Table of Content

01 October 2024, Volume 57 Issue 19

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CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS

Breeding of the Fusarium Head Blight (FHB)-Resistant Wheat Cultivar Lunxuan 20 Using the Dwarf-Male Sterile Wheat Molecular Strategy in the Yellow and Huai River Valley Winter Wheat Region

MAI ChunYan, LIU YiKe, LIU HongWei, LI HongJie, YANG Li, WU PeiPei, ZHOU Yang, ZHANG HongJun

Scientia Agricultura Sinica. 2024, 57(19):  3719-3729.  doi:10.3864/j.issn.0578-1752.2024.19.001

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【Objective】To develop high-yielding and FHB-resistant wheat cultivars in the Yellow and Huai River Valley Winter Wheat Zone (YHWZ), simultaneously improving of yield and resistance was conducted in this study.【Method】Using the elite parent dwarf male sterile (DMS) wheat combined with double haploid (DH) technology and molecular marker assisted selection (MAS) of Fhb1 (DMS wheat molecular breeding strategy), DH lines were developed using Sumai 3 as a donor (FHB- resistant parent) and Zhoumai 16’s DMS wheat, Zhoumai 16, Lunxuan 136 and Lunxuan 6 as recipient parents. The agronomic traits (plant height, heading date, yield, etc.) and FHB resistance were evaluated for these DH lines.【Result】A total of 51 Fhb1-DH lines characterized by facultative growth habit, semi-dwarf and white grains were selected using this strategy. The average number of infected spikelets of 51 lines were 5.7 and 7.3 at the 2020Henan and 2020Beijing sites, respectively, and average disease severities were 27.7% and 35.2%, which is not different from moderately susceptible control Huaimai 20. There was no significant difference in grain yield per hm² between the mean performance of the 51 lines and the control Zhoumai 18. DH116 (Lunxuan 20), a promising line from the 51 lines, was further evaluated for FHB resistance and agronomic traits in multiple environments. The resistance of Lunxuan 20 to FHB was significantly improved, and no significant difference was found in the number of infected spikelets or disease severity between Lunxuan 20 and moderately or highly resistant controls at four sites. Lunxuan 20 showed slightly greater grain yield per hm², and significantly higher number of spikelets per spike and thousand grain weight (P<0.05), earlier heading date and shorter plant height (P<0.05) than the control Zhoumai 18 in two environments. The grain yield per hm² of Lunxuan 20 was 4.6% and 1.7% higher than the control cultivar Bainong 207 in the two list trials of Henan Province, and 3.5% higher than Bainong 207 in the demonstration trial. Resistance of Lunxuan 20 to FHB ranged from moderate susceptibility to moderate resistance in two-year list tests using the single-floret injection and spray inoculation methods. Lunxuan 20 carries the semi-dwarfing gene Rht-D1b at the Rht-D1 locus, and the recessive alleles vrn-A1, vrn-B1 and vrn-D1 associated with the winter growth habit at the Vrn-A1, Vrn-B1 and Vrn-D1 loci. Based on the wheat 660K single nucleotide polymorphisms (SNPs), 64.7% of the SNPs were shared by Lunxuan 20 and its parents, and the direct genetic contributions of Zhoumai 16, Lunxuan 136, Lunxuan 6 and Sumai 3 to Lunxuan 20 were 69.8%, 12.6%, 6.1% and 11.5%, respectively.【Conclusion】A high-yielding and FHB-resistant wheat cultivar Lunxuan 20 was bred using the DMS wheat molecular breeding strategy.

Comprehensive Evaluation on Production Performance and Nutritional Quality of Different Varieties of Forage Oat in the Qinghai Lake Area

WANG XiaoJun, WANG JinLan, JU ZeLiang, LIANG GuoLing, JIA ZhiFeng, LIU WenHui, MA Xiang, MA JinXiu, LI Wen

Scientia Agricultura Sinica. 2024, 57(19):  3730-3742.  doi:10.3864/j.issn.0578-1752.2024.19.002

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【Objective】In order to explore the adaptability of 10 oat (Avena sativa) varieties in the Qinghai Lake area and screen out the high-yield and high-quality oat varieties suitable for planting in this area, so as to provide data support for high-yield and high-quality forage production in this area and similar areas. 【Method】In this study, 10 oat varieties (Avena sativa cv. Qinghai No.444, A. sativa cv. Baiyan No.7, A. sativa cv. Qingyan No.4, A. nuda cv. Qingyou No.3, A. sativa cv. Qingyin No.2, A. sativa cv. Qingyan No.3, A. sativa cv. Lena, A. sativa cv. Qinghai, A. sativa cv. Qingyan No.1 and A. sativa cv. Longyan No.1) commonly cultivated in Qinghai province were used. The experiment was established using a randomized complete block design. Three experimental blocks, located at least 3 m away from each other, were randomly chosen. Each block contained 10 different oat varieties plots, for a total of 30 plots. The area of each plot was 3 m × 5 m. The experiment was sown in strip. The row spacing was 25 cm and the sowing depth was 3-4 cm. According to the thousand seed weight, purity and germination rate of each variety, the seeding rate of each variety was calculated according to the seedling protection number of 6.75 million plants/hm². Moreover, the diammonium phosphate (150 kg·hm^-2) and urea (75 kg·hm^-2) were used as base fertilizer. The seeds were sown on May 16, 2022 and May 19, 2023, and field observations and sample collection were conducted on September 23, 2022 and September 26, 2023, respectively. The production performance and nutritional quality of oat varieties were analyzed, and the piecewise structural equation model was used to explore how varieties, planting years and their interactions affected nutritional quality via agronomic traits and yield traits. Furthermore, the multi-criterion decision model-TOPSIS (Technique for order preference by similarity to an ideal) was used to comprehensively evaluate the various indexes of the tested oat varieties. 【Result】Our results demonstrated that A. sativa cv. Qingyan No.3 had the highest plant height (89.4-92.5 cm), and the lowest acid detergent fiber (34.8%-34.9%) and neutral detergent fiber (51.8%-53.4%). The A. sativa cv. Qingyan No.4 had the most tillers number (2.7-3.6/plant) and the lowest crude ash content (10.9%-11.3%). The highest of forage yield, crude protein, relative feeding value were found in A. sativa cv. Qingyan No.3 and A. sativa cv. Qingyan No.4. while the stem/leaf ratio of the A. sativa cv. Qingyan No.3 and A. sativa cv. Qingyan No.4 were significantly lower than that of other varieties. The highest crude fat was found in A. sativa cv. Qingyan No.1 (3.8%-3.9%). The Pearson correlation analysis showed that the oat yield was positively correlated with crude protein content and relative feeding value, but negatively correlated with acid detergent fiber and crude ash. The stem/leaf ratio was positively correlated with acid detergent fiber and neutral detergent fiber, but negatively correlated with crude protein and relative feeding value. The structural equation model analysis showed that the varieties, planting years and their interactions had direct effects on the oat nutritional quality, and indirectly affected the nutritional quality by affecting plant height, tillering number, stem/leaf ratio and hay yield. The total effect value of stem/leaf ratio was the highest, which was -0.37.【Conclusion】The comprehensive evaluation of TOPSIS model showed that A. sativa cv. Qingyan No.4 and A. sativa cv. Qingyan No.3 could not only maintain higher production performance, but also have higher nutritional quality, which are ideal oat varieties for planting in Qinghai Lake area.

