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    16 April 2021, Volume 54 Issue 8
    CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS
    Polymorphism Analysis Among Chromosomes of Dasypyrum villosum 6V#2 and 6V#4 and Wheat 6A and 6D Based on Wheat SNP Chip
    XU ZhiYing,WANG BaiCui,MA XiaoLan,JIA ZiMiao,YE XingGuo,LIN ZhiShan,HU HanQiao
    Scientia Agricultura Sinica. 2021, 54(8):  1579-1589.  doi:10.3864/j.issn.0578-1752.2021.08.001
    Abstract ( 416 )   HTML ( 69 )   PDF (1234KB) ( 273 )   Save
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    【Objective】 The polymorphism among the chromosomes of Dasypyrum villosum 6V#2 and 6V#4 and their wheat homeologous 6A and 6D in the DNA level was compared in this study based on big data to provide a theoretical basis for precision designing breeding of wheat-Dasypyrom villosum targeted chromosome translocation.【Method】A 6V#4(6D) alien disomic substitution line RW15 was crossed with a 6V#2(6A) alien disomic substitution line Nan 87-88, and their F2 plants were detected using 6V#4S/6V#2S/6AS/6DS/6VL specific molecular markers, and the new types of substitution lines were confirmed in F3 employing the above-mentioned markers again as well as genomic in situ hybridization (GISH) technique. Subsequently, polymorphism among the targeted chromosomes in the new types of the substitution lines and their parents Nan 87-88 and RW15 were detected by the probes of 6A and 6D-specific in the wheat 55K chip, and combined with the results of SNP analysis of two accessions of D. villosum with 6A and 6D probes in 660K chip to screen 6V specific SNPs. 【Result】 GISH analysis showed that 19EL124 and 19EL134 had 42 chromosomes including two complete foreign chromosomes in the somatic cells of root tips. Identification using molecular markers showed that 19EL124 had 6V#4S/6DS and missed 6V#2S/6AS specifically amplified bands, while 19EL134 displayed 6V#2S/6AS distinctive bands and lacked 6V#4S-specific bands; both 19EL124 and 19EL134 contained 6VL specific amplified bands, which confirmed that 19EL124 is a 6V#4(6A) disomic substitution line, and 19EL134 is a 6V#2(6D) disomic substitution line. The results of 55K chip showed that the detected efficiency of the key chromosome probes in the alien substitution lines was significantly lower than that of other chromosome probes, and the detected efficiency of different lines in the same type of substitution was also different. Among the 1 177 probes of 6A, the 6A substitution line Nan87-88 and 19EL124 could not be genotyped by 63.21% and 68.90% of the probes, respectively. And the polymorphism between 6V#2 and 6V#4 was detected by 22.51% of the probes. Among the 479 probes of 6D, 49.48% of the probes could not identify the 6D substitution line RW15, and 53.44% could not genotype the 6D substitution line 19EL134. The polymorphism between 6V#2 and 6V#4 was indicated by 16.70% of the 6D probes, there were 23 and 42 probes that could only detect 6V#2 and 6V#4 chromosome, respectively. Twenty-two and fifteen 6V specific SNPs were screened from 395 6A and 231 6D probes through integrating the same genotypes detected by probes which were shared by 55K and 660K chips in this study, and three of them showed polymorphism between 6V#2 and 6V#4 chromosomes. 【Conclusion】 The detection efficiency of the wheat chromosome probes was greatly reduced when the target wheat chromosome was replaced by the alien chromosome, and the proportion of NA genotyping was greatly increased, and most of the NA genotypes showed polymorphism between the two different alien chromosomes in the same type of substitution. The detection efficiency of probe for two alien chromosomes in the same type of alien substitution lines was different, wheat 6A probe could detect 6V#2 better, and wheat 6D probe could detect 6V#4 better. Thirty-seven SNP markers of 6V specific were obtained base on the association analysis of 6V genotyping between D. villosum and the alien substitution lines.

    CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS
    F1 Performance Prediction of Upland Cotton Based on Partial NCII Design
    QIN HongDe, FENG ChangHui, ZHANG YouChang, BIE Shu, ZHANG JiaoHai, XIA SongBo, WANG XiaoGang, WANG QiongShan, LAN JiaYang, CHEN QuanQiu, JIAO ChunHai
    Scientia Agricultura Sinica. 2021, 54(8):  1590-1598.  doi:10.3864/j.issn.0578-1752.2021.08.002
    Abstract ( 290 )   HTML ( 24 )   PDF (1253KB) ( 185 )   Save
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    【Objective】Develop the method for predicting F1 performance of upland cotton, and reduce breeding cost and improve breeding efficiency. 【Method】A partial NCII population composed of 60 parents and 180 F1 crosses, and three prediction methods with parent additive effect (A), general combining ability (GCA) and mid-parent value (MP) were employed to predict F1 performance on yield and fiber quality traits. 【Result】The Heterosis of lint yield of upland cotton was obvious. All crosses showed 19.63% average mid-parent heterosis and 8.47% average over-parent heterosis. 97.78% and 79.44% crosses showed positive mid-parent heterosis and positive over-parent heterosis respectively. Three prediction methods showed different prediction effects for F1 performance. Prediction with additive effect of parents showed the highest prediction accuracy (pearson correlation coefficient, 0.738-0.928) for seven target traits,lint yield, boll numbers, boll weight, lint percentage, fiber length, fiber strength and micronaire value. The additive variance component of target traits and the crossing times for every parent influenced the prediction effect. The higher additive variance component, the higher the prediction accuracy of three methods; with the increase of crossing times of each parent, the prediction accuracy of A and GCA increased, but did not change for MP prediction. 【Conclusion】 The performance of upland cotton F1 can be effectively predicted by using additive effect of parents based on partial NCII design, and ‘large parent population and less crossing times’ is the preferable strategy to maintain reasonable prediction effect and reduce the workload.

    CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS
    QTL Mapping for Lateral Branch Angle Related Traits of Cultivated Peanut (Arachis hypogaea L.)
    MENG XinHao,DENG HongTao,LI Li,CUI ShunLi,Charles Y. CHEN,HOU MingYu,YANG XinLei,LIU LiFeng
    Scientia Agricultura Sinica. 2021, 54(8):  1599-1612.  doi:10.3864/j.issn.0578-1752.2021.08.003
    Abstract ( 410 )   HTML ( 26 )   PDF (4162KB) ( 351 )   Save
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    【Objective】Cultivated peanut (Arachis hypogaea L.) is an important oil and economic crop in worldwide. Plant type is a typical quantitative trait and an important agronomic trait, which is closely related to yield and mechanized harvesting in peanut. Genetic analysis, QTL mapping and identifying tightly linked molecular markers of plant type, will be conducive to the germplasm protection and cultivar identification, and provide an important theoretical basis for the molecular breeding of plant type in cultivated peanut. 【Method】In the present study, a RIL population as research material was established, which consisted of 321 families and derived from Jihua 5 with erect plant type and M130 with prostrate type. Two parents and RIL population were planted at Hainan city, Handan city, Baoding city and Tangshan city during the growing season (May to September) from 2016 to 2018. The phenotypic data of plant type related traits, such as lateral branch angle, main stem height, lateral branch length, index of plant type and extension radius, were investigated at harvesting season under seven environments. Meanwhile, SSR, AhTE, SRAP and TRAP were used to identify genotypic data of parents and RIL that was applied to construct the molecular genetic linkage map. Later, we combined phenotypic data of seven environments, and identified QTLs for plant type related traits using ICIM of QTL Icimapping V4.2. 【Result】A molecular genetic linkage map containing 363 polymorphism sites was constructed, and all markers were assigned to 20 chromosomes and an unknown linkage group. The total length of the map covered 1 360.38 cM of the whole genome, and the average distance between the markers was 3.75 cM. The length of a single linkage group was 39.599-101.056 cM, including 4-50 molecular markers. Subsequently, 30 additive QTLs for plant type related traits were detected by ICIM-ADD method, which were distributed on A04, A05, A06, A08, A09, B02 and B09 chromosomes. Among these QTLs, 5 QTLs for LBA with PVE was 3.48%-11.22%, 15 QTLs for MSH with PVE was 3.58%-10.05%, 2 QTLs for LBL with PVE was 6.03%-8.56%, 4 QTLs for IOPT with PVE was 4.68%-15.08%, 4 QTLs for ER with PVE was 3.30%-9.33%. Of these, qLBAA05.1, qLBAA05.2, qMSHA04.2, and qIOPTA05.1 were main-effect QTLs, explaining 11.22%, 10.59%, 10.23%, 10.05% and 15.08% of the phenotypic variance, respectively. In addition, 59 pairs epistatic QTLs were detected by ICIM-EPI method. Among them, 6 pairs of epistatic QTLs for LBL with PVE were 2.23% to 2.78%, 50 pairs of epistatic QTLs for IOPT with PVE were 0.25% to 1.44%, and 3 pairs of epistatic QTLs for ER with PVE were 7.28% to 12.25%. Finally, we also found 3 QTL clusters for LBA, MSH, IOPT and ER on GM1867-AHGS1967 interval of A04, me14em5-116-PM418 interval of A05 and HBAUAh177-AhTE0658 interval of A08, respectively. 【Conclusion】In brief, we constructed a molecular genetic linkage map containing 363 loci, and identified 30 additive QTLs and 59 pairs of epistatic QTLs for plant type related traits, and found 3 QTL clusters.

    TILLAGE & CULTIVATION·PHYSIOLOGY & BIOCHEMISTRY·AGRICULTURE INFORMATION TECHNOLOGY
    Effects of Different Sowing Dates and Planting Densities on the Yield and Stem Lodging Resistance of Rapeseed
    YUAN Yuan,WANG Bo,ZHOU GuangSheng,LIU Fang,HUANG JunSheng,KUAI Jie
    Scientia Agricultura Sinica. 2021, 54(8):  1613-1626.  doi:10.3864/j.issn.0578-1752.2021.08.004
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    【Objective】Stem lodging is an important factor that restricts the increase of rapeseed production efficiency in China. The purpose of this study was to investigate the changing discipline and physiological mechanism of the lodging resistance of rapeseed stem treated with different sowing dates and densities, so as to provide theoretical and technical supports for high-yield and lodging resistance of rapeseed cultivation. 【Method】A split-plot experiment with two canola varieties (Huayouza 62 and Fengyou 520), two sowing dates (September 25, October 25) and four densities (15×104, 30×104, 45×104, and 60×104 plants/hm2)was performed to investigate yield and yield composition, stem mechanical strength, lodging index, microstructure, the main components of stem and key enzyme activities of lignin synthesis. 【Result】(1) At the sowing date of September 25 (T1), yield per plant, effective pods per plant and seeds per pod decreased with the density increased from 15×104 hm-2 to 60×104 hm-2, and the highest yield with the lowest lodging index was observed at density of 45×104 hm-2; Under the delayed the sowing date to October 25 (T2), the population yield, yield per plant, effective pods per plant and seeds per pod decreased significantly compared with those under T1, but the above-ground fresh weight decreased more significantly, resulting in the decrease of lodging index and the increase of lodging resistance under delayed sowing dates; (2)When sowing at the appropriate time, both the plant height and stem dry weight decreased significantly, while the lodging index was with a tendency of increasing firstly and then decreasing with increased densities, and the susceptible lodging part was transferred from the upper part to the base part of rapeseed stem. The parameters such as vascular bundle length/the thickness outside the pith and vascular bundle area/stem cross-sectional area gradually increased, and the content of lignin and cellulose of stem increased first and then decreased. With sowing dates delayed from September 25 to October 25, plant height, stem dry weight, stem lignin and cellulose content decreased significantly, but the above-ground fresh weight decreased more, resulting in the decreased lodging index. Stepwise regression analysis showed that lignin was the key index to improve the structure of the transport tissue, coordinate the lodging index and the population yield. The higher lignin content and the population lignin content of the stem could simultaneously obtain stronger lodging resistance of the stem and a higher population yield. (3) When the density increased from 15×104 hm-2 to 60×104 hm-2, the activities of peroxidase (POD), cinnamyl alcohol dehydrogenase (CAD), phenylalanine ammonia lyase (PAL) and 4-coumaryl: CoA ligase (4CL) under T1 increased, and those decreased significantly with sowing date delayed from T1 to T2. 【Conclusion】Optimizing planting density under different sowing dates could significantly increase the population yield, and the decreased yield at the delayed sowing date could be compensated by increasing planting density. The ability of lignin synthesis enhanced, and the lignin content increased under delaying sowing dates with higher plant densities, and coordinated the contradiction between high yield and lodging resistance in rapeseed.

    Advance on the Methylglyoxal Metabolism in Plants Under Abiotic Stress
    ZHAO JingJing,ZHOU Nong,CAO MingYu
    Scientia Agricultura Sinica. 2021, 54(8):  1627-1637.  doi:10.3864/j.issn.0578-1752.2021.08.005
    Abstract ( 590 )   HTML ( 65 )   PDF (600KB) ( 362 )   Save
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    Because plants grow steadily, they cannot escape adversity by moving. Most of plants live in environments where they are constantly exposed to one or combinations of various abiotic stressors, such as extreme temperatures, salinity, drought, and excessive light, which can severely limit plant distribution, growth and development, quality, yield and even survival. Plants can only adapt to the environment by changing their morphological structure and physiological and biochemical reactions, or by releasing chemical substances to affect the growth and development of other surrounding plants, so as to change the microenvironment and make the environment more suitable for their growth. Methylglyoxal (MG) as a normal physiological metabolites, is formed from various metabolic pathways in plants, among them the glycolysis pathway provides the most important source, including elimination of phosphate groups from glycolysis intermediates dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. MG is mostly detoxified by the combined actions of the enzymes glyoxalase I and glyoxalase II that together with glutathione make up the glyoxalase system, and it converts to D-lactate finally. Under normal growth conditions, basal levels of MG remain low in plants; However, when plants are exposed to abiotic stress, MG can be accumulated to much higher levels. Stress-induced MG, as a toxic molecule, inhibited different developmental processes, including seed germination, photosynthesis and root growth, destroyed cell proliferation and survival, controlled of the redox status of cells, and many other aspects of general metabolism. The increase of MG content eventually leads to the destruction of biological macromolecule proteins, DNA, RNA, lipids and biological membranes. Thus, MG is now considered as a potential biochemical marker for plant abiotic stress tolerance, and is receiving considerable attention by the scientific community. The aim of this review was to summarize the mechanisms of MG in plants under abiotic stress. In this review, the recent findings regarding MG synthesis and degradation metabolism in plants under abiotic stress was summarized.

