Table of Content

16 November 2025, Volume 58 Issue 22

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CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS

Foliar Spraying TaSBEIIbs-dsRNA to Increase Amylose Content in Wheat

LI LinYan, ZHANG GaoYang, FENG XianYang, GU ShiLong, HUANG YeNan, SUN ZhongKe, LI ChengWei

Scientia Agricultura Sinica. 2025, 58(22):  4557-4569.  doi:10.3864/j.issn.0578-1752.2025.22.001

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【Objective】Wheat starch mainly consists of amylose and amylopectin. Long-term consumption of refined flour products increases the risk of chronic diseases such as diabetes, whereas consuming flour with a high content of resistant starch has a positive effect on regulating blood glucose levels. Given the generally positive correlation between resistant starch and amylose, increasing the amylose content in wheat germplasm has become a goal for quality improvement breeding research. 【Method】Four gene fragments of starch branching enzyme (TaSBEIIb) were selected to successfully construct a high-efficiency dsRNA expression vector. A gradient optimization based on culture medium components significantly enhanced dsRNA yield. Based on this, the effects of naked dsRNA and dsRNA encapsulated with the nanocarrier hydroxypropyltrimethyl ammonium chitosan chloride (HACC) on wheat starch metabolism were explored through foliar spraying. Utilizing a wheat seedling culture system, the impact of dsRNA spraying on the amylose content in wheat seedlings and the expression of starch-related genes was observed. Furthermore, a field trial analyzed the effects of dsRNA spraying on the amylose content in mature wheat grains. The protective effect of chitosan quaternary ammonium salt-coated dsRNA and its influence on amylose content in mature wheat grains were also investigated. 【Result】Four recombinant plasmids (pRNAI-TaSBE1-pRNAI-TaSBE4), expressing dsRNA were successfully constructed. The optimized fermentation medium increased the dsRNA yield from 26.54 mg·L^-1 to 50.65 mg·L^-1, representing a 91% increase compared to the initial medium. Spraying dsRNA interfered with the expression of the target genes, with the highest interference efficiency observed on day 7 for the TaSBEIIb1 fragment. After interference with the four fragments, the expression of TaSBEIIb was reduced by an average of 47.73%. Additionally, the interference of TaSBEIIb affected the expression of other genes in the starch synthesis pathway, including TaSSII, TaSSIV, and TaSBEIIa1 with peak interference efficiencies occurring on days 3, 7, and 3, respectively. Their expression levels decreased by an average of 54.53%, 59.94%, and 47.64%. The 2023 field trial indicated that spraying naked dsRNA increased the amylose content in wheat grains by 17.2%-36.5% after 7 days of treatment, although the effect diminished to 0.2%-8.3% by the maturity stage. In the 2024 field trial, multiple applications of both naked dsRNA and chitosan quaternary ammonium salt-coated raised the amylose content in mature wheat grains from 27.72% to 30.37%, about 10% increase compared to the control. 【Conclusion】Exogenous spraying of TaSBEIIbs-dsRNA effectively increases the amylose content in starch.

Construction and Application of DNA Fingerprint Database for Known Varieties in Upland Cotton DUS Testing

WANG LiYuan, WANG Hui, WANG MuMu, WANG DongJian, LI RuYu, ZHENG YongSheng, ZHANG Han

Scientia Agricultura Sinica. 2025, 58(22):  4570-4588.  doi:10.3864/j.issn.0578-1752.2025.22.002

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【Objective】DUS (Distinctness, Uniformity, and Stability) testing provides important technical data for cotton variety approval and intellectual property protection, while the selection of similar varieties is a critical step in the process. Based on the revision of “NY/T 2469-2013, Protocol for the identification of cotton variety-SSR marker method”, this study aims further to construct a DNA fingerprint database for known upland cotton varieties for DUS testing in China and determine a genetic similarity threshold for rigorous and precise similar variety selection. 【Method】 Firstly, preliminary screening and subsequent re-screening were conducted on both the SSR primers from NY/T 2469-2013, and newly collected ones to identify a set of primers with stable PCR amplification, high polymorphism and clear peak patterns, replacing the original SSR primer set in the standard. Secondly, the selected SSR primers were employed to genotype the known cotton varieties to construct a DNA fingerprint database for those varieties. Finally, relationships between phenotypic and genetic differences of cotton varieties were investigated and a genetic similarity threshold for similar variety selection was determined. 【Result】 42 pairs of SSR primers covering all 26 chromosomes of upland cotton were selected, including 12 markers from the original standard. Among the selected 42 pairs of primers, 25 pairs amplified two loci, of which 23 revealed only one polymorphic locus, while the remaining two primer pairs (NAU1167 and HAU1413) had both loci showing polymorphism. A total of 164 alleles were detected from the 44 polymorphic loci, with the number of alleles at each locus ranging from 2 to 7. The polymorphism information content (PIC) of the loci varied from 0.15 to 0.66. 2 100 cotton varieties were genotyped using these primers. A DNA fingerprint database of known cotton varieties was constructed with a data integrity of 98.85%. Pairwise comparisons using the DNA data of 648 protected varieties, 843 approved varieties and 2 100 known upland cotton varieties revealed the distribution patterns of genetic differences between varieties among the three categories. More than 90.00% of comparisons in each category were concentrated in the range of 40.00%-70.00%. The proportion of variety pairs with genetic similarity exceeding 80.00% was 0.28%, 0.31% and 0.31% for the protected, approved and known varieties, respectively. Relationship analysis was conducted between phenotypic and genetic differences for 177 upland cotton varieties and their corresponding similar varieties. It was found that for the 54 cotton applications to which similar varieties with genetic similarity exceeding 90.00% existed, 18 (33.33%) failed to meet the distinctness requirement. In contrast, all 123 cotton applications with genetic similarity to their similar varieties below 90.00% could be clearly distinguished, exhibiting clear trait differences, thus fulfilling the distinctness criteria. The genetic similarity threshold for similar variety selection in upland cotton DUS testing based on these SSR markers could be set at 90.00%. For the variety pairs showing genetic similarity above this threshold, further field-based phenotypic evaluations are required. 【Conclusion】 In summary, NY/T 2469-2013, the standard for identifying upland cotton varieties using SSR markers, was revised. A DNA fingerprint database was constructed to support DUS testing of upland cotton in China. Furthermore, a genetic similarity threshold of 90.00% based on these SSR markers was established for selecting similar varieties, enhancing the precision and reliability of this process within DUS testing.