Research on the Production Potential of Main Sugarcane Varieties Under Different Planting Modes in Hilly and Mountainous Areas

ZHAO Yong, AI Jing, WANG YuTong, ZHANG ZhongFu, YANG HongQi, LI JiaQun, GUO ZhaoJian, LIU HaiJun, QIN Wei, DENG Jun, ZHANG YueBin

Scientia Agricultura Sinica. 2024, 57(19):  3743-3757.  doi:10.3864/j.issn.0578-1752.2024.19.003

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【Objective】The research focused on the production potential of sugarcane main varieties under the different planting modes in mountainous and hilly regions, in order to explore the relationship between the varieties and mechanical adaptation, screen the adaptable varieties for mechanical production, and clarify the main characteristics of the varieties, thus to provide the theory for promoting the completely mechanical production of sugarcane in mountainous and hilly regions.【Method】Two different ecological types of sugarcane production regions in mountainous regions of Yunnan were selected to study the production potential of four main planting varieties, including LC05-136, YZ05-49, YZ05-51, and YZ08-1609, under two different planting modes (the completely mechanical or manual production).【Result】The yield and sucrose content of sugarcane varied between the two factors of varieties and planting modes, and the yield was significantly affected. Under the mechanical production, the yield of YZ08-1609 was significantly higher than that with the treatment of artificial production (P<0.01), while YZ05-51 showed the opposite trend (P<0.01), under the mechanical production, the sucrose content of YZ08-1609 is slightly higher than that with the artificial production (P>0.05), while these of LC05-136 and YZ05-49 were slightly decreased (P>0.05). The planting density and the emergence rate of sugarcane varied with both varieties and production modes. The planting density under the machinery production was generally higher than that under the artificial production, while the emergence rate was generally lower than that with the treatment of the artificial production. The emergence rate of YZ05-51 under the machinery production significantly decreased (P<0.05), while the emergence rate of YZ08-1609 remained relatively stable (P>0.05). The main agronomic traits of sugarcanes varied with both varieties and the production modes. The millable stalks of YZ08-1609 under the machinery production were significantly higher than that under the artificial production (P<0.05); the industrial characteristics were mainly influenced by the variety. YZ08-1609 owned higher growth potential under the machinery production, with significantly higher yield and millable stalks compared with these under the artificial production, and slightly higher sucrose content; YZ05-51 owned higher growth potential under the artificial production with the performance of higher yield under the artificial production. 【Conclusion】Different sugarcane varieties owned the different adaptabilities to the machinery production, like some sugarcane varieties were suitable for the machinery production and some were suitable for the manual production. During pushing forward the progress of the application of mechanical production in sugarcane planting, the adaptability of varieties should be fully considered. Overall, sugarcane varieties suitable for the machinery production should own higher sugarcane yield and more millable stalks, and the sucrose content should be less affected or even slightly higher than that under the manual production.

TILLAGE & CULTIVATION·PHYSIOLOGY & BIOCHEMISTRY·AGRICULTURE INFORMATION TECHNOLOGY

Coordinated Effects of Maize Ear Type and Planting Density on Interspecific Competition in Maize-Peanut Intercropping System

LIU Han, DING Di, WANG JiangTao, ZHENG Bin, WANG XiaoXiao, ZHU ChenXu, LIU Juan, LIU Ling, FU GuoZhan, JIAO NianYuan

Scientia Agricultura Sinica. 2024, 57(19):  3758-3769.  doi:10.3864/j.issn.0578-1752.2024.19.004

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【Objective】The interspecific competition during the late coexisting period of maize (Zea mays L.) intercropping with peanut (Arachis hypogaea L.) (maize||peanut) is the main problem of inhibition to further the intercropping advantage. The aim of this study was to investigate coordinated effects and mechanism of maize ear type and planting density on interspecific competition in maize||peanut, and to determine the reasonably variety and planting density of intercropped maize, so as to provide a theoretical basis and technical support for achieving high yield of maize||peanut production. 【Method】The experiment was conducted on the farm of Henan University of Science and Technology from 2022 to 2023, utilizing a field randomized block design. The selected maize varieties were ‘Zhengdan 958’ for medium-ear type and ‘MC4520’ for large-ear type, with two planting densities of 5.00×10⁴ plants/hm² (D1) and 4.55×10⁴ plants/hm² (D2) for intercropping. The effects of maize ear type and planting density on interspecific competition and intercropping advantage of maize||peanut were studied. 【Result】The results of the 2-year experiment showed that compared with D1 density medium-ear type ‘Zhengdan 958’ intercropped with peanut (M_D1ZD||P), D2 density medium-ear type ‘Zhengdan 958’ intercropped with peanut (M_D2ZD||P) and D2 density large-ear type ‘MC4520’ intercropped with peanut (M_D2MC||P) could enhance the canopy daily light intensity of peanut during the late coexisting period, and increase the leaf area index and maximum growth rate of peanut. M_D2MC||P increased dry matter accumulation in maize and peanut populations, especially in maize grains and peanut pods by 26.41%-30.11% and 8.06%-8.25%, respectively. M_D2ZD||P and M_D2MC||P could improve the interspecific competitiveness of peanut, and the interspecific competitiveness index A_p, CR_p and K_p were increased by 7.55%-19.10%, 4.23%-9.12% and 9.05%-9.60%, respectively, during M_D2MC||P later coexistence. M_D2ZD||P decreased maize yield by 8.09%-8.19%, decreased the yield of intercropping system and intercropping advantage by 2.23%-2.58% and 7.55%-13.29%, respectively. M_D2MC||P could increase the yield of maize and peanut, and the yield of intercropping system and intercropping advantage were significantly increased by 6.28%-7.45% and 11.85%-27.18%, respectively (P<0.05). 【Conclusion】In this experimental area, compared with the medium-ear type ‘Zhengdan 958’ with a density of 5.00×10⁴ plants/hm² intercropping with peanut, the large-ear type ‘MC4520’ with a density of 4.55×10⁴ plants/hm² intercropping with peanut could exert the individual production potential of maize and ensure that the yield of maize was not reduced. Simultaneously, it was more conducive to the coordination of interspecific light competition during the latter coexisting period of maize||peanut, which improved the interspecific competitiveness of peanut, and increased the yield of peanut. Therefore, this planting pattern was conductive to increase the yield of intercropping system and intercropping advantages.

