Scientia Agricultura Sinica

Genotyping by Target Sequencing (GBTS) and Its Applications

XU Yunbi,YANG QuanNü,ZHENG HongJian,XU YanFen,SANG ZhiQin,GUO ZiFeng,PENG Hai,ZHANG Cong,LAN HaoFa,WANG YunBo,WU KunSheng,TAO JiaJun,ZHANG JiaNan

Scientia Agricultura Sinica. 2020, 53(15): 2983-3004. doi:10.3864/j.issn.0578-1752.2020.15.001

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Genotyping technology, via molecular markers, has been playing a key role in many biological fields including genetic improvement. Genotyping has been widely applied in multi-national seed companies due to their high-throughput, automatic, large-scale and shared genotyping platforms. Genotyping has moved from its third generation (G3), dominated by expensive DNA chips and random genotyping by sequencing (GBS), to the fourth generation (G4), characterized by low-cost, less facility-demanding and performed via genotyping by target sequencing (GBTS). In this article, we first introduced two GBTS protocols, GenoPlexs, based on multiplexing PCR, and GenoBaits, based on sequence capture in-solution (also called liquid chip). For both protocols, multiple single-nucleotide-polymorphisms (mSNP) or multiple dispersed nucleotide polymorphisms (MNP) can be generated to reveal the genetic variation hidden within each amplified locus (amplicon). Compared to DNA chips and GBS, GBTS has several advantages, including wide applicability to genotyping facilities, very flexible marker types, highly efficient genotyping, sharable and accumulative marker data, less required information management and support, and wide suitability in biological applications. With the same marker panel (for example, 40K maize mSNPs), three types of genotyping (40K mSNPs, 260K SNPs, and 754K haplotypes) can be achieved, and multiple panels with various marker densities (1K to 40K mSNPs) can be generated by sequencing at different depths. Applications of GenoPlexs and GenoBaits in biology were then reviewed, including biological evolution, germplasm evaluation, genetic map construction, gene mapping and cloning, marker-trait association (genome-wide association study and bulked sample analysis, BSA), progeny testing, gene introgression, gene pyramiding, variety right protection, variety quality monitoring, transgenic event and gene editing detection, and bioassay. More 50 marker panels have been developed so far for more than 20 plant, animal and microorganism species and applied in some of the fields described above. Lastly, we prospected for future GBTS by looking insights into carry-on, automatic, high-throughput and intelligent genotyping platforms, multi-functional marker panels with various marker densities designed to meet specific requirements, integration with other technologies such as KASP, high-density DNA chips and BSA strategies, and open-source breeding by sharing germplasm and breeding materials and information. The development in these fields will greatly facilitate the applications of GBTS in genetic improvement and other fields of animals, plants and microorganisms.

Screening and Analysis of Key Metabolic Pathways in Foxtail Millet During Different Water Uptake Phases of Germination

YU AiLi,ZHAO JinFeng,CHENG Kai,WANG ZhenHua,ZHANG Peng,LIU Xin,TIAN Gang,ZHAO TaiCun,WANG YuWen

Scientia Agricultura Sinica. 2020, 53(15): 3005-3019. doi:10.3864/j.issn.0578-1752.2020.15.002

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【Objective】 Foxtail millet (Setaria italica L.) is a hardy cereal and known for its tolerance against drought and barrenness, which originated in China. In this study, the transcriptome of foxtail millet was analyzed in germinating seeds during different uptake of water phases, which is to get a lot of differently expressed genes, and find the important metabolic pathways, key genes and metabolites of regulating germination in foxtail millet. 【Method】The cDNA libraries of Jingu 20 were constructed in germinating seeds during rapid initial uptake of water, plateau phase, further increase in water uptake for the transcriptome analysis. The cluster analysis of gene expression was performed by K-Means. Differential expression analysis used DESeq. The functional annotations of differentially expressed genes were obtained by using COG, GO, KEGG, and so on. The key metabolism pathways and genes that regulated germination in foxtail millet were found during different water uptake phase through KEGG enrichment of DEGs. The reliability of sequencing results was confirmed by qRT-PCR. The contents of metabolites were assayed by HPLC.【Result】The genome-wide gene expression profile was obtained by RNA-sequencing during rapid initial uptake of water, plateau phase and further increase in water uptake. A total of 33 643 genes were expressed. Nine co-expression clusters with distinctive patterns were identified. There were 3 893, 4 612 and 8 472 DEGs in rapid initial uptake of water and plateau phase, plateau phase and further increase in water uptake, rapid initial uptake of water and further increase in water uptake, respectively. The KEGG pathway enrichment analysis showed that these DEGs of the three comparisons were significantly associated with phenylpropanoid biosynthesis,phenylalanine metabolism, starch and sucrose metabolism. The DEGs between rapid initial uptake of water and plateau phase, between rapid initial uptake of water and further increase in water uptake were also enriched in plant hormone signal transduction. And the number of genes enriched in the metabolism pathway of phenylpropanoid biosynthesis and phenylalanine metabolism was the largest in the three comparisons. The peroxidase genes had the highest proportion among these genes of the two pathways. The results obtained from four phenylpropanoids-related genes tested by qRT-PCR were consistent with the trend of regulation identified by gene expression profile. In dormant seeds of foxtail millet, there was the preformed mRNAs of 4-coumarate-CoA ligase 3, which displayed firstly decreasing, then increasing and finally declining patterns during the uptake of water stage. The contents of phenylpropanoids-related metabolites in germination seeds of foxtail millet indicated that large amounts of sinapic acid were stored in dormant seeds. During uptake of water by a dry seed, the content of sinapic acid gradually decreased, but those of p-coumaric acid, caffeic acid and ferulic acid increased first and then decreased. 【Conclusion】DEGs of foxtail millet were significantly related with phenylpropanoid biosynthesis and phenylalanine metabolism in germinating seeds during different uptake of water stages. The upstream and downstream genes of phenylpropanoid biosynthesis, such as 4-coumarate-CoA ligase 3 and peroxidase, played regulation roles in response to water during germination of foxtail millet. Sinapic acid participated in the dormancy and germination of millet seeds, which were the medium product of phenylpropanoid biosynthesis pathway.

