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    01 November 2018, Volume 51 Issue 21
    CONTENTS
    Scientia Agricultura Sinica. 2018, 51(21):  0. 
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    CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS
    The Research Progress of Plant RNA Binding Proteins
    ZaiBao ZHANG,WanJie LI,JiuLi LI,Chi ZHANG,MengHui HU,Lin CHENG,HongYu YUAN
    Scientia Agricultura Sinica. 2018, 51(21):  4007-4019.  doi:10.3864/j.issn.0578-1752.2018.21.001
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    In eukaryotes, RNA-binding proteins (RBPs) are an important class of post-transcriptional regulators that direct and regulate the RNA metabolism. RBPs together with RNA to form ribonucleoprotein complexes have been reported to play critical roles in many RNA processes, including translocation, modification, translation and degradation. RBPs are widely present in animals, plants and microorganisms, accounting for about 2%-8% of the proteins encoded by eukaryotic genes. In recent years, the researches on RNA-binding proteins have become a hot topic. RBPs have been reported to involved in many human diseases by mutation and genetic analysis. The large number of RBPs in plants has also been reported, and they played similar important functions in plant RNA metabolism. However, our understanding of the roles and mechanisms of action of plant RBPs is less well studied than in animals. In this review, we will discuss recent progresses of multiple RBP family members that play essential roles in RNA metabolism during plant growth, development and stress responses. Five classes of plant RBP families were discussed, including serine-arginine-rich RNA-binding proteins (SR proteins), glycine-rich RNA-binding proteins (GR-RBPs), pentatricopeptide repeat proteins (PPR proteins), DEAD-box RNA helicase (DEAD-box RHs) and RNA chaperones. The critical roles of these plant RBPs in RNA metabolism during plant growth, development, and stress responses were summarized. Functions as an alternative splicing factor during RNA metabolism, SR proteins play important roles in plant growth and stress response. GR-RBPs family members displayed functional diversity: Many of them regulate plant stress tolerance and various growth and development processes by mediating plant hormone signaling pathways and others mediate abiotic stress response acting as RNA chaperones. PPR proteins are the most widely studied and they mainly involved in RNA metabolism of mitochondria and chloroplasts. As important RNA splicing factors of cell nuclei and organelles, DEAD-box RHs play variety of functions in plant growth, development and abiotic stress response. RNA chaperones are non-specific RBP that maintain the normal function of RNA molecules by facilitate RNA folding via structural rearrangement of misfolded RNAs.

    Analysis on the Trends of Yield and Quality Related Traits for Maize Hybrids Released in China over the Past Years
    XianMin CHEN,XiaoGui LIANG,Xue ZHAO,Zhen GAO,Gong WU,Si SHEN,Shan LIN,LiLi ZHOU,ShunLi ZHOU
    Scientia Agricultura Sinica. 2018, 51(21):  4020-4029.  doi:10.3864/j.issn.0578-1752.2018.21.002
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    【Objective】Simultaneous improvement in yield and quality of maize hybrids is vital to improve maize variety and develop the maize industry. Amounts of studies focused on the trends of crude starch and crude protein concentrations with maize varieties released in different eras, however, the results are different and the relationships between different substances were rarely mentioned. Hence, further exploration of the changing trends of yield and quality related traits for maize hybrids released in China over the past years and their intrinsic connections are needed. 【Method】The information of 770 maize varieties released in China from 1992 to 2017 were collected, and the related traits of yield and quality of the 73.64% common maize hybrids were used for further analysis. 【Result】The results showed that the volume weight, 1000-kernel weight and crude starch concentration were significantly or highly significantly increased with an average annual improvements of 1.87 g·L -1, 0.91 g, and 0.19%, respectively. To the contrast, the crude fat concentration was significantly reduced by 0.03% per year. The annual average of crude protein and lysine concentration remained stable along with time, the averages from 1992 to 2017 were 9.88% and 0.30%, respectively. Correlation analysis was processed and both volume weight and 1000-kernel weight were significantly positively correlated with crude starch concentration, however, they were negatively related to the crude fat concentration to some extent. In addition, both of the crude protein and lysine concentration showed extremely significant negative relationships with the crude starch concentration and 1000-kernel weight. The accumulation trends based on 1000-kernel weight showed that crude starch, crude protein and lysine per 1000-kernel increased to some extent over the released years. The average annual increase for each trait was 1.159 g, 0.092 g and 0.001 g, respectively. Crude fat accumulation still decreased which is similar to its concentration. 【Conclusion】Overall, during the process of maize varieties improvement, the traits related to yield such as 1000-kernel weight and bulk weight are relatively fast improved in China, and the advance of them rely on, to a great extent, the enhancement of the crude starch concentration, however, which may lead to an effect of carbon dilution on the concentrations of crude protein, lysine and crude fat. The carbon dilution effects and the relatively fast improvement in 1000-kernel weight determined the different performances between concentrations and accumulations for crude protein and lysine. Besides, the relationships between the traits of suitable mechanical grain-harvesting and grain quality deserve further attention during the selection of suitable mechanical grain-harvesting variety.

    TILLAGE & CULTIVATION·PHYSIOLOGY & BIOCHEMISTRY·AGRICULTURE INFORMATION TECHNOLOGY
    The Increasing Effect and Influencing Factors of Leguminous Green Manure on Wheat Grain Zn in Weibei Highland
    YiXuan LIU,DongLin HUANG,Na LIU,ZhiYuan YAO,Dan YIN,YuanYong MENG,HuBing ZHAO,YaYun GAO,ZhaoHui WANG
    Scientia Agricultura Sinica. 2018, 51(21):  4030-4039.  doi:10.3864/j.issn.0578-1752.2018.21.003
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    【Objective】Weibei Highland is one of important winter wheat production areas in northwest China, where Zn concentration in wheat grain are generally low due to soil bioavailable Zn deficiency. So we decided to study leguminous green manure-wheat rotations whether could improve wheat Zn concentration in different regions of Weibei Highland, and to explore the factors affecting wheat Zn biofortification by leguminous green manure-wheat rotations.【Method】Two field experiments were carried out during 2016-2017 cropping seasons in Yongshou and Changwu with a completely randomized block design. There were three main treatments: Summer fallow-wheat (FW) as CK, black kidney bean-wheat (KW) and mung bean-wheat (MW) in Yongshou; Summer fallow-wheat (FW) as CK, huai bean-wheat (HW) and mung bean-wheat (MW) in Changwu. Plant samples of legumes, wheat and 0-20 cm soil samples at different wheat growing stages were collected for chemical analysis. 【Result】 In Yongshou, compare with FW, wheat grain yield and biomass were decreased by 19.2%-38.3% under leguminous green manure-wheat rotations,. In Changwu, leguminous green manure-wheat rotations did not significantly reduce wheat grain yield, but reduced wheat glumes and straw biomass by 19.9%-33.2%. KW rotation in Yongshou and HW rotation in Changwu significantly increased grain Zn concentration by 14.2% and 18.6%, respectively, which partially compensated the potential reduction of wheat grain Zn export and aboveground shoot Zn uptake by increasing Zn concentrations of wheat grain and straw Zn concentration due to the shrink of wheat yield and biomass. There was no significant difference of wheat Zn uptake between HW and FW treatments in Changwu. Wheat grain Zn concentration was positively correlated with leguminous manure shoot Zn uptake. Wheat grain Zn concentration increases 0.23 mg·kg -1 when green manure shoot Zn uptake increases 1.0 g·hm -2, correspondingly. Besides, the Zn, N content of black kidney bean and huai bean were both higher than that of mung bean. Moreover, compared with mung bean, C/N of black kidney bean and huai bean were lower by 18.6% and 20.4%, respectively. At wheat maturity stage, soil nitrate of KW and HW rotations were both higher than that of FW by 36.7% and 69.1%, respectively. Leguminous green manure-wheat rotations had rarely significant effect on soil DTPA-Zn concentration during the growth of wheat.【Conclusion】Relatively higher Zn and N content and lower in C/N of black kidney bean and huai bean made them more readily improved subsequently wheat Zn uptake and accumulation than that of mung bean. The positive correlation between wheat grain Zn concentration and leguminous green manure shoot Zn uptake could be the guidance for screening and optimizing green manure varieties for wheat Zn biofortification. Therefore, leguminous green manure-wheat rotations could be an effective biofortification method to enhance wheat Zn nutrition for residents which suffered from Zn deficiency.

