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Table of Content

    16 April 2018, Volume 51 Issue 8
    CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS
    SLAF-marker Development and Its Application in BSA Analysis of Cellulose Content in Pericarp of Maize Kernel
    DU LongGang, WANG MeiXing
    Scientia Agricultura Sinica. 2018, 51(8):  1421-1430.  doi:10.3864/j.issn.0578-1752.2018.08.001
    Abstract ( 437 )   HTML ( 34 )   PDF (1987KB) ( 643 )   Save
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    【Objective】 Reducing cellulose content in pericarp is one of the important goals for quality improvement of sweet maize. However, the research focusing on cellulose content in pericarp of maize is limited and the related gene has not been identified. In order to map the chromosome regions and candidate genes controlling cellulose content in pericarp of sweet maize, specific-locus amplified fragment-sequencing (SLAF-seq) and bulked segregant analysis (BSA) were conducted in this study. 【Method】 A recombinant inbred line (RIL) population was constructed using E327 and G5-1 as parents. In F6 generation of RILs, the cellulose contents in pericarp of each line were detected. According to cellulose content results, the lines with relatively high and low cellulose contents in pericarp were selected for SLAF tags identification and BSA. For BSA, DNA was extracted from two bulked populations as well as two parent lines and digested with HaeⅢ and Hpy166Ⅱ restriction enzymes. After digestion, the 414-464 bp DNA fragments were recovered and used for Illumina library construction which were subsequently sequenced by 2nd generation sequencing technology. Based on the polymorphic SLAF tags developed in this analysis, the SNPs-cellulose content association analysis was performed to map the chromosome regions associated with cellulose content in pericarp of sweet maize. Then, the genes located on the associated regions were found. To further reduce the number of candidate genes, the genes in Arabidopsis homological to these genes were identified and annotated. Based on the published works related on the functional analysis of these Arabidopsis genes, we further identified the candidate genes regulating cellulose content in pericarp of sweet maize. 【Result】The sequencing results of Illumina libraries were consistent with expected. As a result, 73 786 polymorphic SLAF tags were identified, which were uniformly distributed on 10 chromosomes. A total of 523 395 SNPs in those polymorphic tags were identified from the SLAF-sequencing data. Genes responsible for cellulose content in pericarp were mapped onto 6 chromosome regions of maize genome via association analysis and all these 6 chromosome regions were located on the 5th chromosome. In total, 47 gene loci in those regions and 9 genes in these associated regions were identified as candidate genes in further analysis. 【Conclusion】Through SLAF-sequencing based bulking segregated analysis, the cellulose content related genes in pericarp of sweet maize were mapped, suggesting that this method can be applied in gene mapping for other traits.
    Gene Expression Profiling of Foxtail Millet (Setaria italica L.) Under Drought Stress During Germination
    XU BingXia, YIN MeiQiang, WEN YinYuan, PEI ShuaiShuai, KE ZhenJin, ZHANG Bin, YUAN XiangYang
    Scientia Agricultura Sinica. 2018, 51(8):  1431-1447.  doi:10.3864/j.issn.0578-1752.2018.08.002
    Abstract ( 409 )   HTML ( 23 )   PDF (3693KB) ( 853 )   Save
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    【Objective】Foxtail millet (Setaria italica L.) is a drought tolerant crop. The objective of this research is to get a lot of differently expressed genes during germination in response to drought stress by high-throughput sequencing, then to obtain the key gene and the related molecular mechanism at seed germination stage in foxtail millet under drought stress.【Method】Seed of JinGu45 was treated with 18%PEG-6000(PEG-stress)and distilled water germination(control sample) at 1 h, 10 h and 18 h as a test material, and the activities of SOD, POD and CAT were measured, respectively. SOD activity was assayed by nitro blue tetrazolium (NBT) method. POD activity was determined by guaiacol method, and the activity of CAT was measured by colorimetric method. Sample of control and PEG-stress that germinated for 10h and 18h were used to construct cDNA library by gene expression profiling technology. We compared reads to the reference genome by using Bowtie and analysed the result by using RSEM. Differential expression analysis used DESeq. The functional annotation of differently expresse genes were obtained by using NR, Swiss-Prot, KEGG, COG and GO online databases. The key genes that regulate germination in foxtail millet was obtained through analyse DEGs. The reliability of sequencing results was comfirmed by qRT-PCR. 