Integrated Agronomic Management Close the Gap of Yield and Resource Use Efficiency for Maize Production

LI CongFeng,WANG ZhiGang,WANG YongJun,QI Hua,GU WanRong,ZHANG RenHe,ZHOU WenBin,ZHAO Ming

Scientia Agricultura Sinica. 2020, 53(15): 3020-3023. doi:10.3864/j.issn.0578-1752.2020.15.003

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Quantitative Evaluation of the Contribution of Main Management Factors to Grain Yield of Spring Maize in North China

YANG Zhe,YU ShengNan,GAO JuLin,TIAN Tian,SUN JiYing,WEI ShuLi,HU ShuPing,LI RongFa,LI CongFeng,WANG ZhiGang

Scientia Agricultura Sinica. 2020, 53(15): 3024-3035. doi:10.3864/j.issn.0578-1752.2020.15.004

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【Objective】Quantitative analysis of the contribution of main management factors to grain yield is of great importance for narrowing yield gap of maize (Zea mays L.). 【Method】 To clarify the individual contribution rate of main management factors to maize yield, the present study analyzed data from 114 literatures published after 2000, which focused on crowding tolerance of hybrids, plant density, soil tillage method, nutrient management and leaf disease control in spring maize production of North China. Meanwhile, a 2-year field study with an incomplete factorial design with foregoing 5 factors was conducted in tow fixed locations, to verify the result of literature review and furtherly assess the priority of management optimization for reducing yield gap. 【Result】 The results of literature review was consistent with that of management-factor alternative test in field. The priority of 5 management factors was plant density, nutrient management, crowding tolerance of hybrids, and leaf disease control and soil tillage method, which contributed to yield by 12.6%, 9.2%, 6.7%, 6.3% and 5.5%, respectively. Similarly, the contribution rates to PFP_N of them were 16.7%, 4.1%, 3.4%, 3.8% and 3.3%, respectively. Yield gap induced by each management factor was mainly attributed to mass productivity and sink capacity, which were initially increased along with mean leaf area index (MLAI). When MLAI exceeded optimum value, enhancing radiation efficiency and grain producing efficiency by optimizing assimilative capacity was of great importance for closing yield gap.【Conclusion】Concurrent enhancing yield and resource use efficiency by 15% to 20% could be reached easily through optimizing plant density and nutrient management. However, synchronously enhancing yield and resource use efficiency by more than 30% to 50%, four or all five management factors should be optimized systematically.

Effects of Different Agronomy Factors on Yield Gap and Nitrogen Efficiency Gap of Spring Maize Under Rain-Fed Conditions

CAO YuJun,YAO FanYun,WANG Dan,LÜ YanJie,LIU XiaoDan,WANG LiChun,WANG YongJun,LI CongFeng

Scientia Agricultura Sinica. 2020, 53(15): 3036-3047. doi:10.3864/j.issn.0578-1752.2020.15.005

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【Objective】In order to provide a theoretical basis for the further improvement of the yield and nutrient efficiency of different maize production levels, the effects of the increasing and decreasing measures on the yield and nutrient efficiency of maize under different technical modes were explored, and the technical priorities were clarified. 【Method】 By investigating the yield level and technical mode of farmers, high-yield and high-efficiency, as well as super high yield, it was clear that planting density, cultivation measures, nitrogen management and varieties were the main measures to limit the yield and efficiency improvement of maize at different production levels. On the basis, three technical models of super high yield (SH), high-yield and high-efficiency (HH) and farmer household (FP) were set up. According to the measure factors under different technical modes, the split area experiment was carried out, in which the tillage method was the main plot, the variety was sub-plot, the nitrogen fertilizer management was sub-sub-plot, and the density was sub-sub-sub-plot.【Result】Under the FP model, the priority order of technical measures to yield contribution was nitrogen management, planting density, soil tillage, and variety, while the contribution rate to yield was 9.9%, 6.0%, 4.4% and 2.5%, respectively. Under the HH model, the priority order of cultivation measures to yield contribution was planting density, nitrogen management, soil tillage, and variety, with the contribution rate of 7.7%, 5.2%, 4.5% and 3.5%, respectively. Under SH mode, the priority order of cultivation measures to yield contribution was planting density, soil tillage, nitrogen management, and variety, with the contribution rate of 8.9%, 7.3%, 6.5% and 4.3%, respectively. Among the three models, the contribution rate of cultivation technical factors to nitrogen efficiency from high to low was nitrogen management, planting density, soil cultivation and variety. Among them, the contribution rate of nitrogen management, planting density, soil tillage and variety to nitrogen efficiency was 30.5%, 6.0%, 4.4% and 2.5% in FP mode, 19.7%, 7.7%, 4.7% and 4.5% in HH mode, 25.4%, 8.3%, 6.5% and 4.5% in SH mode, respectively.【Conclusion】There was no fixed priority order for the contribution of technical factors to the yield. The formation of yield gap under different management levels was affected by multiple factors, and the technical factors had synergistic effect. Under the management of farmer's level, the contribution rate of nitrogen management to the yield ranked first, while the contribution of planting density and soil tillage to the yield was greater under the higher management level. However, the nutrient efficiency gap was mainly caused by nitrogen management, and the contribution rate of nitrogen management to nutrient efficiency ranked the first at different yield levels.

Regulation Effects of Improved Cultivation Measures on Canopy Structure and Yield Formation of Dense Spring Maize Population

PIAO Lin,LI Bo,CHEN XiChang,DING ZaiSong,ZHANG Yu,ZHAO Ming,LI CongFeng

Scientia Agricultura Sinica. 2020, 53(15): 3048-3058. doi:10.3864/j.issn.0578-1752.2020.15.006

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【Objective】The objective of this study was to clarify the regulating effect of improved cultivation modes on canopy structure and population production of spring maize, and the contribution for grain yield increasing in the future.【Method】Maize cultivar “Zhongdan 909” was used as experimental materials, under 105 000 plants/hm² as planting density, setting four cultivation modes via the combination among different cultivation measures, which were subsoiling tillage (S), wide-narrow rows (W), and chemical regulator (C): (1) Traditional mode (rotary tillage 20 cm, plus 60 cm uniform plant spacing; RU), (2) Tillage improved mode (subsoiling tillage 35 cm, plus 60 cm uniform plant spacing; SU), (3) Canopy improved mode (traditional rotary tillage 20 cm, plus 80+40 cm plant spacing, and foliar spraying ECK; RWC), and (4) Synthetically improved mode (subsoiling tillage 35 cm, plus 80+40 cm plant spacing, and foliar spraying ECK; SWC). Investigated based on the differences of canopy continual sections (vertical), light distribution, photosynthesis, activity of the key sucrose synthetase enzyme, the dry matter accumulation, and grain fulling. 【Result】Compared with the traditional mode (RU; CK), the green leaf weight of tillage improved mode (SU) increased obviously, and the height and ear height of population reduced more than 30 cm under the canopy improved conditions (RWC and SWC), but the regularity degree of maize population were declined markedly; The leaves vertical distribution more uniformly, looked like “spindle type”, were the vertical height of 180-240 cm light interception and was significantly less than traditional mode (8%-37%), while the ear position (120-180 cm) was significantly higher than traditional mode (44%-129%); However, the leaves distribution of RU and SU treatments were more like “funnel type”, leaves were concentrated in the upper layer. Tillage and canopy improving synergistically could improve the canopy vertical structure significantly, promoted the chlorophyll content, net photosynthetic rate of leaves at ear position and under ear position, as well as enzyme activity own in the sucrose metabolic of ear position leaf. Maintaining the physiological activity of canopy leaves in the late growth stage, which would prolong the active accumulation period of dry matter more than 10 days. 【Conclusion】Synthetical improved mode (SWC) significantly increased the light transmission and C metabolic activity of leaves via changing the biomass spatial distribution, which promoted the canopy productivity of the functional leaves meanwhile the grain filling, then the grain yield were enhanced dramatically.

