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    16 May 2020, Volume 53 Issue 10
    CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS
    Analysis of Foreign Gene Copy Number in Transgenic Wheat by Optimized Digital PCR
    JU PengJu,NING Lei,GE LinHao,XU ChengJie,SHI HuaWei,LIANG KaiGe,MA Liang,LIU TaoRan,CHEN Ming,SUN DaiZhen
    Scientia Agricultura Sinica. 2020, 53(10):  1931-1939.  doi:10.3864/j.issn.0578-1752.2020.10.001
    Abstract ( 710 )   HTML ( 73 )   PDF (2395KB) ( 667 )   Save
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    【Objective】In order to explore the analysis technology of wheat gene copy number based on digital PCR, and improve the analysis efficiency of target gene copy number, and promote the research of wheat genome and gene engineering.【Method】Using the transgenic wheat with high resistance to wheat yellow mosaic disease transformed by nib8 gene created by the wheat stress resistance molecular breeding group in the Crop Science Institute of Chinese Academy of Agricultural Sciences as the experimental material, and used the single copy homozygous endogenous gene PIN-D1b (grain hardness gene) in wheat D genome as the internal reference gene, and four pairs of specific primers and corresponding probes were designed according to the sequence of the transformed disease resistance gene nib8, and the optimal concentration of probe and primer, the optimal annealing temperature of the system, to find the most appropriate template concentration were determined through experiments. Then, the copy number of nib8 genes in the nib8 transgenic wheat was detected by digital PCR; the accuracy of the above-mentioned copy number assay results were verified through using methods including the real time PCR and Southern blot; the copy number of the Wx012 (waxy gene) and SSII (starch synthesis gene) were detected by using the PINb-D1b as reference gene, and copy number of nib8 were detected by using the Wx012 and SSII as reference genes in the nib8 transgenic wheat, to verify the accuracy of the results using the PINb-D1b as reference gene; the specific primers were designed in different regions of the nib8 gene to compare the difference of the copy number analysis results of the nib8 transgenic wheat. Finally, a high-throughput assay method based on digital PCR was established to detect the copy number of target gene in wheat genome. 【Result】Finally, the detection system of target gene copy number in wheat genome based on digital PCR was determined. The optimal final concentration of primer and probe was 500 and 250 nmol·L -1, respectively. The optimal annealing temperature was 59℃ and the optimal amount of DNA template was 40 ng. PINb-D1b gene was used as the internal reference gene to detect the copy number of nib8 gene in the nib8 transgenic wheat. The results showed that the copy number of nib8 gene in the 12th, 16th, 17th, 23rd, 29th and 30th lines was 7, 1, 1, 1, 1, 1, 7, respectively. At the same time, the results were consistent with the real-time PCR and Southern blot results. In the nib8 transgenic lines, the PINb-D1b was used as the internal reference gene to detect copy number of the Wx012 and SSII, and we found that the results of copy number of nib8 gene in nib8 transgenic wheat by using these three kinds of internal reference gene analysis were consistent, which indicated that these three internal reference genes were all suitable for digital PCR method; the primers were designed in the upstream, middle and downstream regions of nib8 gene, and the results of copy number using different primer were consistent. 【Conclusion】The method and reaction system to detect target gene copy number in wheat genome based on digital PCR method were optimized, and the detection system to detect target gene copy number in wheat genome based on digital PCR method was established. The results of digital PCR analysis are stable and reliable, and the detection efficiency is significantly improved, which has a certain application prospect.

    Developing SSR Markers of Proso Millet Based on Transcriptome Sequencing
    CHEN XiaoHong,HE JieLi,SHI TianTian,SHAO HuanHuan,WANG HaiGang,CHEN Ling,GAO ZhiJun,WANG RuiYun,QIAO ZhiJun
    Scientia Agricultura Sinica. 2020, 53(10):  1940-1949.  doi:10.3864/j.issn.0578-1752.2020.10.002
    Abstract ( 521 )   HTML ( 24 )   PDF (637KB) ( 466 )   Save
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    【Objective】Based on the data of transcriptome sequencing, 200 pairs of SSR primers were detected using six proso millet accessions with different origin. The aim of this study is to construct a set of molecular markers for assessing the genetic diversity of proso millet germplasms.【Method】Primer Premier 5.0 software was used to design primers, DNA was extracted by modified CTAB method and amplified by PCR. Polyacrylamide gel electrophoresis was used to screen the polymorphism of primers. Genetic diversity parameters were calculated with PowerMarker 3.25 and PopGen 1.32.【Result】Ninety seven and eighty out of 200 pairs of primers were shown monomorphic and polymorphic, respectively. A total of 20 were mono-nucleotide SSRs, among them 10 were polymorphic with repeat motif A (50%) and T (50%). A total of 36 were di-nucleotide SSRs, among them 15 were polymorphic with seven types of repeat motif, AG class was the most frequent, followed by the TC, GC and GA, CA, TA and AC were the least. A total of 144 were tri-nucleotide SSRs, among them 55 were polymorphic with 24 types of repeat motif, GGC, GCG and GCC class were the most frequent, followed by the GAA, GCT, CGC, etc, ACC, AGG, CAG, CGT, AAG, AAC, TCG, CGA, ATT, CAA and CCA were the least. In terms of the mono-, di-, tri-nucleotide SSR, their Rp value ranged from 0.67 to 4.67 (mean 2.07), 1.33 to 4.33 (mean 2.73) and 0.67 to 4.00 (mean 1.83), respectively. Evaluating the distribution frequency based on Rp, 80 SSRs were divided into five intervals viz 0-1, 1-2, 2-3, 3-4 and 4-5, with 17 (21.25%), 36 (45.00%), 11 (13.75%), 14 (17.50%) and 2 (2.50%) markers, respectively. For the mean of the Rp value, the mono-, di-, tri-nucleotide SSR were 0.33 to 0.67 (average 0.51), 0.40 to 0.78 (average 0.59) and 0.33 to 0.83 (average 0.59), respectively. As for mono-nucleotide SSRs, a total of 22 allelic variations were detected with 2-3 alleles (average 2.2000) for each locus; 2 and 3 alleles were identified for 8 and 2 loci, respectively. In terms of di-nucleotide SSRs, a total of 38 allelic variations were generated with 2-3 alleles (average 2.5333) for each locus; 7 and 8 loci produced 2 and 3 alleles, respectively. As for tri-nucleotide SSRs, a total of 136 allelic variations were generated with 2-3 alleles (average 2.4727) for each locus; 29 and 26 loci produced 2 and 3 alleles, respectively. In terms of the polymorphism information content, the mono-, di-, tri-nucleotide SSR were 0.3750-0.5355, 0.2392-0.7438 and 0.2392-0.7438, respectively. In terms of the diversity index, the mono-, di-, tri-nucleotide SSR were 0.6365-1.0776 (average 0.7497), 0.5623-1.0986 (average 0.8339) and 0.5623-1.0889 (average 0.8312), respectively. 【Conclusion】Two hundreds pairs of SSR primers obtained from transcriptome sequencing were used to detect genetic diversity of 6 proso millet accessions. It was shown that 177 (88.5%) out of the 200 pairs primers amplified intact bands, and 80 SSRs appeared polymorphic with the polymorphic rate 40%.