【Result】The SOD activity of PEG-stress sample was higher than that in the control sample, but the activity of POD and CAT were lower than that in the control group. With the time of germination changes, the activity of SOD was increased, but the activity of CAT and POD was gradually decreased. The sequences of gene expression profile was highly consistent with the selected reference genome sequence, and the gene expression was highly heterogeneous. Expression analysis showed a total of 35470 genes, and with the selection criteria of RPKM ≥0.01, there were 24030 and 24 486 genes in the control samples and 24 019 and 23 877 genes in the samples under PEG drought stress, respectively. 456 and 545 DEGs were screened out during millet germination at 10h and 18h under drought stress, in which 87 and 267 DEGs were up-regulated and 369 and 278 DEGs were down-regulated. GO enrichment analysis showed that these DEGs were mainly relating to metabolism process, cell stimulation and response process. The KEGG enrichment analysis showed that these DEGs were associated with phenylpropanoid metabolism and plant hormone signal transduction. The results obtained from five genes tested by RT-PCR agreed with the trend of regulation identified by gene expression profile. 【Conclusion】DEGs were widely involved in the metabolism of biomacromolecule such as sugar, protein, nucleic acid, secondary metabolism and energy metabolism. SnRK2 and PAL genes may regulate seed germination in foxtail millet under drought stress.
    TILLAGE & CULTIVATION·PHYSIOLOGY & BIOCHEMISTRY·AGRICULTURE INFORMATION TECHNOLOGY
    Effects of Topdressing Magnesium and Zinc Fertilizers at Different Periods on Yield and Quality of Japonica Rice with Good Eating Quality
    LI Jun, XIAO DanDan, DENG XianLiang, ZHU DaWei, XING ZhiPeng, HU YaJie, CUI PeiYuan, GUO BaoWei, WEI HaiYan, ZHANG HongCheng
    Scientia Agricultura Sinica. 2018, 51(8):  1448-1463.  doi:10.3864/j.issn.0578-1752.2018.08.003
    Abstract ( 322 )   HTML ( 19 )   PDF (420KB) ( 580 )   Save
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    【Objective】The objective of this experiment was to investigate the effects of topdressing magnesium and zinc microelement fertilizers at different periods on grain yield and quality of japonica rice with good eating quality. The results could provide a theoretical basis for optimum cultivation of japonica rice with good eating quality. 【Method】Japonica rice with good eating quality including Nanjing 9108 and Nanjing 505 were adopted as test materials. The effects of microelement fertilizer (Mg fertilizer, Zn fertilizer, and Mg-Zn mixed fertilizer) topdressing at different fertilizing stages (basic fertilizer stage, tillering stage, and panicle initiation stage) on the grain yield and rice quality were studied, and set no magnesium zinc fertilizer treatment as CK. 【Result】With the delay of fertilization time, the topdressing Mg fertilizer treatments increased rice grain yield, while the treatments of topdressing Zn fertilizer showed the opposite trend, the grain yield of topdressing mixed Mg-Zn fertilizer decreased first and then increased. Compared with CK , the yield were increased significantly when topdressing Mg fertilizer and mixed Mg-Zn fertilizer at panicle initiation stage, and the highest yield were obtained with mixed Mg-Zn fertilizer. Although the spikelets number per panicle and seed setting rate were decreased with mixed Mg-Zn fertilizer topdressing at panicle initiation stage, the 1000-grain weight and the total spikelets were increased. The process quality was improved with Zn fertilizer topdressed at tillering or panicle initiation stage. With Mg fertilizer topdressed at panicle initiation stage, both the process and the appearance quality were significantly increased. And the best tasting quality was achieved with the lowest protein content and setback value, and with the highest amylose content and breakdown value. Compared with the treatment of topdressing Mg at panicle initiation, although the process quality could be improved with the mixed Mg and Zn, the quality of appearance, tasting and nutrition were deteriorated because of the decreased peak viscosity, trough viscosity and breakdown, and the increased setback and pasting temperature.【Conclusion】Therefore, the topdressing of Mg at panicle initiation stage could not only increase the grain yield, but also improve the rain quality of rice, which could be one of the best ways to optimize rice quality in the high yield production.
