【Background】 Glutathione S-transferases (GSTs) are key detoxification enzymes in insects and play important roles in the development of insect tolerance to chemical insecticides. Ethyl formate (EF), characterized by high efficacy, low toxicity, and low residue, is regarded as a promising green fumigant for stored-grain pest control.【Objective】 To elucidate the molecular mechanisms underlying pest tolerance to EF, this study focuses on the important stored-grain pest Cryolestes ferrugineus, aiming to analyze the relationship between GST genes (CfGSTe1 and CfGSTd) and EF tolerance.【Method】 The present study conducted bioassays to determine the susceptibility of C. ferrugineus with three different levels of phosphine resistance to EF. Through synergistic assays with diethyl maleate (DEM), the potential enhancement of EF fumigation efficacy was evaluated, and the effects of EF treatment on GST activity were analyzed. According to the previous transcriptome data of C. ferrugineus, two key GST genes (CfGSTe1 and CfGSTd1) were identified and subjected to amino acid sequence and phylogenetic analyses. The temporal and spatial expression patterns of these two genes, as well as their transcriptional responses to EF fumigation stress, were further analyzed by using real-time quantitative PCR (RT-qPCR). Finally, the effects of CfGSTe1 and CfGSTd1 on EF tolerance were analyzed by individually silencing these genes using RNA interference (RNAi) technology.【Result】 Bioassay results showed that C. ferrugineus with varying levels of phosphine resistance exhibited no significant differences in sensitivity to EF, confirming the absence of cross-resistance between the two fumigants. The synergist DEM significantly enhanced the fumigant toxicity of EF, and the GST activity in insects was markedly increased under EF stress, suggesting that GSTs play an important role in the detoxification metabolism of EF. Sequence and phylogenetic analyses indicated that CfGSTe1 and CfGSTd1 encode 216 and 215 amino acids, respectively, both containing conserved GST catalytic sites and belonging to the Epsilon and Delta families. The RT-qPCR results indicated that both genes were highly expressed at the adult stage, primarily in the midgut, fat body, and Malpighian tubules, and could be significantly induced by EF exposure. After effectively silencing CfGSTe1 and CfGSTd1 via RNAi, the tolerance of C. ferrugineus to EF was significantly reduced, as evidenced by markedly increased adult mortality following fumigation.【Conclusion】 The CfGSTe1 and CfGSTd1 may play important roles in the detoxification metabolism of EF in C. ferrugineus, suggesting a close association with insect tolerance to this fumigant.
【Objective】 Soil organic carbon (SOC) stability is significantly influenced by the functional group composition and carbon-fixing microorganisms. This study compared the differences in soil organic carbon structure and cbbL bacterial community structure under long-term fertilization regimes, so as to provide an understanding of the carbon sequestration potential and mechanisms of yellow-mud paddy soil.【Method】 In this study, the effects of no fertilization (CK), chemical fertilizer (NPK), chemical fertilizer + rice straw returning (NPKS) and chemical fertilizer + organic manure (NPKM) treatments on the characteristics of organic carbon functional groups and the community structure of carbon sequestration bacteria were studied by using 13C nuclear magnetic resonance (NMR) and Fourier transform technology to determine the organic carbon structure and the community structure of carbon-fixing bacteria by high-throughput sequencing technology, respectively.【Result】 The co-chemical fertilizer and manure application (NPKS and NPKM) significantly increased the total SOC content in yellow-mud paddy soil,while the SOC content decreased with increasing soil depth. The contents of MOC and POC, as well as the POC/SOC ratio, decreased with soil depth, while the MOC/SOC ratio showed the opposite trend. NPKS and NPKM treatments also increased the content of aliphatic and aromatic carbon compounds, then enhancing the stability and complexity of soil organic carbon, with subsoil organic carbon being more stable than surface soil. Analysis of the α-diversity index of carbon-fixing bacteria in the soil revealed that fertilization significantly increased the diversity index of carbon-fixing bacteria, with the NPKM treatment showing the best results. The dominant bacterial phylum in paddy soil was Proteobacteria, and fertilization increased the relative abundance of this phylum to varying extents. The dominant genera were Sulfuricaulis and Sulfuritortus. In the subsoil, compared with the surface soil, the relative abundance of photoautotrophic microorganisms was reduced, while the relative abundance of chemo lithotrophic microorganisms was increased.【Conclusion】 Therefore, combined organic amendments (straw or manure) with chemical fertilizers not only enhanced SOC stability but also increased the diversity and abundance of carbon-fixing bacteria, restructuring their communities. NPKM was the most effective in stabilizing SOC and promoting microbial diversity, highlighting its role in sustainable yellow-mud paddy soil management.