Responses of Canopy Radiation and Nitrogen Distribution, Leaf Senescence and Radiation Use Efficiency on Increased Planting Density of Different Variety Types of Maize

BAI YanWen,ZHANG HongJun,ZHU YaLi,ZHENG XueHui,YANG Mei,LI CongFeng,ZHANG RenHe

Scientia Agricultura Sinica. 2020, 53(15): 3059-3070. doi:10.3864/j.issn.0578-1752.2020.15.007

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【Objective】The objective of this experiment was to study the effects of planting density on canopy senescence, radiation use efficiency and yield of different plant types maize, so as to provide technical support for cultivation of high yield and high efficiency of spring maize in northern Shaanxi. 【Method】 In 2017 and 2018, the field experiments were conducted by using two maize hybrids, including Shaandan 609 (compact) and Shaandan 8806 (flat), with four planting density treatments, including 45 000, 60 000, 75 000 and 90 000 plants/hm². Canopy light and nitrogen distribution, leaf senescence, nitrogen accumulation, dry matter production, radiation use efficiency and yield components were determined. 【Result】 Shaandan609 and Shaandan8806 reached the highest grain yield at 90 000 plants/hm² (13 824 kg·hm^-2) and 60 000 plants/hm² (9 566 kg·hm^-2), respectively. Compared with 45 000 plants/hm², the average kernels per ear (17.8% and 30.1%) and 100-kernels weight (15.2% and 19.6%) of Shaandan609 and Shaandan 8806 both reduced under 90 000 plants/hm². The canopy light interception and leaf nitrogen concentration of the two varieties showed: the upper layer>the middle layer>the lower layer. With the increase of planting density, the fraction of the photosynthetically active radiation interception (FIPAR) and leaf nitrogen concentration increased at upper layer, but decreased at the mid and lower layer. When the density increased to 90 000 plants/hm², the FIPAR at middle and upper canopy and the lower canopy of Shaandan 8806 were 8.8% and 70.6% lower than Shaandan 609, respectively, and the leaf nitrogen concentration at middle and upper canopy layer and lower canopy of Shaandan 609 were 16.0% and 40.5% higher than Shaandan 8806. When density increased from 45 000 plants/hm² to 90 000 plants/hm², the relative green leaf area at maturity (RGLA_m) decreased by 36.4% and 63.3%, respectively, while the mean rate of decrease in RGLA (V_m) increased by 40.2% and 34.6%, respectively. The sequence of date of onset of leaf senescence (T_s) was as follows: the lower layer>the upper layer>the middle layer. Compared with Shaandan 8806, the green leaf area was higher in the middle and upper layer of Shaandan 609, and maintained a higher green leaf area at the lower layer during the later growth stage under 90 000 plants/hm². With the increasing of planting density, the nitrogen uptake during the pre-silking (Npre) and post-silking (Npost), and nitrogen harvest index (NHI) increased significantly. When the density increased to 90 000 plants/hm², Npre, Npost and NHI of Shaandan 609 were 23.5%, 43.9% and 12.7% higher than Shaandan 8806, respectively. The biomass yield (BM), intercept photosynthetically active radiation (IPAR) and radiation use efficiency (RUE) increased significantly when improved plant density. When the density increased to 90 000 plants/hm², the BM, IPAR, RUE and HI of Shaandan 609 were 26.1%, 10.2%, 9.1% and 14.8% higher than Shaandan 8806.【Conclusion】Compared with Shaandan 8806, the compact maize Shaandan 609 could improve the spatial distribution of light and nitrogen at the middle and upper layer under high density, increased the light intercept at the middle and lower canopy, delayed leaf senescence at the mid and lower canopy, and promoted the dry matter accumulation after silking, so a higher grain yield and RUE were obtained.

Effects of Tillage and Straw Incorporation Method on Root Trait at Silking Stage and Grain Yield of Spring Maize in Northeast China

JIANG Ying,WANG ZhengYu,LIAN HongLi,WANG MeiJia,SU YeHan,TIAN Ping,SUI PengXiang,MA ZiQi,WANG YingYan,MENG GuangXin,SUN Yue,LI CongFeng,QI Hua

Scientia Agricultura Sinica. 2020, 53(15): 3071-3082. doi:10.3864/j.issn.0578-1752.2020.15.008

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【Objective】In view of the prominent contradiction of straw utility, the optimization of straw returning method is significant for promoting the green and sustainable development of agriculture in northeast China, where is the main production areas of spring maize. In this study, the effects of tillage and straw incorporation approaches on the morphology and distribution characteristics of root, dry matter accumulation and yield of spring maize were investigated to provide a theory basis for optimizing tillage and straw incorporation measures to maintain agricultural production.【Method】The field experiment was carried out in Shenyang, Liaoning province in 2017 and 2018. The two-factor random zone group design was adopted to set up four treatments, including straw incorporation with full-thickness plough tillage (PTS), straw incorporation with strip plough tillage (PSS), straw incorporation with full-thickness rotary tillage (RTS), and straw incorporation with strip rotary tillage (RSS). Under different tillage and straw incorporation methods, the differences of root length, root dry weight, their spatial distribution in soil, dry matter accumulation dynamics and yield characters of spring maize were analyzed.【Result】Tillage and straw incorporation methods had significant effects on root length and distribution as well as dry weight and specific root length of spring maize at silking stage. In the vertical soil layer of 0-30 cm, the root length of PTS treatment was 7.9%-43.2% and 17.3%-41.5% higher than other treatments, respectively. In the vertical soil layer of 30-60 cm, root length under strip straw incorporation (PSS and RSS) treatments was average 20.1% and 20.3% higher than those under full-thickness straw incorporation (PTS and RTS) treatments, respectively. Centering on maize plant, the horizontal distribution of root length in soil showed that PTS treatment was the highest and RTS treatment was the lowest in 0-10 cm away from the plant. The lowest root dry weight was observed from RTS treatment, PTS, PSS and RSS treatments presented 36.5%, 59.6% and 17.3% higher root dry weight in the 0-10 cm soil layer, respectively. PTS treatment obtained the highest specific root length in 0-20 cm soil layers, with 8.7%-73.8% and 14.3%-44.7% more than those under other treatments. The spatial distribution of root surface area was significantly different among treatments. PTS and RSS treatments had higher root surface area in 0-30 cm soil layer and better root surface distribution in horizontal and vertical directions. The effects of tillage and straw incorporation methods on the accumulation of dry matter in shoot of spring maize at jointing, silking and maturity stages were significant. Compared with other three treatments, RTS reduced the average dry matter weight of stem+sheath and total shoot weight by 15.5% to 19.2% at jointing stage. The weight of ear and shoot dry matter in maturity stage under PTS treatment was 3.6%-12.3% and 2.7%-12.4% higher than those under other treatments, followed by PSS and RSS treatments, and RTS treatment was the lowest. Tillage and straw incorporation methods significantly affected the number of ears and grain yield of spring maize. PTS, PSS, and RSS treatments obtained average 8.3%, 7.9%, and 5.8% higher grain yield than that under RTS treatments in 2017 and 2018. RTS significantly reduced the number of ears by 2.9%-9.1% and 7.0%-9.7%, compared with other three treatments.【Conclusion】Proper tillage and straw returning methods were conducive to promoting the morphological development of crop root and its spatial distribution in the tilled soil layer, optimizing of dry matter accumulation and distribution characteristics, and the distribution of dry matter to ear at maturity, so as to increase the yield of spring maize. In summary, the straw strip returning with plough tillage was recommended in the study area.