Screening and Identification of miRNAs in Potato Seedlings in Response to High Temperature Stress

DING Ning, QI EnFang, JIA XiaoXia, HUANG Wei, MA LiRong, LI JianWu, YAN RuNan

Scientia Agricultura Sinica. 2025, 58(22):  4589-4602.  doi:10.3864/j.issn.0578-1752.2025.22.003

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【Objective】The potato is a nutrient-rich and widespread non-grain crop that can be eaten as a staple food or vegetable. To reveal the influence of high temperature stress on the growth and development of potato seedlings, this study screened and identified small RNAs (miRNAs) and potential target genes that specifically respond to heat stress, providing a theoretical basis for research on the thermotolerance mechanism in potatoes. 【Method】The experimental material used in this study was 1-month-old tissue-cultured seedlings of Longshu 7, a potato variety independently bred by the Potato Research Institute, Gansu Academy of Agricultural Sciences. After incubation in plant growth chamber under normal temperature (17 ℃) and high temperature (28 ℃) for 10 days, samples were collected and subjected to small RNA sequencing (sRNA-seq) and transcriptome sequencing (RNA-seq). Based on high-quality sequencing data, we used bioinformatics methods to analyze and screen candidate miRNA-target gene pairs exhibiting negative regulatory relationships. In addition, real-time quantitative PCR (RT-qPCR) and dual luciferase assays (DLR) were used to validate some miRNA-target pairs. 【Result】Under high temperature stress, the abundance of 21 and 24 nt miRNAs in potato seedlings increased significantly, and the overall variation of all miRNA expression levels was 27.0% for PC1, while PC1 and PC2 accounted for 39.4% of the overall variation. According to miRNAs differential expression analysis, a total of 100 miRNAs were obtained, including 62 upregulated and 38 downregulated. Among the upregulated miRNAs, the fold change ranged from 1.02 to 6.94; while the fold change ranged from 1.01 to 7.11 in the downregulated miRNAs list. Transcriptome analysis showed that 579 differentially expressed genes were upregulated and 958 differentially expressed genes were downregulated in potato seedlings under high temperature stress. These genes were mainly involved in responding to biotic stress, external stress stimuli, and other cellular components and developmental processes. Integration of sRNA-seq and RNA-seq analyses revealed that a total of 13 miRNA-target pairs were obtained in the up-regulated miRNAs, while 5 miRNA-target gene pairs were obtained in the down-regulated miRNAs. Then, we selected three pairs of differentially expressed regulatory relationships (miRNA8051-Soltu.DM.10G026540, miRNA8051- Soltu.DM.10G026560, and miR5072-Soltu.DM.04G010170) for RT-qPCR. The results revealed that miRNAs showed opposite expression trends with their potential target genes, consistent with the results of bioinformatics analysis. In addition, we analyzed the regulatory pair of miRNA8051-Soltu.DM.10G026540 or Soltu.DM.10G026560 using DLR assay, confirming that miRNA8051 has a negative regulatory effect on both Soltu.DM.10G026540 and Soltu.DM.10G026560. 【Conclusion】It was revealed that potato seedlings under high temperature stress would regulate the target genes expression in a post-transcriptional regulatory manner, thus producing certain adaptations to the adverse environment. These candidate genes may be involved in biological processes such as transcription factors, wax synthesis and epigenetic regulation of methylation.

TILLAGE & CULTIVATION·PHYSIOLOGY & BIOCHEMISTRY·AGRICULTURE INFORMATION TECHNOLOGY

Analysis of the Production Status and Economic Benefits of Large- Scale Wheat Cultivation in Henan Province

LIU Huan, WANG GaoFeng, HUANG YuFang, ZHAO YaNan, YANG Xue, YE YouLiang

Scientia Agricultura Sinica. 2025, 58(22):  4603-4616.  doi:10.3864/j.issn.0578-1752.2025.22.004

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【Objective】The smallholder farming model has proven inadequate in meeting the demands of modern agricultural development, with large-scale planting emerging as a crucial pathway for agricultural modernization that facilitates sustainable agriculture development. This study aimed to investigate the current situation of wheat fertilization, irrigation, pest control, yield and economic benefits under different planting scales in Henan Province. Furthermore, it elucidated the impact of scale farming on wheat production, thereby providing a theoretical foundation and practical references for achieving sustainable wheat production. 【Method】A field investigation was conducted in Henan Province to examine the current situation of wheat production and its economic benefits from October 2023 to June 2024. By integrating yield with fertilizer use efficiency, this study systematically analyzed the variations in field management, wheat yield, fertilizer efficiency, and economic performance across different cultivation scales. 【Result】There were significant differences in wheat production among farmers with different cultivation scales. The majority of wheat cultivation areas in Henan Province fell within the range of 6.67-20.00 hm², with the Zhengmai wheat series being the predominant cultivar. The most common seeding rate was 225 kg·hm^-2. The application rates of nitrogen and phosphorus as basal fertilizers were lower in farms with 6.67-20.00 hm² compared with other planting scales. Phosphorus and potassium applied during topdressing were also lower than that of other planting scales ≤33.33 hm². Most surveyed farmers irrigated their fields three times, with the highest irrigation frequency observed in farms ranging from 20.00-33.33 hm². Pesticide use primarily involved insecticides and fungicides, with the highest application frequencies for both observed in the 6.67-20.00 hm² scale. Yields and partial factor productivity of fertilizers were significantly higher in farms sized 6.67-20.00 hm² and 20.00-33.33 hm² than in other scales. Wheat yields in these two groups were significantly increased by 3.57%-20.80%, and by 6.03%-23.67%, respectively, compared with other planting scales. Similarly, partial factor productivity improved by 15.87%-43.02%, and by 10.26%-36.10%, respectively. The output-input ratio was significantly higher in fields ≤6.67 hm² compared with other scales, while the highest net returns were observed at farm sizes ≤33.33 hm². 【Conclusion】Substantial variations in wheat management practices were observed across different farm scales in Henan Province. Comprehensive evaluation of yield, fertilizer partial factor productivity, net returns, and output-input ratio identified 6.67-33.33 hm² as the optimal cultivation scale. Substantial efficiency gaps were observed among large-scale wheat growers, indicating considerable potential for optimization through improved management practices. The adoption of more efficient management measures could significantly enhance the operational efficiency of different planting scales.

Effects of ARC Microbial Agent on Alleviating Functional Decline of Peanut Root Nodules Under Dark Stress

ZHAO Ya, ZHANG Wen, WANG Du, ZHANG LiangXiao, ZHANG Qi, HAN Qin, WANG Wei, LI PeiWu

Scientia Agricultura Sinica. 2025, 58(22):  4617-4627.  doi:10.3864/j.issn.0578-1752.2025.22.005

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【Objective】Root nodule senescence is a major factor limiting the symbiotic nitrogen fixation efficiency of leguminous crops. Delaying nodule senescence and prolonging its functional period is considered an effective strategy to improve crop yield and nutritional quality. To address the challenges of low nodulation efficiency, poor nitrogen fixation, and susceptibility to aflatoxin contamination in peanuts, our team has developed the ARC microbial agent, which effectively enhances nodulation while improving yield and quality. This study aims to elucidate the alleviating effects of the ARC microbial agent on peanut root nodules under dark stress, and to investigate its role in extending nodule lifespan and maintaining nitrogenase activity and functional stability. 【Method】A rapid senescence model was established by applying dark stress to peanut plants at the flowering and pegging stage. Root nodule phenotypic traits were dynamically extracted at different stress time points (0, 6, 12, 24, 36, 48, and 72 h) using the YOLOv8s algorithm, while nitrogenase activity was determined via the acetylene reduction assay. Based on both phenotypic and functional parameters, the regulatory effects of the ARC microbial agent on the rapid senescence of root nodules were comprehensively evaluated. 【Result】Compared with the control group, treatment with the ARC microbial agent significantly improved both the structural and functional traits of root nodules: the average diameter increased by 6.34%, and nitrogenase activity per gram increased by 117.11%. In addition, the average surface brightness and chroma of nodules increased by 6.32% and 3.05%, respectively. RGB color analysis further showed that nodules in the treatment group exhibited significantly higher color intensity across all channels, with mean increases of 6.32%, 8.06%, and 9.35% in the red, green, and blue channels, respectively, indicating a more vivid and brighter color appearance. During the dark stress-induced rapid senescence process, the ARC microbial agent effectively mitigated the decline in both nodule color phenotype and nitrogen fixation activity across different time points. In the early stress stage (0-6 h), the average brightness and chroma of control group nodules decreased by 8.36% and 16.85%, respectively, and nitrogenase activity dropped by 82.65%, indicating rapid onset of senescence. In contrast, nodules in the ARC treatment group showed only 1.26% and 11.31% decreases in brightness and chroma, respectively, with nitrogenase activity decreasing by 63.99%, reflecting a clear delay in early senescence and a strong protective effect. By the late stage of stress (72 h), the ARC treatment group exhibited brightness and chroma reductions of only 4.85% and 20.96%, significantly lower than the 13.82% and 23.15% declines observed in the control group, effectively alleviating the trend of color deterioration. At the same time, nitrogenase activity in the treatment group remained at 22.36% of its initial level, while nodules in the control group had already lost activity by 48 h, further confirming the sustained regulatory effect of ARC on nodule senescence. In addition, Pearson correlation analysis revealed that the red channel intensity was significantly positively correlated with nitrogenase activity (r=0.573, P=0.0003), while the green channel showed a weaker correlation and the blue channel showed no significant correlation. Notably, chroma, as a composite indicator of color variation, exhibited the strongest correlation with nitrogenase activity (r=0.736, P<0.001). Furthermore, with the progressive decline in nitrogenase activity, root nodules showed a gradual color transition from bright red to dark red and brown. 【Conclusion】The ARC microbial agent not only promotes peanut nodulation and nitrogen fixation, but also effectively delays the rapid senescence of root nodules induced by dark stress, thereby maintaining structural stability and sustaining nitrogen-fixing function. This contributes to enhanced symbiotic nitrogen fixation efficiency in leguminous crops. Moreover, nodule chroma was found to reflect changes in physiological activity and can serve as an early warning indicator of functional decline. These findings provide new perspectives and methodological support for advancing the study of nodulation and nitrogen fixation in legumes.