    Validation of an Unmanned Aerial Vehicle Hyperspectral Sensor and Its Application in Maize Leaf Area Index Estimation
    CHEN PengFei, LI Gang, SHI YaJiao, XU ZhiTao, YANG FenTuan, CAO QingJun
    Scientia Agricultura Sinica. 2018, 51(8):  1464-1474.  doi:10.3864/j.issn.0578-1752.2018.08.004
    Abstract ( 324 )   HTML ( 11 )   PDF (763KB) ( 523 )   Save
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    【Objective】 The objective of this study is to validate the unmanned aerial vehicle (UAV) hyperspectral sensor S185, and then to design a new method for maize leaf area index (LAI) estimation based on its collected image. 【Method】 Taking maize in northeastern China as study material, nitrogen fertilizer experiment was conducted in Gongzhuling city in Jilin province. In the experiment, five nitrogen treatments and three replications were applied. The UAV flight experiment, the ground spectrum and LAI were measured at V5-V6, V11, R1-R2 growth stage (Ritchie growth stage) of maize. At last, 45 groups of data were collected. To validate images from hyperspectral sensor S185, the spectra from S185 image and from ground spectral device were extracted and compared in the same scale. On the one hand, the correlation analysis was taken to analysis the relationship between S185 data and ground measured spectra, which were from the same target; On the other hand, 15 commonly used spectral indices were selected, and then they were calculated from S185 date and from the ground spectra, respectively, and at last, the relationship between them during the whole maize growth stage was analyzed to show their change trend consistency. 30 groups of data were randomly selected from the collected 45 groups of data, artificial neural network method (ANN) was used to establish LAI prediction model by using S185 images, and then the remainder 15 groups of data were used to validate the performance of ANN model. In addition, based on the same calibration and validation dataset, LAI prediction models were designed by using each selected spectral index individually in order to compare with ANN LAI prediction model. 【Result】 In each maize growth stage, S185 spectra had a high relationship with corresponding spectra measured by ground spectral device for the same target, with correlation coefficients higher than 0.99; in the whole maize growth stage, the calculated spectral indices from the S185 images had a high relationship with the corresponding value calculated from ground measured spectra, with correlation coefficient higher than 0.88; During the design of the ANN prediction model for LAI, the model had an R2 value of 0.96, a RMSE value of 0.42 and a RMSE% value of 13.15% during calibration and had an R2 value of 0.95, an RMSE value of 0.54 and an RMSE% value of 16.74% during external validation. The model performed better than models designed by spectral indices. 【Conclusion】 The results showed S185 can be mounted on UAV to measure maize canopy hyperspectra image accurately, and ANN method can be used to design LAI prediction model based on UAV.
    PLANT PROTECTION
    Establishment of Sensitivity Baseline and Evaluation of Field Control Efficacy of Fludioxonil Against Fulvia fulva
    HE LiFei, CHEN LeLe, XIAO Bin, ZHAO ShiFeng, LI XiuHuan, MU Wei, LIU Feng
    Scientia Agricultura Sinica. 2018, 51(8):  1475-1483.  doi:10.3864/j.issn.0578-1752.2018.08.005
    Abstract ( 334 )   HTML ( 7 )   PDF (407KB) ( 261 )   Save
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    ObjectiveTomato leaf mold is one of the most devastating diseases that endanger greenhouse tomato. At present, Fulvia fulva has been reported to develop resistance to common fungicides, and thus the control efficacy reduced. It is necessary to develop and introduce the new and effective alternative fungicides. The objective of this study is to investigate the sensitivity of F. fulva to fludioxonil, establish baseline toxicity and definite the effect of disease prevention of fludioxonil in the field.