Effects of Chemical Regulation and Nitrogen Fertilizer on Radiation, Heat and Water Utilization Efficiency and Yield of Spring Maize Under Dense Planting Condition

LIU XiaoMing,GU WanRong,LI CongFeng,ZHANG LiGuo,WANG MingQuan,GONG ShiChen,CHEN XiChang,LI CaiFeng,WEI Shi,LI WenHua

Scientia Agricultura Sinica. 2020, 53(15): 3083-3094. doi:10.3864/j.issn.0578-1752.2020.15.009

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【Objective】 This experiment was conducted to study the effects of chemical regulation and nitrogen fertilizer on photosynthesis, grain filling characteristics and radiation, heat and water utilization efficiency of maize under high planting density, so as to provide a theoretical basis for stress resistance, high yield and high efficiency of maize under high planting density. 【Method】 The experiment was performed with three nitrogen rates, N100 (100 kg·hm^-2), N200 (200 kg·hm^-2) and N300 (300 kg·hm^-2) under high planting density (90 000 plants/hm²) during the maize growth season from 2017 to 2018. Chemical control (Yuhuangjin, 30% amine fresh ester·ethyl hydroxide) was sprayed at the seventh leaf stage. The effects of chemical control and nitrogen application on maize growth and development and radiation (RUE), heat utilization efficiency (HUE) and water utilization efficiency (WUE) under high density were studied. 【Result】 With the nitrogen application increasing, the 8 indexes, including net photosynthetic rate (P_n), max photochemical efficiency (F_v/F_m) of maize leaves, the content of grain endogenous hormones, grain filling rate, radiation utilization efficiency, heat utilization efficiency, water utilization efficiency and yield, first increased and then decreased, and all reached the maximum at 200 kg·hm^-2 nitrogen rate. Compared with the single application of nitrogen fertilizer, the combination of chemical control and nitrogen fertilizer significantly increased P_n and F_v/F_m in leaves, endogenous hormone content in grains, grain filling rate, RUE, HUE, and WUE, which further increased grain yield. The correlation analysis showed that the grain filling rate was positively correlated with indole-3-acetic acid (IAA), cytokinin (CTK), gibberellin (GA) and abscisic acid (ABA), while the yield was positively correlated with RUE, HUE and WUE.【Conclusion】 Under high planting density, 200 kg·hm^-2 nitrogen application and chemical control significantly improved the photosynthetic characteristics, promoted the grain filling process, improved radiation, heat and water utilization efficiency, and significantly increased the yield.

Allelopathy of Six Organic Acids on Wheat Sheath Blight in the Soil of Winter Wheat-Summer Maize Double Cropping Straw Returning System

ZHAO XuSheng,QI YongZhi,YAN CuiMei,ZHEN WenChao

Scientia Agricultura Sinica. 2020, 53(15): 3095-3107. doi:10.3864/j.issn.0578-1752.2020.15.010

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【Objective】Double cropping of winter wheat and summer maize is the most extensive farming system in northern China. Straw returning has been widely used in wheat and maize production. But, the effect of straw returning on the occurrence of soil borne diseases of wheat has been controversial. The objective of this study is to analyze the main chemical constituents in the cultivated-layer soil with different straw returning years and the allelopathic effects of the main organic acids on the growth of wheat seedlings, the mycelium growth and sclerotia formation of Rhizoctonia cerealis and occurrence of wheat sheath blight (WSB), and to understand the relationship between straw returning and WSB incidence.【Method】Gas chromatography-mass spectrometry (GC-MS) technique was used to analyze the types and contents of chemical substances in the extract of ethyl acetate from the cultivated soil where maize straw returning, and the effects of 6 organic acids with higher content on the root activity, root cell membrane permeability, superoxide dismutase (SOD) and peroxidase (POD) activity of wheat seedlings were determined with the TTC reduction, electrical conductivity, NBT photoreduction and guaiacol colorimetry methods, respectively. Meanwhile, the effects of these organic acids on the mycelium growth, sclerotium formation and occurrence of WSB were determined by the conventional methods.【Result】The relative contents of organic acids, alkanes, alcohols, amides, and aldehydes in the cultivated-layer soil and straw returning were 45.45%, 17.70%, 17.08%, 6.12%, and 5.44%, respectively. Organic acids mainly included o-hydroxybenzoic acid (9.24%), 3-phenyl-2-acrylic acid (4.12%), p-hydroxybenzoic acid (3.21%), 4-hydroxy-3,5- dimethoxybenzoic acid (2.26%), heneicosanoic acid (1.88%), 4-methoxy-anthranilic acid (1.73%), 8-octadecanoic acid (0.76%) and 3-(4-hydroxy-3- methoxyphenyl)-2-acrylic acid (0.52%). 4-methoxy-anthranilic acid and 3-(4-hydroxy-3-methoxyphenyl)-2-acrylic acid at 0.08-10.0 μg·mL^-1had significant promoting effects on mycelial growth (expect 4-methoxy-anthranilic acid at 10.0 μg·mL^-1), dry weight and sclerotium formation of R. cerealis, and the relative content of the two substances increased with the extension of straw returning years. O-hydroxybenzoic acid significantly promoted mycelial growth and sclerotia formation of R. cerealis at 0.4 and 0.08 μg·mL^-1. In contrast, 50.0 μg·mL^-1 of o-hydroxybenzoic acid and 4-hydroxyl-3-methoxy-benzoic acid, and 0.4-50.0 μg·mL^-1 of benzoic acid all showed inhibitory effects. All 6 organic acids at concentrations of 2.0-5.0 μg·mL^-1 inhibited the growth of wheat seedlings in a dose-dependent manner. The inhibitory effect of p-hydroxybenzoic acid was the strongest, followed by o-hydroxybenzoic acid, and 4-methoxy-anthranilic acid was the weakest. 3-(4-hydroxy-3-methoxyphenyl)-2-acrylic acid, 4-methoxyanthranilic acid, o-hydroxybenzoic acid, and p-hydroxybenzoic acid at concentrations of 0.4-50.0 μg·mL^-1 aggravated the occurrence of WSB. 3-(4-hydroxy-3-methoxyphenyl)-2-acrylic acid had the strongest enhancing effect, with the incidence and disease index increased by 49.0% and 46.7%, respectively. Benzoic acid and 4-hydroxy-3-methoxyphenyl-benzoic acid had no significant effect on the occurrence of WSB.【Conclusion】Organic acids, esters, hydrocarbons, amides, and aldehydes were found in the cultivated-layer soil in the winter wheat-summer maize double cropping straw returning system. Organic acids were the most abundant of those chemicals. 3-(4-hydroxy-3-methoxyphenyl)-2-acrylic acid, 4-methoxyanthranilic acid, o-hydroxybenzoic acid, and p-hydroxybenzoic acid at a certain concentration can promote the occurrence of WSB. 3-(4-hydroxy-3-methoxyphenyl)-2-acrylic acid has the strongest stimulatory effect, while benzoic acid and 4-hydroxy-3-methoxy-benzoic acid have no effect on the occurrence of WSB. Allelopathic effects of organic acids in the cultivated-layer soil, including the promotion on pathogen growth and inhibition on physiological activity and growth of wheat roots, may be one of the main reasons for the serious occurrence of WSB in the straw returning region in northern China.