Exogenous Brassinolide Alleviates the Inhibitory Effect of SPVD on Sweet Potato Storage Roots Germination

DU TaiFeng, ZHOU YuanYuan, QIN Zhen, LI AiXian, WANG QingMei, ZHANG LiMing, HOU FuYun

Scientia Agricultura Sinica. 2025, 58(22):  4628-4637.  doi:10.3864/j.issn.0578-1752.2025.22.006

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【Objective】This study aimed to elucidate the mechanisms by which exogenous brassinolide (BR) alleviates the inhibitory effects of sweet potato virus disease (SPVD) complex on storage roots germination in sweet potato, so as to provide the theoretical insights for mitigating SPVD impacts on sweet potato seedling production. 【Method】SPVD-infected ‘Jishu 25’ storage roots were subjected to seed soaking treatments with varying BR concentrations (0.1, 0.5 and 1.0 μmol·L^-1) or brassinazole (BRZ, 0.1 μmol·L^-1). Germination kinetics, soluble sugar content, antioxidant enzyme activities (SOD, POD and CAT), reactive oxygen species (ROS) accumulation, malondialdehyde (MDA) content, and expression levels of BR biosynthesis genes (CYP85A1 and CYP90D1) were quantified to characterize BR/BRZ-induced alterations in cellular redox homeostasis and transcriptional regulation. 【Result】SPVD significantly suppressed storage roots germination. 7 d, 14 d, and 28 d after seedling cultivation, the storage roots of sweet potato infected with SPVD germinate later, and the number and quality of seedlings were reduced. During germination initiation stage (7-14 d): Lower BR concentrations (0.1-0.5 μmol·L^-1) enhanced quality of germination, elevated soluble sugar content, upregulated antioxidant enzyme activities, reduced ROS and MDA accumulation, and stimulated CYP85A1 and CYP90D1 expression. BRZ treatment exacerbated germination inhibition, suppressed antioxidant defenses, and increased oxidative damage. During the period of rapid germination (21-28 d), 0.1 μmol·L^-1 BR sustained germination promotion, reduced soluble sugar content, maintained high antioxidant activity, minimized oxidative stress, and further amplified BR biosynthetic genes expression. BRZ consistently impaired germination, antioxidant capacity, and BR genes expression. 【Conclusion】The germination of sweet potato storage roots infected with SPVD was significantly inhibited. The appropriate concentration of BR (0.1 μmol·L^-1) significantly alleviated the inhibition of SPVD on sweet potato storage roots germination by activating the antioxidant defense system, optimizing the dynamic balance of sugar metabolism, and enhancing the expression of endogenous BR synthesis genes.

Spatiotemporal Analysis of Cropland Cropping Intensity in Hubei Province from 2000 to 2021 by Integrating Multi-Scale Remote Sensing Imagery

HU Jie, MA HaiRong, LUO ZhiQing, CHEN PingTing, ZHENG MingXue, GUAN Bo, XU BaoDong, SONG Qian

Scientia Agricultura Sinica. 2025, 58(22):  4638-4655.  doi:10.3864/j.issn.0578-1752.2025.22.007

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【Objective】 To address the uncertainty in the extraction and dynamic monitoring of cropland cropping intensity (CI) caused by frequent cloud cover, fragmented farmland, and multi-cropping systems in southern China, this study aimed to fully leverage the advantages of multi-scale remote sensing observations to achieve efficient and accurate CI mapping for Hubei Province from 2000 to 2021, and to analyze the spatiotemporal evolution of regional agricultural production patterns. 【Method】 Time-series NDVI data from 250 m MODIS and 30 m Landsat were integrated using four representative spatiotemporal fusion algorithms: STARFM, ESTARFM, STNLFFM, and GF-SG. Fusion performance was comprehensively evaluated based on spectral fidelity (AD, RMSE) and spatial detail accuracy (Edge, LBP). The optimal algorithm was used to generate a 30 m/8-day NDVI dataset for 2000-2021. Cropland CI was extracted using a phenology-based peak detection method, and then its spatiotemporal variation was analyzed. 【Result】Compared with the other three spatiotemporal fusion algorithms, the GF-SG algorithm demonstrated the best performance in both spectral fidelity and spatial detail accuracy (|AD|<0.021, RMSE<0.111; |Edge|<0.55, |LBP|<0.10). The reconstructed NDVI time series using this algorithm improved the accuracy of cropland CI extraction by 0.02%-5.53%. Based on ground samples, the overall classification accuracy of cropland CI in Hubei Province reached 86.60%. From 2000 to 2021, approximately 20%-25% of croplands in the study area experienced CI transitions every five years, with the most significant changes occurring between 2005-2010 (25.79%) and the least between 2010-2015 (20.07%). The dominant transition type shifted from 'single-cropping to double-cropping' (13.49%) in the early years to 'double-cropping to single-cropping' (9.35%) and 'single-cropping to fallow' (4.90%) in the later years. 【Conclusion】Over the past two decades, Hubei Province has developed a diversified cultivation pattern dominated by single cropping, with coexistence of double cropping and fallow practices. The evolution of cropland CI has been jointly driven by policy guidance, labor force changes, resource input, and adjustments in cropping structure. By integrating multi-scale remote sensing data from MODIS and Landsat, this study constructed a high spatiotemporal resolution NDVI dataset, which enabled efficient and accurate extraction of long-term cropland CI in complex agricultural landscapes. The findings offered the critical support for agricultural production management and the development of cropland protection policies.

