Scientia Agricultura Sinica

Influence of Expressing OsNRAMP5 Under the Driving of the OsLCT1 Promoter on Cadmium Migration to Rice Seeds

XIONG JiaNi, LI ZongYue, HU HengLiang, GU TianYu, GAO Yan, PENG JiaShi

Scientia Agricultura Sinica. 2025, 58(7): 1259-1268. doi:10.3864/j.issn.0578-1752.2025.07.001

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【Objective】Cadmium (Cd) is the predominant pollutant in China’s arable land, with rice cultivated on these contaminated soils being a significant dietary source of Cd for the population. This study aims to tissue-specifically express OsNRAMP5, a transporter responsible for the majority of Cd uptake in rice, to investigate strategies for developing low-Cd rice varieties and provide a reference for molecular design breeding to cope with Cd pollution. 【Method】To drive the expression of OsNRAMP5 in rice, we utilized a 2 500 bp sequence upstream of the OsLCT1 start codon as the promoter. The red fluorescent protein mRFP was fused to the C-terminus of OsNRAMP5 to visualize its tissue localization. After obtaining independent homozygous transgenic lines, the transcripts of the OsNRAMP5 were first detected using qRT-PCR, and its tissue localization in roots and nodes was observed via laser confocal microscopy. Subsequently, the accumulation and tolerance of Cd were evaluated in transgenic and wild-type rice under varying concentrations of Cd treatment. Furthermore, plants were grown in Cd-contaminated paddy soil, and the accumulation of Cd and other mineral elements in seeds and leaves, as well as related yield traits, were measured. 【Result】Under the drive of the OsLCT1 promoter, OsNRAMP5 was expressed mainly in the epidermis, exodermis and stele of roots, as well as in the phloem area of enlarged vascular bundles and diffuse vascular bundles in nodes, differing significantly from the native expression pattern of OsNRAMP5 in rice. Compared to wild-type rice, the transgenic lines exhibited increased Cd accumulation in roots, decreased Cd accumulation in shoots, and enhanced tolerance to Cd stress during the seedling stage. When cultivated in Cd-contaminated paddy soils, plant height and grain yield were unaffected by the ectopic expression of OsNRAMP5, while Cd accumulation in seeds and leaves significantly decreased in the transgenic lines. The Cd content in seeds decreased by over 80%, with a greater reduction ratio compared to that in leaves. Although the Mn content in seeds and leaves slightly decreased, the expression of OsNRAMP5 had little impact on the accumulation of other mineral elements such as Fe, Zn, and Cu. 【Conclusion】The expression of OsNRAMP5 driven by the OsLCT1 promoter greatly decreases the Cd migration toward rice seeds by reducing Cd transport to the aboveground parts from roots and increasing the Cd transporting to leaves at nodes. Therefore, the expression of OsNRAMP5 under the control of the OsLCT1 promoter is an effective strategy to reduce Cd accumulation in rice seeds.

Molecular Characteristics and Resistance Evaluation of Transgenic Maize LD05 with Stacked Insect and Herbicide Resistance Traits

YUE RunQing, LI WenLan, DING ZhaoHua, MENG ZhaoDong

Scientia Agricultura Sinica. 2025, 58(7): 1269-1283. doi:10.3864/j.issn.0578-1752.2025.07.002

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【Objective】To clarify the molecular characteristics and the effectiveness of target traits of transgenic maize LD05 with composite insect and herbicide resistance, and to provide data basis, technical support and product reserve for industrial application.【Method】Using biological information analysis, we designed and modified the proprietary insect-resistant fusion gene m2cryAb-vip3A, and selected BC₄F₃, BC₄F₄ and BC₄F₅ generations of the newly created transgenic hybrid insect-resistant and herbicide-tolerant maize LD05 to carry out experimental research. Specific PCR and Southern blot were used to analyze the stability of genomic integration. qRT-PCR and ELISA were used to analyze the expression stability. The resistance to target pests was evaluated by bioassay and field trials, and the herbicide tolerance was tested by field spraying of glufosinate. 【Result】A new insect-resistant fusion gene m2cryAb-vip3A with independent property right was discovered and designed, and a multivalent insect-resistant and herbicide resistant maize transformant LD05 was created. The exogenous T-DNA was integrated into the maize genome in the form of a single copy. The qRT-PCR results indicated that m2cryAb-vip3A and bar were both expressed in various tissues and organs across three generations, and the variation trend of expression quantities was largely consistent. Specifically, the expression level of m2cryAb-vip3A was the highest in the leaves at the seedling stage of the three consecutive generations, with an average expression quantity of 36.73, while the expression level was the lowest in the cob at the mature stage, with an average of merely 0.91. The expression pattern of bar was similar to that of m2cryAb-vip3A, with the highest expression level in the leaves at the seedling stage, averaging 7.35, and the expression level decreased after the jointing stage. The ELISA results demonstrated that M2CryAb-VIP3A could stably accumulate in different organs and at different periods in the three generations, and the protein accumulation amounts in different generations were similar. Among them, the accumulation amount was the highest in the leaves at the seedling stage of different generations, all exceeding 19.67 μg·g^-1 fresh weight. The expression of the targeted protein at a relatively high level could be detected in different tissues of the PAT transgenic plants of three consecutive generations, and there was no significant difference in the expression quantity between different generations. Among them, the expression level was the highest in the leaves at the seedling stage of different generations, with an average content of 16.61 μg·g^-1 fresh weight, while the accumulation amount was the lowest in the roots at the mature stage, with an average content of 0.30 μg·g^-1 fresh weight. The bioassay result showed that the corrected mortality of Ostrinia furnacalis, Spodoptera fragiperda and Mythimna separata reached 100% after feeding on V5 maize leaf tissue of LD05 for 96 h, which was a high resistance level. The results of field trials showed that LD05 transformants had high resistance to Ostrinia furnacalis at V5 stage and silking stage, to Mythimna separata at V5 stage, and to Helicoverpa armigera at silking stage. The results of glufosinate tolerance test showed that transgenic maize LD05 could tolerate 4-fold glufosinate. Agronomic character investigation showed that there was no difference between transgenic maize LD05 and control maize Zheng 58.【Conclusion】A novel insect-resistant fusion gene m2cryAb-vip3A with independent property rights was developed, and a transgenic hybrid insect-resistant and herbicide-tolerant maize LD05 was created with clear molecular characteristics, genetic stability and outstanding functional traits.