【Objective】 The zinc (Zn) uptake and translocation in wheat have a significant impact on grain Zn concentration. The aim of this study was to understand the relationships between pre-anthesis and post-anthesis Zn uptake and translocation, grain Zn concentration, and Zn rates across different regions, so as to provide the support to the scientific application of Zn fertilizer and the enhancement of wheat grain Zn biofortification.【Method】 Based on the location-fixed field experiment initiated in major wheat-growing regions of China at 2022, including Yongshou of Shaanxi, Baixiang of Hebei, Zitong of Sichuan, and Shucheng of Anhui, soil available Zn concentration, wheat yield, biomass, yield components and Zn concentration in various plant parts were analyzed to understand the regions varies for grain Zn concentration, pre-and post-anthesis Zn uptake, and transportation responses to different Zn application rates during the wheat growing season of 2023-2024.【Result】 The grain yield and yield components did not change with Zn application, but grain Zn concentration increased significantly. The biofortified target of 40.0 mg·kg-1 and the highest of 51.7 and 80.7 mg·kg-1 was achieved respectively in Sichuan and Anhui, but Shaanxi and Hebei could not, with the highest of 32.2 and 34.5 mg·kg-1, respectively. For each 1.0 kg Zn·hm-2 input, Zn uptake in pre-anthesis increased by 9.8, 7.4, 3.0, and 3.0 g·hm-2 at Anhui, Sichuan, Hebei, and Shaanxi, respectively; Zn uptake in post-anthesis increased by 9.8, 8.3, and 0.97 g·hm-2 at Sichuan, Anhui, and Hebei, respectively, but no significant increase was found in Shaanxi; Zn translocation in post-anthesis increased by 5.6 and 2.5 g·hm-2 at Anhui and Shaanxi, respectively, and decreased by 1.6 g·hm-2 at Sichuan, but no significant increase was found in Hebei. For the Zn uptake and translocation efficiencies, with each 1.0 kg Zn·hm-2 input, the increase of 0.71, 0.53, 0.47, and decrease of 0.40 percentage points in pre-anthesis Zn uptake efficiency was observed at Shaanxi, Hebei, Anhui and Sichuan, respectively; the decrease of 0.71, 0.53, 0.47, and increase of 0.40 percentage points in post-anthesis Zn uptake efficiency was observed at Shaanxi, Hebei, Anhui and Sichuan, respectively; the decrease of 1.41 and 0.44 percentage points in post-anthesis Zn translocation efficiency was observed at Sichuan and Hebei, respectively, but not decrease in Shaanxi and Anhui; the decrease of 0.06 and 0.13 in anthesis Zn transfer index from root to shoot was observed at Sichuan and Anhui, respectively, but not decrease in Shaanxi and Hebei. 【Conclusion】 Wheat grain Zn biofortification was collectively influenced by Zn uptake and translocation processes. Compared with efficiencies of the pre-anthesis and post-anthesis Zn uptake, Zn uptake contribution, post-anthesis Zn translocation, and translocation contribution, the pre-anthesis and post-anthesis Zn uptake and translocation exhibited greater impacts by regions variations of wheat grain Zn concentration. Zn fertilization increased pre-anthesis and post-anthesis Zn uptake, while its effect on post-anthesis Zn translocation varied with regions. Compared with post-anthesis Zn translocation, the pre-anthesis and post-anthesis Zn uptake, exhibited greater impacts in wheat grain Zn concentration, particularly the post-anthesis Zn uptake. Therefore, enhancing the soil Zn supply capacity in main wheat production regions of China and promoting Zn uptake in wheat, particularly post-anthesis Zn uptake, could effectively increase grain Zn concentration and achieve the wheat Zn biofortification target.
