SH3 domain-containing proteins (SH3Ps) in plant are members of a broad protein category and have been proved to be involved in several processes in cells. In Arabidopsis thaliana, the interaction network of SH3P2 is extensive and plays an important role in endocytosis, vesicle trafficking, binding ubiquitin, cell division and autophagosome formation. Up to now, little has been reported about SH3P2 in rice (Oryza sativa). In this study, the rice OsSH3P2 gene was introduced into protoplasts for transient expression. It was observed that OsSH3P2 was mainly localized in clathrin-coated vesicle, and partially localized in the trans-Golgi network/early endosome. Meanwhile, the subcellular localization results showed that OsSH3P2 could co-localize with protein ELC-like (ELC), a member of vesicle transport complex. Further, various means such as yeast two-hybrid (Y2H), co-immunoprecipitation (Co-IP) and bimolecular fluorescence complementation (BiFC) were used to confirm the interaction between OsSH3P2 and ELC, indicating they were highly correlated. Subsequently, by extracting the RNA from various tissues of rice and conducting qRT-PCR detection, the results showed that the expression level of OsSH3P2 was relatively stable at all stages of rice growth, and was the highest in stem. The PlantCARE online analysis revealed that there were multiple light-responsive regulatory elements in the promoter region of OsSH3P2. A dual-luciferase reporter assay was designed to confirm that the expression of OsSH3P2 was affected by light and was induced under dark conditions. This study revealed the cellular localization, protein interaction and expression pattern of OsSH3P2 in rice, laying a preliminary theoretical foundation for further exploration of the biological functions of OsSH3P2.

HvSWEET4, a member of the sugars will eventually be exported transporters (SWEET) sugar transporter family in hulless barley (Hordeum vulgare var. nudum), is involved in hexose transport regulation during grain development. To elucidate the transcriptional regulatory mechanism of HvSWEET4, approximately 2 000 bp of promoter sequences in upstream of the coding region were systematically analyzed from 2 hulless barley cultivars with significantly different grain sizes: 'Beiqing 3' (large grain) and '3917' (small grain). Sequence comparison revealed that the HvSWEET4 promoters from these 2 cultivars were highly conserved, containing only 10 SNPs and 5 InDels, which accounted for merely 0.75% of the total nucleotides. Cis-element analysis indicated that the HvSWEET4 promoter contained regulatory elements associated with stress responses, sugar and starch metabolism and developmental regulation of pollen, endosperm and aleurone layers. Promoter activity assays identified the core functional region at -310~-212 bp. Further analyses suggested that differences in promoter activity could be attributed to suppressor effects within the region from -1 999~-310 bp, as well as SNP variations in the region from -212~-1 bp, potentially altering the types and sites of transcription factor binding. These findings provide essential theoretical foundations for further dissecting the molecular mechanisms underlying HvSWEET4 role in grain size regulation and highlight its potential applications in crop breeding.

Sea Island cotton (Gossypium barbadense) is renowned for its superior fiber quality and high economic value. However, drought stress significantly impacts its yield and fiber quality. Identifying drought stress-related genes as candidate genes for molecular design breeding is one of the key strategies to enhance drought resistance in Sea Island cotton. In this study, the gene GbJMJ25 (JMJ: Jumonji), encoding a histone demethylase in Sea Island cotton, was cloned using RT-PCR. Bioinformatics analysis, expression profiling, and subcellular localization of GbJMJ25 were conducted, and its potential role in drought stress response was preliminarily validated using virus-induced gene silencing (VIGS) technology. Bioinformatics analysis revealed that GbJMJ25 encoded a protein of 1 055 amino acids, containing 2 conserved domains, RING and JmjC. The promoter region of GbJMJ25 contained multiple cis-acting elements related to light response, growth and development, and stress response. Expression pattern analysis demonstrated that GbJMJ25 was responsive to abscisic acid (ABA) and polyethylene glycol (PEG) treatments. Subcellular localization analysis indicated that GbJMJ25 protein was localized in the nucleus. Functional validation via VIGS technology showed that under normal conditions, GbJMJ25-silenced cotton plants exhibited no significant phenotypic differences compared with the control (pTRV2) plants. However, under drought stress, GbJMJ25-silenced plants displayed enhanced tolerance, with a higher survival rate than the control, suggesting that GbJMJ25 might play a role in cotton's drought stress response. The results of this study provide candidate genes for improving drought resistance in Sea Island cotton through molecular design breeding.