    PLANT PROTECTION
    Response Characteristics of Plant SAR and Its Signaling Gene CsSABP2 to Huanglongbing Infection in Citrus
    ZHAO Ke,ZHENG Lin,DU MeiXia,LONG JunHong,HE YongRui,CHEN ShanChun,ZOU XiuPing
    Scientia Agricultura Sinica. 2021, 54(8):  1638-1652.  doi:10.3864/j.issn.0578-1752.2021.08.006
    Abstract ( 343 )   HTML ( 32 )   PDF (2007KB) ( 250 )   Save
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    【Background】Plant systemic acquired resistance (SAR) plays an important role in citrus against Huanglongbing (HLB). The signal exchange between salicylic acid (SA) and methyl salicylate (MeSA) is a key signaling for activating SAR, but its roles in HLB is still unclear. 【Objective】In order to understand regulation mechanisms of citrus SAR in HLB, response characteristics of SAR and its key enzyme gene CsSABP2 (salicylic acid binding protein 2) in ‘Candidatus Liberibacter asiaticus’ (CLas) infection were compared among citrus varieties with different HLB disease-tolerance. 【Method】The response characteristics of citrus SAR in CLas, SA, and MeSA inoculation were determined based on the expression of SAR Marker gene CsPR1, CsPR2, CsPR5, levels of reactive oxygen species (H2O2) and starch. Furthermore, according to comparative transcriptome data between HLB-susceptible variety Jincheng orange (JC, Citrus sinensis) and HLB-tolerant variety Sour pomelo (SP, C. grandis), the differentially expressed genes of CsSABP2s were screened and cloned. The biological function of selected genes was predicted by bioinformatics analysis. qRT-PCR was further used to analyze expression profiles of CsSABP2s induced by Clas infection in JC, SP and other HLB-tolerant variety Kaffir lime (KL, C. hystrix) and induced by exogenous SA and MeSA in JC variety. 【Result】qRT-PCR analysis showed that CsPR1, CsPR2 and CsPR5 were up-regulated in response to CLas infection, and the expression level of CsPR2 and CsPR5 in SP and KL, especially in mesophyll, was significantly higher than that in JC. On the contrast, the expression level of CsPR1 in vein was significantly higher than that in mesophyll, and its expression level in JC vein was significantly higher than that in SP and KL vein. Hormone treatment showed that MeSA treatment obviously induced up-regulated expressions of CsPR1, CsPR2 and CsPR5 in treated and non-treated sites compared to SA treatment. Exogenous MeSA induced H2O2 accumulation in non-treated sites, which was stronger than that of SA treatment. MeSA significantly reduced the accumulation of starch in Clas-infected leaves during five weeks of hormone treatment. Transcriptome data and bioinformatics analysis showed that CsSABP2-1, CsSABP2-2, CsSABP2-3, and CsSABP2-4 had significantly different expressions in response to CLas infection, and their encoded proteins contained conserved domains necessary for SABP2 hydrolysis activity. qRT-PCR showed CsSABP2-1 and CsSABP2-4 were significantly up-regulated by CLas in the HLB-resistant varieties SP and KL, and the expression level in vein was higher than that in mesophyll. The expression levels of CsSABP2-2 and CsSABP2-3 did not change significantly. Hormone induction experiments show that CsSABP2 was mainly induced by MeSA, and MeSA significantly up-regulated CsSABP2-2 expression (>10 times), but significantly down-regulated expressions of CsSABP2-1 and CsSABP2-4 (down to 15%-55% of the expression at 0 h). 【Conclusion】The SAR response to CLas infection in the HLB-tolerant varieties Sour pomelo and Kaffir lime is significantly stronger than that in HLB-susceptible variety Jincheng orange, and MeSA plays a positive role in regulating citrus SAR against HLB. Its key enzyme genes CsSABP2-1 and CsSABP2-4 play an important role in SA and MeSA signal transduction responding to CLas infection, and their high-level expressions are closely related to citrus HLB tolerance; and CsSABP2-1, CsSABP2- 2, CsSABP2-4 may play a key synergistic role in the signal conversion between SA and MeSA responding to CLas infection.

    Mechanisms and Applications of Plant-Herbivore-Natural Enemy Tritrophic Interactions Mediated by Volatile Organic Compounds
    WANG Bing,LI HuiMin,CAO HaiQun,WANG GuiRong
    Scientia Agricultura Sinica. 2021, 54(8):  1653-1672.  doi:10.3864/j.issn.0578-1752.2021.08.007
    Abstract ( 571 )   HTML ( 30 )   PDF (620KB) ( 399 )   Save
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    The complicated interaction among plant, herbivore, and natural enemy is widespread in an agroecosystem. Volatile organic compounds (VOCs) play an important role in tritrophic interactions. Herbivores precisely distinguish and locate host plant through the emission of chemical cues. It is a research priority and highlight that herbivore-induced plant volatiles (HIPVs) acting as a key chemical cue play an indispensable role in regulating interactions. Moreover, floral attractants are the chemical cues used by pollinators to locate flowers and the food reward such as pollen and nectar that flowering plants offer, and they help to increase the probability of pollination and their development and fecundity. Over the last four decades, the novel research concepts and techniques are rapidly developed with the deep progress of traditional chemical ecology, especially improving in method and sensibility of chemical analysis, and widespread penetration of electrophysiological techniques. In tritrophic interactions, a large of chemosensory genes of insects involve in the process of chemoreception to VOCs. Hence, the discovery of putative chemosensory genes and further functional characterizations give the way for elucidating the molecular basis of chemoreception, and developing high-efficiency behavior regulation products for reasonable and environmentally friendly control of agricultural pest. It matters a great deal to the agroecosystem protection. This article summarized behavioural effects of herbivore, natural enemy and pollinator to VOCs, and illustrated mechanism and research status of tritrophic interactions mediated by VOCs, and reviewed applications in environmentally friendly prevention and control of insect pests. The last part is to look into the future of key issues.