【Objective】 Sugars regulate the nitrogen transformation process in soil. However, the effects of different polymerization degrees of dextran on nitrogen conversion were not clear. This study focused on investigating the transformation characteristics of dextran-modified urea with varying degrees of polymerization in calcareous fluvo-aquic soil, analyzing the relationship between the polymerization degree of dextran and nitrogen efficiency, and revealing its functioning mechanism. It aimed to provide a solid theoretical basis for the application of different polymerization degrees of dextran in urea. 【Method】 Four types of modified urea were prepared by incorporating 1% glucose (monosaccharide, GU), maltose (2-poly, MU), oligomeric dextran (≈5-poly, OU), and polydextrose (≈20-poly, PU) into molten urea. Six treatments were designed: no fertilization (CK), conventional urea (U), and four dextran-modified urea treatments. A soil incubation experiment was conducted to evaluate nitrogen transformation, and 13C nuclear magnetic resonance (13C-NMR) spectroscopy was used to characterize the chemical structures, to uncover the urea conversion mechanism affected by dextran polymerization degree and structural changes. 【Result】 (1) Dextran-modified urea contained a Schiff base structure. (2) Compared with U, dextran-modified urea slowed urea hydrolysis and increased soil amide N content by 15.3%-37.1%, with the highest value in OU. (3) After the application of nitrogen fertilizer, the urease activity of the U treatment peaked at 1 d, whereas the urease activity of the dextran-modified urea treatment peaked at 2 d of incubation. (4) Ammonia volatilization accumulation of dextran-modified urea was reduced by 2.2%-12.9% compared with U. With the increase of the degree of polymerization of dextran, the accumulation of ammonia volatilization showed a gradually increasing trend. (5) At the end of the incubation, NO3--N content of dextran-modified urea was increased by 14.1%-30.5%. As the degree of polymerization of dextran increased, the nitrate nitrogen content first increased and then decreased. When the degree of polymerization was 3.35, the nitrate nitrogen content was the highest. 【Conclusion】 Dextran-modified urea effectively delayed urea hydrolysis, reduced ammonia volatilization, and enhanced soil NO3--N content. GU had the best impact on reducing ammonia volatilization, while dextran with a medium polymerization degree (3.35) had the best effect on improving the NO3--N content in the soil.
【Objective】 This study was conducted to perform a genetic analysis of tomato blossom-end scar size, with the aim of enhancing selection efficiency for this trait, accelerating the breeding process for small scar tomatoes, and laying a foundation for discovering key genes controlling blossom-end scar size and the genetic improvement of tomato varieties.【Method】 A small blossom-end scar line (P1) was crossed with a large blossom-end scar line (P2) to obtain F1. The F1 was self-pollinated and backcrossed with both parents to generate F2, BC1P1 and BC1P2 populations. Blossom-end scar size and blossom-end scar index were evaluated across the six generations during the red ripening period of fruits. Genetic analysis was performed using the major gene plus polygene mixed inheritance model. The SEA software was employed to fit 24 potential genetic models. Candidate models were selected based on the maximum likelihood value (MLV) and Akaike’s information criterion (AIC), and the optimal genetic model was determined by integrating the results of the Smirnov test, Kolmogorov test, and homogeneity test. Finally, based on the optimal model, the least squares method was used to estimate genetic parameters, including additive effects, dominance effects, epistatic effects, as well as major gene heritability and polygene heritability.【Result】 The differences in blossom-end scar size between the parental lines were extremely significant. At 55 days after the peak flowering period, the average blossom-end scar size and scar index of the large blossom-end scar parent (P2) were 9.44 times and 8.29 times greater than those of the small blossom-end scar parent (P1), respectively. The average blossom-end scar size and blossom-end scar index of the F1 population were intermediate between the two parental lines. The BC1P1, BC1P2 and F2 populations exhibited continuous variation in both blossom-end scar size and blossom-end scar index, with a wide range of variability and coefficients of variation ranging from 61.2% to 87.4%. The frequency distribution of the F2 population showed a continuous skewed normal distribution. Genetic model analysis suggested that the optimal model for tomato fruit blossom-end scar size was a mixed two major gene plus polygene inheritance model with additive-dominance-epistatic effects (MX2-ADI-ADI). The heritability of major genes in the F2 population was 93.05%, while the polygenic heritability was 4.68%. The two major genes mainly control this trait through negative additive effects. In terms of dominance, the first major gene tended to decrease the fruit blossom-end scar size, whereas the second major gene tended to increase it. These two genes showed partial dominance, with the second having a stronger dominant effect. Additive × additive and dominance × additive interactions increased blossom-end scar size, whereas dominance × dominance and additive × dominance interactions decreased it. 【Conclusion】 Tomato blossom-end scar size is a quantitative trait principally controlled by two major genes.