Characteristics and Immune Response of Prophenoloxidase Genes in Sogatella furcifera

ZHANG DaoWei,KANG Kui,YU YaYa,KUANG FuPing,PAN BiYing,CHEN Jing,TANG Bin

Scientia Agricultura Sinica. 2020, 53(15): 3108-3119. doi:10.3864/j.issn.0578-1752.2020.15.011

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【Objective】Phenoloxidase (PO) is an immune protein and plays an important regulatory role in insects. It exists as the form of inactive prophenoloxidase (PPO). In this study, based on the three PPO sequences of Sogatella furcifera by transcriptome sequencing, the biological functions of SfPPOs were further explored by analyzing the developmental and tissue expression patterns of different SfPPOs, suppressing the expression of SfPPOs and inducing by pathogenic bacteria.【Method】Taking three SfPPO sequences as research objects, the protein structure and homology with other insects were analyzed by bioinformatics method. In addition, dsRNA injected with green fluorescent protein (GFP) was used as a control group, and the inhibitory effect of target gene was detected by quantitative real-time PCR (qRT-PCR) after injecting with dsSfPPO. In order to investigate the role of SfPPO in the growth and immune response of S. furcifera, qRT-PCR was used to detect the expression of SfPPO at different developmental stages and in different tissues, as well as the induced expression of three SfPPOs after injecting with Escherichia coli and Bacillus subtilis.【Result】The open reading frames (ORF) of SfPPO1, SfPPO2-1, and SfPPO2-2 are 2 079, 999, and 2 070 bp in length, respectively, which encoding 692, 332, and 689 amino acids, respectively. The predicted protein molecular weights are 79.84, 37.67, and 79.53 kD, and the isoelectric points are 6.43, 9.56, and 6.20, respectively. Bioinformatics analysis showed that the three PPO proteins of S. furcifera had high homology and were closely related to Nilaparvata lugens. SfPPO1, SfPPO2-1 and SfPPO2-2 were all highly expressed on the 3rd day of adult, and the developmental expression patterns of SfPPO2-1 and SfPPO2-2 were similar. The tissue expression results showed that SfPPO1 and SfPPO2-1 were highly expressed in the epidermis and fat body, while SfPPO2-2 was highly expressed in the wing and fat body, and the expressions of all the three genes were relatively low in head, foot, midgut and Malpighian tubule. Compared with the dsGFP group, injecting with the dsRNA of target gene could significantly silence the expression of target gene, and when the SfPPO2-1 expression was silenced, SfPPO1 appeared to be significantly overexpressed, indicating that there might be compensatory function among different PPOs. The expression levels of SfPPO2-1 and SfPPO2-2 were significantly increased after 12 h of induction with E. coli, while the expression of SfPPO1 was significantly increased after 24 h of induction. In addition, the expression levels of SfPPO1, SfPPO2-1 and SfPPO2-2 were all significantly increased after 24 h of induction with B. subtilis.【Conclusion】The expression of SfPPO1, SfPPO2-1 and SfPPO2-2 has tissue and development specificity, and there are differences in immune response under the induction of different pathogenic bacteria. The results are helpful to explore the potential molecular mechanism of phenoloxidase in insect development and immunity.

Spatial Regional Variability and Influential Factors of Soil Fertilities in the Major Regions of Maize Production of China

ZHAO QingYue,XU ShiJie,ZHANG WuShuai,ZHANG Zhe,YAO Zhi,CHEN XinPing,ZOU ChunQin

Scientia Agricultura Sinica. 2020, 53(15): 3120-3133. doi:10.3864/j.issn.0578-1752.2020.15.012

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【Objective】 The objective of this study was to analyze the current status, regional spatial variation and its influencing factors of soil fertility in the major regions of maize production in China, so as to provide the guidance for soil nutrient management and fertilization strategy. 【Method】 Large-scale soil sample collection and farmer surveys were carried out throughout the major regions of maize production during the harvest season of maize in 2017. By using geostatistics and geographical information systems (GIS) methods, the regional variability and distribution patterns of soil fertilities were determined and mapped. According to relevant grading standards, the current status of soil fertilities in major regions of maize production was evaluated. Correlation analysis and variance analysis were used to assess the effects of major factors (soil texture, climate and fertilization) on soil fertility status.【Result】 The median values of soil pH, organic matter, total nitrogen (N), Olsen phosphorus (Olsen-P), and NH₄OAc extractable potassium (available K) in the major regions of maize production were 6.9, 21.0 g·kg^-1, 1.5 g·kg^-1, 22.4 mg·kg^-1, and 164.5 mg·kg^-1, respectively, and their coefficient of variation were 12.7%, 48.5%, 50.0%, 83.6% and 52.0%, respectively, which all belonged to moderate variation. The concentrations of soil organic matter, total N, Olsen-P, and available K were mainly varied at middle to extremely high level, which occupied 93.5% area of the whole production regions. Significant regional variation was observed for soil fertilities. The concentrations of soil organic matter, total N and Olsen-P were the highest in the Northeast region (with median of 32.0 g·kg^-1, 2.2 g·kg^-1, 32.3 mg·kg^-1, respectively) and the lowest in the Northwest region (with median of 17.2 g·kg^-1, 1.2 g·kg^-1, 16.2 mg·kg^-1, respectively). The concentration of soil available K was the lowest in the Southwest region but showed no significant difference in the other regions. On the national scale, soil pH had strong spatial dependence (nugget to sill ratio <25%) and its spatial variation was likely caused by natural factors (soil texture and precipitation). Soil Olsen-P had weak spatial dependence (nugget to sill ratio >75%) and its spatial variation was probably affected by anthropic activities (fertilization). Soil organic matter, total N and available K had moderate spatial dependence (nugget to sill ratio 25%-75%), which likely was due to the combined effect of natural factors and anthropic activities. 【Conclusion】 Due to high soil fertility, the fertilization rate should be reduced to save fertilizer cost in the maize production of Northeast region. For North China Plain with moderate soil fertility, the amount of nitrogen and phosphorus fertilizer should be strictly controlled to improve fertilizer use efficiency and to reduce environmental pollution. For Northwest region with inadequate soil fertility, the fertilizer input should be appropriately increased to further improve maize yield. The soil fertility in the Southwest region varies greatly. Appropriate fertilization management should be adopted in each subregion to improve soil nutrient preserving capability and maize yield.