The KNOX (KNOTTED1-like homeobox gene) family serves as a core regulatory factor family for plant growth and development. Abnormal functions of this family can result in phenotypes such as organ malformations in plants. Therefore, analyzing its regulatory mechanism is of great significance for basic plant research and crop genetic improvement. In this study, the BlKNOX7 gene from Betula luminifera was cloned and its sequence characteristics were analyzed using bioinformatics methods. The expression patterns of the gene in various tissues and during tension wood formation were examined through qRT-PCR. Additionally, the phenotypic characteristics of transgenic Arabidopsis thaliana overexpressing BlKNOX7 were evaluated. The results showed that the BlKNOX7 gene (GenBank No. PV577094) consisted of 5 exons and 4 introns, with a cDNA sequence length of 1 095 bp and an ORF of 891 bp, encoding 296 amino acids. Evolutionary tree analysis revealed that BlKNOX7 clustered with the reported secondary wall development-associated transcription factor, Populus trichocarpa KNAT7. The expression of the BlKNOX7 gene in stems increased with the progress of lignification and reached the highest level in leaves. During the induction of tension wood, the expression level of BlKNOX7 in tension wood was significantly lower than that in the control. Overexpression of BlKNOX7 in Arabidopsis thaliana resulted in wavy curled plant leaves; Cross-sections of inflorescence stems stained with toluidine blue showed lighter coloration and a smaller range of coloration than the wild type, suggesting less lignin deposition. qRT-PCR results showed that BlKNOX7 overexpression significantly suppressed the expression of the Arabidopsis secondary wall synthesis pathway key enzyme genes phenylalanine ammonialyase (AtPAL)、 4-coumarate-COA ligase (At4CL)、irregular xylem (AtIRX) and cellulose synthase (AtCesA). This study provides a theoretical basis for further investigation of the molecular mechanisms of secondary wall development in Betula luminifera.

Phytosterols play crucial roles in plant growth, development, and stress resistance, with C24-sterol methyltransferase (SMT) serving as a key rate-limiting enzyme in phytosterol biosynthesis pathway. Cynanchum auriculatum is rich in phytosterol-based bioactive components, to investigate the function of SMT in phytosterol biosynthesis and abiotic stress response in C. auriculatum, the genes encoding SMT were cloned based on transcriptome data, bioinformatic analysis was carried out, and the gene expression levels under abiotic stresses were detected by qRT-PCR. The results showed that 2 genes with ORF of 1 041 and 1 080 bp were cloned and named CaSMT1 (GenBank No. PV701631) and CaSMT2 (GenBank No. PV701632), respectively. CaSMT1 encoded a protein consisting of 346 amino acids, with a relative molecular weight of 38.65 kD and a theoretical isoelectric point of 6.33. CaSMT1 was a hydrophilic protein without signal peptide. CaSMT2 encoded a protein consisting of 359 amino acids, with a relative molecular weight of 40.16 kD and a theoretical isoelectric point of 6.42. CaSMT2 protein contained transmembrane domain and was localized to the plasma membrane. Both CaSMT proteins contained canonical sterol C-24 methyltransferase C-terminal domains, with secondary structures predominantly composed of alpha helix and random coil. Phylogenetic analysis revealed that CaSMT1 and CaSMT2 clustered into distinct clades, both exhibiting the closest phylogenetic relationship with their corresponding SMT in Calotropis procera. qRT-PCR analysis revealed distinct tissue-specific expression patterns for both genes, with significantly higher expression levels in roots and leaves compared with stems. Furthermore, steroidal glycoside accumulation exhibited concordant variation patterns with gene expression levels across all examined tissues. Under various abiotic stresses including salt, drought, heat, and cold, CaSMT1 exhibited significant upregulation across all tested conditions. In contrast, CaSMT2 showed distinct stress-responsive patterns: Marked upregulation under heat and cold stress, downregulation under drought stress, and no significant response to salinity. This differential regulation demonstrated the involvement of CaSMT paralogs in adaptive responses of C. auriculatum to diverse abiotic stresses. This study provides theoretical insights for improving stress resistance and enhancing the content of active component in medicinal plants.