    Effects of Different Sowing Dates on Emergence Rates and Seedling Growth of Cotton Under Mulched Drip Irrigation in Xinjiang
    LongLong SUI,JingShan TIAN,HeSheng YAO,PengPeng ZHANG,FuBin LIANG,Jin WANG,WangFeng ZHANG
    Scientia Agricultura Sinica. 2018, 51(21):  4040-4051.  doi:10.3864/j.issn.0578-1752.2018.21.004
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    【Objective】Dry soil seeding with drip irrigation are widely used in field cotton in Xinjiang. And only seedling temperature has to be considered under dry soil seedling with drip irrigation. In this paper, according to the temperature requirement of cotton seedling, cotton was sown under different temperature conditions and different sowing dates. The emergence rate, seedling growth condition and yield of cotton under mulched drip irrigation were observed. The effects of sowing conditions on emergence rate, growth states and development of yield in harvest period were analyzed to provide theoretical basis for cultivating strong seedlings of cotton under mulched drip irrigation.【Method】According to the changes of air temperature and soil temperature, 3-4 sowing dates were set up. The earliest sowing date was based on the biological low temperature for cotton seeds germination and emergence. The morphological indexes, such as emergence rate, seedling height, cotyledon node height and total dry weight, were determined to demonstrate the effects of sowing under different temperature conditions on seedling indexes in cotton.【Result】Under “5 cm deep-18.7℃-3 days before sowing” condition, the germinate rate and emergence rate were the highest. Compared with early sowing at lower temperature treatment, the plant height under “5 cm deep-24.7℃-late sowing” condition was 3.52%-8.64% higher during seedling to three -leaf stage, the height of cotyledon section was 8.82%-20.59% higher, the total root length was 1.79%-6.59% longer, and the root length was 14.84%-25.93% greater. Under “5 cm deep-9.5℃-3 days before sowing” condition, the root dry matter accumulation was higher, and the ratio of root to shoot was the maximum.【Conclusion】Under “5 cm deep-3 days before sowing” condition steady passage 13.8℃-15.7℃, if the average temperature was in 16℃-18℃ after sowing 1 week, the cotton seedling emergence rate could be more than 90%. But if the air temperature was in 6.7℃-14.1℃ under “5 cm deep-9.5℃-17.6℃-3 days before sowing” condition, the average temperature was 18.5℃-19.5℃ and air accumulated temperature was in 110℃-120℃ during the emergence stage to trilobites stage, the unit of cotton seedling dry matter accumulation would be larger with stronger root and increasing boll number and single boll weight. Therefore, earlier sowing could support a good foundation for cotton yield.

    PLANT PROTECTION
    MdWRKY40 Mediated Improvement of the Immune Resistance of Apple and Arabidopsis thaliana to Botryosphaeria dothidea
    QianQian ZHOU,HuaRong QIU,XiaoWen HE,XianPu WANG,XiuXia LIU,BaoHua LI,ShuJing WU,XueSen CHEN
    Scientia Agricultura Sinica. 2018, 51(21):  4052-4064.  doi:10.3864/j.issn.0578-1752.2018.21.005
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    【Objective】The objective of this study is to clone MdWRKY40 from ‘Fuji’ apple, research its expression pattern under salicylic acid (SA)-induced conditions and its role in the disease resistance pathway of Botryosphaeria dothidea, and to provide a theoretical basis for further revealing the disease resistance mechanism of apple. 【Method】 The full-length CDS sequence of MdWRKY40 was cloned from ‘Fuji’ apple, and its bioinformatics analysis was carried out. Fluorescence quantitative PCR (qRT-PCR) was used to analyze the expression level in different apple tissues and the response to abiotic stress SA, to study the effect of exogenous SA treatment on apple leaves inoculated with pathogenic fungi B. dothidea, and to detect the expression of pathogenesis-related protein gene by qRT-PCR. MdWRKY40 was expressed heterologous in Arabidopsis thaliana, and the stably expressed A. thaliana seedlings were treated with B. dothidea to observe the degree of disease and the number of infected leaves. The expression of disease-associated genes was analyzed by qRT-PCR. The root length of A. thaliana seedlings was measured and the expression of auxin-related genes was detected by qRT-PCR. 【Result】 MdWRKY40 contains a complete open reading frame of 858 bp in length and encodes 286 amino acids. The predicted molecular weight is 32.088 kD and the isoelectric point is 8.15. Phylogenetic tree analysis showed that MdWRKY40 has the highest similarity with the PbWRKY40 sequence, and its genetic relationship is closest. MdWRKY40 and AtWRKY40 locate in different branches, and its genetic relationship is far from that of AtWRKY40. The multiple sequence alignment analysis of MdWRKY40 and AtWRKY40 by using DANMAN software revealed that both MdWRKY40 protein and AtWRKY protein contain a WRKYGQK conserved domain, but similarity is only 29.78%. qRT-PCR analysis showed that the expression level of MdWRKY40 was the highest in root and lowest in leaf. SA induced MdWRKY40 expression in root, stem and leaf, and the expression all increased first and then decreased, reached the highest level at 6 h. Exogenous SA enhanced the resistance of apple leaves to B. dothidea, the incidence of untreated leaves reached 92.59%, and the incidence after SA treatment decreased to 79.26%, and significantly increased the expression of disease-associated protein genes MdPR2 and MdPR5. Compared with the wild type, the overexpression of MdWRKY40 in A. thaliana significantly increased the resistance of A. thaliana leaves to B. dothidea. The incidence of wild type A. thaliana reached 77.5%, while the incidence of two transgenic A. thaliana lines was only 21.5% and 17.4%, and significantly increased the expression of PR1, PR3, and PR4 genes associated with disease progression. The root growth of A. thaliana plants with overexpression of MdWRKY40 was inhibited. After 7 days of culture, the length of main root of transgenic A. thaliana was 39.9% and 43.1% respectively of that of wild type A. thaliana. After 10 days of culture, the length of main root of transgenic A. thaliana was 58.5% and 55.4% respectively of that of wild type A. thaliana. The expression level of the auxin synthesis-related gene AtTAA1 and auxin transport-related genes AtPIN1 and AtPIN2 was significantly lower in the MdWRKY40 overexpression lines than in the wild type.【Conclusion】The expression of MdWRKY40 was induced by the infection of SA and the pathogenic fungi B. dothidea. MdWRKY40 is an important disease resistance gene in apple. The overexpression of MdWRKY40 significantly increased the resistance to B. dothidea. MdWRKY40 has the function of regulating the growth and development of plant roots, which may affect the growth and development of plant roots by down-regulating the expression of auxin transport-related genes.