Research Progress of Microbial Deodorization in Livestock and Poultry Wastes Composting

WEI QiHang,REN YanFang,HE JunYu,LI ZhaoJun

Scientia Agricultura Sinica. 2020, 53(15): 3134-3145. doi:10.3864/j.issn.0578-1752.2020.15.013

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With the intensive and industrialized development of livestock and poultry breeding industry in the world, a large number of breeding wastes are generated, which seriously affect the ecological environment and threaten the health of human and livestock. Now, the resource utilization of livestock and poultry breeding waste has been widely concerned. Aerobic composting technology is the most favorable treatment method at present. However, the odor produced by composting makes the popularization of aerobic composting technology face huge problems. In the process of composting, the odor gases not only threaten human health, but also bring a series of environmental problems. Therefore, it is very important to remove odor gases adequately and effectively in composting process. The paper summarized the emission characteristics of odor gases and the transformation characteristics of source materials, and analyzed the influencing factors of odor gases during composting in detail. Moreover, the biological treatment technology and the mechanism of microbial control of odor gases were discussed from two aspects: in-situ removal technology and ectopic removal technology. The following conclusions were drawn: odor gases (NH₃, H₂S and volatile organic compounds) were mainly generated in the heating period and high temperature period of the composting process; the optimum temperature was 55-60℃, with the moisture of 50%-60%, pH of 7.5 to 8.5, C/N for 25-30, oxygen concentration of 10%-18%, and organic matter content of 50%-80%; at the same time, it could choose the right means of compost, optimal frequency of turning heaps and adding exogenous microorganisms to make odor gases minimize; a deodorizing microbial strain usually only had high removal efficiency of one odor gas component, and it was difficult to remove a variety of odor gases at the same time; compound deodorizing microbial agents could remove a variety of odor gas components at the same time, but the removal efficiency was relatively low. It was suggested that the transformation of organic matter, the change of microbial community and the rule of odor gases production should be further studied, so as to reduce the production of odor gases as much as possible during the heating period and high temperature period of compost. Research should focus on development of composite deodorizing microbial agents, and explore the effect and mechanism of microbial deodorization.

Screening of Interacting Protein of Tomato SIVQ6 by GST Pull-Down

YUAN GuiBo,MO ShuangRong,QIAN Ying,ZANG DongNan,YANG Fan,JIANG HongLiang,WU Yuan,DING HaiDong

Scientia Agricultura Sinica. 2020, 53(15): 3146-3157. doi:10.3864/j.issn.0578-1752.2020.15.014

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【Objective】The VQ motif-containing (VQ) proteins are plant-specific proteins with a conserved “FxxhVQxhTG” amino acid sequence, which regulate plant growth and development. SlMPK1 plays an important role in the process of high temperature stress, but its downstream target proteins are poorly understood. Although yeast double hybridization (Y2H) showed that SlVQ6 protein could interact with SlMPK1, there was no further experimental evidence. Therefore, it is especially important to verify the interaction between SlMPK1 and SlVQ6 and to study the interaction network of SIVQ6. 【Method】First, pGEX-4T-1-PC-VQ6 plasmid was constructed. Through design of specific primers based on the SlVQ6 gene sequence, and using the plasmid containing the target gene SIVQ6 as a template to amplify the target gene sequence, the obtained target gene VQ6 was cloned between the restriction sites of BamHⅠ and NotⅠ of the vector pGEX-4T-1, and the pGEX-4T-1-PC-VQ6 was transferred into a TOP10 clone strain. The target protein PC-VQ6 was obtained by IPTG (isopropyl thiogalactoside) inducing expression and the expected GST-SlVQ6 fusion protein was obtained by affinity purification by GST column. Phosphorylation was performed in vitro to further determine whether SlVQ6 is the substrate of SlMPK1. GST-SlVQ6 was used as the bait protein to fix on GST Sepharose Beads and incubated with tomato leaf total protein, and then the incubated fusion material was eluated. The eluate was collected and verified by SDS-PAGE gel electrophoresis. LC-MS/MS was used to detect the candidate proteins that could interact with SlVQ6, and the screened SIVQ6 interacting proteins were used for bioinformatics analysis by GO, KEGG, and protein interaction networks. 【Result】The results showed that we successfully constructed the recombinant gene expression plasmid pGEX4T-1-PC-SlVQ6 and obtained the GST-SlVQ6 fusion protein with a GST tag, of which the molecular weight is about 54 kD. GST-SlVQ6 and His-SlMPK1 were subjected to phosphorylation experiments in vitro. SlVQ6 could be phosphorylated by SlMPK1, however the GST could not be phosphorylated by SlMPK1 as a negative control and there was no autophosphorylation phenomenon in SlVQ6, indicating that SlVQ6 has an interaction with SlMPK1, and SlVQ6 is a downstream substrate of SlMPK1. GST-SlVQ6 fusion protein was used as bait protein (GST as negative control), and the protein bound to SlVQ6 protein in tomato leaf tissue protein was screened by pull-down test. Through SDS-PAGE, liquid chromatography-tandem mass spectrometry (LC-MS/MS) identification, and Mascot protein database search, 37 SlVQ6-bound proteins were identified, including protein kinase Receptor for Activated C Kinase 1B (RACK1B). Bioinformatics analysis of GO, KEGG, and protein interaction networks indicated that these proteins are involved in a variety of signaling pathways, and eight of the ribosomal proteins may be closely related to high temperature stress. 【Conclusion】This research shows that SlVQ6 is a substrate protein of SlMPK1 and 37 proteins may interact with SlVQ6. These proteins are closely related to the stress response and may play an important role in the high temperature tolerance of tomato plants.