PLANT PROTECTION

Transcriptome Analysis of Vicia faba Response to Alternaria alternata Infection and Validation of the Disease Resistance Function of VfPR4

WANG Fan, LIU ChenWei, LU HongChen, XU RenChao, BIAN XiaoChun

Scientia Agricultura Sinica. 2025, 58(22):  4656-4672.  doi:10.3864/j.issn.0578-1752.2025.22.008

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【Objective】Leaf spot is a new type of fungal disease that restricts the production of broad beans, and Alternaria alternata is one of its main pathogens. The objective of this study is to explore the molecular pathways of broad beans in response to the infection of A. alternata through transcriptome analysis and disease resistant gene function verification. 【Method】Broad bean cultivar CD-006 was used as the experimental material, transcriptome sequencing was performed on the leaves inoculated with A. alternata at 0, 6, 12 and 24 h to screen differentially expressed genes (DEGs). GO (gene ontology) functional annotation and KEGG (Kyoto encyclopedia of genes and genomes) metabolic pathway enrichment analysis were conducted to excavate disease resistance related genes. qRT-PCR (quantitative real-time PCR) was conducted to validate transcriptome sequencing results, and VfPR4 was screened for genetic transformation in tobacco. To complete disease resistance function analysis of VfPR4, invasive inoculation method was used. 【Result】At 6, 12 and 24 h after infection, 3 537, 3 152 and 2 947 DEGs were up-regulated in broad beans, while 1 181, 1 453 and 1 319 DEGs were down-regulated. GO enrichment analysis revealed that DEGs were mainly enriched in oxidoreductase activity and some biosynthetic (metabolic) processes, and KEGG enrichment analysis found that DEGs were mainly enriched in pathways such as biosynthesis of secondary metabolites, phenylpropanoid biosynthesis, and flavonoid biosynthesis. At 6 h after infection, KEGG was also enriched in MAPK (mitogen-activated protein kinase) signaling pathway and plant-pathogen interaction pathway, which together enriched 32 DEGs. A total of 52 PR genes were discovered in transcriptome analysis, among which 35 genes were differentially expressed. Six DEGs were selected for qRT-PCR validation, and the validation results were consistent with the transcriptome sequencing results. Among the above genes, the VfPR4 remained up-regulated within 24 h after infection with A. alternata. Tobacco genetic transformation results indicated that the lesion symptoms of transgenic lines were milder, with lesion areas of 128.94, 110.57 and 92.92 mm², respectively, which were significantly smaller than that of the wild type, with a lesion area of 174.32 mm². 【Conclusion】In this study, transcriptome analysis revealed that broad beans might mainly respond to the infection of A. alternata through pathways including oxidative burst, secondary metabolites biosynthesis, phenylpropanoid biosynthesis, MAPK signaling, and plant-pathogen interaction. VfPR4 could positively regulate tobacco’s resistance to A. alternata.

Structure and Herbicidal Activity of 3,5-Dimethylorsellinic Acid-Derived Meroterpenoids from Penicillium sclerotiorum HY5

CAI HaiLin, HUANG XinRong, ZENG WeiAi, PENG ZhiXin, ZHAO AJuan, BO ChunBin, XIANG ShiPeng, GAO PengCheng, ZHANG ChengSheng, ZHAO DongLin

Scientia Agricultura Sinica. 2025, 58(22):  4673-4687.  doi:10.3864/j.issn.0578-1752.2025.22.009

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【Background】3,5-Dimethylorsellinic acid (DMOA)-derived meroterpenoids exhibit diverse chemical structures and broad biological activities. However, their agricultural bioactivities have rarely been reported. Chemical herbicides often lead to increased weed resistance and environmental problem, highlighting the urgent need for the development of novel bioherbicides. 【Objective】The objective of this study is to explore DMOA-derived meroterpenoids from Penicillium sclerotiorum HY5, evaluate their herbicidal activity, and preliminarily elucidate their modes of action, thus to provide lead molecules for the development of novel natural-product herbicides. 【Method】DMOA-derived meroterpenoids were isolated using normal/ reversed-phase silica gel column chromatography (CC), thin-layer chromatography, Sephadex LH-20 gel CC, and semi- preparative high-performance liquid chromatography (HPLC). The structures of the compounds were identified via nuclear magnetic resonance (NMR) and mass spectrometry (MS). The herbicidal activity of the compounds was assessed using a seed germination inhibition assay against Eleusine indica, Setaria viridis, Amaranthus retroflexus, and Trifolium repens. The EC₅₀ values for the bioactive compounds were determined by nonlinear regression analysis of dose-response curves. The herbicidal mechanism of the bioactive compounds was analyzed using a combined transcriptomic-metabolomic approach. 【Result】Nine DMOA-derived meroterpenoids were obtained from the HY5 fermentation extract, namely berkeleydione (1), preaustinoid A3 (2), miniolutelide B (3), 22-epoxyberkeleydione (4), 22-deoxyminiolutelide B (5), miniolutelide C (6), berkeleyacteal (7), preaustinoid A2 (8), and berkeleyacetal A (9). Compound 4 showed a significant promoting effect on the growth of the dicotyledonous weed T. repens, with the promotion rates of radicle and germ growth reaching 30.5% and 55.1%, respectively. Compounds 3, 4, 5, and 6 exhibited significant inhibitory effects on the germination of monocot weed seeds (S. viridis and E. indica). Notably, compound 6 showed the strongest herbicidal activity, with EC₅₀ values of 19.1 and 17.9 μg·mL^-1 for radicle and germ elongation of E. indica respectively, even stronger than the positive control glyphosate (30.9 and 66.2 μg·mL^-1). Additionally, the compounds demonstrated more potent herbicidal activity against radicle elongation compared to germ. Further studies revealed that compound 6 inhibits weed germination through dual regulatory mechanisms: (1) degrading JAZ proteins in the jasmonic acid signaling pathway, thereby promoting abscisic acid synthesis, and (2) disrupting the tryptophan metabolism pathway, leading to abnormal serotonin accumulation and ultimately suppressing weed growth. 【Conclusion】DMOA-derived meroterpenoids possess novel structures and exhibit significant inhibitory effects on monocot weeds, showing promise for development as new natural-product herbicides.

SOIL & FERTILIZER·WATER-SAVING IRRIGATION·AGROECOLOGY & ENVIRONMENT

Optimizing Wheat Nitrogen and Phosphorus Fertilizer Rates Based on Apparent Nitrogen and Phosphorus Balance in a Long-Term Location Fixed Field Experiment

ZHANG Feng, XU JunFeng, GAO ZhiYuan, DANG HaiYan, GUO RongBo, SHE WenTing, LI WenHu, LIU JinShan, WANG ZhaoHui

Scientia Agricultura Sinica. 2025, 58(22):  4688-4702.  doi:10.3864/j.issn.0578-1752.2025.22.010