Study on the Correlation Between Cold Resistance of Maize and Its Ability of Optimizing Sugar Composition at Low Temperature

GU YinHe, ZHAO WenQing, SHI DaiWei, HU Wei, WANG ShanShan, ZHOU ZhiGuo, WANG YouHua

Scientia Agricultura Sinica. 2024, 57(19):  3770-3783.  doi:10.3864/j.issn.0578-1752.2024.19.005

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【Objective】The low temperature in maize seeding stage will lead to reduction of photosynthesis, phosphorus and other nutrient absorption, which will lead to changes in metabolites, growth retardation, or even death. This study aimed to clarify the mechanism of cold-tolerance formation in maize seedlings, and to explore the changes of saccharides and their relationship with phosphorus uptake among different maize varieties at low temperatures, it provides theoretical basis for stress-resistant cultivation of maize. 【Method】Demeiya 3 (DMY3, cold-tolerant variety) and Hongdan 6 (HD6, cold-sensitive variety) that have similar phenotype were screened out in the previous study, which were selected as research materials in this study. Maize seedlings were cultivated in pots, and 28/22 ℃ (day/night) and 15/8 ℃ (day/night) were set as control and low temperature, respectively. Samplings were taken at 0, 1, 3, 5, and 7 d after treatment starts. The changes of phenotypic indexes, such as plant biomass, and growth-related indexes, such as leaf anthocyanin phosphorus absorption rate and carbohydrate content, were measured. 【Result】(1) After low-temperature treatment, morphological indexes, such as plant height, root length and fresh weight of the two varieties, were significantly lower than those of the control. However, the root-shoot biomass ratio increased continuously. The significant differences associated with low-temperature-tolerance were found between the two varieties emerged under 3-5 days of treatment. (2) Low temperature significantly increased the content of sucrose, glucose and starch in the leaves, and most of the increasement in the low-temperature-sensitive variety Hongdan 6 were higher than that in the low-temperature-tolerant variety Demeiya 3, but the increase of glucose content was lower than that in the low temperature tolerant varieties. (3) With low temperature stress, the seedling had a tendency to increase the non-reducing/reducing sugars ratio, soluble sugar/amino acid ratio (C/N ratio) and starch/soluble sugars ratio. The increasement of temporary-storage-sugar (starch/sucrose) and monosaccharide/disaccharide ratios (glucose/sucrose) were higher in the low-temperature-tolerant variety than that in the low-temperature-sensitive variety. (4) With low temperature treatment, the rate of phosphorus uptake by roots decreased significantly, and the rate of uptake was slower in the low-temperature-sensitive variety than that in the low-temperature-tolerant variety. (5) As the treat prolongated, the Pi content gradually decreased, while the anthocyanin content kept increasing. And a negative correlation between the content of Pi and anthocyanin was observed, and the correlation was stronger in the low-temperature-sensitive variety than in the low-temperature-tolerant variety. (6) The increase of sucrose or the decrease of starch would probably reduce the accumulation of biomass, meanwhile, which induced the production of anthocyanin, as well as lots of secondary metabolites. 【Conclusion】Low temperature led to the inhibition of phosphorus uptake in maize seedlings, and the content and composition in leaves were significantly altered. At low temperature condition, the increase of sucrose content was an important cause led to anthocyanin accumulation. The increase of starch and glucose was likely active responses of plants to adapt to low temperature, while the increase of sucrose, soluble sugar, fructose and anthocyanin seemed to be passive responses. The decrease of Pi content and the morphological growth indexes on seedlings were the adverse consequences of low temperature stress, and its magnitude associated with the low-temperature-tolerance of the maize varieties.

Effects of Saline-Fresh Water Rotation Irrigation on Photosynthetic Characteristics and Leaf Ultrastructure of Tomato Plants in Greenhouse

XIN Lang, SONG JiaWen, FU YuanYuan, TANG MaoSong, JING LingKun, WANG XingPeng

Scientia Agricultura Sinica. 2024, 57(19):  3784-3798.  doi:10.3864/j.issn.0578-1752.2024.19.006

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【Objective】Revealing the mechanisms of saline-fresh water rotation irrigation that affected the leaf ultrastructure and photosynthetic characteristics of tomato plants was benefit to optimize the strategy of saline water irrigation of greenhouse tomatoes.【Method】In this paper, the local conventional tomato variety “Mingzhu” was used as the material to carry out a two-year trial of saline and fresh water irrigation in greenhouses in the southern Xinjiang region from 2022 to 2023. The four treatments consisted of rotation irrigation with four times saline-fresh water (W1), rotation irrigation with two times saline water and two times fresh water (W2), rotation irrigation with two times fresh water, four times saline water, and two times fresh water (W3), and freshwater irrigation as a control (CK). The three rotation patterns had the same amount of saline water and fresh water. The effects of saline and alkaline stress produced by saline and freshwater rotational irrigation on the ultrastructure, chlorophyll content, stomatal characteristics, gas exchange parameters, and yield of facility tomato leaves were mainly investigated.【Result】The results indicated that the saline-alkali stress introduced by saline water significantly reduced the gas exchange parameters of tomato leaves and water use efficiency at the leaf scale, and both stomatal and non-stomatal factors played a key role in limiting leaf gas exchange. Tomato leaves not only adapted quickly to salinity stress by reducing individual stomatal openings, but also improved gas exchange efficiency by regulating stomatal density and stomatal shape through long-term stomatal differentiation and development. Compared with the CK treatment, the stomatal density of the leaves under W1, W2, and W3 treatments increased by 22.8%, 43.0% and 13.8%, respectively, and the stomatal width was reduced by 54.6%, 77.8%, and 13.7%, respectively; under the influence of soil salinity stress, compared with CK treatment, the chloroplast granular lamellae structure was disrupted in tomato leaves under W1 and W2. Compared with CK, W1 and W2 decreased leaf chlorophyll content by 6.2% and 11.8%, net photosynthetic rate by 16.3% and 26.2%, and yield by 45.3% and 52.5%, and the maximum leaf area index was 20.8% and 27.5% lower than that in the same period of CK treatment, respectively. In contrast, W3 presented a relatively intact mesophyll cell structure and relatively high chlorophyll content and photosynthetic efficiency, W3 only increased the average single fruit weight by 6.5%, fruit diameter by 6.0% and the yield by 0.7%, with no significant differences compared with CK. 【Conclusion】By comprehensive analysis of physiological changes and yield of tomato, irrigation of saline water in the flowering and fruiting period-fruit expansion period of tomato, the other reproductive stages of freshwater irrigation, to mitigate the adverse effects of saline water irrigation on the growth of tomato, W3 treatment was recommended as a facility in the southern Xinjiang region of the synergistic use of saline-fresh water irrigation of tomato.