    Effects of CMV-Infected Tobacco on the Performance, Feeding and Host Selection Behavior of Myzus persicae
    CHEN Xi,LIU YingJie,DONG YongHao,LIU JinYan,LI Wei,XU PengJun,ZANG Yun,REN GuangWei
    Scientia Agricultura Sinica. 2021, 54(8):  1673-1683.  doi:10.3864/j.issn.0578-1752.2021.08.008
    Abstract ( 307 )   HTML ( 30 )   PDF (571KB) ( 240 )   Save
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    【Objective】The objective of this study is to investigate the effect of cucumber mosaic virus (CMV) infection on the growth, development and behavior of Myzus persicae on tobacco, and the role of 2b gene in the interaction between M. persicae and CMV.【Method】The host plants to be used were healthy tobacco, CMV-infected and CMV 2b gene deletion mutant (CMVΔ2b) infected tobacco. The Y-shaped olfactometer was used to determinate the selection tendency of M. persicae to different host tobacco plants, and electrical penetration graph (EPG) was used to monitor M. persicae feeding behavior combined with the detection of total sugar and free amino acid content in the host plant. The aphid life table was recorded through a single micro-cage to analyze the effects of CMV-infected tobacco on the growth and reproduction of M. persicae.【Result】The total sugar content of CMV-infected tobacco was significantly lower than that of healthy tobacco, while the content of free amino acids was significantly higher than that of healthy tobacco and CMVΔ2b-infected tobacco. The contents of threonine, glutamic acid, glycine, tyrosine, histidine, arginine and proline in the tobacco infected by CMVΔ2b were significantly higher than those of other treatments. The contents of valine and lysine in CMVΔ2b-infected tobacco were significantly higher than those of healthy tobacco. The content of aspartic acid in CMV-infected tobacco was significantly lower than that of other treatments, and its cystine content was significantly higher than that of CMVΔ2b-infected tobacco. Compared with CMV-infected tobacco, M. persicae had a stronger selection trend to healthy tobacco and CMVΔ2b-infected tobacco. There was no significant difference in the host selection behavior of M. persicae between healthy tobacco and CMVΔ2b-infected tobacco. CMV infection adversely affected the growth and feeding behavior of M. persicae. M. persicae had the most frequent probing behavior (pd wave) and the shortest duration of phloem feeding (E2 wave) on CMV-infected tobacco, and the frequency of xylem feeding (G wave) was significantly higher than that of healthy tobacco and CMVΔ2b-infected tobacco. The results indicated that CMV-infected tobacco was not suitable for aphid feeding. The growth characteristics of the M. persicae showed that CMV infection significantly prolonged the duration of the nymph aphid, increased the mortality rate of the 2nd-instar aphid, reduced the longevity of the aphid, and significantly reduced the fecundity of the aphid. The intrinsic rate of increase (r) and finite rate of increase (λ) of M. persicae on CMV-infected tobacco were significantly lower than those of healthy and CMVΔ2b-infected tobacco. CMV infection was not conducive to population growth of M. persicae.【Conclusion】CMV infection changes the composition of tobacco nutrients. The presence of CMV 2b gene increases the probing frequency and reduces the phloem feeding behavior. CMV infection results in prolonging pre-adult period, increasing mortality, decreasing longevity and fecundity of M. persicae. CMV infection reduces the host fitness of M. persicae, promotes aphids to transfer to new host plants, thus promoting the spread of the CMV.

    SOIL & FERTILIZER·WATER-SAVING IRRIGATION·AGROECOLOGY & ENVIRONMENT
    Nitrogen Cycling in the Crop-Soil Continuum in Response to Elevated Atmospheric CO2 Concentration and Temperature -A Review
    ZHANG JinYuan,LI YanSheng,YU ZhenHua,XIE ZhiHuang,LIU JunJie,WANG GuangHua,LIU XiaoBing,WU JunJiang,Stephen J HERBERT,JIN Jian
    Scientia Agricultura Sinica. 2021, 54(8):  1684-1701.  doi:10.3864/j.issn.0578-1752.2021.08.009
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    In geochemical element cycling, nitrogen is one of the most important and active nutrient elements, determining grain yield and quality of crop. With the increase of atmospheric CO2 concentration and temperature in global climate change, the changes in crop-soil nitrogen cycle may significantly affect crop production in agro-ecosystem. Therefore, studying the response of crop-soil continuum nitrogen cycle under elevated atmospheric CO2 concentration and temperature could provide a theoretical basis for the scientific and reasonable prediction of crop nitrogen demand in farmland ecosystem and the guarantee of stable supply of crop yield under the future climate conditions. Effects of elevated CO2 and temperature on nitrogen uptake and distribution in crop and soil nitrogen turnover were reviewed in this paper, and the interaction between elevated CO2 and temperature on crop-soil nitrogen cycling processes in previous studies was systematically summarized. Under elevated atmospheric CO2 concentration, although the transpiration of crop decreased, the photosynthetic rate, biomass, root branches and root surface area increased, and the root nodule nitrogen fixation ability of legume crop also increased. Thus, these factors overall promoted crop nitrogen uptake and increased crop yield and grain nitrogen allocation, but the average nitrogen concentration across crop decreased. Furthermore, the high CO2 concentration increased soil enzyme activity, enhanced soil organic nitrogen mineralization, nitrification and denitrification, and accelerated soil nitrogen turnover. Warming and increasing CO2 concentration interactively affected crop-soil nitrogen cycle. Warming and high CO2 concentration synergistically promoted crop biomass, photosynthesis, underground nitrogen distribution and root branching and root surface area. Elevated temperature enhanced the elevated CO2-induced inhibition on crop transpiration and nitrogen concentration. Warming inhibited the positive effect of high CO2 concentration on nitrogen distribution, nitrogen absorption and yield of crop grains. Elevated temperature enhanced soil enzyme activity and mineralization at high CO2 concentration, but molecular mechanisms of their interactive effect on soil nitrification and denitrification were still not clear. Research on soil microbes in relevant to aboveground crop, in particular, nitrogen cycling processes and their feedback mechanisms on global change remained unknown. The 16 S rRNA, DGGE, T-RFLP, qPCR, RT-PCR techniques, proteomics and in situ studies of stable isotope probes could be used to investigate the microbial species composition and physiological functions in complex environments. This review highlighted further investigations on the potential interaction between elevated CO2 and temperature on nitrogen cycle in the crop-soil continuum in farming soils, and the microbial community in the rhizosphere that were involved in soil nitrogen cycle. Thus, the nitrogen cycle in agricultural ecosystem under climate change could be predicted, by which the adaptability of the ecosystems to climate change could be enhanced effectively.

    Effects of Different Fertilization Methods on Restoration of Eroded and Degraded Cultivated-Layer in Slope Farmland
    SONG Ge,SHI DongMei,JIANG GuangYi,JIANG Na,YE Qing,ZHANG JianLe
    Scientia Agricultura Sinica. 2021, 54(8):  1702-1714.  doi:10.3864/j.issn.0578-1752.2021.08.010
    Abstract ( 269 )   HTML ( 22 )   PDF (539KB) ( 322 )   Save
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    【Objective】The soil erosion of purple soil slope farmland is serious. The study on the effects of soil management measures on the restoration of soil erosion of different erosion degree of cultivated-layer in slope farmland could provide a theoretical and practical basis for the quality control and sustainable utilization of purple soil slope farmland. 【Method】One way ANOVA was used to test the significance of the differences of each index, and the effects of soil management measures on the restoration of soil properties in the cultivated-layer of slope farmland with five degrees of erosion was studied. 【Result】(1) With the increase of erosion degree, the content of sand increased from 38.1%-42.4% to 44.2%-46.4%, while the content of clay decreased from 12.6%-14.8% to 9.6%-11.0%. Compared with S-0, S-5 and S-10, soil bulk density of S-15 and S-20 increased significantly. Under the S-10 erosion degree, the total porosity and capillary porosity decreased the most, and the shear strength of soil was the largest, between 8.71 and 9.56 kg·cm-2. Under the treatments of F and BF, the stable infiltration rate and average infiltration rate of S-15 erosion degree soil decreased the most. (2) There was significant difference in soil properties of cultivated-layer under different management measures. The soil bulk density of 0-10 cm and 10-20 cm soil layers under fertilization treatments was significantly lower than that under CK (P<0.05), and the soil total porosity and capillary porosity were significantly higher than that under CK and F. Under BF treatment, the initial infiltration rate, stable infiltration rate and average infiltration rate were the largest, while those under CK treatment are the smallest. Compared with CK treatment, the shear strength of BF treatment increased significantly. (3) With the increase of erosion degree, K value of soil erodibility decreased significantly. Compared with S-0, K value of soil erodibility was decreased by 0.1960%-0.2192% under S-20 erosion degree. Under BF treatment, K value of soil erodibility was the highest, followed by F treatment, and CK was the lowest. Compared with CK, the increase of F and BF in S-10 was the largest, with an increase of 0.0684% and 0.1404%, respectively. 【Conclusion】The application of biochar and chemical fertilizer was more effective than that of chemical fertilizer alone in improving soil material composition, structural characteristics and soil water storage and permeability. Applying biochar and chemical fertilizer could effectively reduce the soil erosion in the cultivated-layer of purple soil slope farmland, and the effects of soil improvement in the cultivated-layer (0-20 cm) of 10 cm slope was the best.