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【Objective】This study aimed to explore the relationship of nitrogen (N) and phosphorus (P) fertilizer rates to wheat grain yield and protein content, the N and P removal and their balance in soil in a long-term and location fixed field experiment, for scientifically reducing fertilizer inputs, and wheat grain yield increase and nutritional quality improvement. 【Method】A long-term and location-fixed field experiment was initiated in 2004 in the Loess Plateau, and samples were collected during the cropping years of 2022-2024, to investigate effects of N and P fertilization on wheat yield, yield components, grain protein content, grain P and potassium (K) content, soil nutrient total accumulation, and apparent N and P balances. 【Result】Wheat yield and grain protein content were found quadratically correlated with the N rate, and the highest yield of 6 905 kg·hm^-2 and highest protein content of 145.4 g·kg^-1 were attained at 196 kg·hm^-2 and 276 kg·hm^-2 of N, respectively. When the fertilizer N input was equal to the wheat N removal at the N rate of 162 kg·hm^-2, wheat yield and protein content were 6 853 kg·hm^-2 and 135.9 g·kg^-1, respectively, which were 0.8% and 3.4% lower than those when the yield was maximized, and 3.7% and 6.6% lower than those when the grain protein was maximized, but the N rate was reduced by 17.3% and 41.4%, and soil nitrate N total accumulation was reduced by 21.7% and 51.6%, respectively. As the grain production target was set to realize the medium-strong grain gluten protein of 130 g·kg^-1, the N rate could be reduced by 19.2% to 131 kg·hm^-2 with the yield as high as 6 713 kg·hm^-2 and the soil nitrate N total accumulation reduced by 22.7%, compared with that when the fertilizer N input was equal to the wheat N removal. Wheat yield and grain protein content were quadratically correlated with the P rate, and the highest yield of 6 565 kg·hm^-2 and the lowest protein content of 121.8 g·kg^-1 were attained at 55.3 and 87.3 kg·hm^-2 of P rate, respectively. When the fertilizer P input was equal to the wheat P removal at the P rate of 11.4 kg·hm^-2, wheat yield and protein content were 4 678 kg·hm^-2 and 142.6 g·kg^-1, respectively, and the P rate was reduced by 79.5%, protein content was increased by 11.0%, but yield was reduced by 28.7%, and soil N total accumulation was increased by 82.3%, compared with that when the yield was maximized. With the grain production target was set to realize the medium-strong gluten grain protein of 130 g·kg^-1, the P rate could be increased by 329.5% to 48.8 kg·hm^-2 and the yield could be increased by 39.4% to 6 523 kg·hm^-2, and also nitrate N total accumulation was reduced by 41.1% in soil, compared with that when the fertilizer P input was equal to the wheat P removal. 【Conclusion】For balancing wheat yield and grain protein increase as well as soil nutrient balance in calcareous soils at the northwestern dryland area, the wheat production should aim at high yields and medium-strong grain gluten protein, and N application rates should be 0.64-0.88 times of the aboveground N accumulation, as well as the P application rates should be 2.02-2.22 times of the aboveground P accumulation under the growing system when the crop straw was returned.

Effects of Continuous Reduction Fertilization on the Stability of Spring Wheat Grain Quality in the Yellow River Irrigation Area of Ningxia

QIAN ZhiJin, WANG XiNa, TIAN HaiMei, WANG YueMei, HAO WenYue, ZHOU Hui, TAN JunLi

Scientia Agricultura Sinica. 2025, 58(22):  4703-4717.  doi:10.3864/j.issn.0578-1752.2025.22.011

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【Objective】The objective of this study was to investigate the effect of continuous reducing chemical fertilizer on the stability of spring wheat grain quality, to evaluate the sustainability of chemical fertilizer reduction techniques, and then to provide a theoretical basis for the rational reduction of chemical fertilizers in spring wheat in the Yellow River Irrigation Area of Ningxia. 【Method】A positioning field experiment on the reduction of nitrogen, phosphorus, and potassium fertilizers was conducted with Ningchun 4 cultivar of spring wheat as the test crop from 2019 to 2023. According to the fertilizer response functions established in previous experiments on nitrogen, phosphorus, and potassium application rates, the treatments of the upper limit of fertilizer reduction (RF1: applying 180 kg of N, 45 kg of P₂O₅, and 30 kg of K₂O per hectare) and the lower limit of fertilizer reduction (RF2: applying 225 kg of N, 75 kg of P₂O₅, and 45 kg of K₂O per hectare) were designed. Additionally, a conventional fertilization treatment (CF: applying 270 kg of N, 150 kg of P₂O₅, and 75 kg of K₂O per hectare) and a no- fertilization (CK) were set as controls. The variations in the SPAD value, leaf area, dry matter allocation, and photosynthetic capacity of spring wheat flag leaves from 2021 to 2023 were measured to analyze the stability of grain quality. 【Result】During 2022-2023, compared with the conventional fertilization treatment, the SPAD value and leaf area under RF1 treatment did not decrease significantly (P>0.05), and those under RF2 treatment tended to increase, yet without reaching a significant level. As for the photosynthetic indicators, in 2021 and 2023, the net photosynthetic rate, stomatal conductance, and transpiration rate were the highest under RF2 treatment, which was increased by 29.8% and 25.0%, 6.1% and 6.9%, 5.4% and 16.6%, compared with CF treatment, respectively, while RF1 treatment showed a certain degree of decline compared with CF treatment. From 2021 to 2023, the total dry matter of spring wheat presented the order of RF2 > CF > RF1 > CK. In 2021, CK treatment significantly decreased by 53.3%, 50.1% and 56.6% compared with CF, RF1 and RF2 treatment, respectively, and significantly decreased by 67.2%, 57.9% and 67.3% in 2023, respectively. In 2021 and 2023, the distribution rate of dry matter to grain was the highest under RF2, which was 38.3% and 38.6%, respectively. For grain quality, appropriate reducing chemical fertilizer application (RF2) was conducive to and continuously increases the crude protein content and soluble sugar content in spring wheat grains; compared with the CF treatment, in 2021 and 2023, they increased by 6.3% and 1.0%, 14.4% and 1.8%, and 12.2% and 1.4%, respectively. However, excessive reduction of chemical fertilizer application (RF1) led to a decreasing trend in crude protein and soluble sugar content. Except for the no-fertilization control, the RF1 treatment resulted in the lowest protein yield, which was significantly reduced by 46.68% and 38.72% compared to the CF treatment in 2021 and 2023, respectively.The protein stability index was based on the lowest RF2. There was little difference in the soluble-sugar stability index among different treatments, and the starch stability index was the minimum under RF1 treatment. The crude protein content in grains was extremely significantly and positively correlated with the SPAD value, leaf area, net photosynthetic rate, stomatal conductance, intercellular CO₂ concentration, transpiration rate, dry matter accumulation in vegetative organs and grains of the flag leaves during the grain-filling period of spring wheat. Meanwhile, there were extremely significant positive correlations among the SPAD value, leaf area, net photosynthetic rate, stomatal conductance, transpiration rate, and dry matter accumulation in vegetative organs and grains at the maturity stage. The soluble sugar content in grains was positively correlated with stomatal conductance, intercellular CO₂ concentration, and transpiration rate. Through principal component analysis, it was found that the load values of grain dry matter mass, transpiration rate, net photosynthetic rate, stomatal conductance, crude protein content, starch content, and leaf area were relatively high, and the quality of spring wheat under RF2 treatment was the best. 【Conclusion】In the Yellow River Irrigation Area of Ningxia, the continuous and appropriate reduction of chemical fertilizers (applying 225 kg N·hm^-2, 75 kg P₂O₅·hm^-2 and 45 kg K₂O·hm^-2, with a 17.0% reduction in N, a 50.0% reduction in P₂O₅, and a 40.0% reduction in K₂O compared to CF) could, to a certain extent, increase the SPAD value and leaf area of the flag leaves during the grain-filling stage of spring wheat, improve its photosynthetic efficiency, and promote dry matter accumulation and its translocation and distribution to grains, thereby stably improving the quality of grain, especially for the content of crude protein and soluble sugar.