The adrenoceptor beta 3 (ADRB3) gene encodes a G protein-coupled membrane surface receptor with multiple roles in regulating lipolysis and energy expenditure. The aim of this study was to investigate the single nucleotide polymorphisms (SNP) in the untranslated region (UTR) of the ADRB3 gene by Sanger sequencing of PCR products and MassARRAY, and analyze their association with reproductive traits in Hu sheep (Ovis aries). The results showed that 4 SNP loci were identified in the UTR of the ADRB3 gene: g.42G>T, g.2377G>T, g.2501T>A and g.2622G>A. Each of these loci exhibits 3 genotypes and the dominant genotypes were all TT except for g.2622G>A whose dominant genotype was GA. All SNPs showed middle polymorphism (0.25<PIC<0.5) except g.42G>T (PIC<0.25). χ² tests indicated that the genotype distributions of all SNPs were in Hardy-Weinberg equilibrium (P>0.05). Association analysis revealed that for the g.42 G>T locus, the litter size in parity 1 was significantly higher in individuals with the GG genotype than those with the TT genotype, which was significantly higher than the GT genotype (P<0.05). The litter size in parity 2 was significantly higher in individuals with the GG genotype than those with the GT and TT genotypes. The lambing interval of parity 2~3 in individuals with the GT and TT genotypes was significantly shorter than those with the GG genotype (P<0.05). For the g.2377G>T locus, the lambing interval of parity 2~3 in individuals with the GT genotype was significantly shorter than those with the TT genotype (P<0.05). At the g.2501T>A locus, the litter size in parity 4 was significantly higher in individuals with the TT and TA genotype than those with the AA genotype (P<0.05). For the g.2622G>A locus, the litter size in parity 3 was significantly higher in individuals with the GA genotype than those with the AA genotype (P<0.05). Diplotype analysis showed that 5 haplotypes and 15 diplotypes were detected at 4 SNPs loci in the experimental population. Association analysis of 9 (frequency>0.05) haplotypes with reproductive traits revealed that the individuals with the H5H5 (TTTTTTGG) diplotypes had higher litter size. In conclusion, the ADRB3 gene can be used as a candidate gene for molecular markers of reproductive traits. This study provides a reference for accelerating the breeding process of high fertility Hu sheep.

The testis is an important reproductive organ in male animals, capable of secreting hormones and producing sperm. Cellulase is a key factor in breaking down plant cell walls and enhancing the nutrient absorption ability of ruminants. The level of energy supply directly affects testicular development, so it is hypothesized that the addition of cellulase may have an effect on the testicular development of Hu sheep (Ovis aries) lambs. In order to study the effect of adding cellulase to starter feed on testicular development and spermatogenesis of male lambs of Hu sheep before sexual maturity, 24 10-day-old male Hu sheep lambs of similar body weight were selected. They were randomly divided into 2 groups of 12 lambs each. 0.1% cellulase was added to the feed of the experimental group, and 6 lambs were slaughtered at random in each group at 60 and 120 d of age. Testicular tissues were collected for gross dissection, histological observation, hormone determination, and the expression of hormone secretion-related genes and reproductive marker genes detection. The results showed that the testicular indices of Hu sheep lambs at 60 and 120 d of age were extremely significantly higher than those of the control group after the addition of cellulase to the starter diet (P<0.01), and the diameters of the seminiferous tubules increased extremely significantly (P<0.01), and the lumens of the fine tubules appeared by the 120 d of age, with a lumen area that was significantly larger than the lumen area of the control group (P<0.05). Spermatogenic epithelial thickness was extremely significantly (P<0.01) higher than that of the control group at 60 d of age, and the difference was not significant by 120 d of age. Androgen levels in the testes of lambs in the 60 and 120 d of age test groups were extremely significantly higher than those in the control group (P<0.001), and the mRNA expression levels of androgen synthesis-related genes (steroidogenic acute regulatory protein (StAR) and cytochrome P450 family 17 subfamily A member 1 (CYP17A1)) showed the same trend; and the mRNA expression levels of the germ cell marker genes of testis of lambs at 60 and 120 d of age DEAD-box helicase 4 (DDX4) and the gene of deleted in azoospermia like (DAZL) were the same as that of the other genes. Both genes exhibited significantly higher protein and mRNA expression levels compared to the control group (P<0.05). The above results showed that the addition of cellulase to the starter diet could promote the testicular development of male lambs of Hu sheep before sexual maturity, promote the development of the varicose fine tube, increase the level of androgen secretion, and then be able to improve the ability of spermatogenesis. This study provides a theoretical basis for the application of cellulase in open diets for young ruminants.