Construction of Near Infrared Spectrometry Model for Flavonoids Content of Peanut with Red and Black Testa

LI XinYu, HOU MingYu, CUI ShunLi, LIU YingRu, LI XiuKun, LIU LiFeng

Scientia Agricultura Sinica. 2025, 58(7): 1284-1295. doi:10.3864/j.issn.0578-1752.2025.07.003

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【Objective】Flavonoid content is one of the critical quality indicators for peanut seed. Near-infrared spectroscopy (NIR) is an effective method for rapid detection of flavonoid content in peanut. However, the differences of testa color may affect the accuracy of the detection results. Therefore, the construction of NIR prediction models for peanuts with red and black testa can provide a guarantee for efficient and rapid detection of flavonoid content in special peanut kernels. 【Method】In this study, 232 peanut germplasms with different testa colors were selected as materials, including 108 peanut with red testa and 124 peanut with black testa. The total flavonoid content was determined by aluminum chloride chromogenic method, with rutin serving as the standard (RT: rutin). Using the Swedish Broadcom DA7250 Diode Array Analyzer for spectral acquisition, within a scanning spectral range of 950-1 650 nm. Employing the Unscrambler X10.4 modeling software, various calibration models were established through both single and composite processing, utilizing diverse derivative and scattering spectral preprocessing methods, based on full-band partial least squares (PLS) modeling. By comparing the correlation coefficients and errors among different models, the optimal processing method was selected to establish a prediction model for flavonoid content in both red and black peanut kernels. For model external validation, materials were derived from a recombinant inbred line population derived from the parents of Silihong and Jinonghei 3, with 30 lines with red testa and 30 lines with black testa each undergoing external cross-validation.【Result】The results showed that flavonoid content of peanut with red testa was between 60.33-122.49 mg RT/100 g, with an average of 94.34 mg RT/100 g. The flavonoid content of peanut with black testa was between 64.98-121.55 mg RT/100 g, with an average of 95.59 mg RT/100 g. The best spectral pretreatment method of the peanut with red testa prediction model was “Derivative Savitzky-Golay+ SNV+Detrend”, yielding a correction correlation coefficient (Rc) of 0.9022, a root means square error of cross validation (RMSECV) of 1.9101, a prediction correlation coefficient (Rp) of 0.9021, and a root mean square error of prediction (RMSEP) of 1.9606 mg RT/100 g. The external validation correlation coefficient (R²) was 0.923, with a prediction model deviation range of -4.86-8.47 mg/100 g. The best spectral pre-treatment method for the peanut with black testa prediction model was “Derivative Savitzky-Golay+SNV+Deresolve”,resulting in an Rc of 0.9521, an RMSECV of 1.6978, the correlation coefficient (Rp) of the peanut with black testa prediction model was 0.915, and RMSEP of 2.292 mg RT/100 g, the correlation coefficient R² of external verification was 0.907, with a prediction model deviation range of -4.56-2.87 mg/100 g. Cross-validation was carried out with non-corresponding color models, and the correlation coefficient was between 0.0015-0.0975. 【Conclusion】The testa color strongly affected the accuracy of detection, and the near-infrared prediction models constructed in this study are suitable for the detection of flavonoid content in peanuts with red and black testa,which provide an important selection method for breeding characteristic peanuts with high flavonoids.

Effects of Plant Type Improvement on Root-Canopy Characteristics and Grain Yield of Spring Maize Under High Density Condition

ZHAO Yao, CHENG Qian, XU TianJun, LIU Zheng, WANG RongHuan, ZHAO JiuRan, LU DaLei, LI CongFeng

Scientia Agricultura Sinica. 2025, 58(7): 1296-1310. doi:10.3864/j.issn.0578-1752.2025.07.004

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【Objective】Increasing planting density is a key agronomic strategy to enhance maize yield; however, excessive density may result in an imbalanced population structure, reduced utilization efficiency of limited resources (e.g., light), and suppressed yield potential. Gene editing can optimize canopy architecture through targeted improvement of maize plant type, thereby enhancing adaptability to high-density planting and boosting yield. Elucidating the effects of plant type improvement on root-shoot characteristics, grain yield, and density response in spring maize, as well as the underlying mechanisms, will provide theoretical and technical foundations for optimizing plant type and achieving high-yield dense planting in spring maize.【Method】The field experiment was conducted at Gongzhuling farm in Jilin, China. In this study, two maize hybrids, includding Jingke 968 and the improved plant types Jingke Y968, were grown with 60 000 plants/hm² (D1), 75 000 plants/hm² (D2) and 90 000 plants/hm² (D3) in 2019 and 2020, respectively. The effects of two plant types of spring maize of the same genetic background on the root-canopy characteristics and yield of spring maize were studied.【Result】Under normal density conditions (D1), there were no significant differences in leaf area index (LAI), net photosynthetic rate (Pn), PAR utilization (PUE), dry matter accumulation and grain yield between the two different plant types spring maize cultivars. However, compared with Jingke 968, under D3 conditions, the improved plant type Jingke Y968 had a relatively high number of main roots (7.2%) and a relatively large weight of root dry matter (6.0%), which promoted the absorption of nutrients; furthermore, under D2 and D3 conditions, Jingke Y968 significantly improved the canopy structure of maize, so that the upper, middle and lower parts had relatively low leaf angles, higher leaf orientation and LAI, and the excellent canopy structure increased the Pn of mid-to-late ear leaves of (7.5% (D2) and 7.7% (D3)) and PUE (4.3% (D2) and 10.8% (D3)). The structural equation results showed that higher leaf direction values and LAI could positively and directly increase the accumulation of dry matter in the aboveground, thereby increasing grain yield (8.7% (D2) and 11.2% (D3)).【Conclusion】In summary, the improvement of plant type enabled Jingke Y968 to have higher main root number and larger root dry matter weight under high-density conditions, which was conducive to nutrient absorption in the underground part. Meanwhile, its leaves were more compact, Pn was significantly increased, PUE was effectively improved, and root-canopy characteristics were more reasonable, which promoted dry matter accumulation in the above-ground part. Thus, the relatively high grain yield could be obtained.