PLANT PROTECTION

BnJAZ7 Promotes Sclerotinia sclerotiorum Infection by Affecting the Antioxidant Pathway in Brassica napus

YANG HaoRong, JIA Fan, HU Xu, MU Rong, LIU WeiNa, LIU ChangYun, WANG ShanZhi, SUN XianChao, MA GuanHua, CHEN GuoKang

Scientia Agricultura Sinica. 2024, 57(19):  3799-3809.  doi:10.3864/j.issn.0578-1752.2024.19.007

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【Objective】Rapeseed sclerotiniose caused by Sclerotinia sclerotiorum, is the first major disease affecting rapeseed production. The objective of this study is to obtain BnJAZ7 from Brassica napus via molecular cloning, and to clarify the expression characteristics of BnJAZ7 and its role in the process of S. sclerotiorum infection by means of bioinformatics, cell biology and molecular biology, so as to lay a theoretical foundation for molecular breeding of sclerotiniose resistance in rapeseed.【Method】The full-length of BnJAZ7 was amplified using molecular cloning techniques, and the protein characteristics encoded by BnJAZ7 were analyzed through bioinformatics. The genetic relationship tree of BnJAZ7 was constructed using MEGA X. Real-time fluorescence quantitative PCR technology was employed to analyze the tissue-specific expression of BnJAZ7 in rapeseed, as well as its expression during S. sclerotiorum infection. A fusion expression vector of BnJAZ7 and GFP was generated and transiently transformed via Agrobacterium-mediated delivery into Nicotiana benthamiana leaf cells. Subsequently, the subcellular localization of the fusion protein was observed under a confocal laser microscope. The fusion protein was transiently expressed in N. benthamiana leaves and inoculated with S. sclerotiorum to assess the impact of BnJAZ7 expression on S. sclerotiorum infection. The BnJAZ7-OE overexpression transgenic rapeseed lines were developed. Following the generation of transgenic plants with high BnJAZ7 expression, the leaves of the T2 generation were inoculated with S. sclerotiorum to evaluate its effect on S. sclerotiorum infection. Biochemical techniques were utilized to measure the activities of antioxidant enzymes SOD, POD, CAT and PAL after S. sclerotiorum infection in BnJAZ7-OE rapeseed. Furthermore, real-time fluorescence quantitative PCR technology was applied to analyze the expression of antioxidant pathway-related genes BnSOD, BnPOD, BnPAL and BnCAT after S. sclerotiorum infection in BnJAZ7-OE rapeseed.【Result】BnJAZ7 spans 801 bp, encoding a protein of 266 aa, characterized by two structural domains, TIFY and CCT_2. Its molecular formula is C₁₂₄₄H₂₀₀₀N₃₅₈O₃₈₄S₁₃, with an isoelectric point of 9.57 and a theoretical molecular weight of 28.53 kDa. Notably, it lacks a transmembrane domain. Phylogenetic analysis revealed the closest genetic affinity of BnJAZ7 with B. napus TIFY 7. Expression profiling indicated a descending trend of BnJAZ7 expression in rapeseed tissues, from stems to roots, leaves, and flowers. However, after infection with S. sclerotiorum, BnJAZ7 expression showed a sequential increase at 6, 12, 24, and 48 hours post-infection. Subcellular localization studies demonstrated that eGFP:BnJAZ7 localized within the cell membrane and nucleus. Heterologous transient expression of eGFP:BnJAZ7 in N. benthamiana leaves enhanced the invasion of S. sclerotiorum. Additionally, overexpression of BnJAZ7 in transgenic rapeseed (BnJAZ7-OE) promoted S. sclerotiorum infection. After S. sclerotiorum infection in BnJAZ7-OE plants, the transcription levels of BnPOD, BnSOD and BnPAL were significantly reduced, and the transcription level of BnCAT was significantly increased, thereby reducing POD, SOD and PAL activities and increasing CAT activity.【Conclusion】The expression of BnJAZ7 in B. napus is stimulated by S. sclerotiorum infection. Moreover, the overexpression of BnJAZ7 significantly accelerates the infection rate of S. sclerotiorum. The pathogen further expedites infection by upregulating BnJAZ7 expression, subsequently diminishing the transcription levels of BnPOD, BnSOD and BnPAL, and increasing the transcription level of BnCAT, thereby regulating the activity of antioxidant enzymes to further accelerate the infection.

Complete Genome Sequence Analysis and Infectious Clone Construction of Mume Virus A Peach Isolate pp in Xinjiang

REN CaiXia, LIU Lin, LIU ShengXue, BU Fang DI, XIANG BenChun, ZHENG YinYing, CUI BaiMing

Scientia Agricultura Sinica. 2024, 57(19):  3810-3822.  doi:10.3864/j.issn.0578-1752.2024.19.008

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【Objective】Mume virus A peach isolate (MuVA pp) is a newly discovered virus infecting peach (Prunus persica) trees, and its complete genome sequence study has not been reported in China. Therefore, the purpose of this study is to analyze the genome, phylogenetic evolution and pathogenicity of MuVA pp isolate, and to investigate its prevalence in peach in Xinjiang, China, so as to provide scientific basis for the prevention and control of MuVA.【Method】RT-PCR was used to detect MuVA in field samples of peach, and 5′/3′ rapid amplification of cDNA ends (RACE) technology was used to determine the complete genome sequence of MuVA pp isolate. The genome organization and phylogenetic relationships were analyzed by bioinformatics methods. A full-length infectious cDNA clone was constructed using Gibson assembly, and its infectivity was tested by inoculation with Agrobacterium tumefacieas.【Result】The results of RT-PCR showed that 10 of the 30 suspected viral disease samples were infected with MuVA, and the infection rate of different peach varieties was from high to low, namely ‘July flat peach’ (4/9), ‘flat peach on August 1’ (5/13) and ‘medium mature August 1’ (1/8), respectively. The genome of MuVA pp is 7 647 nt in length and consists of 5′ UTR, 3′ UTR and two overlapping open reading frames (ORFs), encoding methyltransferase (Met), RNA helicase (Hel), RNA-dependent RNA polymerase (RdRp), coat protein (CP) and movement protein (MP). The sequence analysis showed that MuVA pp isolate has 80.8% and 82.7% identities with previously reported MuVA isolate pm14 at the nucleotide sequence and amino acid sequence levels, respectively. The polyproteins encoded by these two isolates differ by 406 amino acid residues, which are distributed in Met (21), Hel (29), RdRp (19), CP (18), and other (319). In addition, the 5′ UTR of the genome was more different, and the identity of the nucleotide sequence was only 74.6%. Of these encoded proteins, MP has the greatest variability (80.9% identity), while RdRp is the most conserved (94.0% identity). Phylogenetic analysis showed that MuVA pp isolate was closely related to MuVA pm14 isolate, and MuVA showed a tendency of host and geographic specificity. Plum isolates and mume isolates clustered into clusters, while peach isolates formed clays alone. The Chenopodium amaranticolor plants could be systemically infected by MuVA infectious clone, but symptomless. Tobacco (Nicotiana tabacum var. Samsun NN) agroinfiltrated showed allergic necrosis in inoculated leaves, but no systemic infection occurred. Other indicator plants, including C. quinoa, N. glutinosa, N. occidentalis, N. benthamiana, or Solanum lycopersicum, Cucumis sativus and Cucurbita moschata, could not be infected by MuVA infectious clones.【Conclusion】The complete genome sequence of the peach isolate pp of MuVA was sequenced successfully. The genome is a single-stranded positive-sense RNA with a length of 7 647 nt, lacking a 3′ terminal poly(A) tail, and encoding two ORFs. Mechanical inoculation proved that MuVA could not infect herbaceous plants. pCB301-MuVA, a MuVA pp full-length cDNA infectious cloning vector, was constructed. It could infect C. amaranticolor systematically and N. tabacum var. Samsun NN locally. The results provide a reference for further study of the pathogenic molecular mechanism of MuVA.