    Control Efficacy of Pyraclostrobin and Triazole Fungicides Against Tomato Crown and Root Rot
    HaiChao CAO,XiuHuan LI,XiaoKun WANG,HaiXiu BAI,Wei MU,Feng LIU
    Scientia Agricultura Sinica. 2018, 51(21):  4065-4075.  doi:10.3864/j.issn.0578-1752.2018.21.006
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    【Objective】 The objective of this study is to evaluate the potential efficacy of pyraclostrobin as well as tebuconazole, propiconazole and flusilazole against tomato crown and root rot, which caused by Fusarium oxysporum f. sp. radicis-lycopersici. 【Method】The laboratory toxicity of 4 fungicides to mycelial growth, conidial germination and germ tube elongation of F. oxysporum f. sp. radicis-lycopersici were determined by mycelial growth rate and conidial germination methods. In greenhouse pot experiment, the root-irrigation method was used to treat tomato seedlings. At 7 d and 15 d after treatment, the increment of plant height and stem diameter was measured to evaluate the safety of fungicides on tomato seedlings. In the laboratory, the method of first inoculating and then applying fungicides was adopted. The control efficacy of fungicides on tomato crown and root rot at 7, 15 and 30 d after application was investigated, respectively. In field experiment, same root-irrigation method was used to investigate the incidence of tomato crown and root rot at 60, 90 and 150 d after treatment and the tomato yield statistic, and to evaluate the efficacy of fungicides against the disease in field and the effect on yield of tomato.【Result】Pyraclostrobin showed high toxicity to mycelial growth, conidial germination and germ tube elongation of F. oxysporum f. sp. radicis-lycopersici, with EC50 of 0.055, 0.012 and 0.010 μg·mL -1, followed by propiconazole, EC50 was 0.058, 0.060, 0.011 μg·mL -1, respectively. The inhibitory effect of tebuconazole on mycelial growth was strong, and the toxicity of tebuconazole to conidial germination and germ tube elongation was relatively low, with EC50 of 0.075, 0.255 and 0.455 μg·mL -1, respectively. The toxicity of flusilazole to conidial germ tube elongation was high, but the toxicity of flusilazole to mycelial growth and conidial germination was low, with EC50 of 0.013, 0.078 and 0.457 μg·mL -1, respectively. Tebuconazole at 25, 50 mg a.i./plant, propiconazole at 5, 10 mg a.i./plant and flusilazole at 5, 10 mg a.i./plant significantly inhibited tomato seedling height and increased stem diameter, but pyraclostrobin at 30, 60 mg a.i./plant had no effect on tomato seedling. In greenhouse pot experiment, 30 days after treatment (DAT), pyraclostrobin had the highest control efficacy (87.12%) on this disease at 60 mg a.i./plant, followed by propiconazole 10 mg a.i./plant, the control efficacy was 82.17%, and the control efficacy of tebuconazole at 50 mg a.i./plant and flusilazole at 10 mg a.i./plant was 79.40% and 71.67%, respectively. At 90 DAT in field experiment, the best control efficacy on the disease was 90.36% by pyraclostrobin at 60 mg a.i./plant, followed by tebuconazole at 50 mg a.i./plant and propiconazole at 10 mg a.i./plant, which was 84.20% and 82.55%, respectively. The lowest one was flusilazole at 5 mg a.i./plant (65.06%). At 150 DAT, the control efficacy of each treatment on the disease significantly declined, just tebuconazole at 50 mg a.i./plant was 40.40%. According to field statistics, pyraclostrobin at 60 mg a.i./plant had the highest yield in field, the increased production rate was 50.63%, and propiconazole at 5 mg a.i./plant was the lowest, with the rate of -2.61%. 【Conclusion】Pyraclostrobin, tebuconazole, propiconazole and flusilazole had similar toxicity on F. oxysporum f. sp. radicis-lycopersici mycelial growth, however, pyraclostrobin had higher toxicity on the conidial germination and germ tube elongation of the pathogen than the other three fungicides. Pyraclostrobin, propiconazole and tebuconazole could be used to control tomato crown and root rot in greenhouse. The control efficacy of pyraclostrobin at 60 mg a.i./plant was the highest, which was safe and increased production significantly. However, the dosages of propiconazole and tebuconazole should be concerned before application.

    SOIL & FERTILIZER·WATER-SAVING IRRIGATION·AGROECOLOGY & ENVIRONMENT
    Fertilization Effect of Wheat Under Different Soil Fertilities
    Xia XU,YaNan ZHAO,YuFang HUANG,JunYing YAN,YouLiang YE
    Scientia Agricultura Sinica. 2018, 51(21):  4076-4086.  doi:10.3864/j.issn.0578-1752.2018.21.007
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    【Objective】The data of 1 247 “3414” field experiments, conducted in Henan Province during 2005-2013, were collected in this paper to evaluate effects of nitrogen (N), phosphorous (P) and potassium (K) fertilizer application on wheat yields, economic benefits and fertilizer use efficiencies under different soil fertilities, aiming to clarify the response of fertilization effect to different soil fertilities, and to provide reference for reasonable fertilizer application. 【Method】The five treatments, including N0P0K0, N0P2K2, N2P0K2, N2P2K0 and N2P2K2, were chosen. According to the wheat grain yield of N0P0K0 treatment, the soil fertilities were classified into four grades: <3.0 t·hm -2, 3.0 to 4·5 t·hm -2, 4.5 to 6.0 t·hm -2, and >6.0 t·hm -2. The yield responses to N, P and K fertilizers, gross income, fertilizer profit, input-output ratio, and the agronomic efficiency (AE), partial factor productivity (PFP), the fertilizer contribution rate (FCR) and contribution rate of soil fertilities were also compared. 【Result】Compared with N0P0K0 treatment, the wheat yields with fertilizer application were increased significantly, and the yield increase and yield increase rate showed a significant downtrend with increasing soil fertilities. The yield increase rate of NPK and N, P, and K fertilizers application were 126.07%, 75.98%, 24.93% and 17.73% under soil fertilities <3.0 t·hm -2, respectively, while which were 24.35%, 15.39%, 10.36% and 8.70% under soil fertilities >6.0 t·hm -2, respectively. The gross income, fertilization cost, fertilizer profit and input-output ratio of all treatments increased with the improvement of the soil fertilities level. The gross income and fertilizer profit of NPK treatment was the highest under the soil fertilities >6.0 t·hm -2, which were 19.64×10 3 yuan/hm 2 and 18.24×10 3 yuan/hm 2, respectively; the gross income and fertilizer profit of -N treatment was the lowest, under the soil fertilities <3.0 t·hm -2, which were 8.52×10 3 yuan/hm 2, 7.87×10 3 yuan/hm 2, respectively. In terms of fertilizer use efficiency, the AE and FCR generally decreased when the soil fertilities improved. The contribution rate of soil fertilities was 63.72% averagely, and increased with the increase of the soil fertilities, the contribution rate of soil fertilities of different soil fertilities (<3.0 t·hm -2, 3.0 to 4.5 t·hm -2, 4.5 to 6.0 t·hm -2, and >6.0 t·hm -2) were 43.57%, 57.80%, 70.29%, and 80.34%, respectively. The FCR of the NPK treatments or N, P and K fertilizers decreased with the increase of wheat yield under the CK treatment or corresponding fertilizer omission treatment, indicating that enhancing soil indigenous fertilities could reduce the yield dependence on chemical fertilizers. 【Conclusions】Improving soil fertilities could increase the yield and income of wheat, reduce the dependence of wheat yield on chemical fertilizer application. Therefore, it was important to improve the soil fertilities, and apply fertilization rationally according to soil fertilities levels to ensure high and stable yield, increase fertilizer profit, and improve the fertilizer use efficiency in wheat production in Henan Province.