Effects of Nitrogen Fertilizer Reduction and Green Manure Mixed Cropping on Soil Quality and Wheat Yield

ZHANG MingLong, CHAI Qiang, HAN Mei, FAN ZhiLong, YIN Wen, FAN Hong, HE Wei, SUN YaLi, HU FaLong, TAN Yan

Scientia Agricultura Sinica. 2025, 58(22):  4718-4731.  doi:10.3864/j.issn.0578-1752.2025.22.012

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【Objective】This study aimed to screen a suitable green manure configuration mode to reduce the amount of nitrogen applied to wheat, so as to provide the theoretical basis and technical support for improving soil quality and increasing wheat yield and stable yield in Qinghai Plateau. 【Method】The split plot experiment was carried out in the experimental base of the Academy of Agricultural and Forestry Sciences of Qinghai University from 2022 to 2023. The main plot was set as nitrogen application level, namely 225 kgN·hm^-2 (N2, customary nitrogen application), and 158 kgN·hm^-2 (N1, 30% reduction of customary nitrogen application); the split plot was divided into four green manure configuration modes, namely common vetch monoculture (CV), common vetch and highland barley mixed cropping (CV×CH), common vetch and hairy vetch mixed cropping (CV×HV), and common vetch and rapeseed mixed cropping (CV×RS). 【Result】The soil bulk density and pH of common vetch mixed with highland barley were 8.6%-10.5% and 3.3%-3.4% lower than those of common vetch monoculture, 3.3%-5.8% and 1.6%-1.9% lower than those of common vetch mixed with hairy vetch, and 4.9%-7.5% and 2.3%-2.4% lower than those of common vetch mixed with rape, respectively. The soil organic matter, total nitrogen, total phosphorus, total potassium, nitrate nitrogen, ammonium nitrogen, available phosphorus and available potassium in the mixed cropping of common vetch were 20.0%-22.3%, 8.3%-9.7%, 11.2%-15.6%, 14.6%-16.4%, 14.8%-20.1%, 16.8%-18.9%, 16.4%-19.4%, 23.8%-24.6% higher than those in the single cropping of common vetch, 12.6%-13.8%, 3.9%-4.2%, 3.1%-4.9%, 4.8%-5.5%, 7.5%-8.2%, 8.2%-8.7%, 6.1%-7.7%, 6.9%-10.7% higher than those of common vetch mixed with hairy vetch, and 26.7%-35.4%, 14.3%-14.4%, 7.0%-8.2%, 7.9%-9.8%, 23.5%-28.0%, 23.5%-25.3%, 11.5%-12.3%, 16.5%-19.6% higher than those of common vetch mixed with rape, respectively. The activities of soil sucrase and catalase were increased by 3.2%-4.1% and 4.5%-4.9%, respectively, in the 30% nitrogen reduction treatment compared with the conventional nitrogen application. In addition, the activities of soil sucrase, urease, catalase, and alkaline phosphatase were increased by 20.1%-24.7%, 10.5%-14.4%, 12.2%-16.9%, and 12.2%-12.6%, respectively, in the common vetch mixed cropping of highland barley compared with the common vetch monoculture, 12.0%-12.5%, 4.2%-5.2%, 6.4%-7.6%, 4.7%-6.2% compared with the common vetch mixed with hairy vetch, and 28.1%-33.6%, 15.2%-19.1%, 18.7%-24.2%, 5.7%-5.8% compared with the common vetch mixed with rape, respectively. The soil quality index of 30% nitrogen reduction treatment was 17.1% higher than that of the traditional nitrogen application. Furthermore, the soil quality index for the intercropping of common vetch with hulless barley and the intercropping of common vetch with hairy vetch were 142.3% and 76.6% higher, respectively, compared with the single cropping of common vetch. Under the condition of a 30% reduction in nitrogen fertilizer application, the wheat grain yield of the treatment with common vetch intercropped with hulless barley did not show significant difference compared to traditional nitrogen application. However, it was 10.4%-12.5% and 4.8%-5.9% higher, respectively, than that of single cropping of common vetch and intercropping of common vetch with hairy vetch. The Mantel test indicates that soil quality index and wheat yield were significantly positively correlated with soil organic matter, total nutrients, available nutrients, and enzyme activity, while they were significantly negatively correlated with soil bulk density and pH. Additionally, the random forest model identified soil organic matter, total nitrogen, ammonium nitrogen, nitrate nitrogen, sucrase activity and urease activity were the key factors influencing wheat yield. 【Conclusion】The 30% nitrogen reduction treatment combined with common vetch mixed with highland barley could improve soil quality and wheat yield by improving soil physical and chemical properties and soil enzyme activity, which could be used as a suitable production mode in Qinghai Plateau.

HORTICULTURE

Functional Analysis of CaIAA8, An Interacting Protein of the Autophagy-Related Protein CaATG8c, in the Heat Tolerance of Pepper

LI NiFei, YANG QiaoMin, YANG KeCheng, XING YuTeng, WANG MengYuan, ZANG TianBao, LU MingHui

Scientia Agricultura Sinica. 2025, 58(22):  4732-4745.  doi:10.3864/j.issn.0578-1752.2025.22.013

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【Objective】Autophagy is a conserved protein degradation system in eukaryotic cells and plays an important role in plant stress tolerance. Previous studies showed that silencing the autophagy-related gene CaATG8c reduces heat tolerance in pepper. A candidate interacting protein of CaATG8c, the auxin responsive protein CaIAA8, was identified via yeast two-hybrid screening. This study aimed to analyze the sequence characteristics of CaIAA8, confirm its interaction with CaATG8c, and investigate its role in regulating heat tolerance in pepper, thereby providing insights into the molecular mechanisms of heat tolerance and supporting the breeding of heat-tolerant cultivars. 【Method】 Protein sequence alignment, phylogenetic analysis, and conserved motif identification of CaIAA8 were conducted using BLASTP (NCBI), IQ-TREE, and MEME, respectively. Protein physicochemical properties, signal peptide, transmembrane domain, and phosphorylation sites were predicted using ExPASy, SignalP-6.0, TMHMM-2.0, and NetPhos, respectively. Protein secondary and tertiary structures were predicted using SOPMA and SWISS-MODEL respectively. The interaction between CaATG8c and CaIAA8 was validated by yeast two-hybrid and luciferase complementation assays. Subcellular localization of CaIAA8 was examined by transient expression in tobacco leaves. The expression pattern of CaIAA8 gene in pepper was analyzed using real-time quantitative PCR (RT-qPCR), and its function in heat tolerance was further evaluated by virus-induced gene silencing (VIGS) and transient overexpression in pepper. 【Result】CaIAA8 encodes a 349-amino-acid protein with a molecular weight of 37.48 kDa and an isoelectric point of 7.52. It contains 65 predicted phosphorylation sites and is a basic, hydrophilic, and structurally unstable protein. CaIAA8 localizes primarily to the cytoplasm and co-localizes with chloroplasts. Its expression is highest in stems, followed by fruits and flowers, and lowest in leaves. Under 45 ℃ heat stress, CaIAA8 transcript level decreases. Silencing CaIAA8 enhances pepper heat tolerance, as evidenced by reduced leaf wilting, lower relative electrolyte leakage, decreased cell death and H₂O₂ accumulation, and increased expression levels of heat shock protein and heat shock transcription factor genes. In contrast, transient overexpression of CaIAA8 reduces pepper heat tolerance, leading to more severe heat-induced damage and elevated levels of cell death and H₂O₂. 【Conclusion】CaIAA8 physically interacts with CaATG8c and functions as a negative regulator of heat tolerance in pepper.