Effects of Low Temperature at Seedling Stage on Cotton Floral Bud Differentiation and Cotton Plant Yield

ZHAO YuXuan, MIAO JiYuan, HU Wei, ZHOU ZhiGuo

Scientia Agricultura Sinica. 2025, 58(7): 1311-1320. doi:10.3864/j.issn.0578-1752.2025.07.005

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【Objective】This study aimed to investigate the harm of low temperature in the cotton (Gossypium hirsutum L.) seedling stage on floral bud differentiation and the effect on seedcotton yield, to analyze the change characteristics of cotton floral bud differentiation phenotypes and terminal buds endogenous hormones under low temperature, so as to provide the theoretical basis for the high-quality and high-efficiency cultivation technology of cotton under low temperature.【Method】Using the early-maturing and high-quality cotton variety Zhong 425 as the material, a pot experiment was conducted in the smart greenhouse of the Pailou Experimental Station of Nanjing Agricultural University from 2022 to 2023 to simulate the daily average temperature environment during the cotton seedling stage in Aksu, southern Xinjiang. Two temperature treatments were set up: the control (CK, with a daily average temperature of 27 ℃, and daily maximum and minimum temperatures of 32 and 22 ℃, respectively) and the low-temperature treatment (LT, with a daily average temperature of 20 ℃, and daily maximum and minimum temperatures of 25 and 15 ℃, respectively). The number, size, and morphological anatomical structure of cotton flower bud differentiation were investigated, and the changes in endogenous hormones in shoot apices under low temperature during the seedling stage were analyzed. Additionally, the changes in cotton bolls and their component biomass, as well as relevant indicators of seed cotton yield, were examined after the removal of low temperature stress during the seedling stage.【Result】During the differentiation of cotton flower buds, the increase in Indole-3-acetic acid (IAA) content and the decrease in trans-Zeatin-riboside/Gibberellin A₃(ZR/GA₃ ) ratio in the terminal buds of cotton under low temperature during the seedling stage inhibited flower bud differentiation. Meanwhile, the content of abscisic acid Abscisic Acid (ABA), GA₃, and ZR increased in response to the adverse effects of low temperature. Changes in endogenous hormones in the shoot tips caused by low temperature during the seedling stage slowed down the process of flower bud differentiation. When the flower buds of the first fruit node on the first fruit branch differentiate from the bract differentiation stage to the sepal differentiation stage, petal-stamen differentiation stage, pistil differentiation stage, and sexual organ formation stage, the leaf age increased by 16.6%-19.4%, 26.5%-31.3%, 17.6%-29.0%, 16.6%-23.3%, and 26.6%-30.0%, respectively; the number of flower buds at the 4-leaf-1-heart, 5-leaf-1-heart, and 6-leaf-1-heart stages of cotton seedlings decreases by 33.3%-55.2%, 24.0%-53.1%, and 26.8%-32.9%, respectively. Due to the slow growth and development of cotton seedlings under low temperature during the seedling stage, the number of flower buds in cotton seedlings exposed to the same number of days of temperature treatment decreased more significantly, with reductions of 66.7%-85.7%, 74.0%-87.8%, and 70.7%-81.7% compared with the control group at the 4-leaf-1-heart, 5-leaf-1-heart, and 6-leaf-1-heart stages, respectively; the sizes of flower buds at these stages also decreased by 33.3%-36.4%, 70.7%-71.6%, and 44.6%-48.3%, respectively. After the removal of low temperature stress during the seedling stage, the development of cotton bolls was still affected, with significant reductions in boll and its component biomass. Specifically, the biomasses of boll shell, fiber, and cottonseed decreased by 64.6%, 65.5%, and 66.7%, respectively. The number of cotton bolls decreased by 65.4%, ultimately leading to a 65.5% reduction in seed cotton yield.【Conclusion】Under low temperature conditions during the seedling stage, the increased IAA content and decreased ZR/GA₃ ratio in the apical buds of cotton inhibited the differentiation of pre-summer peach flower buds. Low temperature during the seedling stage retarded the reproductive development of cotton by delaying flower bud differentiation, which reduced the biomass of cotton bolls. Low temperature at this stage also decreased the number of flower buds, ultimately leading to a reduction in the number of cotton bolls and lower seed cotton yield.

Research on the Estimation Model of Calcium Content in Guanxi Honey Pomelo Leaves Based on Spectral Index

LI FangLiang, KONG QingBo, ZHANG Qing

Scientia Agricultura Sinica. 2025, 58(7): 1321-1332. doi:10.3864/j.issn.0578-1752.2025.07.006