    Effects of Combined Application of Various Organic Materials and Chemical Fertilizer on Soil Nitrogen Formation and Greenhouse Gas Emission Under Equal Nitrogen Rates from Purple Soil
    Rong HUANG,Ming GAO,JiaCheng LI,GuoXin XU,FuHua WANG,Jiao LI,ShiQi CHEN
    Scientia Agricultura Sinica. 2018, 51(21):  4087-4101.  doi:10.3864/j.issn.0578-1752.2018.21.008
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    【Objective】In this study, the effects of agricultural organic materials combined with chemical fertilizer on soil nitrogen species (ammonium nitrogen, nitrate nitrogen, available nitrogen and total nitrogen) and greenhouse gases (carbon dioxide (CO2), methane (CH4) and nitrous oxide (N2O)) emission were investigated to provide knowledge for chemical fertilizer reduction and friendly environment according to the same nitrogen rate addition. From the aspect of greenhouse gases mitigation, theoretical suggestions were also provided for dry land fertilization strategy. 【Method】 A vegetable rotation cropping experiment (lettuce-cabbage-chili rotation) with six treatments was carried out on purple soil in the field, and the treatments were control without any chemical fertilizer and organic material (CK), conventional fertilizing (F), straw returning with chemical fertilizing (SF), mushroom dregs returning with chemical fertilizing (MF), biochar returning with chemical fertilizing (BF), and cow dung returning with chemical fertilizing (CF). The variations of mineral nitrogen, available nitrogen and total nitrogen contents and the emissions of CO2, CH4 and N2O from soil were monitored from November 2016 to September 2017.【Result】 In the condition of equal nitrogen rates, organic materials could change the formation of soil nitrogen. The contents of ammonium nitrogen increased after organic materials application in SF and MF treatments. CF treatment could increase the content of ammonium nitrogen during lettuce and cabbage growth seasons, and BF treatment increased the content of nitrate nitrogen and available nitrogen during chili growth season. Seasonal dependent CO2, CH4 and N2O emission was found and high emission mainly occurred during summer. Fertilization and irrigation events also increased CO2, CH4 and N2O emission. Compared with F treatment, N2O emission reduced by 7.5% under BF treatment, but significantly increased by 233.5% under CF treatment. Combined application of organic materials and chemical fertilizer could increase the CO2 emission. Significant increases of CO2 emission were found under MF and CF treatments, which increased the mean CO2 emission flux of 35.6% and 31.3% compared with F treatment, respectively. However, biochar returning delayed the emission peak of CO2 and increased the CO2 emission during summer. Negative CH4 emission was found in most treatments, indicating a CH4 adsorption by soil itself. Moreover, the emission of CH4 fluctuated during the chili growth season and the highest CH4 emission (668.7 μg·m -2·h -1) appeared under BF treatment with the condition of high temperature and intensive precipitation. However, the mean CH4 emission flux under SF, MF and BF increased significantly by 104.85%, 175.2% and 77.5%, respectively. Moreover, SF and MF treatment could promote CH4 consumption and reduce mean CH4 emission flux of -0.1 and -1.3 kg·hm -2, respectively. Compared with CK and F treatment, organic materials combined with chemical fertilizer application could increase the global warming potential (GWP) by 26.7%-52.4% and18.1%-42.0% respectively. The lowest GWP was found under SF treatment.【Conclusion】Combined application of organic materials and chemical fertilizer had different greenhouse gases emission characters and soil nitrogen speciation. Biochar and straw returning combined with chemical fertilizer could reduce the greenhouse gases emission and increase available nitrogen content. However, cow dung returning with chemical fertilizing increased greenhouse gases emission.

    HORTICULTURE
    Identification and Expression Analysis of LBD Gene Family in Grape
    HongHong HE,ZongHuan MA,YuanXia ZHANG,Juan ZHANG,ShiXiong LU,ZhiQiang ZHANG,Xin ZHAO,YuXia WU,Juan MAO
    Scientia Agricultura Sinica. 2018, 51(21):  4102-4118.  doi:10.3864/j.issn.0578-1752.2018.21.009
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    【Objective】The objectives of this research are to identify the Lateral Organ Boundaries Domain(LBD) family genes from grape(Vitis vinifera) genome, to know the profile of LBD family such as gene number, gene structure and abiotic stress expression characteristics in grape, and to provide theoretical basis for exploring what roles the LBD transcription factors play in abiotic stress of grape.【Method】The LBD genes in grape genome-wide were identified by BLAST software in the grape genome database based on LBD genes have been reported from Arabidopsis. DNAMAN5.0, Clustalx, MapInspect, MEME, GSDS2.0, ExPASy and MEGA5.0 software were used for carry out various bioinformatics analysis of LBD. The expression profile chip was draw by the data comes from Affymetrix Gene Chip 16K in PLEXdb. The qRT-PCR was used to detect the expression of grape LBD gene family in abiotic stress.【Result】Total of 30 LBD genes were identified from grape genome, which can be divided into ClassⅠand ClassⅡ. The classⅠcan be divided into 5 subtribe which are namedⅠa,Ⅰb,Ⅰc,Ⅰd andⅠe. ClassⅡ can be divided into Ⅱa and Ⅱb. The number of amino acids is between 127 and 386, and the theoretical equivalence point is between 4.77 and 9.28 in the VvLBD gene family by physicochemical analysis. The analysis of the gene's location on the chromosome revealed that the 30 genes in this family were distributed on 11 of the 19 chromosomes of the grape, and the chromosome 13 contains 7 genes. Multiple sequence alignments and Motif analysis showed that the VvLBD gene family has three conserved domains, namely the sinusoidal structure, the leucine zipper structure, and the Glycine-alanine-serine (GAS) structure. The VvLBD gene family is mainly expressed in chloroplast, mitochondria and nucleus through the sub-cellular location analyzing. The alpha-helix and irregular curl are the main structure of the secondary structure in the VvLBD gene family. The expression of the most gene was increased under the salt and PEG stress, and the expression characteristic was changed by the stress time change in the chip expression profile. The qRT-PCR results showed the expression of VvLBD8, VvLBD11, VvLBD12, VvLBD15, VvLBD16, VvLBD17 were raised to the 3, 1, 8, 4, 5, and 13 times compared the control under the 400 mmol .L -1NaCl treatment. The expression of VvLBD12 and VvLBD19 was raised to the 4 and 26 times compared the control under 10% PEG treatment.【Conclusion】30 LBD gene family members were identified from the grape genome. These members distributed on 11 of the 19 chromosomes of the grape and the evolution of VvLBD is highly conservative. The LBD gene family is related to the abiotic stress, but the different expression were existed between the different members when the encountered the adversity environments.