【Objective】 The purpose of this study is to investigate the differences in agronomic traits, nutritional quality, and metabolic components between Tibetan bunching onion and chive to elucidate their unique nutritional and metabolic characteristics, thereby exploring their distinctive resource value. These findings will provide a scientific basis for the exploitation of the unique resource value of Tibetan bunching onion, as well as for the promotion of the high-value utilization and varietal improvement of specialty vegetables in Xizang.【Method】 The materials used in this study were Tibetan bunching onion and chive. A systematic comparison of their agronomic traits and nutritional quality was conducted through physiological and biochemical analyses. The metabolic profiles of the subjects were analyzed using widely targeted metabolomics, with differential metabolites being identified using the criteria of VIP>1 and FC≥2 or FC≤0.5. Subsequent KEGG pathway enrichment analysis was performed to elucidate the impacted metabolic pathways.【Result】 The results showed that there were significant differences between the two species in terms of pseudostem morphology, epidermal color, plant height, pseudostem length, leaf length, and single plant weight. Nutritional quality analysis showed that the dry matter, free amino acid, pyruvic acid, and crude fiber contents of Tibetan bunching onion were higher than those of chive, while the soluble solids, soluble sugars, vitamin C content, and sugar-acid ratio were significantly lower. Utilizing the widely targeted metabolomics-based technology, a total of 2 014 metabolites from 13 distinct classes were identified, predominantly comprising flavonoids, lipids, and alkaloids. Then 1 012 differential metabolites were identified by multivariate statistical analysis, of which 583 were up-regulated and 429 were down-regulated. KEGG pathway enrichment analysis demonstrated that the differential metabolites were significantly enriched in pathways associated with flavonoid biosynthesis, primarily encompassing the biosynthesis of flavones and flavanols, anthocyanin glycosides, and the biosynthetic pathways of kaempferol aglycone Ⅰ and quercetin aglycone Ⅰ. By constructing a flavonoid anabolic network and comparing the relative contents of metabolites, significant interspecific divergence in downstream flavonoid metabolite accumulation between Tibetan bunching onion and chive was revealed. The diversity was mainly due to the branch transformation of the common precursor dihydrokaempferol, which led to the synthesis and accumulation of different secondary metabolites, including flavonols (kaempferol and quercetin) and anthocyanins (cyanidin and delphinidin).【Conclusion】 Significant differences were observed in the agronomic traits, nutritional quality, and metabolite accumulation of the two Allium species. The Tibetan bunching onion had a high dry matter and amino acid content, a spicy and rich flavor, a coarse texture, and good storage resistance. In contrast, the chive was rich in sugars and vitamin C, and tasted sweet and fresh. Differentiation of the flavonoid synthesis pathway is mainly due to the transformation of dihydrokaempferol branches, which produce various metabolites such as flavanols and anthocyanins.