The Ningxiang pig (Sus scrofa domesticus), a geographically indicated agricultural product of China, is characterized by excessive backfat thickness due to oversized lipid droplets, which represents a major disadvantage. To investigate the regulatory mechanisms of lipid droplet size and develop a more market-competitive strain of Ningxiang pigs, this study utilized PLIN2 (perilipin-2) knockout Ningxiang pigs as experimental models. Transmission electron microscopy was employed to observe lipid droplet size and fusion events, while qRT-PCR and Western blot were used to verify the expression of downstream target genes. Additionally, lipid droplet size was assessed using staining techniques such as boron-dipyrromethene (BODIPY) and Oil Red O. Following gene expression screening via qRT-PCR and validation through Western blot, CIDEa (cell death-inducing DNA fragmentation factor alpha-like effector A) was identified as a key regulator of lipid droplet size. A CIDEa overexpression vector was constructed and transfected into PLIN2-knockout Ningxiang pig cells, and lipid droplet size was subsequently evaluated. Furthermore, bioinformatics tools were utilized to predict the binding sites between PLIN2 and CIDEA. The results showed that PLIN2 knockout significantly reduced lipid droplet fusion and markedly decreased CIDEA expression. Overexpression of CIDEa compensated for the lipid droplet reduction caused by PLIN2 deletion. AlphaFold predictions revealed multiple binding sites between PLIN2 and CIDEa. These findings suggest that PLIN2 may regulate lipid droplet size through CIDEA-mediated lipid droplet fusion, providing novel theoretical insights into the regulatory mechanisms of lipid droplet size and a scientific basis for breeding more market-competitive Ningxiang pig strains.

Stem cells have the ability of self-replication and multidirectional differentiation, which is of great significance in the field of regenerative medicine. As a unique species adapted to extreme environments, the induced pluripotent stem cells (iPSCs) of the bactrian camel (Camelus bactrianus) contain a special differentiation and control mechanism. This study centered on the optimal culture protocol for the osteogenic induced differentiation of bactrian camel induced pluripotent stem cells (bciPSCs). The optimal culture protocol for osteoblast induced differentiation were screened, and then explored the optimal drugs and concentrations for constructing inflammation models using osteoblast (OB) cells obtained from this protocol. The pluripotency and differentiation ability of bciPSCs kept in the laboratory were firstly determined by qRT-PCR, immunofluorescence and karyotyping; then bciPSCs were induced and differentiated by 2 direct and indirect induction methods and 2 different culture mediums. The OB obtained were tested by qRT-PCR, immunofluorescence, Western blot, etc. to determine the effect of osteogenic differentiation and compare the purity of the OB obtained to determine the optimal induction method; The cells obtained by the optimal method of osteogenic induction were selected to construct the inflammation model, and the OB were treated with lipopolysaccharide (LPS) and Interleukin-1β (IL-1β), and then screened the optimal drug and concentration by CCK-8, qRT-PCR, and Western blot. The results showed that compared with bactrian camel embryonic fibroblasts (CEF), the expression of pluripotency genes in laboratory-preserved bciPSCs was extremely significantly up-regulated (P<0.01). During the passaging process, the cells did not undergo morphological and pluripotency changes, indicating their ability to differentiate into three blastomeres and their suitability for subsequent studies. BciPSCs were directionally induced to differentiate via 2 methods: Direct induction and indirect induction. After induction, the expression of osteogenic marker genes—including osteocalcin bone gamma-carboxyglutamate protein (BGLAP), collagen typeⅠ α2 (COL1A2), runt-related transcription factor 2 (Runx2), and secreted phosphoprotein 1 (SPP1)—was significantly up-regulated (P<0.05). Additionally, the expression levels of osteopontin (OPN) and Runx2 were also significantly increased (P<0.05), and both were positively expressed in immunofluorescence staining and osteogenesis-specific staining. Through intergroup comparisons, the group of cells exhibiting the most pronounced osteogenic characteristics was selected for subsequent studies; The cell viability was significantly decreased after 24 h of LPS and IL-1β treatment (P<0.01), and the expression levels of the inflammatory factors tumor necrosis factor-α (TNF-α), interleukin-6 (IL6) and IL-1β were significantly increased (P<0.05), with 70 ng/mL of IL-1β up-regulated inflammatory factors more significantly, which could be used as a condition for constructing an inflammation model. This study provides biological materials and reference data for the study of osteoarthritis disease pathogenesis and drug screening.