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

Effect of Long-Term Straw Returning on the Soil Organic Carbon Bound to Iron Oxides in Black Soil and Fluvo-Aquic Soil

SUN Yue, REN KeYu, ZOU HongQin, GAO HongJun, ZHANG ShuiQing, LI DeJin, LI BingJie, LIAO ChuQian, DUAN YingHua, XU MingGang

Scientia Agricultura Sinica. 2024, 57(19):  3823-3834.  doi:10.3864/j.issn.0578-1752.2024.19.009

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【Objective】 The purpose of this study was to explore the differences in the content and properties of different types of iron oxide bound organic carbon in different dryland soils and their responses to long-term straw returning. 【Method】 Based on the long-term fertilization experiments of black soil and fluvo-aquic soil in Gongzhuling, Jilin Province and Zhengzhou, Henan Province since 1990, soil samples under no fertilization (CK), single application of chemical fertilizer (NPK) and chemical fertilizer +straw returning (NPKS) for 29 years were collected, and the complex iron (Fe_PP), amorphous iron oxides (Fe_HH) and crystalline iron oxides (Fe_DH) were obtained through continuous extraction method. The differences in the content of different forms of iron oxides and their bound organic carbons were explored. Combined with ultraviolet-visible spectroscopy, the differences in the properties of iron oxide-bound organic carbon were also accessed. 【Result】 Compared with CK and NPK treatments, the soil organic carbon content under NPKS treatment in black soil was significantly increased by 18.1% and 14.9%, respectively, and fluvo-aquic soil was significantly increased by 46.1% and 13.3%, respectively. The content of Fe_PP in the two soils were lower than those under Fe_HH and Fe_DH, but the content of complexed iron-bound organic carbon (OC_PP) were significantly higher than those of amorphous iron oxide-bound organic carbon (OC_HH) and crystalline iron oxide-bound organic carbon (OC_DH), accounting for 71.4%-93.2% of the total iron oxide-bound organic carbon content, indicating that OC_PP was the main form of iron oxide-bound organic carbon. Compared with NPK treatment, the Fe_PP content in black soil was significantly reduced by 65.6%. Fe_HH content was significantly reduced by 17.8%, and Fe_DH content was not significantly changed under NPKS treatment, while the Fe_PP content in fluvo-aquic soil was significantly increased by 15.9%. Fe_HH content was not significantly changed, and Fe_DH content was significantly increased by 24.6%. Compared with NPK treatment, NPKS treatment significantly reduced the content of total iron oxide bound organic carbon in black soil, among which OC_PP decreased by 11.3%, while OC_DH increased by 53.7%, and OC_HH did not change significantly, but had no significant effect on the content of total iron oxide bound organic carbon in fluvo-aquic soil. Further analysis of the binding organic carbon content per gram of iron oxides showed that compared with NPK treatment, NPKS treatment significantly increased the binding capacity of three iron oxides in black soil, but had no significant effect in the fluvo-aquic soil. Fe_PP was mainly combined with macromolecular organic carbon with strong hydrophobicity and weak aromaticity while Fe_HH was mainly combined with small molecule organic carbon with strong hydrophobicity and aromaticity, and Fe_DH was mainly combined with some macromolecular compounds with poor hydrophobicity and aromaticity. 【Conclusion】 Comparing with the single application of chemical fertilizer, the content of crystalline and amorphous iron oxide bound organic carbon in black soil and fluvo-aquic soil increased significantly after long-term straw returning to the field, while the content of complexed iron bound organic carbon in black soil decreased significantly and did not change in fluvo-aquic soil. This was one of the important mechanisms for the different increases of soil organic carbon levels in different soil types after straw returning.

Effects of Long-Term Fertilization on Soil Aggregate Stability and Its Driving Factors in Black Soil and Brown Soil

LI TianJiao, ZHANG NaiYu, SHEN WenYan, SONG TianHao, LIU HongFang, LIU XiaoYan, ZHANG XiuZhi, PENG Chang, YANG JinFeng, ZHANG ShuXiang

Scientia Agricultura Sinica. 2024, 57(19):  3835-3847.  doi:10.3864/j.issn.0578-1752.2024.19.010

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【Objective】 The effects of long-term different fertilization practices on aggregate distribution and stability of two typical soils (black soil and brown soil) in the black soil area were compared, and the key factors affecting aggregate stability were explored to provide the theoretical support for alleviating soil structural degradation. 【Method】 Based on a 34-year long-term positioning experiment in black soil and a 45-year long-term positioning experiment in brown soil, soil samples of 0-20 cm were collected by selecting the common fertilization treatments: no fertilization (CK), chemical fertilizer (NPK), and chemical fertilizer combined with organic fertilizer (NPKM), as well as the special treatments of chemical fertilizer plus straw (NPKS) in black soil and manure (M) in brown soil. Soil chemical properties were determined and soil aggregates were sieved by wet-sieving procedure. 【Result】 Compared with CK, the proportion of microaggregates (>0.25 mm) under the NPK treatment in black soil and brown soil was significantly reduced by 8.6% and 11.3%, respectively, and the mean weight diameter (MWD) of aggregates was significantly reduced by 18.6% and 10.7%, respectively, indicating that the stability of aggregates in black soil and brown soil was significantly reduced by chemical fertilizer alone. The MWD under the NPKM and M treatments in brown soil increased significantly by 44.2% and 17.9%, respectively, whereas that had not noticeably changed under the NPKM treatment and significantly increased by 11.8% under the NPKS treatment in black soil, indicating that the application of pig manure in brown soil and straw return in black soil were important measures to improve aggregate stability. The stability of soil aggregates was mainly affected by soil chemical properties. NPK resulted in the decrease of pH and exchangeable Ca and Mg ions in black soil and brown soil, which hindered the formation and stability of microaggregates. The application of organic fertilizers (M, NPKM, NPKS) inhibited the decrease of soil pH, increased the content of particulate organic carbon (POC) and mineral-associated organic carbon (MAOC), and promoted the association of organic carbon with Fe/Al oxides and Ca/Mg ions, which accelerated the aggregation process of soil particles, maintaining and improving the stability of aggregates. The comprehensive analysis showed that the factors affecting the stability of black soil aggregates were chelated Al (Al_p), Ca²⁺, POC, pH and MAOC in order from the largest to the smallest, among which the stability of black soil aggregates was significantly negatively correlated with Al_p and positively correlated with other factors. The key factors affecting the stability of brown soil aggregates were POC, chelated Fe (Fe_p), pH, Mg²⁺, Ca²⁺, amorphous Al (Al_o), Al_p and amorphous Fe (Fe_o) in order from the largest to the smallest, among which the stability of brown soil aggregates was significantly negatively correlated with Al_o and Fe_o and positively correlated with other factors. 【Conclusion】 The application of organic fertilizers (pig manure, straw return) could mainly increase the contents of soil POC and Ca²⁺, inhibit soil acidification, and improve the stability of aggregates in black soil and brown soil, which were important fertilization practices to delay the degradation of soil structure in black soil areas.