    Improvement of the Resistance Against Sclerotinia sclerotiorum in Ogu CMS Restorer in Brassica napus Using Wild B. oleracea as Donor
    WAN HuaFang,DING YiJuan,CHEN ZhiFu,MEI JiaQin,QIAN Wei
    Scientia Agricultura Sinica. 2020, 53(10):  1950-1958.  doi:10.3864/j.issn.0578-1752.2020.10.003
    Abstract ( 407 )   HTML ( 21 )   PDF (1292KB) ( 312 )   Save
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    【Objective】Sclerotinia stem rot (SSR) is a devastating disease in rapeseed (Brassica napus L.). The resistance breeding is greatly limited due to the lack of resistance resources in rapeseed. Ogu CMS is one of the most effective pollination control systems in Brassica. Up to date, however, there has no Ogu CMS restorer with high resistance against SSR reported in rapeseed. High resistant source was reported in a wild B. oleracea which carried two major resistant loci, C1-and C9-QTL. The objective of this study is to transfer the resistance loci from the resistant B. oleracea and develop resistant Ogu CMS rapeseed restorer.【Method】Resynthesized rapeseed line was developed via interspecies cross between a SSR-resistant wild B. oleracea (namely B. incana) and B. rapa by embryo rescue and chromosome doubling. The hybridization was carried out between the resynthesized line and a rapeseed Ogu CMS line, followed by successive self-pollination. Marker assisted selection (MAS) was carried out in each generation to select individual plants carrying resistant loci from B. incana. These plants were screened for resistance level to SSR by inoculation test on detached branches. The plants which exhibited significant lower susceptibility (S) than rapeseed cultivar Zhongshuang 9 (the tolerant control) were then chosen for seed quality analysis. Fertile plants with relatively lower erucic acid and glucosinolate content were self-pollinated for the next generation development. Finally, self-pollinated lines carrying resistant QTL, exhibiting high resistant to SSR, performing good seed quality and yield were considered as SSR-resistant rapeseed Ogu CMS restorer.【Result】Resynthesized rapeseed line AC1-4 was successfully developed from the resistant B. oleracea and B. rapa, which showed significantly higher resistance level to SSR than the tolerant control Zhongshuang 9. From 146 F1 plants derived from AC1-4 and a rapeseed Ogu CMS line 4Q292, two individual plants (4C99 and 4C115) were selected to develop F2 generation, which carried resistant QTLs (C1- and C9-QTL) and exhibited normal seed setting and low susceptibility values (0.66 and 0.40, respectively). Among 124 F2 plants, eight individual plants with good self-fertility were selected to develop 982 F2:3 plants, from which a total of 50 individual plants were identified to carry resistant QTL C1-QTLand C9-QTL. Especially, two fertile plants (5X82-2 and 5X82-7) exhibited ‘single low’ seed quality (6.5% erucic acid in 5X82-7, and 45.38 μmol·g-1 meal glucosinolate in 5X82-7) and low susceptibility to SSR (S = 0.48 and 0.33, respectively).【Conclusion】It successfully improved SSR-resistance in Ogu CMS rapeseed restorer using wild B. oleracea as donor, which will be helpful for developing Ogu CMS restorer with high resistance against SSR and good seed quality in rapeseed.

    TILLAGE & CULTIVATION·PHYSIOLOGY & BIOCHEMISTRY·AGRICULTURE INFORMATION TECHNOLOGY
    Effects of Tillage, Nitrogen Application, Planting Density and Their Interaction on Soil Moisture and Yield Formation of Spring Maize in Dryland
    LI Ao,ZHANG YuanHong,WEN PengFei,WANG Rui,DONG ZhaoYang,NING Fang,LI Jun
    Scientia Agricultura Sinica. 2020, 53(10):  1959-1970.  doi:10.3864/j.issn.0578-1752.2020.10.004
    Abstract ( 579 )   HTML ( 44 )   PDF (480KB) ( 777 )   Save
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    【Objective】The semi-humid and arid areas on the Loess Plateau belong to rain-fed agricultural areas, in which water deficit is the main limiting factor for agricultural production. Therefore, a comprehensive cultivation mode of spring maize suitable for semi-humid and drought-prone areas on the Loess Plateau was screened to improve maize water use efficiency and yield. 【Method】During the 2017 and 2018 maize growing season, the effects of different tillage methods (conventional tillage, no-tillage, and subsoil tillage), nitrogen application (0, 150, and 225 kg·hm-2) and planting density (52 500 and 67 500 plants/hm2) on soil water dynamics, water use efficiency, crop growth, yield and its components in spring maize field were analyzed to improve the comprehensive utilization of maize. 【Result】 (1)The soil water storage (0-200 cm) of spring maize before sowing in 2017and 2018 showed the trend of NT>ST>CT, which under NT and ST increased by 6.1% and 4.1% than that under CT, respectively. In addition, water use efficiency was the highest under STH2 treatment during two consecutive years. (2) Compared with conventional tillage treatment, subsoiling and no-tillage treatment significantly increased leaf area index (LAI) and dry matter accumulation. LAI and dry matter accumulation of spring maize increased with the increase of density and nitrogen rates. The tillage×density interaction had significant effect on LAI at jointing and tasseling stages, and the effect of density×fertilization interaction had significant effect on LAI at filling stage. Compared with other treatments, the dry matter accumulation of STH2 treatment in maturity stage increased significantly by 3.3% -32.9%. (3)There was a significant positive correlation between nitrogen application and the number of grains per ear and 100-grain weight; the maize yield under subsoiling and no-tillage treatment was significantly higher than that under conventional tillage, among which the yield under STH2 treatment was the highest, and compared with other treatments, the maize yield under STH2 treatment increased by 1.4%-63.3% in 2017 and 2.9%-39.6% in 2018. 【Conclusion】Under the condition of subsoiling tillage, applying 150-225 kg·hm-2 nitrogen and 67 500 plants/hm2 planting density could not only improve water use efficiency of spring maize, but also obtain higher yield benefit, no-tillage, applying 225 kg·hm-2nitrogen and 67 500 plants/hm2 planting density could get the high economic benefit.