Rhesus proteins (Rh) belong to the ammonia transporter gene family and play an important role in the ammonia excretion of animals. Sinonovacula constricta, as a main economic shellfish in mixed culture in confined ponds, often faces severe high ammonia nitrogen environment due to its buried lifestyle. In this study, the gene family of Rh transporters in S. constricta (Sco-Rhs) was identified at the genome-wide level by bioinformatics methods. Subsequently, their expression patterns under ammonia stress, gene functions, and correlation with ammonia-nitrogen tolerance were investigated. The results showed that 2 Sco-Rhs genes (Sco-Rhbg-1和Sco-Rhbg-2) were identifed from the S. constricta genome. Moreover, the homology between Sco-Rhs was 57.96%. The tissue expression results showed that Sco-Rhs genes expressed in all tissues, with the highly specific expression in gills (P<0.01). And Sco-Rhs had different expression patterns under ammonia stress. With the increase of stress time, the expression of Sco-Rhbg-1 gene in gills showed a downward trend and had a stronger response to ammonia stress. The expression of Sco-Rhbg-2 gene firstly down-regulated and then increased, and finally approached to the initial level. In addition, heterologous yeast complementation was used to study the function of Sco-Rhs. The results showed that the Sco-Rhbg-1 protein is a functional ammonia transporter, and its ability to transport NH₄⁺ was not affected by external pH. And 15 SNPs in the CDS region of Sco-Rhbg-1 were significantly associated with the ammonia tolerance of S. constricta. These results indicated that the Sco-Rhbg-1 gene played an important role in the regulation of ammonia stress in S. constricta. This study provides an important theoretical basis for further exploration of the molecular regulation of Sco-Rhs in high ammonia nitrogen adaptation and molecular marker-assisted breeding.

As a common soil-borne fungal disease, wheat sharp eyespot has emerged as a crucial factor constraining wheat (Triticum aestvum) yield. In this study, a polyethylene glycol (PEG)-mediated genetic transformation system was employed to transform green fluorescent protein (GFP) into Rhizoctonia cerealis and obtained a transformant that was genetically stable; The GFP-labeled R. cerealis was inoculated to wheat seedlings, and the colonization pattern of R. cerealis in wheat was investigated by section observation. It was found that positive transformants were attained by PEG-mediated transformation onto protoplasts and screened in PDA medium containing 100 mg/mL hygromycin. PCR detection and fluorescence observation indicated that the plasmid carrying GFP had been successfully transfected into the R. cerealis. After successive passaging culture, the positive transformants were able to express stably and their pathogenicity was consistent with that of the wild type. On the fourth day after inoculating the transformants to wheat, distinct fluorescence could be observed in the xylem of the wheat root, and on the seventh day, a fluorescence phenomenon emerged in the phloem of the wheat coleoptile. This experiment successfully obtained R. cerealis with GFP labeling which could be stably inherited and preliminarily elucidated the colonization pattern of R. cerealis in wheat, it provide theoretical basis for further in-depth research on the pathogenic mechanism of R. cerealis and the use of microbial fertilizers to control wheat sharp eyespot.