    Breeding for Grape Germplasm Involved in Seedlessness with Cold-Resistant Using Embryo Rescue
    YaNan ZHAO,QiangWei LUO,YueJin WANG
    Scientia Agricultura Sinica. 2018, 51(21):  4119-4130.  doi:10.3864/j.issn.0578-1752.2018.21.010
    Abstract ( 423 )   HTML ( 7 )   PDF (2386KB) ( 426 )   Save
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    【Objective】 The objective of this study is to obtain new cold-resistant seedless grape germplasm and improve the breeding efficiency through embryo rescue in vitro. 【Method】 The different genotypes, basal culture media (MM3 and ER), amino acids (2.5 mmol·L -1cysteine, asparagine, glutamine and serine were added in MM3 medium) were used to investigate the effect on the embryo development rates and seedling rates from ovules of 7 cross combinations. From the parents, the molecular markers GSLP1-569 and SCC8-1018 for seedlessness and the marker S241-717 for cold-resistance were used to identify the strains with specific bands, and then the three markers were used to screen the hybrids by recognizing the specific bands. 【Result】 Of the 7 combinations, 1 168 ovules cultured in MM3 were obtained 331 embryos and 97 hybrid strains. Among these crosses, ‘Ruby Seedless’ × ‘Beichun’ got the highest embryo development rate and seedling rate, which were 46.00% and 17.33%, respectively. Compared with female parents, for ‘Shuangyou’ as male parent and ‘Blush Seedless’ ‘Thompson Seedless’ ‘Kunxiang Seedless’ as female parents, ‘Kunxiang Seedless’ × ‘Shuangyou’ had the highest embryo development rate and seedling rate, which were 45.39% and 16.31%, respectively. For ‘Beibinghong’ as male parent and ‘Blush Seedless’ ‘Beauty Seedless’ ‘Thompson Seedless’ as female parents, ‘Blush Seedless’ × ‘Beibinghong’ had the highest embryo development rate and seedling rate, which were 33.52% and 6.59%, respectively. Different male parents also influenced seedlings formation. The embryo development rates of ‘Blush Seedless’ × ‘Beibinghong’ and ‘Blush Seedless’ × ‘Shuangyou’ were 33.52% and 39.00%, and the seedling rates of them were 6.59% and 9.50%, respectively. The embryo development rates of ‘Thompson Seedless’ × ‘Beibinghong’ and ‘Thompson Seedless’ × ‘Shuangyou’ were 7.64% and 19.26%, and the seedling rates of them were 1.91% and 8.15%, respectively. The rate of embryo development and seedling of ovule cultured in MM3 medium was higher than that of ER medium. For ‘Kunxiang Seedless’ × ‘Shuangyou’, the ovules cultured in MM3 medium with 2.5 mmol·L -1 glutamine showed the highest embryo development rate, which was 59.69%. The seedling rate of ovules cultured in basal media with asparagine and glutamine was 21.43% and 20.93%, respectively. The effect of promoting seedlings development was significant. For ‘Ruby Seedless’ × ‘Beichun’, the ovules in media with 2.5 mmol·L -1 asparagine showed the highest embryo development rate, which was 55.71%. The seedling rate of ovules cultured in basal media with asparagine and glutamine was 21.43% and 22.22%, respectively. The effect of promoting seedlings development was significant. By using the molecular markers GSLP1-569 and SCC8-1018 for seedlessness and the marker S241-717 for cold-resistance, 83 hybrid strains from 6 cross combinations were detected, and there were 49 strains with specific bands of the molecular markers linked to the seedlessness, 55 strains with specific bands of the markers S241-717 for cold-resistance. Importantly, 36 strains with both seedlessness and cold-resistance were identified in this study. 【Conclusion】 ‘Kunxiang Seedless’ and ‘Blush Seedless’ are suitable as the female parents for embryo rescue in vitro of seedless grapes. The embryo rescue efficiency of ‘Shuangyou’ as the male parent is higher than that of ‘Beibinghong’. MM3 is suitable for embryo rescue in vitro, and the addition of amide acids to MM3 culture medium is helpful to the seedling formation. Among the 83 hybrid strains detected by molecular markers, 36 strains may have the seedless and cold-resistant characteristics.

    FOOD SCIENCE AND ENGINEERING
    Effects of Microwave Treatments on Rice Quality and Lipase Activity
    Jian YUAN,Teng ZHAO,Chao DING,ChangRui XING,Bin ZHANG,ShangBing CHEN,Rong HE,XingRong JU
    Scientia Agricultura Sinica. 2018, 51(21):  4131-4142.  doi:10.3864/j.issn.0578-1752.2018.21.011
    Abstract ( 311 )   HTML ( 9 )   PDF (1672KB) ( 245 )   Save
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    【Objective】This paper researched the influence of the different microwave conditions on water migration, quality, lipase activity, internal structure of paddy, and it aimed to improve the drying rate of rice, shorten the drying time and select the optimum conditions of microwave drying, realizing fast and efficient drying. 【Method】 In this paper, rice was treated to 50℃, 60℃ and 70℃with additional tempering or no-tempering by the microwave dosage of 0.69, 1.29, 1.92 W·g -1. The quality as well as physical and chemical indexes of paddy were determined, and the changes of water migration and internal structure were observed by LF-NMR and SEM. 【Result】 Microwave dose and rice temperature were key factors that affected rice quality. The processing quality of paddy rice was better under the condition of 1.29 W·g -1, 60℃. The rate of crack, broken rice, brown rice, head rice were 8.65%, 6.76%, 83.9%, and 68.07%, respectively. Whereas, the processing quality of paddy rice had no significant difference with hot air drying. Meanwhile, at the same time, microwave had obvious inhibitory effect on lipase activity. The activity of lipase was lowest (5.65 U) in the tempered condition of 1.92 W·g -1, 70℃, and the results showed that the lipase activity in this condition was 4.65 U less than that under control. By using the membership degree comprehensive scoring method to judge the quality after drying, in the condition of 1.29 W·g -1, 60℃-tempering, the rice score ranked third, therefore, with considering the heating rate and the quality score, it was the most suitable microwave treatment condition. The results of LF-NMR and SEM showed that the water content of paddy rice decreased and gradually moved towards the left. It suggested that water was more tightly bonded to other components. The results of SEM showed that the rupture of the endosperm cells and the degree of starch exposure were increased, the structure of radioactive arrangement gradually disappeared, the composite starch gradually disintegrated, and the single grain and the internal cracks in rice increased. 【Conclusion】 Microwave drying had a significant effect on the heating rate, quality and enzyme activity of rice. The firmness of the combination of water and other components was stronger after microwave. During drying process the loss of water could cause the change of the internal structure of paddy to different degree. Compared with control group, the internal cracks of microwave treated sample were smaller.