    Benefits and Marginal Effect of Dry Matter Accumulation and Yield in Maize and Soybean Intercropping Patterns
    ZHAO DeQiang,LI Tong,HOU YuTing,YUAN JinChuan,LIAO YunCheng
    Scientia Agricultura Sinica. 2020, 53(10):  1971-1985.  doi:10.3864/j.issn.0578-1752.2020.10.005
    Abstract ( 685 )   HTML ( 71 )   PDF (926KB) ( 981 )   Save
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    【Objective】The study was carried to explore the intensity and extent of marginal effect in the maize-soybean intercropping system and the effect on two crops, and to investigate the aggressivity between two crops and the marginal effects of each line, so as to provide a base for the mechanism of maize-soybean intercropping in the increase of land productive on dry matter accumulation and land equivalent ratio (LER). 【Method】 Field experiment consisted of maize-soybean intercropping system (6M6S, 6M3S, 3M6S, and 3M3S), sole maize (CKM) and sole soybean (CKS), and each row (marked I, II and III) of 4 intercropping treatments has been sampled. The dry matter accumulation and the composite of biomass and yield were measured, and the marginal effects of each crop lines were also calculated. 【Result】A two-year experiment showed that the maize dry matter accumulation of intercropping treatments including 6M6S, 6M3S, 3M6S, and 3M3S was higher than that of CKM, and the soybean dry matter accumulation of 6M6S and 3M6S was higher than that of CKS. Compared with the monoculture treatment, intercropping treatment increased the dry matter accumulation rates of the whole growth period of maize and the early-branching, late branching, filling and maturity stage of soybean. During these two years, the yield of maize under 6M6S, 6M3S, 3M6S and 3M3S accounted for 73.9%, 88.7%, 52.8% and 65.5% of CKM and the yield of soybean accounted for 26.1%, 11.3%, 47.2% and 34.5% of CKS, respectively; The LERYMS of them were 1.31, 1.23, 1.33 and 1.13, and the aggressivity of maize relative to soybean were 0.44, 0.47, 0.45 and 0.46 in average, respectively. The I, II, and III rows in intercropped maize and the II and III rows in intercropped soybean produced more dry matter accumulation and rate than the respective monoculture of maize and soybean, but the intercropping treatment decreased the dry matter accumulation and rate of I row soybean. During these two years, the per plant yield of maize in I, II, and III rows were 151.43%, 138.51% and 130.83% of CKM, respectively, and the per plant yield of soybean in I, II, and III rows were 90.22%,104.16% and 109.03% of CKS, respectively. The stem and leaf dry matter of crops reached the maximum at silking stage (flowering stage) in two years experiment, the remobilization of maize leaf, maize stem, and soybean stem and leaf were 15.70 g/plant, 27.64 g/plant and 7.43 g/plant, and the efficiency rates of them were 22.80%, 44.23%, 19.61%, and the conversion rates were 14.99%, 26.28%, and 27.79%, respectively. 【Conclusion】The intercropping of maize and soybean increased the dry matter accumulation and yield of crops, the remobilization of leaf and stem, and increased LER, in which maize contributed more than soybean. The marginal effect of intercropping was affected by different row ratios. The dry matter accumulation and yield of I, II, and III rows in intercropped maize were decreasing, and it was increasing in intercropped soybean. In this experiment, the 3M6S intercrop pattern was the most lucrative system, and it had the highest LER and marginal advantage.

    PLANT PROTECTION
    Noncontact Inhibitory of Volatile Organic Compounds from Rice Root Bacteria on Rhizopus microsporus
    WANG EnZhao,FAN FenLiang,LI YanLing,LIU XiongDuo,LU YuQiu,SONG ALin
    Scientia Agricultura Sinica. 2020, 53(10):  1986-1996.  doi:10.3864/j.issn.0578-1752.2020.10.006
    Abstract ( 435 )   HTML ( 18 )   PDF (1380KB) ( 472 )   Save
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    【Objective】The objective of this study is to identify new bacterial volatile organic compounds (VOCs) with potential antimicriobial function by analyzing the composition of VOCs of rice root bacteria and their inhibitory effects on Rhizopus microsporus.【Method】The inhibition rate of different bacteria to R. microsporus was determined by a double Petri dish assay. The VOCs of bacteria were collected by headspace-solid phase microextraction and detected by gas chromatography-mass spectrometry (GC-MS). The detected VOCs were compared and identified in the NIST/EPA/NIH database. According to the inhibitory effect and the peak area of each substance in mass spectrum, the correlation analysis was conducted. The VOCs which have a significant correlation with inhibition rate were purchased and the inhibitory effect of these pure substances on R. microsporum was detected in vitro.【Result】All strains could release VOCs and had different inhibitory effects through non-contact of VOCs. These strains produced a total of 90 VOCs of 14 types, which were esters, olefins, alkanes, ketones, acids, thiophenes, aldehydes, naphthalenes, sulfides, phenols, alcohols, pyrazines, pyrroles and benzenes. All strains could release alcohols. Through correlation analysis, 6 VOCs (2-heptanone, 5-methyl-2-hexanone, 2-nonanone, 5-methyl-3-hexanone, 3-methyl-butanoic acid and methyl isobutyl ketone) were found to have potential antimicriobial activity, there was a significant positive correlation between the release amount and inhibition rate. Among them, 3 of these VOCs (2-heptanone, 2-nonanone and methyl isobutyl ketone) had been reported to have antimicriobial activity in previous studies. The other 3 VOCs (5-methyl-2-hexanone, 5-methyl-3-hexanone and 3-methyl-butanoic acid) had not been reported. In vitro fumigation test was conducted by purchasing two of them (3-methyl-butanoic acid and 5-methyl-2-hexanone). It was found that 3-methyl-butanoic acid seriously inhibited the growth of R. microspores, and 5-methyl-2-hexanone was lethal to the R. microspores. Among them, 5-methyl-2-hexanone was mainly released by the strains of Paenisporosarcina macmurdoensis, Brevibacillus brevis and Bacillus aryabhattai. 5-methyl-3-hexanone was mainly produced by the strains of Pseudomonas, B. aryabhattai and P. macmurdoensis. 3-methyl-butanoic acid was mainly released by strains of Enterobacter kobei, Pseudomonas and B. aryabhattai. 【Conclusion】Rice root bacteria can release a variety of VOCs, some of which have a significant inhibitory effect on rice pathogen R. microsporum. Through correlation analysis and in vitro validation, two new antimicriobial VOCs (5-methyl-2-hexanone and 3-methyl-butanoic acid) and one possible antimicriobial VOC (5-methyl-3-hexanone) were identified. All these strains and VOCs have the potential as biological resources for new drugs and antifungal metabolites.