Cloning, Expression and Electrophysiological Function Analysis of Potassium Channel Gene VviSKOR in Grape

SHEN JingYuan,TANG MeiLing,YANG QingShan,GAO YaChao,LIU WanHao,CHENG JieShan,ZHANG HongXia,SONG ZhiZhong

Scientia Agricultura Sinica. 2020, 53(15): 3158-3168. doi:10.3864/j.issn.0578-1752.2020.15.015

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【Objective】The aim of this study was to isolate and characterize potassium (K⁺) channel gene SKOR (stelar K⁺ outward rectifier) from grape genome, based on the analysis of the tissue-specific expression patterns of VviSKOR gene and response to K⁺ depletion, K⁺ excess, NaCl and ABA treatments, as well as function study by patch clamping electrophysiological technology.【Method】By carrying out homology-based cloning, a putative K⁺ channel gene VviSKOR was screened and isolated from grape genome. The details of VviSKOR gene were analyzed, and the protein encoded via utilizing bioinformatical analysis software. With the help of MEGA7.0 software, a phylogenetic tree was constructed by multiple alignments of SKOR proteins from grape, Arabidopsis, rice, maize, sorghum, slender false brome, soybean, tomato, cucumber, poplar, peach, pear, strawberry, apple, papaya, citrus, banana and pineapple. Using quantitative real-time PCR (qRT-PCR), the expression profiles of VviSKOR gene and its response to K⁺ depletion, NaCl and ABA treatments were analyzed. Preliminary analized the physiological function of VviSKOR gene by using patch clamping.【Result】VviSKOR was isolated and determined from grape genome. VviSKOR contained the functional domains of cyclic nucleotide-binding domain, ion channel transmembrane, ankyrin repeats and KHA domain, which belonged to the classic plant potassium channels. The amino acid sequences of SKOR protein from 18 plants shared an overall identity of 58.92%. Phylogenetic tree analysis showed that VviSKOR was closely clustered with homolog of CsaSKOR from cucumber. SKOR members from 4 grass family plants (maize, rice, slender false brome and sorghum) were prone to be clustered together, while SKOR members from Rosaceae, including strawberry, apple, pear and peach, were prone to be clustered together. VviSKOR was mainly localized in plasma membrane, which contained 6 transmembrane domains (TMs), and the theoretical isoelectric point (PI) was 6.24. Twelve kinds of cis-acting regulatory elements, including stress response, hormone response and cell cycle regulation, were found in the promoter region of VviSKOR gene. Database expression profile analysis showed that VviSKOR gene was expressed in different tissues or organs in grape, and the highest percentage was predicted in roots, followed by leaves and xylem. qRT-PCR analysis showed that VviSKOR gene was mainly expressed in roots of both 7-year-old ‘Marselan' and young seedlings. Moreover, VviSKOR gene was more sensitive to K⁺ depletion, ABA and NaCl treatments, whose expression were decreased under K⁺ depletion and ABA treatments, but induced under NaCl treatment in all tested tissues, including roots, and roots. Expression of VviSKOR gene had no response to NaCl treatment. Preliminary evidence of patch clamp analysis revealed that outward current was recorded in HEK293-T cells transfected with pTracer-CMV3-SKOR plasmid, and the amount of released K⁺ was reduced with elevated external-K⁺, indicating that VviSKOR was an outwardly rectifying K⁺ channel. In addition, the recorded current increased with the enhancement of voltage, indicating that VviSKOR was also a voltage dependent K⁺ channel.【Conclusion】VviSKOR gene was mainly expressed in grape roots (mature trees and seedlings). VviSKOR was closely clustered with homolog of CsaSKOR from cucumber in phylogenetic tree. Expression of VviSKOR gene was prone to be regulated by K⁺ depletion, ABA, and NaCl treatment. VviSKOR was an outwardly rectifying K⁺ channel that dominated the K⁺ release in grape roots.

Prediction and Verification of MicroRNAs Targeting Porcine Endoplasmic Reticulum Stress Pathway

ZHU JingJing,ZHOU XiaoLong,WANG Han,LI XiangChen,ZHAO AYong,YANG SongBai

Scientia Agricultura Sinica. 2020, 53(15): 3169-3179. doi:10.3864/j.issn.0578-1752.2020.15.016

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【Objective】 This study was aimed to predict and validate microRNAs (miRNAs) that targeting key genes in the porcine endoplasmic reticulum (ER) stress pathway, so as to provide a theoretical basis for regulation of porcine ER stress signaling pathway by miRNAs. 【Method】 Firstly, the differential expression of miRNAs in PRV-infected PK15 cells in porcine ER were determined by high-throughput sequencing. TargetScan was used to predict miRNAs that targeting the genes ATF6, IRE1, PERK, GRP78, and XBP1 of ER stress pathways. Recombinant plasmids were constructed, including candidate miRNA target sites, and the regulation of ATF6, IRE1, PERK, GRP78, and XBP1 by candidate miRNAs was validated by co-transfecting dual-luciferase reporter gene vector construct with miRNA-mimics into BHK-21 cells. Then, the candidate miRNAs were over-expressed in PK15 cells, and then fluorescent quantitative PCR and Western blot were used to detect the effect of miRNAs on key genes expression at mRNA and protein levels. 【Result】 MiRNA sequencing results showed that 35 differential expression of miRNAs were determined in PRV-infected PK15 cells. The results of TargetScan prediction showed that the intersection of miRNAs targeting four or more genes of ATF6, IRE1, PERK, GRP78, and XBP1 were miR-142-5p, miR-145-5p, miR-150, and miR-199a-5p, and these intersections of miRNAs were selected as candidate miRNAs. The dual-luciferase reporter plasmids psiCHECK-2-ATF6-m142-3'UTR, psiCHECK- 2-ATF6-m145-3'UTR, psiCHECK-2-ATF6-m150-3'UTR, psiCHECK-2-ATF6-m199-3'UTR, psiCHECK-2-IRE1-m150-3'UTR, psiCHECK-2-IRE1-m142/145/199-3'UTR, psiCHECK-2-PERK-m145/150-3'UTR, psiCHECK-2-XBP1-m142/145/150/199- 3'UTR, psiCHECK-2-GRP78-m145/199-3'UTR were successfully constructed. The luciferase assay experiments showed that miR-142-5p mimics could significantly inhibit luciferase activity of psiCHECK-2-ATF6-m142-3'UTR dual-luciferase reporter recombinant vector. psiCHECK-2-IRE1-m142/145/199-3'UTR was co-transfected with miR-142-5p mimics, miR-145-5p mimics, and miR-199a-5p mimics, respectively. The luciferase activity of the over-expressing group were significantly lower than the negative control group. In addition, psiCHECK-2-XBP1-m142/145/150/199-3'UTR was co-transfected with miR-142-5p mimics and miR-199a-5p mimics, respectively, and the luciferase activity of the over-expressing group were also significantly lower than the negative control group. The luciferase assay experiments showed that miR-145-5p mimics could significantly inhibit luciferase activity of psiCHECK-2- PERK-m145/150-3'UTR dual-luciferase reporter recombinant vector. The results indicated that miR-142-5p, miR-145-5p and miR-199a-5p might targeted the genes ATF6, IRE1, and XBP1. Among them, miR-142-5p might target these three key genes to regulate the ER signaling pathway. Overexpression of miR-142-5p significantly down-regulated the levels of mRNA and protein by qRT-PCR and Western blot methods. The results showed that miR-142-5p might participate in the regulation of ER signaling pathway by targeting ATF6.【Conclusion】 In this study, miR-142-5p was validated to target the key gene ATF6 of the porcine ER stress pathway. Thereby, these results laid a foundation for further study of regulation of ER stress pathway through miR-142- 5p-ATF6 gene axis.