    SPECIAL FOCUS: SWINE FEVER AND AFRICAN SWINE FEVER
    The Impact of Classical Swine Fever and African Swine Fever on Pig Industry
    Qin WANG
    Scientia Agricultura Sinica. 2018, 51(21):  4143-4145.  doi:10.3864/j.issn.0578-1752.2018.21.012
    Abstract ( 366 )   HTML ( 46 )   PDF (261KB) ( 347 )   Save
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    Dynamic Distribution of Classical Swine Fever Virus in vivo After Infection by Intermediate Virulent Strains
    JunXiang SUN,QianYi ZHANG,HeMin XU,TuanJie WANG,Lu XU,XingQi ZOU,YuanYuan ZHU,Cui LI,YingJu XIA,Yuan XU,Kai CHEN,YuJie ZHANG,QiZu ZHAO,Qin WANG
    Scientia Agricultura Sinica. 2018, 51(21):  4146-4156.  doi:10.3864/j.issn.0578-1752.2018.21.013
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    【Objective】At present, the infection type of classical swine fever in China is mainly subacute or chronic infection. The aim of this study was to investigate the differences and distribution of RNA and protein expressions in pigs infected with the intermediate virulent strain of classical swine fever virus. The results could help to elucidate the replication and distribution of the subacute disease virus and to provide technical support for the early diagnosis and prevention of classical swine fever.【Method】Using the medium virulence strain (HEBHH1/95), we successfully established a subacute CSF infection animal model. Duodenum, spleen, kidney, lung, pancreas and ileocecal samples were collected from pigs for viewing in situ hybridization (ISH), immunohistochemistry (IHC) and hematoxylin-eosin staining (HE) at 1 day post infection (dpi), 3 dpi, 6 dpi, 10 dpi, 13 dpi, 20 dpi, 24 dpi and 28 dpi. The ViewRNA ISH was used to study the dynamic distribution of viral RNA in infected tissues. Immunohistochemistry (IHC) and HE staining were used to detect the distribution of viral proteins in infected tissues and their contribution to tissue damage.【Result】The clinical score increased rapidly from 6 dpi to 10 dpi, then from 11 dpi to 26 dpi the score remained at approximately 15, until at 28 dpi clinical score peaked at 20 points. The body temperature showed an upward trend from 8 dpi to 10 dpi, and then livestock suffered from continuous fever which persisted at about 40℃ from 13 dpi to 24 dpi, thereafter, the body temperature began to fall back to about 39.5℃ before the animal died. Viral RNA were detected in duodenum, pancreas, ileocecal valve and kidney at 1 dpi and in the lungs of the bronchioles, spleen oval body at 3 dpi; Viral RNAs widely distributed in each tissue at 28 dpi, and were mainly observed in the spleen artery around the lymphatic sheath, pancreatic acinar, renal tubular with secretion function. The IHC and HE staining were used to verify the results of ViewRNA ISH in similar fields of vision. The positive signals of viral proteins and the corresponding histopathological changes of duodenum, pancreas and kidney were also detected at 1 dpi, but the viral protein and tissue pathological changes were detected in the ileocecal valve, spleen and lung at 3 dpi. Viral RNA and protein localization tended to be the same in each tissue after 3 dpi.【Conclusion】All the results showed that CSFV had an increasing virus load from 1 dpi to 28 dpi detected by ViewRNA ISH, which was in consistent with the result of IHC. Moreover, CSFV was firstly tropism to secretory cells such as pancreatic acinar, renal tubular and spleen artery at the beginning of infection (1 dpi-3 dpi), and then showed pantropically infectious to all the tissues during the infection period (6 dpi-13 dpi), and during the final stage CSFV was accumulated both around lymphocytes and secretory cells (20 dpi-28 dpi).

    Screen of MicroRNAs in Classical Swine Fever Virus-Infected PK-15 Cells and the Regulation of Virus Replication by miR-214
    ShaoFeng DENG,ZuoDong YE,ShuangQi FAN,JinDing CHEN,JingYuan ZHANG,MengJiao ZHU,MingQiu ZHAO
    Scientia Agricultura Sinica. 2018, 51(21):  4157-4168.  doi:10.3864/j.issn.0578-1752.2018.21.014
    Abstract ( 307 )   HTML ( 8 )   PDF (785KB) ( 169 )   Save
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    【Objective】 In this study, differential expression of miRNAs in CSFV-infected PK-15 cells were determined by miRNA expression array, and further explore the function of miRNAs in the pathogenic of Classical swine fever virus (CSFV), and provide some new basis for the prevention and control of Classical swine fever (CSF) .【Method】 In order to investigate the changes of the miRNAs expression in CSFV-infected PK-15 cells, we synthesized probes of 326 miRNAs of pig according to the miRBase database version 19.0, and screening of differential expression of miRNAs in CSFV-infected PK-15 cells using miRNA expression array. Then, miR-214, the most obvious difference in expression of CSFV-infected PK-15 cells, was selected as the further study object to investigate the function of miR-214 in the infection process of CSFV. We detected mRNA expression of miR-214 in CSFV-infected PK-15 cells using qRT-PCR. In order to further study the effect of miR-214 of CSFV infection, we synthesized miR-214 analog and inhibitor and transfected into PK-15 cells respectively, follow with CSFV infection at 24 h post-transfection, and then detected CSFV titers and quantity of CSFV genomic copies. In order to further explore the mechanism of miR-214 participate in the regulation of CSFV replication, we predicted the target protein of miR-214 using bioinformatics software and confirmed it by luciferase reporter gene system. Given TRADD can specific interacts with TNFR1 intracellular dead zones and participate in the programmed cell death, we assume that miR-214 influencing apoptosis of PK 15 cells by influencing expression level of target protein TRADD. PK 15 cells transfected with miR-214 and inhibitor respectively, follow with CSFV infection at 24 h post-transfection. At 48 h post-infection, the expression levels of TRADD were detected, and the effect of miR-214 on the apoptosis of CSFV-infected PK-15 cells was detected by flow cytometry. 【Result】 69 miRNAs with different expressions were screened by miRNA expression array in CSFV-infected PK-15 cells. Among which the expression changes of miR-214 were most obvious and up-regulated, and confirmed it by qRT-PCR. After transfected with miR-214 to PK-15 cells, CSFV titers and quantity of CSFV genomic copies decreased significantly, while transfected with miR-214 inhibitor, CSFV titers and quantity of CSFV genomic copies were increased significantly, which suggested that miR-214 promoted the replication of CSFV. In order to further explore the mechanism of miR-214 promoting CSFV replication, we confirmed TRADD is the target protein of miR-214. After transfected miR-214 to PK-15 cells, mRNA and protein expression of TRADD were increased significantly, while transfected with miR-214 inhibitor, that were decreased significantly, suggesting that miR-214.inhibits the expression of TRADD. And then, we verified that CSFV infection inhibits apoptosis of PK-15 cells, and miR-214 inhibits apoptosis of CSFV-infected PK-15 cells. 【Conclusion】 The expression of miR-214 in cells was up-regulated after CSFV infected PK-15. miR-214 inhibits the apoptosis of PK-15 cells and promote the replication of CSFV in cells by targeting inhibits the expression of TRADD protein.

    Eradication of Classical Swine Fever in China: Is It Far Away?
    Yuan SUN,HuaJi QIU
    Scientia Agricultura Sinica. 2018, 51(21):  4169-4176.  doi:10.3864/j.issn.0578-1752.2018.21.015
    Abstract ( 439 )   HTML ( 12 )   PDF (878KB) ( 250 )   Save
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    Classical swine fever (CSF), caused by classical swine fever virus (CSFV), is one of the most devastating diseases to the pig industry worldwide from both economic and sanitary points of view. CSF is notifiable to the World Organization for Animal Health (OIE) and classified as a List-I animal infectious disease in China. The National Middle- to Long-Term Plan for the Prevention and Control of Animal Infectious Diseases (2012-2020) has listed CSF as one of five priority animal diseases to be prevented. C-strain, a lapinized attenuated live vaccine developed by Chinese scientists in the 1950s, is highly safe and efficacious, which plays an important role in the control and eradication of CSF all over the world. Up to now, CSF has been eradicated in more than 30 countries and regions in the world. CSF has a high consequence on the pork industry in China. Therefore, we must be committed to eradicate CSF in China. In the middle of the last century, China had put forward a strategy to eradicate CSF. Now sixty years have passed, however, CSF is still endemic in most parts of China. Some factors seriously restrict the eradication of CSF in China, such as vast territory, complex pig-raising environment, diversified pig-raising modes, various animal diseases, and low-level biosecurity measures, etc. At present, China has entered a new era, the overall national strength has grown considerably, the breeding industry is developing towards a scientific, large-scale and environment-friendly direction, and the scientific research is being gradually improved. The current prevalence of CSF is pretty low and the vaccine manufacturing technologies are being improved. New CSF marker vaccines and companion differential diagnosis techniques have been developed. Some pig-raising enterprises in China are also aware of the importance and significance of the eradication of CSF. The government is also actively taking actions to formulate corresponding regulations and policies and to organize farmers, pig-raising enterprises and veterinary administrative departments to carry out local CSF eradication. All these make the eradication of CSF possible and practical. This review summarized the recent situation concerning CSF in China and the world. The importance of the eradication of CSF in China and the favorable and unfavorable conditions for the eradication of CSF were discussed. The cost and benefit of the eradication of CSF in China was also analyzed. This review also summarized the eradication experiences of CSF in Europe and USA and discussed the ideas and plans for the eradication of CSF in China. Considering its historical inevitability, strategic necessity, realistic possibility, and technical feasibility, we believe that the eventual eradication of CSF in China is never so far away from us.