    A New Method to Improve the Accuracy of Digital Elevation Model in Northeast China by Using Terrain, Soil and Crop Information
    MA YuYang,GUAN HaiXiang,YANG HaoXuan,SHAO Shuai,SHAO YiQun,LIU HuanJun
    Scientia Agricultura Sinica. 2021, 54(8):  1715-1727.  doi:10.3864/j.issn.0578-1752.2021.08.011
    Abstract ( 345 )   HTML ( 18 )   PDF (2248KB) ( 189 )   Save
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    【Objective】SRTM DEM is a publicly available DEM accessible at no cost. However, it is well known that SRTM DEM has a large vertical deviation. In order to improve the accuracy of SRTM DEM in cultivated land, the effects of topography on the temporal and spatial distribution of soil physical and chemical properties and crop growth were analyzed to mine the factors of interaction with topography, so as to obtain a digital elevation model for precision agriculture. 【Method】This paper took Helen Dongxing Agricultural Machinery Cooperative in Heilongjiang Province as the study area, the actual ground elevation data were collected, and SPOT-6, Sentinel-2A remote sensing images and SRTM DEM were obtained. The variables, such as normalized differential moisture index (NDMI), normalized difference vegetation index (NDVI), Tasseled Cap Brightness (TCB) and potential solar radiation (PSR), were extracted, and the effects of topography on them were analyzed. Extreme Learning Machine (ELM) and back Propagation Neural Network (BPNN) were used to improve horizontal spatial resolution and vertical accuracy of SRTM DEM. The accuracy was verified with the actual ground elevation point and compared with the DEM generated by the UAV and the ZY-3. 【Result】The correlation degree between SRTM, NDVI, NDMI, TCB and the improved elevation were more than 90%, which were important factors for improving SRTM DEM. In the whole study area, the RMSEP of BPNN method was 0.98, the RMSEP of R2P was 0.98, and the RMSEP of Elm was 1.00, R2P was 0.90. In the flat area, the RMSEP of BPNN method was 0.84 and the RMSEP of ElM was 1.00. In the fluctuation area, the RMSEP of BPNN method was 0.99, and the RMSEP of ELM was 0.94. The vertical accuracy of DEM obtained by this method was higher than that of DEM generated by ZY-3. It provided a new idea for improving the spatial resolution of SRTM. 【Conclusion】 The introduction of auxiliary information of SRTM, NDVI, NDMI and TCB was helpful to improve the spatial resolution and vertical accuracy of SRTM DEM, so as to obtain high accuracy DEM. The accuracy of digital elevation model obtained by BPNN method improved by SRTM DEM was higher than that obtained by ELM method as a whole. In addition, the further research showed that the BPNN method was more suitable for the acquisition of high-precision DEM in the flat area, and the ELM method was more suitable for the acquisition of high-precision DEM in the undulating area. The research results improved the accuracy of the existing DEM and provided data support for accurate farmland management zoning and digital soil mapping.

    HORTICULTURE
    Effects of Simulated Acid Rain Conditions on Plant Photosynthesis and Disease Susceptibility in Tomato and Its Alleviation of Brassinosteroid
    LI JianXin,WANG WenPing,HU ZhangJian,SHI Kai
    Scientia Agricultura Sinica. 2021, 54(8):  1728-1738.  doi:10.3864/j.issn.0578-1752.2021.08.012
    Abstract ( 343 )   HTML ( 29 )   PDF (1443KB) ( 253 )   Save
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    【Objective】 In the era of climate change, the acid rain deposition has become a global environmental issue, which seriously affects the yield, quality and disease incidence of vegetables and other crops. Brassinosteroids (BRs) are a group of plant hormones widely existing in plants, which regulate plant resistance to broad-spectrum environment stresses. The aim of this study was to investigate effects of BRs on plant photosynthesis and disease susceptibility in tomato under simulated acid rain conditions and its alleviation effect, so as to provide guidance for safety production of vegetable crop. 【Method】In this study, using tomato (Solanum lycopersium L.) cultivar ‘Hezuo 903’ as material, the effects of exogenous BR foliar spray on the photosynthetic characteristics and the incidence of bacterial leaf spot disease caused by Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) were studied under two levels of simulated acid rain including simulated acid rain 1 (SiAR2): NH4NO3 (1.3 g·L-1),MgSO4·7H2O (3.1 g·L-1),Na2SO4 (2.5 g·L-1),KHCO3 (1.3 g·L-1),CaCl2·2H2O (3.1 g·L-1), pH (3.0, adjusted by 1N H2SO4 ) and simulated acid rain 2 (SiAR2): rain from Hanzhou area in spring, pH (3.0, adjusted by 1N H2SO4), and spraying leaves with H2O as control condition. The alleviation of exogenous BR were studied through spraying exogenous BR on tomato leaves under two levels of simulated acid rain and control conditions. To reveal the underlying mechanism of BR induced stress alleviation, the transcript abundance of photosynthesis-related genes (e.g. FBPase, SBPase, rbcS), defense-related genes (e.g. PR1 and NPR1), and the activity of antioxidant enzymes were measured. 【Result】The results showed that the phytotoxic effect of simulated acid rain on photosynthesis in tomato was mainly reflected by the decrease of net photosynthesis rate (Pn), the photosystem II photochemical efficiency (ΦPSII), and the photochemical quenching coefficient (qP). The simulated acid rain increased tomato susceptibility to Pst DC3000, resulting in a significant increase in disease incidence and leaf bacterial population. However, the exogenous BR was able to enhance the leaf photosynthetic capacity and decrease the susceptibility of tomato to Pst DC3000 by reducing the leaf bacterial population under two levels of simulated acid rain and control conditions Furthermore, the exogenous BR treatment was able to protect plant photosynthesis and pathogen resistance from the damages caused by simulated acid rain. The BR pretreatment not only significantly increased the transcript abundance of photosynthesis-related genes (e.g. FBPase, SBPase, and rbcS) and defense-related genes (e.g. PR1 and NPR1), but also reduced the content of malondialdehyde and enhanced the activity of G-POD and CAT in tomato plants under simulated acid rain treatments. Thus, the exogenous BR alleviated the inhibition of simulated acid rain on tomato photosynthesis and disease resistance. 【Conclusion】It was concluded that exogenous BR could increase leaf photosynthesis, transcript abundance of photosynthesis, defense-related genes, and the activity of antioxidant enzymes under simulated acid rain, and could also improve the resistance of tomato plants to bacterial leaf pathogen.