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【Objective】By constructing the estimation model for calcium (Ca) content in honey pomelo leaves based on spectral analysis, it could provide a theoretical basis for monitoring and rapid non-destructive diagnosis of Ca content in honey pomelo leaves.【Method】The original spectral and first-order derivative spectral characteristic bands and spectral characteristic indices (difference spectral index (DSI), ratio spectral index (RSI), and normalized difference spectral index (NDSI)) were analyzed and extracted. Single variable estimation model, partial least squares estimation model (PLS), backpropagation neural network estimation model (BPNN), random forest estimation model (RF), and support vector machine estimation model (SVM) for honey pomelo leaf calcium content were established, and the optimal spectral estimation model for honey pomelo leaf calcium content was evaluated and verified. 【Result】There was a significant multi band correlation between the original spectrum and first-order derivative spectrum of pomelo leaves and calcium content. Based on the correlation coefficients of the original spectrum and first-order derivative spectrum, the maximum wavelengths were 553, 714 nm and 528, 699, 602 nm, respectively. The spectral indices with significant correlation between the original spectrum, first-order derivative of pomelo leaves and calcium content were DSI_790,1040, RSI_910,990, NDSI_900,990 and NDSI′_350,580, DSI′_560,570, RSI′_350,580. The polynomial estimation model constructed with spectral indices such as RSI_910,990, NDSI_900,990, NDSI′_350,580, DSI_790,1040, DSI′_560,570, RSI′_350,580, DSI′_528,602 as independent variables had relatively high determination coefficient R² (R²>0.60). A hyperspectral estimation model for calcium content in honey pomelo leaves was established using the above four machine learning methods. The R²of PLS, BPNN, RF and SVM estimation models were 0.79, 0.82, 0.85 and 0.84, respectively, and the root mean square errors (RMSE) were 4.33, 4.11, 3.81 and 3.93, respectively; the R² of the validation models were 0.77, 0.80, 0.87 and 0.83, respectively, and the RMSE were 4.50, 4.28, 3.67 and 3.90, respectively. The order of estimating the accuracy of the model was RF>SVM>BPNN>PLS.【Conclusion】The accuracy comparison analysis of four models for calcium content in honey pomelo leaves showed that the RF estimation model had better predictive performance than the other three estimation models. This result could provide a new method for rapid diagnosis of calcium content in honey pomelo leaves for reference.

Widespread Non-Targeted Metabolomics Reveals Metabolites of Chloroplasts in Wheat Responses to Stripe Rust

WU Yu, QU XiangRu, YANG Dan, WU Qin, CHEN GuoYue, JIANG QianTao, WEI YuMing, XU Qiang

Scientia Agricultura Sinica. 2025, 58(7): 1333-1343. doi:10.3864/j.issn.0578-1752.2025.07.007

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【Objective】The purpose of this study is to elucidate the changes of chloroplast metabolites during wheat resistance to stripe rust, and to clarify the role of key chloroplast metabolites in wheat resistance to stripe rust.【Method】Wheat cultivar Suwon11 was used as the experimental material, wheat chloroplasts were extracted 48 and 72 h after spraying sterile water (control group) and Puccinia striiformis f. sp. tritici CYR23 (experimental group), and a widespread non-targeted metabolomics analysis was conducted via ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS). Principal component analysis (PCA) and correlation analysis were employed to visualize inter-group and intra-group differences and associations. VIP (variable importance in projection) values derived from the orthogonal partial least squares discriminant analysis (OPLS-DA) model were employed to identify differential chloroplast metabolites between the disease-resistant and control groups. Pathway enrichment analysis was performed on differential chloroplast metabolites using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database to investigate key metabolic pathways in chloroplasts during the resistance response of wheat to stripe rust.【Result】The metabolomics data exhibited high quality, with good intra-group reproducibility and significant inter-groups variability validated by multivariate statistical analyses. A total of 1 496 metabolites were detected, which predominantly included lipids and lipid-like molecules, organic acids and their derivatives, organic heterocyclic compounds, phenylpropanoids and polyphenolic compounds, organic oxides, and benzoid compounds. The metabolite species were generally similar between different groups, but significant variations in metabolite levels were observed at different infection time points. At 48 hours post infection (hpi) compared to the control group (CK), 121 differential metabolites were identified, including 21 up-regulated and 100 down-regulated metabolites; At 72 hpi compared to the CK, 58 differential metabolites were detected, with 35 up-regulated and 23 down-regulated; Between 72 and 48 hpi groups, 53 differential metabolites were observed, of which 33 were up-regulated and 20 were down-regulated. Four differential metabolites were shared between the 48 hpi vs CK and 72 hpi vs CK comparisons. KEGG analysis revealed that the differential metabolites were enriched in multiple metabolic pathways. Among them, the expression of linoleic acid which in the linoleic acid metabolic pathway exhibited 2.75- and 2.93- fold increases after inoculation 48 and 72 h, respectively, indicating that linoleic acid was continuously synthesized induced by P. striiformis f. sp. tritici.【Conclusion】The chloroplast metabolites associated with wheat resistance to stripe rust primarily include lipids, fatty acids and their derivatives, organic acids and their derivatives, organic oxides, as well as phenylpropanoids and polyphenolic compounds. Notably, linoleic acid may play a crucial role in the sustained resistance of wheat against stripe rust.

Effects of Wild Populations for Hybridization and Outdoor Population Exercise on the Parasitic Ability of Aphidius gifuensis and Enzymes Related to the Growth and Development

HE JiaoPing, HAN Meng, ZHOU Ting, GU Gang, LAI YuFei, LAI RongQuan

Scientia Agricultura Sinica. 2025, 58(7): 1344-1354. doi:10.3864/j.issn.0578-1752.2025.07.008

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【Objective】Aphidius gifuensis is the dominant parasitic wasp of Myzus persicae, however there are some problems in the process of artificial or large-scale production and propagation of A. gifuensis, such as the body size becoming smaller and the parasitism decreasing. The purpose of this study is to analyze the effects of different regulation methods on the parasitism rate, body size and the content and activity of enzymes related to the growth and development of offspring A. gifuensis, so as to improve the population rejuvenation technology of the degraded A. gifuensis and ensure the scale and stability of artificial reproduction.【Method】Indoor selection of A. gifuensis, which showed significant population degradation after 10 generations, was conducted for wild population hybridization rejuvenation, indoor exercise rejuvenation, and outdoor exercise rejuvenation. The formation of stiff aphids was observed every 12 h. After the formation of stiff aphids (during the yellow brown period of stiff aphids), the number was counted and the parasitism rate of A. gifuensis was calculated. After the metamorphosis of the aphid, six representative female adults A. gifuensis that could reflect the overall characteristics of each treatment group were randomly selected and the metathoracic tibia length was measured under a stereomicroscope as an indicator of body size. The stiff aphids with different treatments were collected and placed separately. The standard curve was made by Coomassie brilliant blue G-250 method within 12 h after emergence, and the content and activity of growth and development related enzymes were determined by ultraviolet spectrophotometer colorimetry.【Result】Wild population hybridization rejuvenation and outdoor exercise rejuvenation had good regulatory effects on the parasitic ability of A. gifuensis (P<0.05), but indoor exercise rejuvenation had no significant regulatory effect on the parasitic ability of A. gifuensis (P>0.05). The hybridization between wild female A. gifuensis with the indoor degraded male one (1﹕1) had the best rejuvenation effect on the parasitic ability. Compared with the control group, the parasitism rate and offspring body size were increased by 17.83% and 18.98%, respectively. The contents of carboxylesterase, phenoloxidase and trehalase in the offspring and the activity of phenoloxidase were significantly increased (P<0.05). After six generations of outdoor exercise, compared with the 0 generations, the parasitism rate and body size of offspring A. gifuensis were increased by 24.67% and 49.33%, respectively, and the contents of carboxylesterase, phenoloxidase, superoxide dismutase (SOD) and trehalase in the offspring were increased significantly (P<0.05), the activities of phenoloxidase and SOD were also significantly increased (P<0.05).【Conclusion】In production, the field hybridization rejuvenation and outdoor exercise rejuvenation of the degraded A. gifuensis population can improve the parasitic ability of A. gifuensis to M. persicae.

