA) is a small RNA virus belonging to the genus Senecavirus that causes vesicular disease in pigs (Sus scrofa), which is clinically difficult to distinguish from Foot-and-mouth disease, swine vesicular disease, and vesicular stomatitis. In this study, a mutant SVA strain with a 13-nucleotide (nt) deletion in the 5' untranslated region (5' UTR) was successfully identified. To investigate the impact of this deletion on SVA's biological characteristics, this study employed reverse genetics technology, using the full-length cDNA infectious clone of the SVA CH/FJ/2017 strain as a template, and a targeted deletion in the genomic 5'UTR sequence was introduced.The recombinant plasmid was then transfected into BHK-21 cells, successfully rescuing a 5'UTR-deleted Seneca virus strain designated rSVA-5UTRM. The recombinant virus was characterized by indirect immunofluorescence assay (IFA) and Western blot, and its genetic stability was analyzed through serial passages in BHK-21 cells. Subsequently, plaque assays and viral growth curves were performed to compare the replication characteristics between the recombinant virus and parental strain in BHK-21 cells. The results demonstrated that the recombinant virus exhibited specific reactivity with SVA antibodies and SVA VP2 antibodies, confirming the successful rescue of the recombinant strain. Genetic stability analysis was performed by serially passaging the recombinant virus in BHK-21 cells for up to 20 generations, indicating excellent genetic stability of the recombinant strain. Virus biological characterization revealed that the recombinant strain exhibited similar replication properties to the parental SVA CH/FJ/2017 strain in BHK-21 cells and maintained a high viral titer. These findings suggested that the 13-nt deletion in the 5' UTR did not affect viral replication in BHK-21 cells. This study successfully rescued a recombinant SVA strain with a 5' UTR deletion, investigated the functions of the deleted region, and provided new scientific data for elucidating the non-coding functions of SVA.