    Function of Citrus Bacterial Canker Resistance-Related Transcription Factor CitMYB20
    YAO LiXiao,FAN HaiFang,ZHANG QingWen,HE YongRui,XU LanZhen,LEI TianGang,PENG AiHong,LI Qiang,ZOU XiuPing,CHEN ShanChun
    Scientia Agricultura Sinica. 2020, 53(10):  1997-2008.  doi:10.3864/j.issn.0578-1752.2020.10.007
    Abstract ( 419 )   HTML ( 18 )   PDF (1174KB) ( 452 )   Save
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    【Objective】The objective of this study is to reveal the response of CitMYB20 to Xanthomonas citri subsp. citri (Xcc) and exogenous phytohormones in different citrus varieties, evaluate the resistance of transgenic plants to citrus bacterial canker (CBC), and to verify the biological function of CitMYB20.【Method】The primer pairs were designed according to the Citrus genome, the open reading frame sequences and promoter sequences of CitMYB20 were cloned from CBC resistant variety Fortunella japonica and CBC sensitive variety Poncirus trifoliata by PCR. The cis-acting elements of promoter sequences were predicted using online software PlantCARE. The detached mature leaves from F. japonica and P. trifoliata were inoculated with Xcc by acupuncture method, and collected at 0, 1, 3, and 5 d after inoculation. The citrus leaves were collected after being treated with exogenous salicylic acid, methyl jasmonate, and ethephon respectively for 0, 12, 24, 36, and 48 h. The expression of CitMYB20 was detected by qRT-PCR using the above treated leaves. The over-expressed vector and RNA interference vector of CitMYB20 were constructed through inserting the target sequences into the pLGNe plasmid. The transgenic plants were obtained through Agrobacterium tumefaciens, and the mature leaves of transgenic lines were randomly selected to conduct resistance evaluation against CBC.【Result】The open reading frames of CitMYB20 in F. japonica (GenBank number MN689609) and P. trifoliata (GenBank number MN689608) were highly conservative, with a similarity of 99%. There were abscisic acid-responsive and methyl jasmonate-responsive cis-acting elements in the promoter sequence of CitMYB20 from two varieties of citrus. However, there was a salicylic acid-responsive element in the promoter sequence from F. japonica (GenBank number MN689611), but not in the sequence from P. trifoliata (GenBank number MN689610). At the 5th day of Xcc in vitro inoculation, the expression of CitMYB20 in mature leaves of F. japonica was increased significantly, reaching 2.5 times of the healthy control, while the expression of CitMYB20 in P. trifoliata did not show significantly change during the whole infection period. During exogenous hormone treatment, the response of CitMYB20 to salicylic acid and methyl jasmonate was similar, that is, it showed the trend of first increasing and then decreasing in F. japonica, while the expression of CitMYB20 in P. trifoliata was decreased firstly and then returned to normal. When the leaves were treated with exogenous ethephon, the expression of CitMYB20 in F. japonica was decreased slightly, while in P. trifoliata, it showed the trend of first increasing and then decreasing. A total of 7 over-expressed plants and 5 RNA-interfered ones were obtained through genetic transformation. There was no significant difference in morphological development between transgenic plants and control plants. The results of disease resistance evaluation showed that the CitMYB20 over-expressed plants could reduce the symptom of CBC, while the RNA-interfered plants were more sensitive to Xcc.【Conclusion】The expression of CitMYB20 was induced by Xcc in CBC resistant variety. CitMYB20 is a positive regulatory transcription factor against Xcc, its function against CBC may require salicylic acid and jasmonic acid signaling pathway. CitMYB20 can be used as a candidate gene for genetically modified breeding against CBC.

    Identification and Expression Analysis of the Halloween Gene Family in Agasicles hygrophila
    LIU YiRan,ZHANG Hong,JIN JiSu,ZHOU ZhongShi,GUO JianYing
    Scientia Agricultura Sinica. 2020, 53(10):  2009-2019.  doi:10.3864/j.issn.0578-1752.2020.10.008
    Abstract ( 568 )   HTML ( 16 )   PDF (1351KB) ( 643 )   Save
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    【Background】Agasicles hygrophila is an effective host-specific biological control agent for the worldwide malignant invasive weed Alternanthera philoxeroides. The population dynamics of A. hygrophila fluctuates during the year. In Hunan Province of China, the population of A. hygrophila decreases dramatically in summer leading to a significant reduction in the control of A. philoxeroides. The Halloween gene family is involved in the synthesis of ecdysone, which affects the growth and reproduction of insects, and then has a certain effect on population dynamics.【Objective】The objective of this study is to reveal the expression patterns of the Halloween gene family at different developmental stages and in different tissues of A. hygrophila, and to provide a theoretical basis for further exploring the roles of Halloween gene family in A. hygrophila and a new idea for the propagation of biological prevention means.【Method】The ovarian transcriptome data were screened with the combination of bioinformatics analysis and Halloween family genes were cloned. Conserved domains were analyzed through NCBI, MEGA6.0 was used for multiple sequence alignment and phylogenetic tree was constructed. Meanwhile, the transcript levels of the six Halloween family genes in A. hygrophila at different developmental stages (egg, larva, pupa, female adult) and in different tissues (head, thorax, midgut, ovary, fatbody) were analyzed by qRT-PCR.【Result】Six members of the Halloween gene family were identified and cloned, namely AhCYP302A1, AhCYP306A1, AhCYP307A1, AhCYP307A2, AhCYP314A1 and AhCYP315A1, respectively. All of them belonged to the P450 superfamily and had certain conservatism. Among them, AhCYP306A1, AhCYP307A1, AhCYP307A2 were grouped into CYP2 group and the other three genes belonged to mitochondrial group. Meanwhile, all of AhCYP306A1, AhCYP314A1, AhCYP315A1 had one transmembrane region at the 5′ end. The six Halloween genes were expressed with different levels at different developmental stages and in different tissues of A. hygrophila. The expression level of AhCYP302A1 was the highest on the 6th to 7th day of pupae stage, but it was lower at the adult stage. The expression level of AhCYP306A1 fluctuated throughout the life cycle of A. hygrophila and it was the highest on the 10th day of larvae stage. Higher expressions of AhCYP307A2 were detected at the late larvae and early pupae stages of A. hygrophila. In brief, these three genes were highly expressed at larvae and pupae stages of A. hygrophila. AhCYP307A1 was highly expressed at the egg stage, and its expression showed fluctuation and decline in subsequent developmental stages. While the expression levels of AhCYP314A1 and AhCYP315A1 were higher in adults and eggs than those in larvae and pupae. Among head, thorax, midgut, ovary and fatbody of A. hygrophila, most of the six Halloween genes were highly expressed in the ovary except AhCYP307A1. AhCYP307A1 had significantly high expression in the head of female adults.【Conclusion】Six Halloween genes were cloned in A. hygrophila, and their expression levels differed at different developmental stages and in different tissues of the insect. By comparing their expression patterns, it is speculated that the Halloween gene family may play important roles in the larval development and adult reproduction of A. hygrophila by affecting its molting and ovary development.

    SOIL & FERTILIZER·WATER-SAVING IRRIGATION·AGROECOLOGY & ENVIRONMENT
    Research on Nitrogen Fertilizer Application Recommended Method Based on Soil Organic Matter in Dryland Wheat Production
    JIANG LongGang,HUANG Ming,SONG QingYun,YUAN AiJing,LI JiaHua,QIU WeiHong,WANG ZhaoHui
    Scientia Agricultura Sinica. 2020, 53(10):  2020-2033.  doi:10.3864/j.issn.0578-1752.2020.10.009
    Abstract ( 406 )   HTML ( 34 )   PDF (501KB) ( 571 )   Save
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    【Objective】 Most of the current existing recommended methods of nitrogen (N) fertilizer application for wheat involved collecting and analyzing a huge quantity of soil samples every year, especially for those based on soil testing, thus these methods were very expensive and difficult to be accepted and applied by the smallholder farmers and junior agricultural technicians. Therefore, it is important to explore an effective N fertilizer recommendation method that does not need testing soil samples each year. 【Method】 A recommended N fertilizer application method that based on the soil organic matter content (SOM) was proposed, the purpose of which was to maintain or improve soil fertility under low SOM condition, and to decrease the environmental risk under high SOM condition in dryland. The N fertilizer application rate was calculated as the N requirement of the target grain yield multiplied by the N fertilizer coefficient (Nf), where Nf was determined by SOM in topsoil. Then a three-year field experiment was conducted to verify this recommended method in six counties of winter wheat planting area in Weibei dryland, with two treatments: FP (Farmers’ practice) and OFP (Optimized fertilization practice based on the method of soil organic matter). 【Result】 The results showed that the average N fertilizer rate of OFP and FP was 161 and 190 kg N·hm-2, respectively, with a significant decrease of 15.3% by OFP. However, the winter wheat yield in OFP was 5 817 kg·hm-2and higher (by 9.0%) than that in FP. The net economic return of OFP was 1 451 yuan/hm2, with an increase of 19.3% when comparing with FP. In addition, compared with FP, OFP decreased 39 kg·hm-2nitrate-N residue in 0-1 m soil layers at harvest stage, with a decrease of 28.3%. 【Conclusion】 The recommended N fertilization method based on soil organic matter decreased N fertilizer input increased the wheat grain yield and economic benefits, and reduced the environmental risk in Weibei dryland. Therefore, this method could be taken into account in the management of N fertilizer input by local farmers in this region.