Water Demand Characteristics of Rice-Oilseed Rape Rotation System in the Middle Reaches of the Yangtze River

WANG Bin, WU PengHao, LU JianWei, REN Tao, CONG RiHuan, LU ZhiFeng, LI XiaoKun

Scientia Agricultura Sinica. 2025, 58(7): 1355-1365. doi:10.3864/j.issn.0578-1752.2025.07.009

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【Objective】This study aimed to clarify the water demand characteristics of the rice-oilseed rape rotation system in the middle reaches of the Yangtze River, so as to provide the theoretical support for water allocation in this cropping system.【Method】This study analyzed the water demand of the rice-oilseed rape rotation system in the middle reaches of the Yangtze River using the single crop coefficient method. The supplementary irrigation amount was calculated based on the effective precipitation, and the water surplus/deficit characteristics of the rotation system were identified according to the crop water surplus-deficit index.【Result】The rice-oilseed rape rotation pattern in the Middle Reaches of the Yangtze River required an average annual water demand of 1 172 mm, with rice accounting for approximately 898 mm (76.6%) and oilseed rape accounting for approximately 274 mm (23.4%). Rice required an average annual supplementary irrigation of 643 mm, while oilseed rape required drainage of 54 mm on average per year. Years with moderate, severe, and extreme drought in the rice accounted for 77.5%, 15%, and 2.5%, respectively. Years with moderate, severe, and extreme flooding in the oilseed rape accounted for 10%, 17.5%, and 2.5%, respectively. Special attention should be paid to the field water conditions during the tillering, joint-booting, and heading and filling stages of rice, and flood prevention measures should be taken during the seedling and maturity stages of oilseed rape. The early rice-late rice-oilseed rape rotation pattern required an average annual water demand of 1 161 mm, with early rice accounting for approximately 550 mm, late rice accounting for approximately 401 mm. The total water demand of the two seasons accounts for 82.0% of the total water demand of the whole rotation system. while oilseed rape accounting for approximately 210 mm (18.0%). Early rice required an average annual supplementary irrigation of 322 mm, late rice requires 272 mm, and oilseed rape requires drainage of 59 mm on average per year. Years with moderate, severe, and extreme drought in the rice season account for 40%, 15%, and 1.3%, respectively. Years with moderate, severe, and extreme flooding in the oilseed rape season account for 12.5%, 17.5%, and 17.5%, respectively. The special attention should be paid to the water conditions during the joint-booting and heading and filling stages of late rice, and flood prevention measures should be taken during the seedling and maturity stages of oilseed rape.【Conclusion】In the middle reaches of the Yangtze River, the water demand of rice-oilseed rape rotation mode was 1 172 mm, and the water demand of rice and oilseed rape accounted for 76.6% and 23.4%, respectively. The water demand of early rice-late rice-oilseed rape rotation mode was 1 161 mm, and the water demand of rice and oilseed rape accounted for 82.0% and 18.0%, respectively. Supplementary irrigation was required to prevent water deficits during the rice season, while drainage measures were necessary during the oilseed rape season. In years with extreme precipitation, special attention should be paid to excess water conditions during the seedling stage of rice and the seedling and maturity stages of oilseed rape, and corresponding measures should be taken. In years with extreme drought, special monitoring of water deficits should be conducted during the tillering, joint-booting, heading and filling stages of rice and the flowering stage of oilseed rape, and the timely supplementary irrigation should be provided.

Effects of Green Manure Returning Combined with Nitrogen Fertilizer Reduction on Hydrothermal Characteristics of Wheat Field and Grain Yield in Oasis Irrigation Area

YIN Bo, YU AiZhong, WANG PengFei, YANG XueHui, WANG YuLong, SHANG YongPan, ZHANG DongLing, LIU YaLong, LI Yue, WANG Feng

Scientia Agricultura Sinica. 2025, 58(7): 1366-1380. doi:10.3864/j.issn.0578-1752.2025.07.010