    Genetic Diversity Evaluation of Castanea in China Based on Fluorescently Labeled SSR
    NIE XingHua, ZHENG RuiJie, ZHAO YongLian, CAO QingQin, QIN Ling, XING Yu
    Scientia Agricultura Sinica. 2021, 54(8):  1739-1750.  doi:10.3864/j.issn.0578-1752.2021.08.013
    Abstract ( 328 )   HTML ( 28 )   PDF (4882KB) ( 377 )   Save
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    【Objective】The characteristics of genetic diversity, relationships and population structure of Castanea plants in China were analyzed with SSR molecular markers, so as to provide a theoretical basis for variety improvement, germplasm innovation and utilization of Castanea plants. 【Method】Firstly, 330 SSR molecular markers were screened among 12 chestnut cultivars from different production areas, and 12 pairs of high-quality SSR primers were obtained from them. Secondly, 96 accessions of Castanea from 4 species were detected by high resolution capillary electrophoresis. Finally, Power Marker 3.25, GenAlEx 6.51, FigTree v1.4.3 and Structure 2.3.3 were used to analyze the population genetic diversity of all accessions. 【Result】A total of 129 alleles were acquired from 96 accessions, and the average variation of each marker was 10.750. The gene diversity (GD) ranged from 0.656 (CmSI0396) to 0.877 (CmSI0930) with an average of 0.800; the observed heterozygosity (Ho) ranged from 0.329 (CmSI0742) to 0.769 (CmSI0702) with an average of 0.615; the expected of heterozygosity (He) ranged from 0.489 (CmSI0742) to 0.789 (CmSI0922), with an average of 0.672; the polymorphic information content (PIC) ranged from 0.586 (CmSI0396) to 0.868 (CmSI0930), with an average of 0.774. Among different Castanea species, in terms of number of alleles (Na), number of effective alleles (Ne) and Shannon diversity index, Castanea seguinii had the highest genetic diversity, followed by Castanea mollissima, and Castanea crenata was the lowest. According to pairwise population Fst values, the genetic differentiation value of Castanea species was 0.077-0.180, showing moderate to high differentiation among the species. Compared with Castanea crenata, both Castanea mollissima and Castanea henryi exhibit relatively high genetic differentiation with Fst values of 0.165 and 0.180, respectively. At the same time, the gene flow (Nm) of the Castanea plants was 1.580>1, suggesting frequent gene exchange among Castanea plants, and which therefore reduced the degree of genetic differentiation among the species caused by genetic drift. The results of analysis of molecular variance (AMOVA) showed that the variation mainly occurred within populations, accounting for 73% of the total variation, and the variation among populations made up 27%. The results of UPGMA cluster analysis, principal coordinate analysis and population genetic structure were consistent, and the genetic background of each accessions had obvious inter-species boundaries. Some accessions inherited the genetic information of different ancestor species in generational inheritance. For example, accession 65, 71 and 82 are mixed types, which contain the genetic background of Castanea seguinii and Castanea crenata, but there is geographic isolation between the two species. Castanea plants in the same ecological region existed extensive gene exchange, and there were no complete reproductive isolation. Accession 48 (‘Guangdongaisheng’) had the genetic background of both Castanea mollissima and Castanea seguinii. In terms of geographical distribution, the native place of this accession was in the overlapping ecological area of Castanea mollissima and Castanea seguinii. 【Conclusion】The 12 pairs of SSR primers screened could accurately assess the genetic diversity of Castanea plants in China. Comprehensive cluster analysis could confirm that the classification of Castanea plants was highly consistent with the inter-species information and there was a certain amount of gene exchange between species.

    Construction of Molecular ID for Tea Cultivars by Using of Single- nucleotide Polymorphism (SNP) Markers
    FAN XiaoJing, YU WenTao, CAI ChunPing, LIN Yi, WANG ZeHan, FANG WanPing, ZHANG JianMing, YE NaiXing
    Scientia Agricultura Sinica. 2021, 54(8):  1751-1760.  doi:10.3864/j.issn.0578-1752.2021.08.014
    Abstract ( 465 )   HTML ( 34 )   PDF (2834KB) ( 558 )   Save
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    【Objective】In order to facilitate the protection and precise management of tea cultivars and avoid the phenomenon of homonyms and synonyms, single-nucleotide polymorphism (SNP) molecular marker database of tea cultivars was established, and the 28 digit molecular identities of tea cultivars were constructed by DNA fingerprinting of SNP loci and the basic information of tea cultivars. 【Method】By mining the expressed sequence tags (EST) of tea plants, a large number of high-quality ESTs were obtained. Then, the ESTs were assembled to develop candidate SNP loci. And, high-quality SNPs for tea plants were screened. Furthermore, the candidate SNP loci were compared with the whole genome of tea plant to confirm their positions on the chromosomes and specific genes. The genomic DNA was extracted from fresh leaves of 103 tea cultivars. Subsequently, the genotyping of accessions was carried out on microfluidic chips. Information index, observed heterozygosity and expected heterozygosity of the candidate SNPs were obtained. The SNP loci were further screened by their polymorphism, obtaining the optimal combination of SNP loci. The molecular identities of tea cultivars were finally constructed by combining the basic information of tea cultivars. 【Result】A total of 1 786 candidate SNP loci were selected from the EST database of Camellia sinensis. According to the sequence conservation, 96 SNP loci were selected. Compared with the latest tea plant genome, the candidate loci were evenly distributed on 15 chromosomes of the whole tea plant genome. The polymorphism information of candidate SNP loci of tea cultivars were analyzed, and 10 non-polymorphic loci were eliminated. The average values of information index, observed heterozygosity, expected heterozygosity, fixed index and minor allele frequency of the remaining 86 loci were 0.517, 0.370, 0.346, -0.036, 0.269, respectively. 24 SNPs, with high polymorphism, were screened out from 86 SNPs to distinguish all tea cultivars. Based on the fingerprint of 24 SNP markers and the basic information of tea cultivars, the tea molecular ID, which composed of 28 digits, was formed finally. 【Conclusion】According to the polymorphism information of SNP markers, the candidate SNP loci were screened. And all tea cultivars were accurately distinguished. Furthermore, The DNA fingerprints of 103 tea cultivars were constructed by the 24 SNP markers and the converted serial codes from information of the tea cultivars, each germplasm thus has a unique molecular identity code, and the bar codes and quick response (QR) codes are generated as the molecular ID card, which can be quickly identified by the code scanning equipment.

    FOOD SCIENCE AND ENGINEERING
    Effects of nuoB on Physiological Properties of Pseudomonas fragi and Its Spoilage Potential in Chilled Chicken
    WANG GuangYu,LI Qing,TANG WenQian,WANG HuHu,XU XingLian,QIU WeiFen
    Scientia Agricultura Sinica. 2021, 54(8):  1761-1771.  doi:10.3864/j.issn.0578-1752.2021.08.015
    Abstract ( 269 )   HTML ( 17 )   PDF (1711KB) ( 183 )   Save
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    【Objective】The effects of nuoB on the properties of Pseudomonas fragi and its spoilage activity on chilled chicken were studied in this work, which could provide a theoretical basis for revealing mechanism of nuoB-mediated chilled chicken spoilage and developing new preservation technologies. 【Method】The nuoB mutant was constructed by inserting a resistance cartridge to analyze the differences of the growth curve, aggregation, motility, and biofilm formation ability between the wild type and the mutant in vitro. The effects on the spoilage characteristics of chilled chicken were studied in situ, including the total viable count, TVBN, pH, and sensory evaluation. This study investigated the influence of nuoB on the physiological properties and spoilage potential of P. fragi. 【Result】The results showed that nuoB did not affect the growth condition, aggregation, and swarming motility of P. fragi. However, the swimming motility and biofilm formation of the mutant were significantly decreased during the incubation period. The in situ assessment of the spoilage ability on chilled chicken showed that there was no significant difference in the total viable count between two groups, which both reached to 10 lg CFU·g -1. The TVBN in the mutant group was significantly lower than that in the wild type group during the whole storage period and exceeded the national standard limit of 15 mg/100 g on the 4th day. The maximum value of TVBN in the mutant group at the end of storage was about half of that in the wild type group. The pH values of both samples were within the normal levels in the first 2 days, while the mutant group was significantly lower than that in the wild type group after the 5th day. Sensory evaluation results showed that slime and odor occurred in both groups on day 4, which could be considered as spoilage, but the spoilage extent of mutant group was slightly weaker than the wild type group. 【Conclusion】The destruction of nuoB did not affect the growth ability of P. fragi, but inhibited its swimming motility, biofilm formation, and spoilage potential in chilled chicken.