    Investigation of Present Fertilization Status and Fertilizer Utilization Rate on Rapeseed in Anhui Province
    BU RongYan,CHENG Yi,WANG Hui,HAN Shang,HU RongGen,YU Zhong,LI Min,CHENG WenLong,SUN YiXiang,WANG JiaBao,WU Ji
    Scientia Agricultura Sinica. 2020, 53(10):  2034-2044.  doi:10.3864/j.issn.0578-1752.2020.10.010
    Abstract ( 522 )   HTML ( 20 )   PDF (1404KB) ( 635 )   Save
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    【Objective】 Winter rapeseed is an important oil crop in China, while the rational scientific fertilization is a crucial measure to ensure the yield of winter oilseed rape. Understanding the current situation and existing problems of fertilization in winter rapeseed planting areas of Anhui Province is important to guide rational fertilization. 【Method】Taking winter oilseed rape planting in Anhui Province as example, this study was conducted from farmers in the main winter rape producing areas from 2017 to 2018. The survey included fertilizer varieties, fertilizer usage, fertilization methods and proportion, planting area, and yield level. Based on the survey results, the current fertilization status of winter rape growing areas in Anhui Province was analyzed by Cate-Nelson method to evaluate the yield efficiency of winter rapeseed in Anhui Province, and the main methods to increase yield and efficiency of winter rapeseed in Anhui Province were explored.【Result】The results showed that chemical fertilizers were applied in all farmlands, only 59.0% farmers used organic matter. The results showed that the average yield of winter oilseed rape in Anhui Province was 2 017 kg·hm-2, and the average nitrogen (N), phosphorus (P2O5) and potassium (K2O) fertilizers were 160, 63, and 65 kg·hm-2, respectively. To sum up, the average use of N, P2O5 and K2O fertilizers becomes more reasonable, however, the results of fertilizer grading show that the phenomenon of either insufficient or excessive use of N, P2O5 and K2O fertilizers does exist. The fertilizer is mainly distributed by hand, as the mechanized fertilization is not prevalent. Cate-Nelson method (cross-over method) was used to evaluate the energy production efficiency of winter rapeseed in Anhui Province, the results showed that only 23.8%, 20.2% and 21.0% farmers of winter rapeseed in Anhui Province achieved high yield and high efficiency of N, P2O and K2O fertilizers. This indicated there was still much room for increasing yield and efficiency of rapeseed in Anhui Province. 【Conclusion】 Referring to the current recommended amount of rapeseed fertilizer, the average amount of chemical fertilizer in winter rapeseed cultivation in Anhui Province became more rational, but did not enough in fertilization methods, nutrient management and organic substitution. Therefore, it needed to take measures to increase of fertilizer efficiency by controlling the total amount, increasing organic fertilizer substitution, promoting the application of special fertilizer, rational nutrient management, encouraging the mechanized fertilization, and so on, to finally achieve high yield of rapeseed yield.

    SPECIAL FOCUS: QUALITY OF AGRICULTURAL PRODUCTS
    Effects of Different Strain Fermentation on Protein Hydrolysis and Lipid Profile of Quinoa
    YAN Sha,XING JieWen,WANG XiaoWen
    Scientia Agricultura Sinica. 2020, 53(10):  2045-2054.  doi:10.3864/j.issn.0578-1752.2020.10.011
    Abstract ( 439 )   HTML ( 21 )   PDF (453KB) ( 441 )   Save
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    【Objective】 This study was performed to compare the differences of the protein hydrolysis and lipid productions during the quinoa fermentation with the different strains, so as to promote the development of quinoa added-value food by the fermentation .【Method】 Yeast, lactobacillus plantarum and the two mixed was used for the fermentation of quinoa, respectively. Proteolysis index, protein composition, lipid profile, the content of total free amino acid and free fatty acid were determined to evaluate which microbe had more advantages in the transformation of nutrients. 【Result】 The proteolysis index increased after the quinoa fermentation by different microbes, and albumin and globulin were mainly hydrolyzed among quinoa protein composition. There was a significant increase in the free amino acid of quinoa after fermentation, especially essential amino acids. The yeast-fermented quinoa was beneficial in gaining smaller molecular compounds that originated from protein hydrolysis. The free fatty acids content increased sharply after fermentation and met a proper ratio of SFA﹕MUFA﹕PUFA close to 1﹕1﹕1. The content of triglycerides and diglycerides were reduced, but the content of functional lipids (phospholipid) was increased. The quinoa with yeast fermentation was more favorable for the above transformation.【Conclusion】Quinoa fermented by yeast or lactobacillus plantarum could result in the hydrolysis of albumin and globulin into amino acids, especially the free essential amino acid levels increased significantly. The free fatty acids also increased after fermentation, wherein the triglyceride and diacylglycerol were reduced, and the content of phospholipid was improved. Quinoa with yeast fermentation had great potential in developing high-value nutritional products in the future.