【Objective】 This study aimed to establish a scientific framework for cucumber fruit quality evaluation by integrating the analysis of nutritional traits and flavor compound profiles across diverse germplasm resources with human sensory evaluation. 【Method】 Twelve cucumber lines/varieties with significant sensory variation (provided by Tianjin Cucumber Research Institute) were investigated. The measurements included three parts: first, nutritional traits of fruits, such as soluble sugar content (SSC), total soluble solids (TSS), and titratable acidity (TA); second, flavor compound content in fruits, such as total volatiles and key aldehydes [(E, Z)-2, 6-nonadienal, (E)-2-nonenal, (Z)-6-nonenal]; third, sensory evaluation of fruit quality, such as sweetness, juiciness, crispness, aroma, non-astringency, and non-bitterness. An integrated correlation analysis was performed to link sensory attributes with physicochemical profiles. 【Result】 In the sensory evaluation, non-astringency and sweetness had the highest weighting coefficients (27.12% and 25.49%, respectively), while the other four attributes (aroma, crispness, non-bitterness, and juiciness) ranged from 10.59% to 12.51%. Higher scores across these six dimensions indicated superior overall sensory quality. Furthermore, sweetness, juiciness, crispness, aroma, and non-bitterness of cucumber fruit sensory evaluation were (extremely) correlated with the SSC, TSS content and ascorbic acid content. In addition, cucumber flavor compounds (Z)-6-nonenal content showed significant correlation with sweetness, aroma, juiciness, non-bitterness and non-astringency in sensory evaluation, and it also had extremely significant positive correlation with the SSC, TSS and ascorbic acid content, respectively, while the ratio of cucumber flavor compounds (E, Z)-2, 6-nonadienal to (E)-2-nonenal was significantly correlated with non-astringency. Among the different cucumber varieties tested, line L2 generally exhibited higher levels of fruit SSC, TSS and ascorbic acid content, as well as the ratio of soluble solid content to titratable acid compared with the other lines. It also achieved the highest composite score across all those six sensory attributes. Consequently, line L2 demonstrated superior overall fruit quality. 【Conclusion】 The SSC, TSS, ascorbic acid content, (Z)-6-nonenal content, and the ratio of (E, Z)-2, 6-nonadienal to (E)-2-nonenal content of cucumber fruits could serve as effective indicators for the rapid evaluation of sensory and flavor quality.
【Objective】 This study aimed to investigate the effect of alcoholic-malolactic co-fermentation mediated by Lactobacillus brevis (LB-21) on the color quality of dry red wine, to provide the data support for optimizing this technology.【Method】 Using Marselan grapes from Turpan, Xinjiang, as raw material, alcoholic-malolactic co-fermentation treatments were designed with mixed inoculation of Saccharomyces cerevisiae SC-19, Pichia fermentans Z9Y-3, and lactic acid bacteria (Lactobacillus brevis LB-21 or Oenococcus oeni SD-2a). Concurrently, traditional sequential alcohol-malolactic fermentation group and single alcohol fermentation group served as the controls. After fermentation, the wines underwent conventional stabilization and were stored until June of the following year. Spectrophotometric methods were used to determine CIElab color parameters (L*, a*, b*, C*ab, hab, etc.) and multiple pigment indicators, including 520 pigment (520P), chromaticity of free anthocyanins (FA), total pigment (TP), polymeric pigment (PP), percentage of polymeric pigments (PP%), total phenols, and tartrate esters. Principal Component Analysis (PCA) was employed to identify key compounds responsible for color differences among the wine samples, and Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA) was used to investigate compounds distinguishing the color characteristics of co-fermented samples.【Result】 Color differences among the wine samples primarily centered on CIElab parameter hue angle (hab) and pigment indicators such as 520P and TP. Compared with single alcohol fermentation, alcohol-malolactic fermentation increased red hue (a*) and color saturation (C*ab) by 3.69%-13.74% and 7.24%-19.77%, respectively, and TP increased by 27.52%-47.41%, and PP by 4.39%-34.12%, respectively, significantly enhancing color stability (P<0.05). However, total anthocyanins and free anthocyanin levels decreased by 20.60%-25.69% and 14.03%-38.59%, respectively. PCA results indicated that under the alcoholic-malolactic co-fermentation treatment, the Lactobacillus brevis LB-21 group significantly outperformed the Oenococcus oeni SD-2a group in key indicators, including a*, C*ab, TP, PP, and PP% (P<0.05). OPLS-DA analysis confirmed that L*, a*, C*ab, hab, ΔEab, and PP% were key color difference indicators in LB-21-mediated co-fermented wine samples, indicating this strain’s significant advantage in maintaining dry red wine color.【Conclusion】 Alcohol-malolactic three-strain co-fermentation, particularly the treatment mediated by Lactobacillus brevis LB-21, significantly enhanced a*, TP, and PP in dry red wine, improved color stability, and outperformed traditional fermentation models in key color parameters. The optimized strain LB-21 and its application technology demonstrated substantial practical value for enhancing dry red wine fermentation processes.