    Changes of Wine Flavor Properties from the Decreased Higher Alcohols Induced by Ultrasound Irradiation
    ZHANG QingAn,XU BoWen,CHEN BoYu,ZHANG BaoShan,CHENG Shuang
    Scientia Agricultura Sinica. 2021, 54(8):  1772-1786.  doi:10.3864/j.issn.0578-1752.2021.08.016
    Abstract ( 247 )   HTML ( 23 )   PDF (1481KB) ( 190 )   Save
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    【Objective】 This research was conducted in order to investigate the effects of the decreased higher alcohols by ultrasound irradiation on the changes of wine flavor properties, so as to provide a theoretical basis for the application of ultrasound technology in decreasing the higher alcohols and improving the flavor properties of red wine. 【Method】 Firstly, the electronic tongue and electronic nose were employed to identify the wine samples treated by ultrasound irradiation, and the data about the flavor changes were analyzed by the principal component analysis (PCA) and discriminant function analysis (DFA), respectively. Thereafter, the gas chromatography mass spectrometer (GC-MS) was used to detect the changes of the volatile compounds in red wine induced by ultrasound irradiation with the optimum conditions being proved to effectively degrade the higher alcohols of red wine. Finally, the effects of the decreased higher alcohols from ultrasound irradiation on the volatile compounds and wine flavors, and the detailed mechanism were investigated by constructing the model wine solutions with the addition of the standards of N-propyl alcohol, isobutanol, isopentanol and n-pentanol. 【Result】The results of single factor experiments from the electronic tongue and electronic nose indicated that no significant influence could be identified on the sensory properties of red wine exposed to ultrasound irradiation, while the varieties of the volatile compounds in red wine tentatively identified by GC-MS could be increased by the optimum ultrasonic treatment. Moreover, the content of esters increased, and the aldehydes and alcohols decreased correspondingly, which was in accordance with the results from the model wine. 【Conclusion】In conclusion, ultrasound treatment could not only degrade the content of higher alcohols, but also had a positive effect on the modification of taste, aroma and flavor properties of red wine by decreasing the higher alcohols, and all the results could provide a theoretical basis for the application of ultrasonic technology in improving the wine quality.

    ANIMAL SCIENCE·VETERINARY SCIENCE·RESOURCE INSECT
    Curcumin Alleviates H2O2-Induced Oxidative Stress in Bovine Mammary Epithelial Cells Via the Nrf2 Signaling Pathway
    JIANG ChunHui,SUN XuDong,TANG Yan,LUO ShengBin,XU Chuang,CHEN YuanYuan
    Scientia Agricultura Sinica. 2021, 54(8):  1787-1794.  doi:10.3864/j.issn.0578-1752.2021.08.017
    Abstract ( 279 )   HTML ( 40 )   PDF (766KB) ( 372 )   Save
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    【Objective】The aim of this study was to investigate whether curcumin alleviated oxidative stress in bovine mammary epithelial cells induced by H2O2 via the nuclear factor E2-related factor 2 (Nrf2) signaling pathway. 【Method】Bovine mammary epithelial cells MAC-T cells were treated with H2O2 (500 μmol·L-1) for 24 h, followed by incubation of curcumin (0, 5, 15 or 30 μmol·L -1) for an additional 3 h; the MAC-T cells were transfected with Nrf2 siRNA for 48 h, followed by incubation of H2O2 (500 μmol·L-1) for 24 h and then treated with curcumin (30 μmol·L -1) for an additional 3 h. Real-time quantitative PCR (qPCR), Western blot (WB) were used to detect the protein abundance of Nrf2 and the mRNA and protein abundance of NAD(P)H quinone oxidoreductase 1 (NQO1) and Heme oxygenase 1 (HO-1), the activity of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT) and the content of malondialdehyde (MDA). 【Result】 (1) Compared with the control group, H2O2 treatment significantly increased MDA content (P<0.01), while it decreased the activity of SOD, GSH-Px, and CAT (P<0.01). Compared with the H2O2 group, the content of MDA in MAC-T cells in the 15 μmol·L-1 or 30 μmol·L -1 curcumin with H2O2 treatment groups was significantly decreased (P<0.01), while the activity of SOD, GSH-Px, and CAT were significantly increased (P<0.05, P<0.01). (2) Compared with the control group, H2O2 treatment significantly decreased Nrf2 protein abundance (P<0.01) and decreased HO-1 and NQO1 their mRNA and protein abundance (P<0.01). However, compared with the control group, curcumin treatment significantly increased Nrf2 protein abundance (P<0.01) and increased HO-1 and NQO1 mRNA and protein abundance (P<0.01). Compared with the H2O2 group, H2O2+curcumin treatment significantly increased Nrf2 protein abundance (P<0.01) and increased HO-1 and NQO1 mRNA and protein abundance (P<0.01). (3) Compared with the control group, si-Nrf2 treatment group significantly decreased Nrf2 mRNA abundance (P<0.01). Compared with si-Control+H2O group, the content of MDA was significantly decreased in si-Control+H2O2+curcumin treatment group (P<0.01), while the activity of SOD, GSH-Px, and CAT were significantly increased (P<0.01). However, compared with si-Control+H2O2+curcum group, the content of MDA was significantly increased in si-Nrf2+H2O2+curcumin treatment group (P<0.01), while the activity of SOD, GSH-Px, and CAT were significantly decreased (P<0.01). 【Conclusion】These results suggested that curcumin could alleviate the oxidative stress of bovine mammary epithelial cells induced by H2O2 through increasing the expression of Nrf2 and inducing the transcription of downstream antioxidant molecules. This study provided a theoretical basis for the prevention and treatment of oxidative damage of mammary epithelial cells caused by metabolic disorderd in perinatal dairy cows.