    Comparative Analysis of Alkylresorcinols Homologue Composition in Whole Wheat Flour and Refined Wheat Flour
    ZOU YanYu,FANG Yong,LI Peng,HUANG QinQin,XIA Ji,LIU Qin,XIE MinHao,HU QiuHui
    Scientia Agricultura Sinica. 2020, 53(10):  2055-2065.  doi:10.3864/j.issn.0578-1752.2020.10.012
    Abstract ( 496 )   HTML ( 14 )   PDF (831KB) ( 369 )   Save
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    【Objective】 In order to realize the authenticity discrimination of whole wheat flour, a high performance liquid chromatography (HPLC) detection and analysis method was established to explore the composition of alkylresorcinols (ARs) homologues in whole wheat flour and refined wheat flour. 【Method】 The HPLC conditions were investigated to determine five kinds of ARs homologues, including 5-heptadecylresorcinol (C17:0), 5-nonadecylresorcinol (C19:0), 5-heneicosylresorcino (C21:0), 5-tricosylresorcinol (C23:0) and 5-hentacosylresorcinol (C25:0), and then a methodological evaluation was processed. The contents and composition of ARs homologues in 47 samples, including whole wheat flour, refined wheat flour and wheat bran from home and abroad, were determined and conducted by heat map and principal component analysis (PCA).【Result】 The chromatographic column of ZORBAX SB-C18 (150 mm×4.6 mm, 5 μm, Agilent ) was used for gradient elution, and the column was eluted with 0.1% formic acid aqueous solution and 0.1% formic acid methanol solution. The ARs homologues could be effectively separated within 35 minutes under the UV detection wavelength at 280 nm. The column temperature was 35℃, and the flow rate was 0.5 mL·min-1with 20 μL injection. The peak shape of each homologue was symmetrical without interference, and the detected resolution was exactly. After methodological evaluation, the method was proved that it had good precision and sensitivity, the linear correlation coefficients of the standard curves were more than 0.999, the detection limit was 0.06-0.19 μg·mL -1, the recovery rate was 81.16%-112.92%, and the precision standard deviation was 0.02%-4.02%. Therefore, this method could be applied to determine the composition of ARs homologues. The composition of ARs homologues and total amount of ARs in different samples were found significantly after thermal cluster analysis (P<0.05). Among the ARs homologues of wheat flour samples, C21:0 was the highest, which accounted for 33%-61%; followed by C19:0, which accounted for 12%-44%; while the ratio of C17:0, C23:0 and C25:0 were the least. The content and abundance of ARs homologues in the whole wheat flour of United States and Canada were similar. The content of ARs homologues in abroad whole wheat flour was higher than the whole wheat flour in domestic. The composition of ARs homologues in domestic whole wheat flour was more abundant than refined wheat flour, except for individual refined wheat flour samples. The composition of homologous and total content of ARs in wheat bran F1 were significantly higher than whole wheat flour and refined wheat flour (P<0.05). The total content of ARs in wheat bran F1 was 1 795.78 μg·g-1, which was the most significantly difference compared with other samples (P<0.05). The results of PCA showed that the main difference index between wheat flour samples was the composition of ARs homologues. As the first principal component, the cumulative contribution rate was 98.40%. The relative dispersion between wheat bran and other wheat flour samples was the farthest, while the dispersion of Chinese refined wheat flour was the largest. Even the whole wheat flour of United States and Canada could nearly overlapped, the whole wheat flour and part of refined wheat flour of China could only partially overlapped.【Conclusion】 The HPLC method could be employed to effectively and rapidly quantify the ARs in whole wheat flour products. Moreover, the abundance of ARs homologues in whole wheat flour was better than that of ARs homologues in refined wheat flour under comparative analysis. In summary, the results could provide a theoretical basis for authenticity discrimination and quality control for whole wheat flour by exploring the composition of ARs homologues.

    Quality Improvement of Rapeseed Meal Based on Static-State Fermented with Mixed Microorganisms
    HAO YiNing,WANG ZhiGao,HE Rong,JU XingRong,YUAN Jian
    Scientia Agricultura Sinica. 2020, 53(10):  2066-2077.  doi:10.3864/j.issn.0578-1752.2020.10.013
    Abstract ( 550 )   HTML ( 11 )   PDF (3030KB) ( 534 )   Save
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    【Objective】A study was conducted to investigate effects of innoculation with a combination of Bacillus subtilis and Actinomucor elegans on the nutritional value and sensory characteristics of double-low rapeseed meal, and to evaluate the safety by in vivo experiment, so as to provide a theoretical basis for the utilization and deep development of rapeseed meal in the food industry. 【Method】The double-low rapeseed meal was used as the research object, and the static-state fermentation was used instead of the traditional stirring-state fermentation. The nutritional components (crude protein, water-soluble protein, small peptide, amino acid and tetramethylpyrazine) and anti-nutrient components (glucosinolates, phytic acid and crude fiber) of rapeseed meal after fermentation were determined, and then based on the odor and taste differences of electronic nose and electronic tongue, the correlation analysis and principal component analysis were used to evaluate the effect of static fermentation of mixed bacteria on quality of double-low rapeseed meal. The effects of fermented rapeseed meal on the growth performance of rats were studied preliminarily. These studies provided a theoretical basis for the application of fermented rapeseed meal products. 【Result】The results showed that soluble protein, peptides and total amino acid were increased by 96.7%, 281.48% and 19.58%, respectively after fermentation. Most rapeseed proteins were degraded into oligopeptides and amino acids with molecular weight between 500 Da and 108 Da. Tetramethylpyrazine, a new nutrient, was detected after fermentation. The concentration of tetramethylpyrazine reached 590 mg?kg-1 on the fifth day of fermentation. The contents of glucosinolates, phytic acid and crude fibers were decreased by 45.26%, 41.37% and 31.16%, respectively. The results of electronic nose and electronic tongue analysis showed that the flavor and taste of rapeseed meal changed significantly before and after fermentation, and the acid taste of rapeseed meal increased significantly after fermentation. Animal experiments showed that the addition of fermented rapeseed meal and other nitrogen instead of 25% soybean meal to rat diet could significantly increase the average daily intake and average daily weight gain of rats, and there was no damage to the kidney, thymus and liver of rats. 【Conclusion】The inoculant and fermentation method in the study could improve the nutritional value and flavor properties, and the fermented rapeseed meal could improve the growth performance of rats.

    Effects of Hot Air-Vacuum Freeze Combined with Drying on Physical Properties and Prebiotic Activities of Brittle Dried Longan
    DENG YuanYuan,YANG Jing,WEI ZhenCheng,ZHANG Yan,LIU Guang,ZHANG RuiFen,TANG XiaoJun,WANG JiaJia,LIAO Na,ZHANG MingWei
    Scientia Agricultura Sinica. 2020, 53(10):  2078-2090.  doi:10.3864/j.issn.0578-1752.2020.10.014
    Abstract ( 414 )   HTML ( 14 )   PDF (527KB) ( 598 )   Save
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    【Objective】 The effects of hot air-vacuum freeze combined with drying (HA-VFCD) on the physical properties and prebiotic activities of high-quality brittle dried longan were investigated in the present study, so as to provide a theoretical basis for the optimal industrialized energy-saving drying mode. 【Method】 In present study, the effect of HA-VFCD on the physicochemical and nutritional characteristics, such as moisture content, water activity, shrinkage rate, water rehydration ratio, total sugar and polysaccharide contents of brittle dried longan, were compared with that of individual hot air drying (HAD) and vacuum freeze drying (VFD) longan samples. The flavor substance changes and the energy consumption were also evaluated by GC-MS and ammeter, respectively. Furthermore, the dried longan was fermented by Lactobacillus plantarum and Lactobacillus acidophilus to investigate the content changes of viable bacteria, total sugar, reducing sugar and short-chain fatty acid, thus evaluating the effect of HA-VFCD on the in vitro prebiotic activities of longan. 【Result】 Longan dried by HA-VFCD showed significantly lower moisture content, water activity and shrinkage rate, but higher rehydration ratio, compared with the HAD counterparts. The content of total sugar, polysaccharide, and the types and amounts of volatile flavor of longan dried by HA-VFCD were lower than those of VFD samples, but higher than that of HAD counterparts. In addition, HA-VFCD process could save 12.16% of drying time and 25.40% of unit energy consumption, compared with VFD process. Both Lactobacillus plantarum and Lactobacillus acidophilus could increase the number of viable bacteria through fermentation of dried longan, which were used the total sugar in dried longan to produce short-chain fatty acids and to reduce the pH of fermentation broth. And the prebiotic activities of dried longan after fermentation were remarkably affected by the drying methods and the strain types of bacteria. After fermentation by the Lactobacillus plantarumfor 48 hours, the viable bacteria number of longan dried by HA-VFCD achieved the highest value of 12.40 lg cfu/mL, which was higher than that of HAD, VFD and fresh longan samples. After fermentation by the Lactobacillus acidophilus for 48 hours, the viable bacteria number of longan dried by HA-VFCD reached 11.84 lg cfu/mL, which was close to that of VFD samples, lower than HAD samples but higher than that of fresh longan. 【Conclusion】 The HA-VFCD process combined both the advantages of HAD and VFD, which could remarkably shorten the drying time, lower the energy consumption, and improve the drying efficiency and quality of dried longan.