【Objective】 The aim of this experiment was to study the effects of different levels of Mytilaria laosensis leaves feeding on the growth performance, plasma biochemical indicators, jejunal mucosal cytokine content, hepatic detoxification and antioxidant indicators, carcass quality and meat quality of Yellow-feathered chickens. This study serves to evaluate the application value of Mytilaria laosensis leaves as a potential forest by-product feed ingredient for yellow-feathered chickens.【Method】 A total of 720 six-day-old fast-growing mahuang Yellow-feathered chickens with similar body weights were selected and randomly assigned to 6 treatment groups using a completely randomized design. The feeding levels of Mytilaria laosensis leaves were 0% (control group), 1.5%, 3.0%, 4.5%, 6.0% and 7.5%, and each group was set up with 6 replicates, with 20 chickens in each replicate (10 males and 10 females each), and the test period was 50 days. 【Result】 with the feeding levels of Mytilaria laosensis leaves increased, 1) During the period of 6 to 21 days of age, body weight, average daily gain, and eurobenefit index were increased quadratically (P<0.05), while feed conversion ratio and cost per kilogram gain decreased quadratically (P<0.05). From 22 to 42 days of age, both feed to gain ratio and weight gain cost were decreased linearly and quadratically (P<0.05). In the final phase of 43 to 55 days of age, body weight, average daily feed intake, feed to gain ratio, and weight gain cost were increased both linearly and quadratically (P<0.05), whereas the eurobenefit index was decreased in both linear and quadratic patterns (P<0.05). During the experiment of 6 to 55 days of age, the body weight, average daily gain, feed to gain ratio and weight gain cost of chickens were increased linearly and quadratically, while the eurobenefit index was decreased linearly and quadratically (P<0.05). 2) At 21 days of age, the plasma concents of TP, IgA, IgG, and the antibody titer against NDV-Ab were increased linearly (P<0.05). In contrast, the activities of ALT and CK, as well as the concentration of MDA were decreased both linearly and quadratically (P<0.05). At 55 days of age, compared with the control group, the TP concent was significantly higher in the 4.5% leaf group (P<0.05), while the TG concent was significantly lower in the 1.5% leaf group (P<0.05). 3) The feeding level of Mytilaria laosensis leaves had no significant influence on the organ indexes of chickens. 4) The contents of sIgA, TNF-α, IFN-γ, and IL-10 in jejunal mucosa were increased linearly and quadratically (P<0.05). No significant difference was observed in the concent of IL-1β in the jejunal mucosa (P>0.05). 5)The activity of CarE in liver was increased linearly and quadratically (P<0.05), while the activities of AchE and MDA content were decreased linearly and quadratically (P<0.05). No significant differences were observed in the activities of hepatic GSH-ST, T-AOC, and T-SOD (P>0.05). 6) The half-eviscerated rate of chickens was decreased linearly and quadratically (P<0.05), and the breast muscle rate was increased linearly and quadratically (P<0.05), and the abdominal fat rate was decreased linearly (P<0.05). 7) Compared with the control group, chickens in the 4.5% leaf group were demonstrated significantly higher redness (a*) values in the breast muscle at both 45 min and 24 h post-slaughter (P<0.05).【Conclusion】 In summary, the suitable of Mytilaria laosensis leaves can improve the growth performance, immune function, antioxidant and detoxification capacity, carcass quality and meat quality of yellow-feathered chickens. However, the feeding level of Mytilaria laosensis leaves requires precise control, as excessively high levels may adversely affect economic returns. According to quadratic regression model, combined with the body weight, average daily gain and feed to gain ratio as the main evaluation indicators, it is estimated that the appropriate feeding level of Mytilaria laosensis leaves for 6 to 55 days of age of fast-growing Yellow-feathered chickens is 2.75% to 2.90%. This study provided a scientific basis and experimental reference for the use of Mytilaria laosensis leaves as an unconventional feed ingredient in the rearing of yellow-feathered chickens.