    African Swine Fever: A Major Threat to the Chinese Swine Industry
    YuZi LUO,Yuan SUN,Tao WANG,HuaJi QIU
    Scientia Agricultura Sinica. 2018, 51(21):  4177-4187.  doi:10.3864/j.issn.0578-1752.2018.21.016
    Abstract ( 713 )   HTML ( 52 )   PDF (1134KB) ( 408 )   Save
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    African swine fever (ASF) is a devastating disease of domestic and wild pigs caused by African swine fever virus (ASFV), and causes signi?cant economic losses to the pig industry in affected countries. Acute disease is characterized by high fever, hemorrhages in the skin and internal organs, and a high mortality rate up to 100%. The continuous spread of the disease through Africa, Europe and Russian Federation keeps the neighboring countries on heightened alert. In August 2018, ASF emerged in China for the first time and subsequently rapidly spread across many regions of China, posing a major threat to the Chinese pig industry. Considering unavailability of ASF vaccines, rapid and early diagnostic assays are urgently needed for on-site field testing. Developing an effective ASF vaccine continues to be challenging due to incomplete understanding of the virus. Recent attempts on attenuated live vaccines and gene-deleted vaccines have been reported with promising efficacy, which have been demonstrated to provide effective homologous protection and partial heterologous protection. This review summarizes the epidemiology, diagnostics, vaccines and control strategies and challenges of ASF.

    RESOURCE INSECT
    Functional Characterization of BmCaspase-8-Like (BmCasp8L) as an Immune Negative Regulatory Molecule in Silkworm (Bombyx mori)
    Jie HU,XinYi WANG,Fei WANG
    Scientia Agricultura Sinica. 2018, 51(21):  4188-4196.  doi:10.3864/j.issn.0578-1752.2018.21.017
    Abstract ( 310 )   HTML ( 5 )   PDF (485KB) ( 110 )   Save
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    【Objective】The objective of this study is to characterize BmCaspase-8-like (BmCasp8L) as an immune negative regulatory molecule in silkworm (Bombyx mori) cell line as well as in B. mori larvae, and to provide a basis for further studies of negative regulation mechanism in insect immunity.【Method】Domain prediction and phylogenetic analysis were performed after cloning of BmCasp8L by RT-PCR. Then fluorescence quantitative PCR was used to investigate the spatial-temporal expression profile of BmCasp8L at different development stages and in different tissues extracted from the 3rd day of 5th-instar larvae and prepupa, as well as the larvae body after bacterial infection. The dsRNA for RNAi was synthesized to silence BmCasp8L in the B. mori larvae, and the effect of BmCasp8L on expression of the anti-microbial peptides was studied. The plasmid for expressing BmCasp8L in cells was constructed. After transfection of BmE cells with the expression constructs or dsRNA, Western blot or quantitative PCR was performed to confirm the over-expression or knock-down of BmCasp8L in cells. Meanwhile, the change in expression of the anti-microbial peptides and cleavage of nuclear transcription factor BmRelish were detected.【Result】BmCasp8L is homologous to Lepidoptera Caspase-6 and mammalian Caspase-8. The similarity between BmCasp8L and N-terminal of BmDredd, DmDredd is 61% and 42%, respectively, but it lacks the C-terminal Caspase domain. Spatial-temporal expression profile showed that in molting larvae BmCasp8L level was higher than in newly exuviated ones, and the BmCasp8L expression level was significantly increased in the prepupa, 7th day of pupa and 1st day of moth stage. The BmCasp8L expression level in the hemocyte was significantly higher than in other tissues in the 3rd day of 5th-instar, but in the prepupa stage, it was mainly expressed in the silk gland. The BmCasp8L expression level in the 3rd day of 5th-instar larvae increased within 1 h post infection of Bacillus bombyseptieus or Serratia marcescens, and then gradually returned to normal. Injection of dsCasp8L or dsEGFP into the 2nd day of 5th-instar larvae, the expression of BmCasp8L was significantly down-regulated in B. mori injected with dsCasp8L, while the expression of antimicrobial peptide BmCecropinA1 was up-regulated after 24 h. Over-expression of BmCasp8L in BmE cells led to a remarkable decrease of the anti-microbial peptide BmCecropinA1. In addition, over-expression of BmCasp8L suppressed the cleavage of the transcription factor BmRelish. Moreover, the expression level of BmCasp8L could be efficiently knocked down by dsRNA, at the same time, the expression level of BmCecropinA1 was significantly up-regulated. 【Conclusion】Phylogenetic analysis, expression features and functional studies in cells as well as in B. mori larvae all indicated that BmCasp8L acts as an immune negative regulatory molecule by suppressing the cleavage of BmRelish and the expression of anti-microbial peptides, thereby negatively regulating the Imd signaling pathway, and down-regulation of BmCasp8L resulted in an increase of anti-microbial peptides which would potentially increase the resistance of B. mori larvae to bacterial infection.