HORTICULTURE

Identification of Ginger (Zingiber officinale Roscoe) NHX Gene Family Members and Characterization of Their Expression Patterns in Silicon Alleviating Salt Stress

YIN JunLiang, LI JingYi, HAN Shuo, YANG PeiHua, MA JiaWei, LIU YiQing, HU HaiJun, ZHU YongXing

Scientia Agricultura Sinica. 2024, 57(19):  3848-3869.  doi:10.3864/j.issn.0578-1752.2024.19.011

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【Objective】 Systematic analysis of the basic features of the ZoNHX family members in ginger and exploration of their expression patterns in different tissues and treatments of ginger can provide a theoretical foundation for further research on the function of ZoNHXs. 【Method】The Arabidopsis thaliana NHX protein sequences were used as reference sequences to compare with the ginger genome to obtain the ginger ZoNHXs. The phylogenetic tree containing Arabidopsis and ginger NHX family members were constructed based on the neighbor-joining method by MEGA 7.0. Physicochemical properties, gene structure, conserved motifs, secondary structure and tertiary structure of ZoNHXs were analyzed by using ExPASy ProtParam software, GSDS, MEME online website, PHYRE 2 and SOPMA, while chromosomal localization, intragenomic covariance and expression patterns of ZoNHXs were analyzed by using TBtools software. The gene expression levels of ZoNHXs in different treatments including CK, NaCl, and NaCl+SiNP100, and different ginger tissues were analyzed using RT-qPCR. The Na⁺ and K⁺ contents of ginger tissues was determined. 【Result】A total of 15 NHX gene family members were identified from the ginger genome, and were named as ZoNHX1-ZoNHX15 based on their chromosomal locations. The ZoNHXs were classified into three subgroups according to phylogenetic relationship and subcellular localization, including vesicular membranes (Vac, vacuole), endosomal membranes in the nucleus (Endo, endosome) and plasma membranes (PM, plasma). Protein characterization analysis showed that the relative molecular weights of ZoNHXs ranged from 26.01 kDa to 163.59 kDa, and the protein lengths ranged from 231 to 1 459 aa. The results of subcellular localization prediction showed that 11 ZoNHXs were distributed on the vacuole, ZoNHX14 were distributed both on the cell membrane and nucleus, ZoNHX1, ZoNHX7 and ZoNHX9 were respectively distributed on cytoplasm, cell membrane, and chloroplast. Signal peptide prediction showed that only ZoNHX1 contained a signal peptide and was a secreted protein, while other ZoNHX family members were non-secretory proteins. Cis-acting element analysis showed that the promoter regions of the ZoNHXs contain growth and development response elements, hormone response elements, and stress response elements. Transcriptome analysis showed that all ZoNHXs were detected to be expressed in ginger leaf and root, among which, ZoNHX12 was highly expressed in all tissues of ginger and under different abiotic stresses, and ZoNHX14 was significantly up-regulated in all growth periods of ginger and at low temperatures. RT-qPCR results showed that, compared with the CK, all genes were significantly up-regulated in roots and leaves after salt stress except for ZoNHX8 and ZoNHX11. Compared with the NaCl treatment alone, NaCl+SiNP100 decreased the expression levels of all genes except for ZoNHX8 and ZoNHX11, the expression levels of which were significantly up-regulated. These indicated that ZoNHXs were involved in the response process of ginger to salt stress and the expression of ZoNHXs were also regulated by exogenous SiNP100. The results of Na⁺, K⁺ ion content, K⁺/Na⁺ ratio and transport selection coefficient S_K⁺_/Na⁺ showed that the Na⁺ content in ginger rhizomes, stems, and leaves under salt treatment was increased compared with CK, and the K⁺/Na⁺ ratio and transport selection coefficient were decreased. NaCl+SiNP100 treatment significantly decreased Na⁺ content in roots, rhizomes, stems and leaves, whereases increased K⁺/Na⁺ ratio and transport selection coefficient. 【Conclusion】 The ginger genome contains 15 ZoNHXs and divides into three subgroups. RT-qPCR results showed that the expression of ZoNHX1-ZoNHX15 were all induced by salt stress. SiNP100 pretreatment could alleviate plant salt stress through reducing the Na⁺ content and increase the K⁺/Na⁺ ratio and K⁺ and Na⁺ content of the ginger roots, rhizomes, stems, and leaves by regulating the expression of ZoNHXs.

Influence of Pre-Harvest Application of Benzothiadiazole on Color and Aroma of Cabernet Gernischt Grapes During Fruit Development

WANG JianFeng, HAN YuQi, WANG Kai, ZHAO Man, LI JiXin, FENG LiDan, ZHANG Bo, ZHAO Yong, JIANG YuMei

Scientia Agricultura Sinica. 2024, 57(19):  3870-3893.  doi:10.3864/j.issn.0578-1752.2024.19.012