Innate Immunomodulatory Effect of Pine Needle Polysaccharide on Chicken Macrophage HD11

CUI XiaoZhen,LUAN Yan,LI TingTing,YANG Yu,GUAN WenChao,ZHANG Kai,WANG FuChuan,SONG XianYi

Scientia Agricultura Sinica. 2020, 53(15): 3180-3186. doi:10.3864/j.issn.0578-1752.2020.15.017

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【Objective】 The innate immunomodulatary effect of pine needle polysaccharide on chicken macrophage HD11 were examined to provide theoretical ground for broiler disease prevention and treatment.【Method】Different concentrations of pine needle polysaccharide were used to treat the macrophage HD11. The experiment was divided into blank control group, positive control group (1 μg·mL^-1 was the final concentration of LPS), and pine needle polysaccharide group (25, 50, 100, 200 and 400 μg·mL^-1 were the final concentration of pine needle polysaccharide). MTT method was used to evaluate the effect of pine needle polysaccharide on the proliferation of HD11 cells. the content of NO in the supernatant of HD11 cells was determined, phagocytic function was detected by neutral red, the release of IFN-α, iNOS, IL-10, IL-6 and TNF-α were detected by ELISA, the levels of iNOS mRNA in the HD11 cells were detected by PCR.【Result】The results of MTT show, there was no significant difference among all groups (25, 50, 100, 200, 400 μg·mL^-1) in proliferation of HD11 cells. It indicated that there was no toxicity to macrophages in the range of 25-400 μg·mL^-1, and the immunoregulation experiment could be carried out. The immunomodulatory test results showed that, the concentration of NO, phagocytic function were higher significantly (P<0.01) than control group. The release of cytokines IFN-α and iNOS were higher significantly (P<0.01 or P<0.05) than control group. The amount of cytokines IL-10 was lower significantly (P<0.01 or P<0.05) than control group. The content of cytokines IL-6 and TNF-α were higher significantly (P<0.01) than control group when the pine needle polysaccharide concentration were 50, 100, 200, 400 μg·mL^-1. the concentration of NO was lower significantly (P<0.01) than positive control group. The release of cytokines IFN-α, iNOS and IL-10 were lower significantly (P<0.01 or P<0.05) than positive control group. The phagocytic function and the content of cytokines IL-6 was lower significantly (P<0.01) than positive control group when the pine needle polysaccharide concentration were 25, 50, 100 μg·mL^-1. The content of cytokines TNF-α was lower significantly (P<0.01) than positive control group when the pine needle polysaccharide concentration were 25, 50, 100, 200 μg·mL^-1.【Conclusion】 The results showed that pine needle polysaccharide can activate macrophages and enhance the innate immune function of macrophages HD11, and there was a dose-dependent effect.

The Mechanism Underlying MicroRNAs-Mediated Nosema ceranae Infection to Apis mellifera ligustica Worker

GENG SiHai,SHI CaiYun,FAN XiaoXue,WANG Jie,ZHU ZhiWei,JIANG HaiBin,FAN YuanChan,CHEN HuaZhi,DU Yu,WANG XinRui,XIONG CuiLing,ZHENG YanZhen,FU ZhongMin,CHEN DaFu,GUO Rui

Scientia Agricultura Sinica. 2020, 53(15): 3187-3204. doi:10.3864/j.issn.0578-1752.2020.15.018

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【Objective】Differentially expressed miRNAs (DEmiRNAs) and their target mRNAs in purified spores of Nosema ceranae and N. ceranae infecting Apis mellifera ligustica worker were systematically analyzed, followed by screening, investigation and exploration of DEmiRNAs and corresponding regulatory networks associated with pathogenic virulence factors and infection factors to reveal the mechanism of N. ceranae infection to A. m. ligustica worker.【Method】A. m. ligustica workers' midguts at 7 d and 10 d post infection (dpi) with N. ceranae and purified spores of N. ceranae (NcCK) were deeply sequenced using small RNA-seq (sRNA-seq), followed by screening out datasets of N. ceranae during the infection process (NcT1 and NcT2) and purified spores through sequential mapping to rRNA database, Apis mellifera genome and N. ceranae genome. Based on the criteria of P≤0.05, |log₂ fold change|≥1, DEmiRNAs in each comparison group were filtered out by comparative analysis. Expression profiling of DEmiRNAs, prediction of target mRNAs, function and pathway annotations of target mRNAs, and construction and survey of regulatory networks were performed by relevant bioinformatic software. The differential expression trend of DEmiRNAs and the reliability of sequencing data were verified by RT-qPCR.【Result】There were 164, 122 and 60 DEmiRNAs in NcCK vs NcT1, NcCK vs NcT2 and NcT1 vs NcT2 comparison groups. Venn analysis showed that 5 up-regulated miRNAs and 6 down-regulated ones were shared by these three comparison groups. In total, 1 885, 1 733 and 1 524 target mRNAs of DEmiRNAs were respectively predicted. These targets were annotated to 27, 25, and 26 functional terms, respectively, with the most abundant annotations being metabolic processes, catalytic activity, cellular processes, binding and cell. These targets were as well respectively annotated to 84, 84, and 84 pathways, among them the largest groups were metabolic pathway, ribosome, and secondary metabolite biosynthesis. In addition, 35, 26 and 12 target mRNAs associated with MAPK signaling pathway were targeted by DEmiRNAs in NcCK vs NcT1, NcCK vs NcT2 and NcT1 vs NcT2, while 49, 40 and 17 target mRNAs related to glycolysis/gluconeogenesis pathway were targeted by DEmiRNAs in the aforementioned three comparison groups. Further analysis revealed that DEmiRNAs of N. ceranae were involved in regulating the expression of genes encoding virulence factors such as ricin B lectin, apoptosis inhibitor, polar tube protein and spore wall protein; and genes encoding infection factors such as hexokinase and ATP/ADP transferase, ABC transporter and transcription factor ste12.【Conclusion】The differential expression profile of miRNAs in N. ceranae infecting A. m. ligustica worker was parsed via in-depth and detailed investigation and exploration. N. ceranae may regulate the expression level of genes encoding virulence factors including ricin B lectin, apoptosis inhibitor, polar tube protein and spore wall protein, as well as genes encoding infection factors such as hexokinase, ATP/ADP transferase, ABC transporter and transcription factor ste12 corresponding miRNAs, by controlling the expression of corresponding miRNAs, thus adapting to the environment within the host cell and promoting its proliferation and infection.

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