Development and Application of A KASP Marker-Based Identification System for Tomato Varieties

SU XiaoMei, YANG ZongHui, LIU ShuMei, ZHANG ZongJie, LÜ HongJun, HOU LiXia

Scientia Agricultura Sinica. 2025, 58(22):  4746-4756.  doi:10.3864/j.issn.0578-1752.2025.22.014

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【Objective】To screen KASP core markers and establish a high-throughput molecular identification system for tomato, providing technical support for tomato variety authentication, purity testing, and plant variety protection. 【Method】Based on re-sequencing data of tomato inbred lines, high-quality SNP loci covering the whole genome were selected for KASP markers design. A total of 360 tomato varieties, collected from different breeding research institutions and private companies, were used to validate the designed KASP markers. According to genotyping data, a set of KASP core markers with high polymorphism information content (PIC), excellent stability, and relatively uniform distribution across chromosomes was screened. Cluster analysis and DNA fingerprinting of the collected tomato varieties were performed, and the core markers were applied to authenticate variety authenticity and assess seed purity. 【Result】Utilizing the SNPs obtained from re-sequencing data analysis of over 300 tomato materials, 600 SNP loci were selected for marker development, averaging 50 per chromosome. Among these, 517 were successfully converted into KASP markers, with a conversion success rate of 86.2%. Based on physical distances on chromosomes, 10-12 markers per chromosome were prioritized, totaling 128 markers, which were validated and refined using diverse tomato varieties. Ultimately, 68 core primer pairs were selected, averaging 4-8 per chromosome. Genotyping analysis of 360 tomato varieties using these 68 core markers revealed similarity coefficients ranging from 0.32 to 1.00 in cluster analysis. For four tomato types (fresh tomatoes, flavor tomatoes, cherry tomatoes and processing tomatoes), the core marker sets were streamlined to 48, 32, 36, and 36 markers, respectively, with PIC values all exceeding 0.35. These markers effectively distinguished different varieties. To validate marker applicability, two flavor tomato varieties were authenticated using core markers, revealing two differing loci, confirming their distinctness. Additionally, two polymorphic markers between parental lines were used to assess the purity of hybrid seeds of Pinfan 4031, yielding 100% purity, consistent with field evaluation results. 【Conclusion】The developed KASP markers are applicable for tomato genetic diversity analysis, DNA fingerprinting, variety authenticity testing, and hybrid seed purity evaluation.

FOOD SCIENCE AND ENGINEERING

Characterization of Non-Volatile Metabolites of White Peony Tea Make of Camellia sinensis Fu’an-dabaicha from Different Origins

LUO Qin, CHEN XieYong, XU YuYing, WEI Han, HUANG Biao, YAO QingHua, YE NaiXing, ZHENG DeYong, YAN MingJuan

Scientia Agricultura Sinica. 2025, 58(22):  4757-4770.  doi:10.3864/j.issn.0578-1752.2025.22.015

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【Objective】 This study aimed to explore the discrimination technology of the origin of small-region white peony tea based on non-volatile metabolite characteristics. 【Method】Ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry was used to determine the composition and content of non-volatile metabolites in white peony tea made of Camellia sinensis Fu’an-dabaicha varieties from three different production areas, including Zhenghe County (ZH), Songxi County (SX), and Fu’an City (FA). Combined with mathematical statistics methods, characteristic compounds were screened, and a discrimination model of the origin of white peony tea was constructed. 【Result】 After screening and filtering, 219 kinds of non-volatile metabolites were identified in the white peony tea from the three regions. Among them, polyphenol metabolites accounted for the highest proportion in the tea samples from the three production areas, with 30.921%, 29.046%, and 29.110% in Fu’an, Songxi, and Zhenghe, respectively. Analysis of variance showed that polyphenols and amino acids and their derivatives had significant differences among the three production areas (the order of polyphenol content from high to low was FA, SX, and ZH, while the order of amino acid and their derivatives content from high to low was ZH, FA, and SX (P<0.01), while organic acids and nucleotides and their derivatives had no significant differences among the three production areas (P>0.05). Among the 219 metabolites, 158 non-volatile metabolites, such as 5-formylsalicylic acid and quercetin 3-D-galactoside, showed significant or extremely significant differences. The orthogonal partial least squares discriminant analysis (OPLS-DA) model did not show overfitting, and the model validation effect was good. The prediction index and discrimination accuracy of the model were 0.859 and 98.89%, respectively. There were 85 characteristic non-volatile metabolites with variable importance in projection (VIP)≥1. Stepwise linear discriminant analysis (SLDA) selected 16 metabolites into the model, and the initial and cross-validation discrimination accuracy of the constructed discrimination model were both 100.00%. 【Conclusion】A discrimination technology of the origin of small-region white peony tea based on non-volatile metabolite characteristics was established. Both OPLS-DA and SLDA discrimination models can effectively discriminate against the origin of white peony tea based on non-volatile metabolites, which could provide the theoretical references for the traceability and brand building of tea origins.

ANIMAL SCIENCE·VETERINARY SCIENCE

Study on the Antioxidant Function and Mechanism of 18β-glycyrrhetinic Acid Using Porcine Intestinal Organoid Model

LI RuiTong, CHEN QingMei, XU JianFang, ZHANG JunMin, SI Wei, ZHANG TieYing

Scientia Agricultura Sinica. 2025, 58(22):  4771-4785.  doi:10.3864/j.issn.0578-1752.2025.22.016

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【Background】Oxidative stress is a key factor compromising intestinal health and impeding the growth and development of pigs. Consequently, the identification and development of natural and effective antioxidant compounds are of great significance for enhancing animal health and production efficiency. 18β-Glycyrrhetinic acid (GA), a pentacyclic triterpenoid extracted from Glycyrrhiza spp., has been recognized for its antioxidant activity. 【Objective】This study aimed to establish an oxidative stress injury model based on porcine intestinal organoids to assess the antioxidant efficacy of GA and to explore its potential underlying mechanisms. The findings were intended to provide a theoretical foundation for the application of GA as a functional antioxidant additive in livestock and poultry nutrition. 【Method】Intestinal crypts were isolated and collected from the jejunal tissues of piglets and used to construct porcine intestinal organoids via a three-dimensional culture system. The structural and functional integrity of the organoids was confirmed by immunofluorescence staining for intestinal epithelial stem cell and differentiation markers, including LGR5, β-catenin, Ki67, PCNA, c-Myc, CDX2, and lysozyme, indicating the presence of typical intestinal epithelial lineages. After 48 hours of passaging, organoids were treated with hydrogen peroxide (H₂O₂) at concentrations of 0, 100, 250, 500, 750, 1 000, 1 500, and 2 000 μmol·L^-1 for three days to induce oxidative stress, respectively. The optimal H₂O₂ concentration for model establishment was determined through comprehensive evaluation of morphological parameters (budding rate, branching coefficient, and bud condition), cellular proliferation activity (EdU incorporation and Ki67 expression), intracellular reactive oxygen species (ROS) levels (detected by fluorescent probes), and expression of key genes in the Wnt/β-catenin signaling pathway (Lgr5, C-myc, Cyclin D1, β-catenin, PCNA, and AXIN2). Subsequently, GA at concentrations of 10, 50, and 100 μmol·L^-1 was applied to the oxidative stress model to assess its protective effect. The antioxidant capacity and underlying mechanism of GA were investigated by analyzing organoid morphology, proliferation activity, intracellular ROS accumulation, and expression of representative proteins associated with the Wnt/β-catenin signaling pathway (β-catenin, PCNA, LGR5, and CDX2). 【Result】After 48 hours of passaging, treatment of porcine intestinal organoids with 1 mmol·L^-1 H₂O₂ for three days successfully established a stable oxidative stress injury model. This was demonstrated by a significant decrease in the organoid budding rate and branching coefficient, accompanied by a marked reduction in the proportion of EdU-positive cells and Ki67 expression. Moreover, oxidative stress induced activation of the Wnt/β-catenin signaling pathway, as evidenced by significant upregulation of C-myc expression. Under normal culture conditions, GA at concentrations of 10, 50, and 100 μmol·L^-1 exerted no adverse effects on organoid growth. However, under oxidative stress conditions, 100 μmol·L^-1 GA exhibited the most pronounced protective effect, significantly enhancing the budding rate and branching coefficient, and promoting cell proliferation as indicated by increased EdU-positive cell proportion and elevated Ki67 expression. Additionally, GA treatment effectively reduced ROS accumulation and inhibited the activation of the Wnt/β-catenin pathway, demonstrated by the downregulation of β-catenin, PCNA, and LGR5 protein expression levels. 【Conclusion】In this study, a porcine intestinal organoid model of oxidative stress was successfully established. Based on this model, the protective role of GA against oxidative damage in organoids was confirmed, and its underlying mechanism was preliminarily elucidated. GA exerted notable antioxidant activity by improving organoid morphological development, promoting cellular proliferation, reducing intracellular ROS levels, and downregulating the Wnt/β-catenin signaling pathway. These findings laid a theoretical foundation for the functional development and practical application of GA in the nutritional regulation and health management of livestock and poultry.