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【Objective】This study aimed to investigate the impact of benzothiadiazole (BTH) on the expression of pigments and related genes in grapevines during fruit ripening, and to elucidate its influence on the composition and concentration of free and bound volatile compounds, thereby exploring the underlying mechanisms of BTH in regulating anthocyanin and aroma biosynthesis in grape berries.【Method】Wine grapes Cabernet Gernischt were used as test material, and the whole grapevine was sprayed with 0.37 mmol∙L^-1 BTH solution containing 0.1% Tween 80 at the swelling period. A control group (CK) was sprayed with an equivalent solution of Tween 80 alone. The impact of BTH treatment on grape quality was evaluated by measuring chlorophyll, carotenoids, total anthocyanins, total proanthocyanidins and anthocyanin, as well as changes in concentration and composition of free and bound aroma in grapes.【Result】BTH application notably decreased chlorophyll a, b, and total carotenoid content while enhancing total proanthocyanidins, anthocyanins, and color index of red grapes, thus facilitating grape color development. In addition, BTH significantly upregulated the expression of anthocyanin synthesis-related genes, including VvCHS1, VvF3H1, VvDFR, VvLDOX, VvUFGT, and VvMYBA. It also significantly decreased the content of free and bound C₆/C₉ compounds, free straight-chain aliphatic compounds, free branched aliphatics compounds, but increased bound straight-chain aliphatic compounds, free and bound aromatic compounds, bound branched aliphatic compounds, free and bound terpenes, and free and bound norisoprenoids concentrations. By defining qualitative and quantitative biomarkers for grape ripening stages, it was found that aroma evolution from two adjacent developmental stages was markedly different, and BTH significantly influenced the trend of aroma evolution in both free and bound. BTH significantly altered the trend of free aroma at 9 weeks post-flowering (wpf) and 11 wpf as well as in the bound aroma of 11 wpf. The six major categories of aroma, free C₆/C₉ compounds, straight-chain aliphatic compounds, and terpenes, and bound C₆/C₉ compounds, straight-chain aliphatic compounds, and terpenes, were the major contributing components that differentiated aroma performance between CK and BTH grapes. A total of 23 active aroma components including 3-hexenal and β-ionone were identified by using the odor activity value>0.1 as threshold. BTH mainly attenuated the vegetal and fatty flavors of grapes and enhances the floral and fruity odors.【Conclusion】BTH promoted grape coloration and changed the aroma content and composition by altering expression pattern of chlorophyll, carotenoid, anthocyanin synthesis and related genes, which in turn improved the quality of grapes.

Comprehensive Analysis of Morphologic Characters and Biochemical Components of Guizhou Dashu Tea Germplasms

YANG Chun, YANG DaiXing, LI Yan, LIANG SiHui, DENG XiaoQiang, QIAO DaHe, CHEN Juan, GUO Yan, LIN KaiQin, CHEN ZhengWu

Scientia Agricultura Sinica. 2024, 57(19):  3894-3916.  doi:10.3864/j.issn.0578-1752.2024.19.013

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【Objective】 The aim of this study was to elucidate the morphological and biochemical characteristics of Guizhou Dashu tea germplasms in different regions, so as to provide the scientific basis for the conservation, development and utilization of Guizhou Dashu tea germplasms.【Method】In this study, 121 Dashu tea germplasms from seven counties in Guizhou were selected as materials to determine their morphological and biochemical traits. Multivariate statistical methods, including correlation analysis, principal component analysis (PCA), and cluster analysis were applied to comprehensively evaluate the genetic diversity of Guizhou Dashu tea germplasms. 【Result】The genetic diversity of biochemical traits was higher than morphological traits in Guizhou Dashu tea. The coefficient of variation (CV) for the 7 morphological traits ranged from 11.30% (length-width ratio) to 145.61% (ovary pubescence), with a mean CV of 41.12%. The index of genetic diversity (H') for them ranged from 0.47 (axillary pubescence) to 2.07 (length- width ratio), with an average value of 1.43. The CV of the 14 biochemical traits ranged from 23.15% (total catechins) to 99.54% (gallic acid), with an average value of 54.71%, and the H' ranged from 1.36 (epicatechin, EC) to 2.07 (total catechins), with a mean of 1.89. Most Guizhou Dashu tea germplasms were characterized by hairy axillary buds, glabrous ovaries, large or middle leaves, and long elliptical or elliptical leaf shape. The biochemical content of Guizhou Dashu tea from different regions were distributed uniform relatively, and 8 biochemical traits, including caffeine, theophylline, catechin (C), epigallocatechin-3-gallate (EGCG), total alkaloids, non-galloylated catechins, galloylated catechins and total catechins, showed a gentle and symmetrical normal distribution trend. Most of the morphological and biochemical traits of Guizhou Dashu tea were significant (P<0.05) or extremely significant (P<0.01) correlated with geographical location and climate factors. With the increase of altitude and latitude and the decrease of annual average temperature, the leaf of Guizhou Dashu tea germplasms were wider and rounder, and the content of purine alkaloids, such as caffeine, theobromine and theophylline, were higher. Based on orthogonal partial least squares discrimination analysis (OPLS-DA), 10 key traits, including total catechins, gallic acid, total alkaloids, gallated catechins, EC, caffeine, epigallocatechin gallate (ECG), EGCG, theobromine and axillary pubescence, with VIP >1 were screened out which could be used to distinguish Guizhou Dashu tea germplasms in different counties. Cluster analysis based on 10 biochemical components showed that the 121 Guizhou Dashu tea germplasms could be divided into 4 categories. There were 20 Guizhou Dashu tea germplasms in Group I, all from Huishui, had extremely significant higher content of EC and ECG than those in other three categories. 16 Guizhou Dashu tea germplasms in Group Ⅱ and the content of ECG was significant lower than that in other three categories. 9 Guizhou Dashu tea germplasms in Group Ⅲ, most of them came from Huishui, and the content of epicatechin-3-gallate (EGC) was significantly higher than that in other three categories. 76 Dashu tea germplasms in Group Ⅳ, mainly from Xishui, Tongzi, Daozhen and Wuchuan, and the content of caffeine, theobromine, theophylline, EGCG, C, and gallocatechin-3-gallate (GCG) were significant or extremely significant higher than those in other three categories. PCA was carried out with 14 biochemical traits, and a total of 3 principal components (PC1, PC2, and PC3) were extracted, with a cumulative contribution of 80.94%. The determinants of biochemical factors EGCG, total alkaloids and ester catechins in the PC1 were the key to evaluate the biochemical qualities of Guizhou Dashu tea.【Conclusion】Guizhou Dashu tea germplasms had abundant genetic diversity in morphological and biochemical traits. There were significant differences in morphological and biochemical characteristics between Dashu tea germplasms from Huishui, which located in southern Guizhou and those from other 6 counties, which located in northern Guizhou. Among them, GT-XS-08, GT-TZ-11, GT-DZ-06, GT-DZ-13, and GT-TZ-03 were Dashu tea germplasms with both superior biochemical quality and excellent biochemical content, which could be used as high potential breeding materials to select new tea varieties.