    Differentially Expressed MicroRNA and Their Regulation Networks During the Developmental Process of Apis mellifera ligustica Larval Gut
    Rui GUO,Yu DU,CuiLing XIONG,YanZhen ZHENG,ZhongMin FU,GuoJun XU,HaiPeng WANG,HuaZhi CHEN,SiHai GENG,DingDing ZHOU,CaiYun SHI,HongXia ZHAO,DaFu CHEN
    Scientia Agricultura Sinica. 2018, 51(21):  4197-4209.  doi:10.3864/j.issn.0578-1752.2018.21.018
    Abstract ( 384 )   HTML ( 7 )   PDF (10339KB) ( 170 )   Save
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    【Objective】MicroRNA (miRNA) is a kind of key regulator for negative regulation of mRNA at post-transcriptional level. The objective of this study is to provide miRNA expression patterns and differential expression information, illuminate the function of differentially expressed miRNA (DEmiRNA) in the development of larval gut by comprehensively investigating the DEmiRNAs and their regulation networks during the developmental process of Apis mellifera ligustica larval gut. 【Method】Deep sequencing of the 4-, 5- and 6-day-old larval guts of A. m. ligustica was conducted using small RNA-seq (sRNA-seq) technology, followed by mapping of the data after quality-control with the reference genome of Apis mellifera, and the mapped tags were then compared to miRBase database. The miRNA expression level was normalized by TPM algorithm, and the expression clustering, prediction of secondary structure of precursor and differential expression analysis were performed using related bioinformatic softwares. TargetFinder software was used to predict target gene of DEmiRNA, which was annotated to GO and KEGG databases using Blast, furthermore, miRNA-mRNA regulation networks were constructed using Cytoscape software. Stem-loop RT-qPCR was used to verify the sequencing data in this study.【Result】High-throughput sequencing of larval gut samples produced 10 841 644, 12 037 678 and 9 230 496 clean tags, respectively. In Am4 vs Am5 comparison group, there were16 up-regulated and 10 down-regulated miRNAs, while Am5 vs Am6 comparison group included 5 up-regulated and 7 down-regulated miRNAs, respectively. Among them, Novel-m0031-3p was shared by both Am4 vs Am5 and Am5 vs Am6, binding 5 target genes associated with ecdysone inducible protein, 25 and 11 DEmiRNAs were specific for the above-mentioned two comparison groups. DEmiRNA in Am4 vs Am5 could bind 5 742 target genes, among them 2 725 targets could be annotated to 46 GO terms in GO database, and the largest ones were binding, cellular process, metabolic process and single-organism process. Similarly, 12 DEmiRNAs in Am5 vs Am6 could link 3 733 target genes, among them 2 725 targets could be annotated to 41 GO terms, and mostly enriched terms were binding, cellular process, single-organism process and metabolic process. In addition, 1 046 and 676 target genes of two comparison groups were related to 116 and 92 KEGG pathways, and the number of DEmiRNA target genes in Am4 vs Am5 was more than that in Am5 vs Am6, which annotated to Wnt signaling pathway, Hippo signaling pathway, purine metabolism and endocytosis. Further analysis demonstrated that up-regulated and down-regulated miRNAs in Am4 vs Am5 could bind 611 and 85 target genes, and ame-miR-6052 linked the most target genes and participated in regulating cytochrome P450 via 5 target genes. miR-281-x could bind 49 target genes and indirectly regulate histidine metabolism, TGF-β signaling pathway and Hippo signaling pathway. In Am5 vs Am6 comparison group, up-regulated and down-regulated miRNAs could bind 43 and 431 target genes, respectively, among them miR-iab-4-x linked the most target genes, and it could participate in regulating growth and development related pathways, such as dorso-ventral axis formation, Hippo signaling pathway, Wnt signaling pathway, FoxO signaling pathway, Notch signaling pathway and mTOR signaling pathway. Regulation network analysis indicated that complex networks formed between DEmiRNAs and target genes, and DEmiRNAs lied in the center while target genes lied in the periphery. Finally, Stem-loop RT-qPCR was carried out to validate the randomly selected three DEmiRNAs, and the result confirmed the reliability of sequencing data. 【Conclusion】The DEmiRNA and corresponding target genes in the A. m. ligustica larval gut were predicted and analyzed at genome-wide level, it was found that A. m. ligustica are capable of regulating the expression of many miRNAs such as ame-miR-6052, miR-iab-4-x, miR-281-x and novel-m0031-3p. The results not only offer the expression pattern and differential expression information of miRNA during the developmental process of A. m. ligustica larval gut, but also lay a foundation for clarifying the molecular mechanisms underlying the larval gut’s development.

    RESEARCH NOTES
    Effect of Root Irrigation of Acetic Acid and Wine on Photoinhibition of Grape Under Seawater Stress
    Hui WANG,YuLu GAO,Meng YU,YuanPeng DU,YongJiang SUN,Heng ZHAI
    Scientia Agricultura Sinica. 2018, 51(21):  4210-4218.  doi:10.3864/j.issn.0578-1752.2018.21.019
    Abstract ( 295 )   HTML ( 10 )   PDF (413KB) ( 158 )   Save
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    【Objective】In order to expand the effective utilization of salinized soil and improve the quality of grape growth and development in salinized soil, the effects of acetic acid and wine on leaf photoinhibition under 15% seawater stress were studied. The result will provide a theoretical basis and technical reference for the improvement of grape cultivation techniques in salinized soil. 【Method】One-year old potted ‘Moldova’ seedlings were grown in plastic pots (with a diameter of 20 cm, and a height of 18 cm). The ratio of soil to substrate in each pot was 1﹕1 and the pots were placed in greenhouse. The culture conditions of the greenhouse were as follows: the average daily temperature was about 25℃, the light transmittance was more than 50% of natural light intensity, the maximum light intensity was 800 μmol·m -2·s -1, the relative humidity was 40%-60%, and the normal fertilizer and water was managed until 8-10 pieces of fully expanded leaves, then the seawater and exogenous acetic acid were used. The plants were irrigated with 15% seawater, 30 mmol·L -1 exogenous acetic acid and 2% wine at the same time, each treatment repeated 5 times, the controls were treated with clear water. Each treatment was irrigated every 2 days and 3 times, the irrigation amount was 2 times of water holding capacity, and about 1/3 of the solution leaked out, chlorophyll fluorescence indicators were determined on the 2nd day of irrigation, after 10 days treatment, the effects of the two methods on malondialdehyde (MDA) content, chlorophyll content, and root activity under seawater stress were analyzed.【Result】15% seawater treatment significantly decreased the chlorophyll content of leaf and root activity, but increased MDA content of root, stem and leaf. Compared with clear water control, the chlorophyll content decreased by 18.5% and root activity decreased by 41.9%. MDA content of root, stem and leaf increased by 1.10, 0.27 and 0.41 times, respectively. However, 30 mmol·L -1 acetic acid and 2% wine treatments significantly decreased the MDA content in grape. The MDA content in root, stem and leaf of grape treated with acetic acid decreased by 29.3%, 20.6% and 15.8%, respectively, compared with that of seawater stress, and the MDA content in root, stem and leaf of grape treated with wine decreased by 29.4%, 20.2% and 25.2%, respectively, compared with that of seawater stress. Irrigated with acetic acid and wine significantly increased the leaf chlorophyll content and root activity. Compared with seawater stress treatment, the root activity increased by 68.4% and 56.9%, and the chlorophyll content increased by 18.8% and 20.3%, respectively. The analysis of chlorophyll fluorescence showed that 15% seawater treatment decreased the levels of photosystem II (PSII) maximum photochemical efficiency (Fv/Fm) and the maximum photo-oxidizable P700 (Pm). The Pm on the 3rd, 6th, and 9th day decreased by 5.0%, 9.6% and 13.0% compared with the control, the levels of photochemical quenching coefficient (qP) and PSII actual photochemical efficiency (YII) decreased gradually, but the quantum yield of regulated energy dissipation Y (NPQ) and the quantum yield of non-regulated energy dissipation Y (NO) increased gradually. The nonphotochemical quantum yield due to PSI donor side limitation Y (ND) changed little, but the nonphotochemical quantum yield due to PSI acceptor side limitation Y (NA) increased gradually, resulting in the decrease of PSI actual photochemical efficiency Y (I). The irrigation of 30 mmol·L -1 acetic acid and 2% wine significantly relieved the stress of 15% seawater. The actual photochemical efficiency of PSI and PSII increased significantly, meanwhile the energy dissipated through heat decreased, then enhanced the efficiency of light energy utilization of grape leaves, in which 2% wine treatment showed a more obvious alleviated effect.【Conclusion】Irrigation of acetic acid and wine significantly improved root activity and chlorophyll content, which alleviated the photoinhibition level and improved the adaptability of grape to saline environment. The results provided a theoretical basis for the use of extract of wine lees to improvement of coastal saline soil.