【Method】The strains of tomato leaf mold were collected from six vegetable production areas, including Ji’nan, Taian, Liaocheng, Weifang, Laiwu and Zibo in Shandong Province. After isolation and purification, 126 strains of F. fulva were obtained. With the methods of inhibition rate on mycelial growth and spore germination, the inhibitory activity of fludioxonil against mycelial growth, spore germination and germ tube elongation at different growth and development stages of 3 representative strains was determined. The sensitivity of 126 strains from different regions in Shandong to fludioxonil was determined by mycelial growth rate method, and the sensitive baseline was also established. Two-year field trials were conducted to evaluate the protective and curative effects of fludioxonil.【Result】Fludioxonil had high inhibitory activity against germ tube elongation and mycelial growth, the average EC50 values were 0.30 and 0.80 μg·mL-1, respectively, but the inhibition of spore germination of F. fulva was weak, the average EC50 values were greater than 100 μg·mL-1. There was no significant difference in the sensitivity of the strains among different populations of tomato leaf mold. Among them, the strains from Weifang had the highest sensitivity to fludioxonil, and the average EC50 value was 0.43 μg·mL-1. The sensitivity of strains from Zibo was relatively low, with an average EC50 value of 0.79 μg·mL-1. There was no significant difference in the sensitivity of F. fulva in Taian, Ji’nan, Liaocheng and Laiwu areas, with average EC50 values of 0.65, 0.75, 0.71, 0.58 μg·mL-1, respectively. EC50 values to fludioxonil ranged from 0.16 to 1.69 μg·mL-1, with a mean of 0.64 μg·mL-1. The average EC50 value of F. fulva to fludioxonil could be regarded as the baseline sensitivity as the sensitivity frequency complied with single peak curve of Gaussian distribution. In 2016-2017, fludioxonil had high control effect on tomato leaf mold, the protective and curative activity of fludioxonil on tomato leaf mold was 72.21%-75.02% and 61.94%-70.65% at the dose of 60.75 g a.i./hm2, which was significantly higher than that of difenoconazole at 100 g a.i./hm2, mancozeb at 700 g a.i./hm2 and thiophanate-methyl at 540 g a.i./hm2, and which had no significant difference with fluopyram at 150 g a.i./hm2. The protective and curative activity of fludioxonil on tomato leaf mold at the dose of 40.50 g a.i./hm2 was significantly higher than that of thiophanate-methyl at 540 g a.i./hm2. In 2017, the protective and curative activity of fludioxonil on tomato leaf mold at the dose of 20.25 g a.i./hm2 was significantly higher than that of mancozeb at 700 g a.i./hm2 and thiophanate-methyl at 540 g a.i./hm2. All the protective effect of fludioxonil against tomato leaf mold was higher than that of the curative ones.【Conclusion】Fludioxonil has good inhibitive activity against germ tube elongation and mycelial growth of F. fulva, but had no effect on spore germination, and the strains from six cities in Shandong Province are relatively sensitive to it. Fludioxonil could be applied before the invasion of the pathogen and has great potential in the control of tomato leaf mold.

    Growth Kinetics and Virulence of Two Beauveria bassiana Strains in Frankliniella occidentalis Under Different Temperatures
    LIU XiaoChen, WU ShengYong, LEI ZhongRen, WANG HaiHong
    Scientia Agricultura Sinica. 2018, 51(8):  1484-1492.  doi:10.3864/j.issn.0578-1752.2018.08.006
    Abstract ( 292 )   HTML ( 9 )   PDF (410KB) ( 480 )   Save
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    【Objective】The objective of this study is to compare the growth kinetics and virulence of two strains of Beauveria bassiana infected Western flower thrips (Frankliniella occidentalis) under different temperatures, analyze the relationship between the proliferation and insecticidal virulence of B. bassiana, and to provide a theoretical support for improving the efficiency of B. bassiana against F. occidentalis.【Method】Firstly, from 1st to 8th day, the mortality of the infected F. occidentalis with two strains of B. bassiana (SCWJ-2 and GZGY-1-3) under 20, 25, and 30 was recorded, and cumulative corrected mortality was calculated while uninfected F. occidentalis was used as control. The mortality of F. occidentalis infected with the two strains at the 3rd day (the mortality of control was 2%-5%) was compared. Secondly, under 20, 25 and 30, the colony diameter of the two strains was recorded continuously from 1st to 8th day. The colony diameter of the two strains at the 3rd day was compared. Finally, the DNA mixture of F. occidentalis infected with fungi from 1st to 3rd day under 20, 25 and 30 was extracted, respectively. The real-time fluorescence quantitative PCR (qRT-PCR) was used to quantify the copy number of fungi within each insect host sample. The gene copy of two strains under the same temperature at the 3rd day was compared. 