【Background】 Follicle development is characterized by a unique hierarchical progression in poultry, regulated by autocrine, paracrine, growth factors, and multiple functional genes. Granulosa cells are the most abundant functional somatic cells within the follicle, and their survival directly determines follicular growth, development, and maturation. Ferroptosis, a newly identified type of regulated cell death, has been closely linked to follicular development. USP18 plays an important regulatory role in ferroptosis, however, its function in duck granulosa cells remains unclear. 【Objective】 The objective of this study was to investigate the molecular regulatory mechanisms of USP18 in the process of ferroptosis in duck granulosa cells of laying ducks, so as to provide a theoretical basis and molecular targets for the genetic improvement of egg production traits in poultry. 【Method】 Four high-yield and four low-yield laying ducks aged 300-day-old were selected. Prehierarchical follicular tissues were collected from each group. The expression level of USP18 was assessed using quantitative real-time PCR (qRT-PCR) and Western blotting in follicles of high-yield and low-yield laying ducks. Granulosa cells isolated from prehierarchical follicles were transfected with the interference fragment of USP18. The effects of USP18 on cell viability were evaluated using the CCK-8 assay and Calcein-AM/PI double staining. The effects of USP18 on intracellular oxidative status were evaluated using the reactive oxygen species (ROS) detection kit and enzyme-linked immunosorbent assay (ELISA). The impact of USP18 on ferroptosis was examined using the mitochondrial membrane potential assay kit, the lipid peroxidation fluorescent probe, FerroOrange iron probe, and Prussian blue staining. Furthermore, the interaction between USP18 and GPX4 protein, as well as GPX4 protein expression, were analyzed by immunofluorescence, co-immunoprecipitation, and Western blotting.【Result】 The expression level of USP18 in prehierarchical follicles of high-yield ducks was significantly higher than that in low-yield ducks (P<0.01); USP18 knockdown significantly reduced granulosa cell viability (P<0.01), inhibited cell survival (P<0.05), promoted ROS accumulation (P<0.01), increased malondialdehyde (MDA) content (P<0.01), and suppressed the activities of superoxide dismutase (SOD) and catalase (CAT) (P<0.01); USP18 knockdown notably elevated lipid peroxidation levels (P<0.05), decreased mitochondrial membrane potential (P<0.05), enhanced mitochondrial membrane permeability (P<0.01), increased intracellular iron levels and iron deposition (P<0.01), increased the expression of ferroptosis suppressor markers ACSL4 and GPX4, and suppressed the expression of TFR1 (P<0.01); the interaction relationship between USP18 and GPX4 was confirmed by co-immunoprecipitation and immunofluorescence assays. Western blotting and co-immunoprecipitation analyses further demonstrated that USP18 knockdown promoted ubiquitination and degradation of GPX4 protein.【Conclusion】 USP18 was highly expressed in the prehierarchical follicles of high-yield laying ducks. USP18 knockdown significantly increased oxidative stress levels and induced ferroptosis in duck granulosa cells. Moreover, USP18 was found to interact with GPX4 protein, and USP18 knockdown promoted ubiquitination and degradation of GPX4 protein. In summary, USP18 modulated ferroptosis in duck granulosa cells by suppressing the ubiquitination and degradation of GPX4.