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【Objective】In order to solve the problems of long-term continuous cropping of wheat and high amount of nitrogen fertilizer in the production process in the arid irrigation area of Northwest China, the effects of green manure returning combined with reduced nitrogen application on soil hydrothermal variation characteristics and yield of wheat were studied, so as to provide the theoretical basis for the optimization of nitrogen application system in this area. 【Method】The field experiment was carried out in Wuwei Oasis Agricultural Experimental Station from 2021 to 2022. The treatments included no green manure (G0) and conventional nitrogen application (N1), as well as three green manure returning treatments (G1, G2, G3, applying green manure 15 000, 22 500, 30 000 kg·hm^-2, respectively) and two nitrogen fertilizer reduction treatments (N2, N3, reducing 15% and 30% compared with conventional nitrogen application, respectively). The effects of green manure returning combined with nitrogen reduction on soil water and heat variation characteristics, leaf area index and yield of wheat field were analyzed. 【Result】The green manure returning combined with nitrogen reduction could increase soil water storage in 0-120 cm soil layer of wheat field. Compared with G0N1, G2N2, G3N2 and G3N3 increased soil water storage by 4.0%-7.8%. Among them, G3N2 maintained higher soil water content in all soil layers during sowing, vegetative, reproductive and harvesting stages. From sowing to jointing stage, the soil temperature under G2N2, G3N2 and G3N3 increased by 0.6-1.3 ℃ and the soil accumulated temperature increased by 24.8-55.3 ℃ compared with G0N1. From the filling stage to the mature stage, the soil temperature of each green manure returning combined with nitrogen reduction treatment was 0.4-1.0 ℃ lower than that under G0N1, and the soil accumulated temperature decreased by 7.9-20.0 ℃. At the same time, the temperature change range under G3N2 in soil warming and cooling stage was smaller than that under other treatments. Green manure returning combined with nitrogen reduction significantly increased the leaf area index of wheat from booting stage to maturity stage, providing sufficient photosynthetic source for dry matter accumulation at late growth stage. Under this condition, compared with G0N1, the biomass and grain yield of wheat increased by 13.7%-28.0% and 11.7%-31.3%, respectively, and the increase under G3N2 was the largest. Correlation analysis showed that grain yield and its components were significantly positively correlated with leaf area index, soil water content and soil temperature in 0-60 cm soil layer. Structural equation model analysis found that soil hydrothermal conditions indirectly affected yield changes by directly affecting leaf area index. 【Conclusion】Green manure returning combined with nitrogen reduction could improve the soil hydrothermal environment of wheat field and increase the leaf area index of wheat, so as to obtain high yield. Therefore, 30 000 kg·hm^-2 green manure+15% nitrogen reduction was the best green manure nitrogen fertilizer application mode to optimize the field hydrothermal environment and obtain high yield in oasis irrigation area.

The Regulatory Effect of Reduced Irrigation and Combined Organic- Inorganic Fertilizer Application on Stay-Green Characteristics in Silage Maize Leaves After Tasseling Stage

CHEN GuiPing, LI Pan, SHAO GuanGui, WU XiaYu, YIN Wen, ZHAO LianHao, FAN ZhiLong, HU FaLong

Scientia Agricultura Sinica. 2025, 58(7): 1381-1396. doi:10.3864/j.issn.0578-1752.2025.07.011

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【Objective】The study investigated the regulatory effects of reduced irrigation and the combined application of organic and inorganic fertilizers on stay-green characteristics in leaves and yield performance of silage maize after tasseling stage, in order to explore the optimal nitrogen application ratio of organic and inorganic fertilizers under reduced irrigation conditions, so as to provide a theoretical basis for high-yield and efficient cultivation practices of silage maize in arid irrigation areas.【Method】From 2021 to 2023, a two-factor split-plot experimental design was employed in the Hexi oasis irrigation area. The main plots consisted of two irrigation levels: reduced 20% irrigation (I1) and conventional irrigation (I2), while the subplots included five ratios of organic and inorganic fertilizer nitrogen fertilization maintaining equivalent nitrogen levels: 100% inorganic nitrogen fertilizer (F1), 75% inorganic nitrogen fertilizer+25% organic fertilizer (F2), 50% inorganic nitrogen fertilizer+50% organic fertilizer (F3), 25% inorganic nitrogen fertilizer+75% organic fertilizer (F4), and 100% organic fertilizer (F5). The study explored the response of stay-green characteristics in silage maize leaves after tasseling stage and fresh and hay yields to different irrigation amounts and organic-inorganic nitrogen fertilizer ratios.【Result】The reduction in irrigation alone resulted in a decrease in leaf stay-green characteristics of silage maize after tasseling stage. However, combining reduced irrigation with the application of both organic and inorganic fertilizers enhanced leaf stay-green characteristics after tasseling stage. Among these combinations, the reduced 20% irrigation combined with 75% inorganic nitrogen fertilizer+25% organic fertilizer (I1F2) showed a significant advantage. I1F2 could increase leaf area index and stay-green in leaves of silage maize after tasseling. Compared with conventional irrigation combined with 100% inorganic nitrogen fertilizer (I2F1), I1F2 could increase leaf area index and stay-green in leaves by 14.3% and 6.8%, respectively. Compared with the I2F1 treatment, I1F2 also increased chlorophyll a and b content in leaves of silage maize by 14.2% and 10.7%, respectively. As the increase in chlorophyll a content was greater than that of chlorophyll b, a higher chlorophyll a/b ratio was achieved. 75% inorganic nitrogen fertilizer+25% organic fertilizer under conditions of 20% reduced irrigation enhanced the reactive oxygen species scavenging capacity in leaves of silage maize after tasseling stage. Superoxide dismutase, peroxidase, catalase, and ascorbate peroxidase activities under I1F2 were increased by 12.0%, 7.8%, 10.7%, and 10.2% than that under I2F1, respectively. Compared with the I2F1 treatment, I1F2 increased proline and solute protein content in silage maize after tasseling stage by 9.8% and 9.7%, respectively, and reduced malondialdehyde content by 8.4%. Therefore, the silage maize under I1F2 could achieve higher fresh and hay yields at the optimal harvest time, increasing by 9.9% and 13.6% compared with I2F1. Comprehensive analysis indicated that I1F2 could significantly improve leaf area index, stay-green characteristics, and chlorophyll content of silage maize by enhancing leaf antioxidant enzyme activity, increasing content of cellular osmotic regulatory substances, and reducing malondialdehyde content after tasseling stage. Consequently, this effectively boosted the yield of silage maize.【Conclusion】Reduced 20% irrigation combined with 75% inorganic nitrogen fertilizer+25% organic fertilizer was an optimal water and nitrogen management strategy for extending the stay-green period of silage maize leaves after tasseling stage and increasing yield in arid irrigation areas.