Regulatory Effect of FTO Gene on Lipid Deposition in Chicken Intramuscular Adipocytes

HUANG HuaYun, SUI YuLe, KONG Yi, LIANG Zhong, YANG MiaoMiao, LIU Xing, HAN Wei

Scientia Agricultura Sinica. 2025, 58(22):  4786-4796.  doi:10.3864/j.issn.0578-1752.2025.22.017

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【Objective】 The fat mass and obesity-associated gene (FTO) is closely related to fat deposition in humans and livestock/poultry. However, its role in intramuscular fat deposition in chickens has not been reported yet. This study aims to clarify the effect of FTO on the proliferation and lipid droplet deposition of chicken intramuscular adipocytes, screen the key pathways and genes responsive to its regulation, and provide a theoretical basis for analyzing the molecular regulatory network of intramuscular fat deposition in chickens. 【Method】In this study, an FTO lentiviral expression vector was constructed, and chicken primary intramuscular adipocytes were transfected with the FTO lentiviral expression vector and a negative control vector. Fluorescence was observed using a fluorescence inverted microscope, and quantitative PCR (QPCR) was performed to detect changes in FTO gene expression in the transfection and negative control groups, thus ensuring successful transfection of the FTO lentiviral expression vector into the cells. Cell proliferation changes in the negative control and transfection groups were then detected using a CCK8 assay at 72 h and 96 h post-transfection with the FTO lentiviral expression vector. At the sixth day of transfection, oil red O staining was performed, lipid droplets were extracted with isopropanol to detect changes in intramuscular adipocyte lipid droplet deposition, and a glycerol assay kit was used to detect changes in glycerol content in the culture medium. Finally, key pathways and genes responding to FTO regulation of intramuscular adipocyte lipid deposition were screened through transcriptome sequencing, bioinformatics analysis, and PPI analysis. 【Result】Following transfection of intramuscular adipocytes with the FTO lentiviral expression vector, QPCR analysis revealed a significant increase in FTO mRNA expression in the transfected group compared with the control group (P<0.01), confirming successful transfection of the FTO lentiviral expression vector into the cells. The proliferation capacity of intramuscular adipocytes was significantly reduced at 72 h and 96 h post-transfection with the FTO lentiviral expression vector, compared with the control group (P<0.05). After six days of transfecting intramuscular adipocytes with the FTO lentiviral expression vector, the transfected group exhibited a significantly higher capacity for lipid droplet deposition than the negative control group (P<0.05), while the glycerol content in the culture medium was significantly lower (P<0.05). Transcriptome sequencing identified a total of 164 differentially expressed genes responding to FTO-regulated intramuscular fat deposition, with 71 genes upregulated and 93 downregulated. KEGG enrichment analysis of these differentially expressed genes revealed significant enrichment in 13 signaling pathways, including focal adhesion, regulation of actin cytoskeleton, MAPK signaling pathway, gap junction, cytokine-cytokine receptor interaction, FoxO signaling pathway, apoptosis, C-type lectin receptor signaling pathway, and TGF-beta signaling pathway (P<0.05), all of which were directly or indirectly related to lipid metabolism. Key pathways and genes responsive to FTO regulation were further screened, and PPI (Protein-Protein Interaction) analysis on the differentially expressed genes in significantly enriched pathways was performed. The results showed that PIK3R2, FGF16, FGF9, RHOA and NGF were key genes for FTO-regulated intramuscular adipocyte lipid deposition in chickens. Among them, PIK3R2, FGF16, FGF9, and RHOA were all enriched in the regulation of actin cytoskeleton pathway, which was identified as the core pathway. 【Conclusion】 The FTO gene inhibited intramuscular adipocyte proliferation, promoted lipid deposition, and suppressed lipolysis in chickens. PIK3R2, FGF16, FGF9 and RHOA (regulation of actin cytoskeleton pathway) have been identified as key genes/pathways that responded to FTO regulation of intramuscular adipocyte lipid deposition.

RESEARCH NOTES

Biochemical Characteristics Analysis and Suitability Evaluation of Liannan Daye Tea Germplasm Resources

TIAN XianXian, FENG ShaoFang, WANG Qing, PAN ChenDong, LI Bo, FANG KaiXing, WU HuaLing, QIN DanDan

Scientia Agricultura Sinica. 2025, 58(22):  4797-4812.  doi:10.3864/j.issn.0578-1752.2025.22.018

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【Objective】 This study aimed to systematically analyze the biochemical composition of Liannan Daye tea germplasm from Guangdong Province and to evaluate its suitability and quality characteristics for green tea and black tea through sensory evaluation. The findings were intended to provide the scientific support for the breeding of new Liannan Daye varieties, the conservation and utilization of local tea resources, and the development of functional tea products. 【Method】A total of 19 representative Liannan Daye tea germplasm resources were selected for biochemical composition analysis. 16 key compounds were quantified, including water extract, tea polyphenols, free amino acids, catechins, and alkaloids. Principal component analysis (PCA) and cluster analysis were employed to systematically evaluate the variability of these components. In addition, sensory evaluation was conducted to assess the quality of green teas and black teas made from each resource. 【Result】Significant differences in biochemical composition were observed among the 19 Liannan Daye tea germplasm resources. Variations were especially notable in amino acids, theanine, tea polyphenols, theacrine, and caffeine content. Most resources exhibited high levels of amino acids and theanine but relatively low content of tea polyphenols and catechins. All resources had phenol-to-amino acid ratios below 8.0, indicating good suitability for green tea production. Sensory evaluation showed that green teas made from Liannan Daye exhibited generally high quality in appearance, liquor color, aroma, and taste. In particular, Liannan Daye 16, 29, and 38 demonstrated superior overall quality. For black tea, Liannan Daye 31 and 38 also exhibited excellent performance. Cluster analysis grouped the 19 Liannan Daye tea resources into three distinct clusters and identified eight resources with unique traits, including two high amino acid content, three high theanine content, two high theacrine content, and one low caffeine content. 【Conclusion】The Liannan Daye tea germplasm resources exhibited significant diversity in both biochemical composition and sensory quality. Differences in the levels of amino acids, theanine, and theacrine directly affected their suitability for green tea or black tea production. High amino acid, high theanine, and low caffeine resources were more suitable for green tea processing, while high theacrine resources showed potential for the development of functional tea products.