    Hc-hrg-2 of Haemonchus Contortus Rescues the Growth of Heme Deficient Yeast Strain
    ZHOU JingRu,WU Fei,CHEN XueQiu,HUANG Yan,SHI HengZhi,DU AiFang,YANG Yi
    Scientia Agricultura Sinica. 2021, 54(8):  1795-1804.  doi:10.3864/j.issn.0578-1752.2021.08.018
    Abstract ( 222 )   HTML ( 16 )   PDF (2902KB) ( 163 )   Save
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    【Objective】In previous study, we have identified a heme responsive gene Hc-hrg-2 in Haemonchus contortus (H. contortus), with a high transcriptional level in the presence of high concentration of heme. However its function in heme regulation is still lacking research. To verify that Hc-hrg-2 was involved in the intracellular heme transport, a hem1 gene knockout strain of Saccharomyces cerevisiae, which was heme deficient, was constructed by homologous recombination technique and then exogenously expressed Hc-hrg-2 of H. contortus to rescue the growth of the knockout strain. 【Method】The genomic DNA of S. cerevisiae BY4741 was used as a template to obtain the upstream and downstream homology sequences of hem1 (SGD: S000002640) gene. The plasmid pYES2-CT was used to obtain the screening marker URA3 sequence. Two overlapping PCR techniques were used to sequentially connect upstream homology sequence, URA3, and downstream homology sequence to form the knockout components which was purified and then transformed into BY4741 competent cells by lithium acetate transformation method, and the transformants were selected on SD /-URA plates supplemented with 250 μmol·L -1 5-aminolevulinic acid (ALA). PCR identification using multiple primer pairs was further performed to verify the correctness of Δhem1 strain. The Hc-hrg-2 sequence (GenBank: MK371241) of Zhejiang strain and its functional domain deleted sequence Hc-hrg-2(Δgst-n) and Hc-hrg-2(Δgst-c) were amplified from the plasmids and inserted into the yeast expression vector pESC-LEU through a seamless cloning kit. The expression vectors, which were identified and sequenced to be correct, were then transformed into Δhem1 competent cells and selected on SD/-URA/-LEU (containing 250 μmol·L -1 ALA) plates. PCR identification was performed to verify the positive exogenous expression strain. By comparing the growth of Δhem1 strain and its exogenous expression strains in SD/-URA/-LEU liquid medium with or without 250 μmol·L -1 ALA, the phenotype of the knockout strain were further verified and the effects of expression vectors on phenotype were excluded. The exogenous expression strains were induced by 2% w/v galactose and then sonicated to identify the protein expression by Western Blot. The induced strains were resuspended to an OD600 of 0.2 and 4 μL of 5-fold serial dilutions of each induced strain was spotted onto 2% w/v galactose plates supplemented with either 250 μmol·L-1 ALA or different concentrations of heme for 2 to 3 days at 28 ℃ to compare the growth of the strains. 【Result】The hem1 gene knockout strain was successfully obtained. Compared with wild strain, Δhem1 cannot synthesis heme in vivo and requires ALA (250 μmol·L -1) or heme (≥10 μmol·L-1) for growth. The phenotypes of the exogenous expression strains were consistent with that of the knockout strain. Western Blot results indicated that 2% w/v galactose could successfully induce the expression of Hc-hrg-2 and its functional domain deleted gene in the knockout strain. Hc-hrg-2 expression allowed S. cerevisiae to import heme from the environment, and rescued the growth of Δhem1 strain. Notably, the deletion of two signature domains of Hc-hrg-2, a thioredoxinlike (GST-N) and a glutathione S-transferase C-terminal domain-like (GST-C), could reduce the effect of rescue. 【Conclusion】Hc-hrg-2 could facilitate heme uptake in cells and its functional domains, GST-N and GST-C, played an important role in this process. This study laid a solid foundation for further exploring heme transport mechanism of H. contortus.

    MicroRNA-Mediated Cross-Kingdom Regulation of Apis mellifera ligustica Worker to Nosema ceranae
    DU Yu,FAN XiaoXue,JIANG HaiBin,WANG Jie,FENG RuiRong,ZHANG WenDe,YU KeJun,LONG Qi,CAI ZongBing,XIONG CuiLing,ZHENG YanZhen,CHEN DaFu,FU ZhongMin,XU GuoJun,GUO Rui
    Scientia Agricultura Sinica. 2021, 54(8):  1805-1820.  doi:10.3864/j.issn.0578-1752.2021.08.019
    Abstract ( 253 )   HTML ( 32 )   PDF (12391KB) ( 143 )   Save
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    【Objective】Nosema ceranae infects Apis mellifera ligustica and causes microsporidiosis. In this study, to reveal the mechanism of miRNA-mediated cross-kingdom regulation of A. m. ligustica worker to N. ceranae, prediction, GO and KEGG database annotation as well as regulatory network analysis of N. ceranae mRNAs and differentially expressed mRNAs (DEmRNAs) targeted by differentially expressed miRNAs (DEmiRNAs) of A. m. ligustica workers’ midguts were conducted by bioinformatic approaches based on previously gained miRNA and mRNA omics data. 【Method】Significant host DEmiRNAs were screened out by comparison of miRNA omics data from A. m. ligustica workers’ midguts at 7 d and 10 d post N. ceranae infection (AmT1, AmT2) and corresponding uninfected midguts (AmCK1, AmCK2). DEmRNAs of pathogen were screened out through comparison of mRNA omics data from N. ceranae infecting A. m. ligustica worker’s midgut (NcT1 and NcT2) and pure fungal spores (NcCK). mRNAs and DEmRNAs of N. ceranae targeted by significant host DEmiRNAs were predicted using TargetFinder software. GO and KEGG database annotations of aforementioned targets were conducted using related bioinformatics tools. On basis of our previous findings, pathogen DEmRNAs associated with spore wall protein, polar tube protein, ricin B lectin, ATP/ADP translocase, ABC transporters and glycolysis/gluconeogenesis, and their target significant DEmiRNAs of host were filtered out, followed by construction and investigation of regulatory network. 【Result】In AmCK1 vs AmT1 comparison group, 48 significantly up-regulated miRNAs and 36 significantly down-regulated miRNAs could respectively target 1 345 and 1 046 mRNAs of N. ceranae; additionally, 47 significantly up-regulated miRNAs and 34 significantly down-regulated miRNAs of host could target 584 significantly down-regulated mRNAs and 265 significantly up-regulated mRNAs in NcCK vs NcT1; these targets were involved in 19 and 22 functional terms as well as 66 and 64 pathways. In AmCK2 vs AmT2 comparison group, 56 significantly up-regulated miRNAs and 51 significantly down-regulated miRNAs could respectively target 1 260 and 1 317 mRNAs of N. ceranae, additionally, 52 significantly up-regulated miRNAs and 49 significantly down-regulated miRNAs could target 587 significantly down-regulated mRNAs and 336 significantly up-regulated mRNAs in NcCK vs NcT2, which were engaged in 20 and 23 functional terms as well as 64 and 65 pathways. Further, eight common significantly up-regulated miRNAs and one common significantly down-regulated miRNA in AmCK1 vs AmT1 and AmCK2 vs AmT2 comparison groups could respectively target 144 common significantly down-regulated mRNAs and 10 common significantly up-regulated mRNAs in NcCK vs NcT1 and NcCK vs NcT2 comparison groups, which could be annotated to 18 and 13 functional terms as well as 38 and seven pathways. Moreover, host significantly up-regulated miRNAs in AmCK1 vs AmT1 and AmCK2 vs AmT2 could target pathogen significantly down-regulated mRNAs in NcCK vs NcT1 and NcCK vs NcT2, associated with RNAi, virulence factors such as polar tube protein, spore wall protein and ricin B lectin, glycolysis/gluconeogenesis and MAPK signal pathway. 【Conclusion】Complex target binding relationship and potential cross-kingdom regulatory relationship exist between host DEmiRNAs and pathogen DEmRNAs during the infection of A. m. ligustica worker with N. ceranae; host DEmiRNAs are likely to inhibit or degrade pathogen DEmRNAs associated with RNAi, virulence factor/infection factor, glycolysis/gluconeogenesis pathway, ATP/ADP translocase, ABC transporters, and MAPK signal pathway to affect N. ceranae infection and proliferation.