ANIMAL SCIENCE·VETERINARY SCIENCE

Analyzing the Molecular Mechanism of Hair Follicle Development in Subo Merino Based on miRNA Sequencing Data

HE JunMin, MAO JingYi, WEI Chen, REN YiFan, ZHANG GuoPing, TIAN KeChuan, LIU GuiFen

Scientia Agricultura Sinica. 2024, 57(19):  3917-3935.  doi:10.3864/j.issn.0578-1752.2024.19.014

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【Background】Wool is produced and controlled by hair follicles (HFs). The structure, function and morphogenesis of HF is a complex biological process. The morphogenesis of HF in the embryonic stage of fine wool sheep determines the wool yield and quality after sheep adulthood. Subo Merino sheep is a new breed of ultra-fine wool sheep for worsted spinning with wool fineness up to 17-19 μm independently bred in China. The miRNAs and their regulatory mechanisms during the morphogenesis of the HF in superfine wool sheep need to be studied in depth. 【Objective】 Analyzing the molecular regulatory mechanisms of miRNAs in the early development of HF in superfine wool sheep was of great significance for a better understanding of the morphogenesis of HF as well as for the breeding of ultrafine wool sheep, and it could provide the reference molecular markers for the analyzing of molecular mechanisms of the molecular regulatory mechanisms of the development of HF in ultrafine wool sheep and for the selection and breeding of high-quality ultrafine wool sheep. 【Method】 In this study, simultaneous estrus treatments and artificial insemination were performed on Subo Merino sheep under the same feeding conditions, with insemination day designated as embryonic day 0 (E0). Skin tissues from embryos were collected by cesarean section after euthanasia of pregnant ewes at embryonic days 65 (E65), 85 (E85), 105 (E105), and 135 (E135), respectively; skin tissues from the left scapular region were collected at 7 (D7) and 30 (D30) days after the birth of the lambs, and three samples were taken at each period. miRNA-Seq were used to identify conserved miRNAs and Novel miRNAs at different periods of hair follicle development, and constructed skin tissue miRNA libraries at different periods of HF development in Super Fine Wool Merino sheep. The target gene prediction and bioinformatics analysis of differentially expressed miRNAs (DE-miRNAs) were also performed, the key miRNAs and candidate genes involved in HF development in Superfine wool sheep were screened, and constructed miRNA-target gene regulatory networks. The targeting of miR-433-3p with NOTCH1 was validated by using RT-qPCR and dual luciferase reporter gene assays. 【Result】 In this study, 18 skin tissue miRNA libraries were constructed at different periods of HF development in Subo Merino sheep, and 87 DE-miRNAs and 446 novel DE-miRNAs were screened. DE-miRNA clustering analysis showed that there were 21 DE-miRNAs in the HF-induced differentiation stage (E65, E85, and E105), in which 5 DE-miRNAs (oar-miR-23b, oar-miR-133, etc.) were key candidate miRNAs; there were 28 DE-miRNAs in the HF-maturation stage (E135, D7, and D30). SOM analysis showed that DE-miRNAs could be clustered into 10 clusters, and miRNAs in each cluster had similar functions. Mixed prediction of target genes and functional enrichment of DE-miRNAs and Novel DE-miRNAs revealed that the main enriched pathways of target genes were AMPK, Notch, and hedgehog pathways. The miRNA-target gene regulatory network associated with HF development was constructed by combining DE-mRNA and target gene with bioinformatics analysis. NOTCH1 wild-type and mutant vectors were cotransfected with miR-433-3p mimics and mimics-NC in 293T cells, and the results showed that NOTCH1 was a target gene of miR-433-3p. 【Conclusion】In summary, this study constructed miRNA expression profiles in different periods of hair follicle development in Subo Merino sheep, analyzed the relationship between miRNAs and their target genes, and built a miRNA-target gene regulatory network, to further understand the roles of miRNAs in different periods of hair follicle development and molecular mechanisms. This study also provided a reference molecular marker for analyzing the molecular regulation mechanism of hair follicle development, as well as selecting and breeding high-quality ultra-fine wool sheep.

Preparation of Monoclonal Antibody Against African Swine Fever Virus p54 Protein and Identification of Its Epitope

FENG ChunYing, ZHANG ZhaoXia, LIU YunFei, HUANG Li, WENG ChangJiang

Scientia Agricultura Sinica. 2024, 57(19):  3936-3944.  doi:10.3864/j.issn.0578-1752.2024.19.015

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【Objective】The aim of this study was to obtain soluble African swine fever virus (ASFV) p54 protein and anti-p54 monoclonal antibodies (mAb), along with the identification of their recognized epitopes, so as to lay a foundation for the study of the structure and function of p54 protein and the development of serological diagnostic reagents. 【Method】To prepare mAb against ASFV p54 protein, the prokaryotic recombinant expression plasmid pET-21a-E183L was constructed and transformed into E.coli BL21 (DE3) cells. The recombinant protein p54 was purified by Ni-affinity chromatography and gel filtration, five weeks old BALB/c mice were immunized with the purified p54 recombinant protein. The immunization protocol involved three rounds at two-week intervals, starting with an emulsion of the antigen and an equal volume of Freund’s complete adjuvant, followed by the same antigen emulsion with an equal volume of Freund’s incomplete adjuvant for the subsequent immunization. After three immunizations, the blood of mice was collected, and the serum antibody titers were detected by indirect enzyme-linked immunosorbent assay (ELISA). The mice with the highest serum titers were selected for a booster immunization. Spleen cells from the mice were fused with SP2/0 cells three days later. The positive hybridoma cells were screened by indirect ELISA with the recombinant p54 protein. The specificity of the selected mAb was further confirmed via Western blot and indirect immunofluorescence assay (IFA). The isotype of the mAb was detected using a subclass identification kit. Subsequently, the p54 protein was systematically truncated and expressed as GST fusion proteins to identify the antigen epitopes recognized by the mAbs by Western blot. 【Result】 The constructed prokaryotic expression plasmid pET-21a-E183L (54-183 aa) was transformed into BL21 (DE3) cells. After induction with IPTG, the p54 recombinant protein was expressed in the supernatant in a soluble form with a molecular weight of about 17 kDa. Immunization of mice with purified p54 recombinant protein results in a serum titer of 1﹕409 600 seven days after the third immunization. Cell fusion was successfully performed. After four rounds of subcloning, a hybridoma cell line named 5B11 that could stably secrete mAb against p54 protein was obtained. The ascites fluid was prepared and the mAbs were purified. Western blot and IFA assay results showed that 5B11 could specifically recognize p54 protein expressed in HEK293T cells and ASFV-infected porcine alveolar macrophages (PAMs). The mAb subclass identification showed that 5B11 was of IgG1 heavy chain and κ light chain. The epitope sequence recognized by 5B11 was ⁸⁰VTPQPGTSKPA⁹⁰.【Conclusion】In this study, the recombinant protein ASFV p54 with amino acid 54-183 was successfully expressed in a soluble form in the prokaryotic system. The development of anti-p54 mAbs and the identification of their recognized epitopes have expanded understanding of p54 protein epitopes and provided the basic materials for the serological detection of ASFV.