Genome-Wide Identification and Analysis of TPS Gene Family and Functional Verification of VvTPS4 in the Formation of Monoterpenes in Grape

YANG CaiLi, LI YongZhou, HE LiangLiang, SONG YinHua, ZHANG Peng, LIU ZhaoXian, LI PengHui, LIU SanJun

Scientia Agricultura Sinica. 2025, 58(7): 1397-1417. doi:10.3864/j.issn.0578-1752.2025.07.012

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【Objective】Based on grape genome information, this study identifies and analyzes the terpene synthase (TPS) gene family expression in grapes using bioinformatics methods. This research lays an important foundation for subsequent studies on the biological functions of VvTPSs and grape breeding. 【Method】The VvTPS gene family was identified using the protein sequences of Arabidopsis AtTPS gene family members and the reported Hidden Markov model (HMM) files PF01397 and PF03936. Various bioinformatics tools, including Expasy, Tbtools, MEME, MEGA, MCScanX, SPOMA, WoLF PSORT, and PlantCARE were used to analyze the physicochemical characteristics, phylogenetic tree, chromosomal distributions, gene structure, subcellular localization, secondary structure of protein, and cis-acting elements in the promoters of gape TPS family genes. Additionally, the expression profiles of TPS genes in two aromatic grape varieties Red Globe (neutral) and Muscat Hamburg (muscat) were analyzed using qPCR. The transgenic tomato overexpressed VvTPS4 was used to study its function in influencing the muscat aroma by metabolomics. 【Result】A total of 65 VvTPS gene family members were identified, encoding proteins ranging from 339 to 840 amino acids, with an average molecular mass of 64.13 kDa and theoretical isoelectric points of 4.93 to 7.65. Most members have 7 exon structures. Phylogenetic analysis classified the grape TPS genes into five subfamilies: TPS-a, TPS-b, TPS-c, TPS-e/f, and TPS-g. Subcellular localization analysis indicated that most VvTPS proteins are localized in chloroplasts and cytoplasm, with 10 members from the TPS-g subfamily found in plastids. Promoter analysis revealed numerous cis-acting elements associated with responses to light, temperature, drought, hormones, and defense mechanisms. Of the 29 VvTPS genes cloned, most showed higher expression levels in the muscat-flavored grape variety compared to the non-aromatic variety. In transgenic tomatoes overexpressing VvTPS4, volatile monoterpenoids were significantly accumulated, with linalool levels increasing 20.73-fold and L-α-terpineol levels rising 14.55-fold compared to the wild type. Flavor characteristics analysis demonstrated these two compounds have floral aroma and are the main characteristic substances affecting the aroma of muscat fragrance.【Conclusion】Sixty-five VvTPS genes were identified in grapes, showing high conservation with some variations. Several TPS genes showed significantly expressed in muscat-flavored grape varieties. Overexpression of VvTPS4 led to a significant increase the accumulation of volatile monoterpenoids, which may play an essential role in developing the muscat aroma in grapes.

Identification and Functional Analysis of Ca²⁺-ATPase Gene Family in Banana

TENG MengXin, XU Ya, HE Jing, WANG Qi, QIAO Fei, LI JingYang, LI XinGuo

Scientia Agricultura Sinica. 2025, 58(7): 1418-1433. doi:10.3864/j.issn.0578-1752.2025.07.013

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【Objective】Ca²⁺-ATPase is a vital Ca²⁺ efflux channel in plants, which plays an important role in maintaining cell homeostasis and inducing plant response to stress. In this study, bioinformatics and molecular biology were used to analyze the members of Ca²⁺-ATPase family in banana A genome, detect the changes of gene expression and verify gene function, so as to provide reference for exploring the mechanism of banana salt tolerance.【Method】BLAST the members of banana A genome in banana genome database by gene family name combined with Arabidopsis and rice Ca²⁺-ATPase sequences, and use Pfam, ExPASy, Cell-PLoc, NCBI, MEGA-X, TBtools and other online websites or software to predict and analyze the protein physicochemical properties, subcellular localization, conservative domain and cis-acting elements of banana Ca²⁺-ATPase. The expression of the family genes was analyzed by qRT-PCR, and the subcellular localization and prokaryotic expression of the key genes were analyzed.【Result】In this study, 20 members of Ca²⁺-ATPase family were identified in banana A genome, including 13 MaACAs and 7 MaECAs; proteins analysis of physicochemical properties showed that Ca²⁺-ATPase contained 6-11 transmembrane structures, encoding amino acids between 857 and 1 103. Subcellular localization predicted that MaACAs might be located on plasma membrane, chloroplast, endoplasmic reticulum and tonoplast, while MaECAs was located in endoplasmic reticulum. The conserved motifs are highly consistent, including four conserved domains peculiar to the family except MaACA13, responses to light, hormone, defense and stress. qRT-PCR results showed that other members of Ca²⁺-ATPase gene family except MaECA3 were up-regulated in Brazilian banana cells treated with 100 mmol·L^-1 NaCl, in which MaACA5 and MaACA10 were up-regulated to a large extent, and the expression of these two genes was affected by Ca²⁺. The results of subcellular localization of tobacco leaves showed that MaACA5 and MaACA10 were located on the plasma membrane, and the recombinant plasmids pET28a-MaACA5, pET28a-MaACA10 and transformed into E. coli BL21 were superior to the control strains under the conditions of 800 mmol·L^-1NaCl, 800 mmol·L^-1 mannitol and 50 ℃. 【Conclusion】 20 Ca²⁺-ATPase family members were identified in banana A genome, the gene structure was highly conservative, including hormone, defense and stress-related responses, MaACA5 and MaACA10 related to banana salt tolerance were screened and prokaryotic expression to verify the gene function, which provides a reference for further study of banana salt tolerance.