    Effects of Protein Supplements on Agronomic Characters and Quality of the Mushroom Agaricus bisporus
    ZHANG WenQiang,CHEN QingJun,ZHANG GuoQing,SHI ShiDa,CAO Na,ABLAT· Tohtirjap,GUO YuXin,LIN WenCai
    Scientia Agricultura Sinica. 2020, 53(10):  2091-2100.  doi:10.3864/j.issn.0578-1752.2020.10.015
    Abstract ( 373 )   HTML ( 11 )   PDF (483KB) ( 284 )   Save
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    【Objective】 The effects of protein supplements on the agronomic characters and quality of mushroom Agaricus bisporus were studied in the study, so as to provide a reference for the development, application and evaluation of Agaricus bisporus protein supplements. 【Method】 Four single protein raw materials were investigated as protein supplements, including soybean meal (SM), extruded soybean (ES), corn protein (CP) and Feather meal (FM). Commercial supplement (MCSubstradd?, MCS) was used as a positive control, and an unsupplemented compost was used as a blank control (CTL). The dosage of each supplement was determined based on their protein content and with a final protein dosage of 0.5% in the compost. Before casing, the supplements were thoroughly mixed with fresh Phase III compost. The mushroom strain was W192. Mushroom cultivation was carried out in accordance with the factory cultivation process mushroom room management. Three flush mushrooms were harvested, and yield, diameter and thickness of pileus, weight of single mushroom, and hardness characters were determined with reference to UPOV. Protein content was determined by kjeldahl method, and amino acid content was determined by amino acid analyzer. 【Result】 SM, ES and CP could effectively increase the mushroom yield. Among the four experimental groups, CP group manifested the highest total yield of 35.24 kg?m-2 with the yield increasing rate of 24.26% towards CTL group. Its biological efficiency was 105.98%, which was significantly higher than that of the other three assayed groups and CTL group, and slightly higher than that of MCS group. CP group demonstrated considerably high sustained-release effect with three flush yield of 48.72%, 35.09%, 16.19%, respectively, which was close to that of MCS group. Economic benefit analysis showed that CP group demonstrated the highest increasing benefit of 785 yuan in one ton compost. ES group showed high yield with significantly high hardness character in the first two flush. All the five protein supplements could significantly enhance the protein content in the fruiting bodies in all the three flush. In all the groups, the three amino acids with the highest content were Glu, Val, and Asp. FM and CP could enhance the content of delicious amino acids, which raised the flavor and taste of mushrooms. 【Conclusion】 Protein supplements could effectively increase mushroom yield, and had specific effects on the agronomic characters and qualities of fruiting bodies. Focusing on specific advantages of various additives, developing mixed and sustained-release supplements could achieve high quality and high yield of A. bisporus, which was the research direction of mushroom supplements in the future.

    ANIMAL SCIENCE·VETERINARY SCIENCE·RESOURCE INSECT
    Effects of Soybean Isoflavones on Growth Performance, Antioxidant Performance and Intestinal Morphology of Early-Weaned Piglets
    LIN XiaJing,CHEN Fang,JIANG ShouQun,JIANG ZongYong
    Scientia Agricultura Sinica. 2020, 53(10):  2101-2111.  doi:10.3864/j.issn.0578-1752.2020.10.016
    Abstract ( 503 )   HTML ( 46 )   PDF (3011KB) ( 509 )   Save
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    【Objective】This experiment was conducted to investigate the effects of soy isoflavone (SI) on growth performance and its antioxidation of early-weaned piglets. 【Method】One hundred and sixty weaned piglets (Duroc×Landrace×Yorkshire) were divided by body weight and sex into five treatments with four replicates of eight piglets in each treatment. The piglets were fed either a control diet or the control diet supplemented with 10, 20, 40 and 80 mg·kg-1 of soy isoflavone, respectively. Two piglets per pen were slaughtered on the first and sixth week. 【Result】The average daily feed intake of 40 mg·kg-1 SI group was significantly higher than that of the control group (P<0.05) on day 8-42 after weaning and the whole experimental period. On day 8-42 after weaning, the feed to gain ratio of 40 mg·kg-1 SI group was significantly lower than that of the control, 10 and 20 mg·kg-1 SI groups (P<0.05). The MDA content of liver under the 40 and 80 mg·kg-1 SI groups was significantly lower than that in the control and 10 mg·kg-1 groups. The serum SOD activity of piglets under the 20 mg·kg-1 SI group was significantly higher than that under the control group, 40 and 80 mg·kg-1 SI groups (P<0.05). The GSH-px activity of liver tissue under the 10 mg·kg-1 SI group was significantly higher than that under the control group and 80 mg·kg-1 SI group (P<0.05). On day 42 after weaning, the SOD of activity serum under the 40 mg·kg-1 SI group was significantly higher than that under the control group, 10 and 80 mg·kg-1 SI groups (P<0.05). The GSH-px activity of liver under 20 mg·kg-1 SI group was significantly higher than that under the control group and 80 mg·kg-1 SI group (P<0.05). On day 42 after weaning, the SOD activity of jejunal under the 40 and 80 mg·kg-1 groups was significantly higher than that under the control group (P<0.05).The GSH-px activity of jejunum under the 40 mg·kg-1 SI group was significantly higher than that under the control group and 10 mg·kg-1 SI group (P<0.05). Dietary SI had a significant effect on MT content in jejunum, and the MT content of jejunum under the 10 mg·kg-1 SI group was significantly higher than that under the control group (P<0.05). On day 7 and 42 after weaning, the duodenal villi of piglets under the control group were arranged in tongue shape, and the top of the villi were dented and shed seriously. Compared with the control group, the damage degree of the duodenal villi in each treatment groups were reduced, the duodenal villi of piglets under the 40 mg·kg-1 group were the most complete villi. The addition of SI had a significant effect on the CD4+ level of the blood, but had no significant effect on lymphocyte conversion, CD8+, CD4+/ CD8+ (P>0.05) on day 8-42 after weaning. The CD4+ level of the 10 and 20 mg·kg-1 SI group was significantly lower than that under the control group and 80 mg·kg-1 SI group (P<0.05). 【Conclusion】 Dietary SI could improve the growth performance, antioxidant levels of 21-day-old weaned piglets, and the optimal level of SI was 40 mg·kg-1.