Cowpea (Vigna unguiculata) root rot is primarily a soil-borne fungal disease caused by Fusarium solani. This disease is particularly prevalent during the seedling stage of cowpea and poses significant challenges for prevention and control, especially in Hainan. Currently, there are no effective microbial agents available for its management. In this study, a strain designated ZF520, exhibiting a notable inhibitory effect on F. solani, was isolated from cowpea rhizosphere soil. Through morphological observation, determination of physiological and biochemical characteristics, and multi-gene phylogenetic tree analysis, this strain was identified as Bacillus velezensis. Antibacterial spectrum analysis revealed that strain ZF520 demonstrated significant antagonistic effects against 6 pathogenic fungi (F. oxysporum, Ascochyta citrullina, Rhizoctonia solani, F. solani, Corynespora cassiicola, Botrytis cinerea). Pot test results indicated a considerable reduction in the disease index of root rot when plants were inoculated with ZF520, achieving a control efficacy of 71.82%. A ZF520 seed coating agent was developed for cowpea seeds. This agent achieves a control effect on cowpea root rot of 76.67% without compromising the emergence rate, outperforming 23% imidacloprid fludioxonil difenoconazole 23% imidacloprid fludioxonil difenoconazole suspension seed coating agent treatment. The effective viable bacterial count of the microbial seed coating agent, formulated using ZF520 fermentation broth, was 1.5×10⁹ CFU/mL. The coated strain maintained a stable rhizospheric colonization level of approximately 2.3×10⁵ CFU/g in cowpea seedlings, demonstrating potential for mitigating pathogenic infection risks during the early growth stage through probable antagonistic mechanisms. In conclusion, strain ZF520 exhibits promising biocontrol potential and application prospects for managing cowpea root rot.

Lignocellulose, one of the primary components of agricultural waste straw, has a complex structure that limits the efficiency of cellulose degradation, resulting in a low utilization rate of straw resources. To enhance the efficient utilization of agricultural waste, this study isolated 17 fungal strains capable of growing on straw powder medium from soil, distiller's grains, and wild macrofungi samples in Yanbian Korean Autonomous Prefecture, Jilin Province. Through preliminary screening using guaiacol medium and secondary screening based on laccase activity, a highly efficient laccase-producing fungus, Coriolopsis trogii LFB-F1, was identified. Whole-genome sequencing and bioinformatics analysis revealed the genomic characteristics of this strain and the distribution of lignin-degrading genes. The results indicated that the LFB-F1 genome had a total length of 38.8 Mb, with a GC content of 54.93%, and contained 7 497 coding genes, of which 134 were carbohydrate-active enzymes (CAZymes), including 1 laccase gene and 5 lignin peroxidase genes. Biological characterization showed that LFB-F1 exhibited optimal growth at 30 ℃ and pH 5.5, with strong biomass accumulation capability. Enzymatic property analysis revealed that the optimal pH for LFB-F1 laccase activity was 4.5, with an optimal temperature of 45 ℃, and it exhibited good thermal stability at 35 ℃. Studies on the effects of metal ions on laccase activity showed that Cu²⁺ and Fe²⁺ had minimal impact, while Mn²⁺ strongly inhibited the enzyme. Additionally, the laccase retained 92.5% of its activity after 90 d of storage at 4 ℃, demonstrating excellent storage stability. In summary, the fungal strain isolated in this study exhibited lignin-degrading potential. The fungi screened in this study for the high efficiency in degrading lignin can provide theoretical and technical support for the resource utilization and high-value utilization of agricultural waste.