CRISPR-Cas12a Gene Editing Technology and Its Application in Agricultural Production

LUO Gang, CHENG YiYi, YANG Wen, XIAO YiMeng, YANG ChengXi

Scientia Agricultura Sinica. 2025, 58(7): 1434-1450. doi:10.3864/j.issn.0578-1752.2025.07.014

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The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein (CRISPR- Cas) gene editing technology has not only revolutionized life sciences but also catalyzed transformative advancements in agriculture. As a critical branch of the CRISPR system, the CRISPR-Cas12a system exhibits unique molecular characteristics and distinct application potential in biological breeding and disease diagnosis compared to the classical CRISPR-Cas9 system. Unlike the Type II Cas9 system, the Type V Cas12a protein possesses a single RuvC-like nuclease domain, contrasting sharply with the dual HNH-RuvC nuclease domains of Cas9. Cas12a generates staggered double-strand breaks (DSBs) in target DNA while retaining the CRISPR RNA (crRNA) and Cas12a-formed "R-loop". The preservation of this R-loop constitutes the the structural basis for the collateral cleavage activity inherent to the CRISPR-Cas12a system, which underpins its utility in developing nucleic acid and small molecule detection technologies. Recognizing thymine-rich protospacer adjacent motifs (PAMs), CRISPR-Cas12a acts as a powerful complement to existing CRISPR-Cas systems. Its crRNA-dependent autonomous processing mechanism, distinct from the tracrRNA-dependent system of Cas9, offers superior advantages in multiplex gene editing. These features have driven breakthroughs in crop genetic improvement, including the successful development of disease-resistant and high-yield commercial crop varieties. In basic research, catalytically inactive Cas12a (dCas12a) fused with transcriptional regulators or epigenetic modifiers enables precise gene expression regulation without inducing DSBs. Furthermore, its integration with isothermal amplification techniques allows for visual disease detection.This review systematically introduced the CRISPR-Cas12a system from multiple perspectives: (1) classification of Type V Cas proteins, (2) mechanistic principles of Cas12a in bacterial immunity, and (3) functional domains of the Cas12a-crRNA complex. A comparative analysis between CRISPR-Cas12a and CRISPR-Cas9 was conducted across four dimensions: crRNA processing mechanisms, structural-functional features of Cas effectors, editing efficiency, and application scenarios. Additionally, the regulatory systems of CRISPR-dCas12a and CRISPR-dCas9 were evaluated regarding gene expression modulation, epigenetic editing, and base editing. The review also elucidated the molecular detection principles of CRISPR-Cas12a in targeting nucleic acids, proteins, and small molecules, as well as its agricultural applications in gene regulation, base editing, pathogen detection, disease diagnosis, and bio-breeding.With the emergence of safer non-DSB- dependent technologies such as prime editing, the CRISPR-Cas12a system was poised to play an increasingly vital role in crop precision breeding, livestock genetic improvement, and rapid clinical diagnostics. These advancemented promise innovative solutions to global food security challenges and infectious disease control, further cementing CRISPR-Cas12a as a cornerstone tool in agricultural biotechnology and molecular medicine.

Effect of Gln on Endoplasmic Reticulum Stress in Retained Fetal Membranes Cows Under Oxidative Stress via the PI3K/AKT Pathway

WANG Wei, LUO ChunHai, JIA HongDou, LIU JiaJin, LI DanYang, FU ShiXin

Scientia Agricultura Sinica. 2025, 58(7): 1451-1462. doi:10.3864/j.issn.0578-1752.2025.07.015

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【Objective】This study aimed to determine whether glutamine could alleviate endoplasmic reticulum stress in the maternal placenta of cows with retained fetal membranes (RFM) caused by oxidative stress through the PI3K pathway, and to explore the protective mechanism of glutamine. 【Method】Three cows with RFM and three healthy cows (NRFM) were selected based on whether the fetal membranes could be normally expelled within 12 hours after parturition. The content of glutamine (Gln) in the serum of RFM and NRFM cows was determined. The expression changes of endoplasmic reticulum stress (GRP78, p-PERK, PERK, p-IRE1α, IRE1α, and ATF6) and key apoptosis factors (Caspase-3, Caspase-8, Caspase-9, P53, Bcl-2, and Bax) in the maternal placenta of the two groups of cows were detected by qRT-PCR and Western blot. In the in vitro experiment, bovine uterine caruncle epithelial cells (UCEs) were stimulated with 400 μmol·L^-1 H₂O₂ to establish an in vitro oxidative stress model of bovine UCEs. On this basis, Gln pretreatment, PI3K inhibitor (LY294002) and glutamine co-pretreatment were performed. The concentration changes of Ca²⁺ in the cytoplasm were detected by immunofluorescence, and the expression changes of endoplasmic reticulum stress and apoptosis-related indicators in bovine UCEs were detected by qRT-PCR and Western blot. 【Result】When cows had RFM, the serum Gln content was significantly decreased (P<0.01), and the expression of endoplasmic reticulum stress-related proteins and genes GRP78 (P<0.01), p-PERK/PERK, p-IRE1α/IRE1α, ATF6 (P<0.05) in the placenta tissue was significantly increased, while the expression of apoptosis-related proteins and genes P53 (P<0.01), Caspase-3, Caspase-8, Caspase-9, Bax/Bcl-2 (P<0.05) was significantly decreased. In the in vitro oxidative stress model, the expression of endoplasmic reticulum stress-related proteins and genes GRP78, p-PERK/PERK, p-IRE1α/IRE1α, ATF6 (P<0.01) and apoptosis-related proteins and genes P53, Caspase-3, Caspase-8, Caspase-9, Bax/Bcl-2 (P<0.01) in UCEs were significantly increased after H₂O₂ stimulation, and the concentration of Ca²⁺ in the cytoplasm was significantly increased (P<0.01). However, compared with the Gln protection group pretreated only with glutamine, Gln could not reduce the expression of endoplasmic reticulum stress and apoptosis-related proteins and genes in bovine UCEs when PI3K was inhibited, and the concentration of Ca²⁺ in the cytoplasm was significantly increased (P<0.01). 【Conclusion】When cows had RFM, the maternal placenta tissue underwent strong endoplasmic reticulum stress and abnormal apoptosis, which led to the damage of normal cell function and was an important cause of the disorder of fetal membrane expulsion. High levels of Gln could regulate the expression of key proteins in endoplasmic reticulum stress through the PI3K/AKT pathway, alleviate the cellular dysfunction caused by endoplasmic reticulum stress, and thereby reduce the placental expulsion disorder caused by endoplasmic reticulum stress induced by oxidative stress.

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