    Establishment and Preliminary Application of Microagglutination Detection Method for Salmonella Abortus Equi
    GUO Kui,WANG Ning,WANG JinHui,CHU XiaoYu,ZHAO YuTing,GUO Wei,LIU DiQiu,HU Zhe,WANG XiaoJun
    Scientia Agricultura Sinica. 2020, 53(10):  2112-2121.  doi:10.3864/j.issn.0578-1752.2020.10.017
    Abstract ( 510 )   HTML ( 18 )   PDF (568KB) ( 671 )   Save
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    【Objective】Salmonella abortus equi was isolated and identified from the tissue of aborted foal. The aim for the study was to establish a sensitive, specific, easy to operate and high throughput microagglutination method by using the isolated Salmonella abortus equi, so as to realize the rapid diagnosis of Salmonella abortus equi antibody. 【Method】Selective medium for Salmonella was used for bacteria reproduction from aborted foal organs, and the purple colonies were selected and identified by Gram staining. The whole genome of bacteria was extracted, and 16S rRNA was used for PCR amplification and sequencing identification. Biochemical identification and serotype identification of the bacteria were carried out to make a comprehensive diagnosis of the etiology. The obtained positive bacteria was used as agglutinating antigen after inactivation, and the standard positive serum of Salmonella was used as positive control. The microagglutination assay was established by optimizing the reaction conditions, and then the specificity, sensitivity and repeatability of the method were verified. Meanwhile, a total of 151 serum samples from three different locations in north and northwest of China were detected with microagglutination. And 30 of these samples were randomly selected to be detected with both microagglutination and test tube agglutination, and the sensitivities of these two methods were compared and analyzed.【Result】A large number of purple target colonies could be obtained from the coloration culture medium of Salmonella. The results of gram staining showed that the isolated strains were gram-negative short bacillus, and 16S rRNA sequencing confirmed that the isolated strains were Salmonella. The biochemical identification results showed that all the 17 isolated strains were in line with the biochemical characteristics of Salmonella abortus equine, but could not ferment rhamnosus compared with C77-1, a strong strain of Salmonella abortus equi identified in China in the last century. Serotype identification confirmed that all isolated strains were Salmonella abortus equi, and the 17 isolated strains were named as ES-1-17 respectively. Therefore, it was confirmed that the abortion of horses was caused by Salmonella abortus equi infection. The agglutination antigen of Salmonella abortus equi was prepared by inactivating E.S-1 strain with formaldehyde, and a rapid and sensitive method of microagglutination was established for the detection of agglutination, in which the optimal concentration of agglutination antigen was 400 million mL-1. The sensitivity was 1 titer higher than that of test tube agglutination, and the specificity was good without any cross reaction with sera against other equine infectious pathogens, and it had good repeatability. Equine sera collected from three epidemic areas in North China were detected with microagglutination for antibodies against Salmonella abortus equi, and the positive rates were 28.6%, 42.9% and 27.3%, respectively, while the positive rate was 0% in three non-epidemic areas in Northwest China. In addition, 30 samples of the above clinical serum were detected by these two methods, 10 of 33 were positive for microagglutination and 3 of 33 were positive for test tube agglutination with positive rates of 33% and 10%, respectively. The sensitivity and specificity of microagglutination were 100% and 74.1%, respectively, and the total coincidence rate was 76.7%.【Conclusion】Seventeen strains of Salmonella equi abortion were isolated and identified in this study. A microagglutinating antibody detection method for Salmonella abortus equi was established. This method was of high sensitivity, specificity and good repeatability, simple, rapid, high throughput, could be widely used in the horse abortion antibody detection of Salmonella in the animal, and was one of the important basis for evaluation of the disease diagnosis and vaccine.

    Investigation of Competing Endogenous RNA Regulatory Network and Putative Function of Long Non-Coding RNAs in Nosema ceranae Spore
    ZHOU DingDing,SHI XiaoYu,WANG Jie,FAN YuanChan,ZHU ZhiWei,JIANG HaiBin,FAN XiaoXue,XIONG CuiLing,ZHENG YanZhen,FU ZhongMin,XU GuoJun,CHEN DaFu,GUO Rui
    Scientia Agricultura Sinica. 2020, 53(10):  2122-2136.  doi:10.3864/j.issn.0578-1752.2020.10.018
    Abstract ( 331 )   HTML ( 12 )   PDF (2655KB) ( 291 )   Save
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    【Background】Long non-coding RNAs (lncRNAs) are a kind of non-coding transcripts with a length of more than 200 nt. LncRNAs can regulate gene expression at transcriptional level and post-transcriptional level via cis effect, trans effect or competing endogenous RNA (ceRNA) mechanism. LncRNAs have been proved to play pivotal roles in many biological processes such as growth and development.【Objective】The objective of this study is to perform detailed investigation and discussion of the regulatory manner of lncRNAs by combining small RNA (sRNA) omics dataset obtained in this work and previously gained lncRNA omics dataset, and to reveal the putative function of lncRNAs in Nosema ceranae spores.【Method】Here, purified spores of N. ceranae were sequenced using small RNA-seq (sRNA-seq), related bioinformatics software was used to conduct quality control of sequencing data. The upstream and downstream genes of N. ceranae lncRNAs were predicted based on the positional relationship between lncRNAs and mRNAs. Annotations of these upstream and downstream genes in GO and KEGG databases were carried out using Blast software. The lncRNA-miRNA and lncRNA-miRNA-mRNA regulatory networks were predicted using Target Finder software and then visualized with Cytoscape v3.7.2 software. The expression of partial lncRNAs, miRNAs and mRNAs within regulatory networks was validated with RT-PCR.【Result】In total, 16 597 883, 15 451 791 and 12 248 316 raw reads were obtained from sRNA-seq of N. ceranae spore samples, respectively, and after quality control, 15 608 370, 14 249 255 and 11 440 684 clean reads with a mean Q30 above 98.58% were gained, respectively. A total of 310 upstream and downstream genes of lncRNAs were predicted. These genes could be annotated to 35 functional terms associated with metabolic process, cell process, catalytic activity, binding and cells, etc. Additionally, these genes could be annotated to 56 metabolic pathways, including material metabolic pathways such as purine metabolism, carbon metabolism, pyruvate metabolism; and energy metabolic pathways including methane metabolism, oxidative phosphorylation, glycolysis/gluconeogenesis and so forth. The results indicated that corresponding lncRNAs could regulate the expression level of upstream and downstream genes through cis effect, thus participating in regulation of material and energy metabolisms as well as cell activities in N. ceranae spore. Furthermore, lncRNA-miRNA regulatory networks were constructed and analyzed, the result showed that MSTRG.3636.1, MSTRG.4498.1 and MSTRG.4883.1 could bind to four miRNAs including nce-miR-7729, nce-miR-7502, nce-miR-8639 and nce-miR-8565, suggesting that these three lncRNAs as ceRNAs could exert potential function in N. ceranae spore. LncRNA-miRNA-mRNA regulatory network analysis demonstrated that complex networks existed among them, miRNAs were located in the center, connecting lncRNAs and mRNAs, nce-miR-7502 and nce-miR-8639 could bind to the most mRNAs (28). MiRNAs targeting MSTRG.3636.1, MSTRG.4498.1 and MSTRG.4883.1 could target several mRNAs, indicating that these three lncRNAs might play a role via ceRNA mechanism, thus affecting the mechanism and vital activity in N. ceranae spore.【Conclusion】LncRNAs are likely to regulate the expression of upstream and downstream genes via cis effect, and indirectly affect the expression of target genes by absorbing miRNAs as ceRNAs, thus controlling basic activities in N. ceranae spore such as material metabolism and energy metabolism.