Dicer-like (DCL) protein is a key enzyme in the RNA interference (RNAi) pathway, responsible for processing double-strand RNA (dsRNA) or precursor miRNA (pre-miRNA) into small interfering RNA (siRNA) or mature miRNA, thereby guiding mRNA degradation or gene expression inhibition. The rice (Oryza sativa) genome encodes 8 DCL proteins (OsDCLs) that exhibit distinct substrate specificity, small RNA (sRNA) product length, and biological functions. This review summarized the research progress on the functional diversification and mechanisms of OsDCLs in the processing of different types of sRNAs, their specialized roles in regulating growth, development, and important agronomic traits, as well as the regulatory mechanisms of their own activities. Moreover, the potential role of OsDCL-mediated RNA silencing pathways in the formation of rice heterosis was analyzed and the application value of OsDCLs in breeding and variety improvement was discussed. In-depth investigation of the OsDCL family would contribute to a comprehensive understanding of the roles of RNA silencing pathways in rice and other cereal crops. This review provides theoretical foundations and technical support for elucidating the mechanisms underlying heterosis formation and creating new germplasm with superior agronomic traits.

The genetic transformation technology of pollen-tube pathway, as a commonly used method for genetic transformation, offers advantages such as simplicity of operation, independence from genotype, and the ability to directly obtain transgenic seeds. Despite being influenced by factors such as external environmental conditions, the natural flowering period of plants, and limited flowering frequency, the pollen tube pathway genetic transformation technique remains highly applicable to a wide range of plants, especially those that are genotype-dependent. This article reviewed the origin, classification, and characteristics of this method, discussed the main factors affecting its genetic transformation efficiency and the progress in understanding its transformation mechanisms, and compareed it with the Agrobacterium-mediated genetic transformation method. Finally, the current applications, existing issues, and future prospects of the pollen tube pathway method were elaborated, providing theoretical references for establishing or optimizing a rapid and highly efficient plant genetic transformation technique via the pollen tube pathway.

The forest musk deer (Moschus berezovskii), a first-class protected animal in China, has musk secreted by its male that holds significant medicinal value. The S100A8 protein, as a member of the calcium - binding protein family, plays a crucial role in the inflammatory response. Therefore, to meet the health - monitoring needs in the artificial breeding of forest musk deer, a double-antibody sandwich ELISA for S100A8 was established to detect S100A8 in the serum samples of forest musk deer.In this study, the prokaryotic expression vector pET28a-B2M was constructed to obtain the S100A8 recombinant protein. Rabbit-derived polyclonal antibodies were obtained by multi-point injection into New Zealand white rabbits (Oryctolagus cuniculus). Mouse-derived S100A8 monoclonal antibodies were prepared through hybridoma technology and used as capture antibodies. The horseradish peroxidase (HRP) labeled rabbit-derived S100A8 polyclonal antibodies were used as detection antibodies. A double-antibody sandwich ELISA quantitative detection method for S100A8 in musk deer was established, and the antibody concentration, optimal coating conditions of the capture antibody and incubation conditions were optimized. The established ELISA method was used to specifically detect 31 serum samples.The results showed that the concentration of the recombinant protein obtained by Ni²⁺ column affinity chromatography was 5 mg/mL, with a purity of over 85%. Indirect ELISA indicated that the titers of both mouse and HRP-rabbit anti-S100A8 antibodies were higher than 1∶50 000. When the concentration of the capture antibody was 4 μg/mL and the dilution ratio of the detection antibody was 1∶2 000, these were the optimal coating concentration of the capture antibody and the working concentration of the detection antibody. The minimum sensitivity of the double-antibody sandwich ELISA method established in this study was 15.625 ng/mL, and it could specifically distinguish between inflammatory samples and normal samples. A double-antibody sandwich ELISA detection method was successfully established to achieve the quantitative detection of S100A8 protein in samples. It is expected to be applied to comprehensively evaluate the health status of forest musk deer and provide support for the early diagnosis and treatment of diseases.

Middle east respiratory syndrome coronavirus (MERS-CoV), first identified in 2012, primarily infects dromedary camels (Camelus dromedarius) and humans (Homo sapiens). Nanobodies have garnered significant attention due to their unique structural and functional advantages. This study focuses on the molecular design, recombinant expression in engineered yeast, purification, and biological activity evaluation of nanobodies targeting MERS-CoV. Based on previously screened nanobodies Nb1 and Nb2, this study amplified the corresponding nanobody genes and constructed eukaryotic expression vectors for monovalent nanobodies (Nb1, Nb2) as well as a bispecific nanobody (Nb3). High-expression yeast (Pichia pastoris) strains were subsequently selected. Considering the physicochemical properties of nanobodies, a purification protocol was established incorporating affinity chromatography, ion-exchange chromatography, and size-exclusion chromatography to obtain tag-free monovalent and bispecific nanobodies.The binding capabilities of the nanobodies to MERS-CoV antigens were evaluated using enzyme-linked immunosorbent assay (ELISA) and biolayer interferometry (BLI). Additionally, pseudovirus-based neutralization assays were conducted to verify antiviral activity. High-purity, tag-free preparations of Nb1, Nb2, and Nb3 were successfully obtained. Among them, Nb3 exhibited the highest antigen-binding activity, with an efficient concentrations of half-maximal (EC₅₀) of 8.419 nmol/L. Notably, Nb2 showed the strongest neutralizing activity in Huh-7 cells, with an inhibitory concentrations of half-maximal (IC₅₀) of 1.126 nmol/L.In conclusion, this study successfully developed 3 kinds of nanobodies targeting MERS-CoV and provides a valuable reference for the optimization of nanobody design and yeast-based production. The findings also lay a technical foundation for the development of nanobody-based therapeutics against MERS-CoV.

Porcine reproductive and respiratory syndrome (PRRS), caused by the Porcine reproductive and respiratory syndrome virus (PRRSV), is an acute infectious disease primarily characterized by reproductive disorders in sows (Sus scrofa) and respiratory symptoms in piglets. PRRSV has long been a serious threat to the swine industry in China. The current national control strategy focuses on eradication, making antibody monitoring and prevention of wild-type virus infection crucial for this goal. There is an urgent need to develop a novel, rapid, sensitive, low-cost and fully automated method for detecting PRRSV antibodies. This study established an indirect chemiluminescence immunoassay (CLIA) based on the prokaryotically expressed recombinant nucleocapsid (N) protein of PRRSV. The recombinant expression plasmid pET-28a-N-SUMO was constructed, and the expressed N protein was verified for reactivity via Western blot. The purified recombinant N protein was used as the coating antigen to develop an indirect CLIA for PRRSV antibody detection. The specificity, sensitivity and repeatability of the method were systematically evaluated. Field serum samples were tested in parallel using the established CLIA and a commercial indirect ELISA kit for PRRSV antibody detection to evaluate the concordance between the 2 methods. The results showed that serum samples with OD₄₃₅ luminous value≥13 438 were considered positive in the CLIA. No cross-reactivity was observed with antisera against other common swine viruses. The repeatability coefficients of variation were all below 7.19%. The detection limit of the CLIA was 4 times higher than that of the commercial ELISA kit. A total of 193 serum samples from Sichuan and Liaoning provinces were tested using both methods, and the Kappa value was 0.924, indicating excellent agreement. These results demonstrated that the established CLIA exhibited high specificity, sensitivity and reproducibility. The entire detection process taked approximately 35 minutes. In conclusion, an indirect CLIA based on the recombinant PRRSV N protein was successfully developed. This method was highly sensitive, reproducible and suitable for rapid detection of PRRSV antibodies in swine serum. This study provides reliable technical support for PRRSV immune monitoring, disease control and eradication efforts.

Currently, the detection of gene editing products mostly relies on methods such as genome sequencing analysis and PCR technology, which are time-consuming, costly, and require large instruments and equipment. In order to establish a rapid, sensitive, visual and suitable method for the detection of gene edited crops, primers and single guide RNA (sgRNA) were designed according to the rice OsSWEET14 gene editing material. Recombinase polymerase amplification (RPA) and CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated protein) 12b technology combined with lateral flow strips (LFS) strip were used to detect gene edited rice (Oryza sativa). The results showed that RPA-CRISPR/Cas12b combined with LFS could make the detection results visualized through the test strips within 33 min at most, and the detection sensitivity was 100 fg/μL. The RPA-CRISPR/Cas12b-LFS detection method developed in this study was simple and rapid, did not need large instruments and equipment. This study provides technical support for the field rapid detection with limited experimental conditions.