【Result】The bioassay results of B. bassiana against F. occidentalis showed that within the tested temperature range (20-30), both strains GZGY-1-3 and SCWJ-2 were highly lethal to F. occidentalis adults. No matter what the temperature or the strain, the dead individuals F. occidentalis appeared from the 2nd day after treatment. On the 8th day, the corrected mortality of F. occidentalis infected by strain SCWJ-2 and GZGY- 1-3 was 83%-91% and 79%-90%, respectively. The corrected mortality of F. occidentalis infected by the two strains on the 3rd day was compared (the morality of control were 2%-5%). the virulence of strain SCWJ-2 was significantly higher than that of GZGY-1-3 under 30 (P<0.05), but there was no significant difference between them under 25 and 20 (P>0.05). Within the tested temperature range from 20 to 30, the colony diameter of two strains increased with time. On the 8th day, the colony diameter of the strains SCWJ-2 and GZGY-1-3 was 31-36 and 28-32 mm, respectively. On the 3rd day, the colony diameter of strain SCWJ-2 was significantly larger than that of strain GZGY-1-3 under all test temperatures (P<0.05). The results of qRT-PCR showed that, with the exception of strain SCWJ-2 at 30, the gene copy number of both strains in F. occidentalis decreased on the 1st day and gradually increased after 2 days at all the three temperatures. The gene copy number of strain SCWJ-2 within F. occidentalis was significantly higher than that of strain GZGY-1-3 under 30 (P<0.05), but there was no significant difference between them under 25 and 20 (P>0.05). 【Conclusion】 The number of fungal gene copies within insect host was affected by strains and temperatures, which is in accordance with the result of bioassay. Compared with B. bassiana strain GZGY-1-3, strain SCWJ-2 is more suitable for controlling F. occidentalis under high temperatures.
    Cloning and Expression of Phosphatidylethanolamine Binding Protein in Helicoverpa armigera
    ZHAO Jie, REN SuWei, LIU Ning, AI XinYu, MA Ji, LIU XiaoNing
    Scientia Agricultura Sinica. 2018, 51(8):  1493-1503.  doi:10.3864/j.issn.0578-1752.2018.08.007
    Abstract ( 310 )   HTML ( 6 )   PDF (3351KB) ( 495 )   Save
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    【Objective】The objective of this study is to clone and analyze phosphatidylethanolamine binding protein (PEBP) gene from Helicoverpa armigera, investigate the temporal and spatial expression profile in H. armigera, and test the expression of HaPEBP after 2-tridecanone treatment in the 6th instar larval midgut of H. armigera. The results will provide a theoretical basis for further studying the function of HaPEBP and select the gene as a molecular target to regulate the population of H. armigera. 【Method】 The cDNA sequence of HaPEBP was obtained from midgut of 6th instar larvae by using RACE technique, and its amino acid sequence and protein structure were analyzed. The recombinant vector pET32a-HaPEBP was constructed and transformed it into Escherichia coli BL21 (DE3) strain. The fusion protein was induced by IPTG and identified by SDS-PAGE to confirm its distribution. the His-HaPEBP was purified using Ni-NTA affinity chromatography. The HaPEBP expression profile at different developmental stages, tissues and under 2-tridecanone treatments was determined by qRT-PCR. 【Result】 The HaPEBP cDNA sequence is 760 bp, and its ORF is 588 bp, encoding 195 amino acids. The predicted molecular weight and isoelectric point of the protein are 21.76 kD and 5.93, respectively. The HaPEBP is a cytoplasmic monomer protein without signal peptide, transmembrane region and disulfide bond, which consists of 4 α-helixes and 9 β-sheets. The soluble fusion protein, which was about 40 kD consistent with predicted 39.1 kD, was synthesized in BL21-pET32a-HaPEBP strain by 1 mmol·L-1 IPTG induced 4 h at 37℃. And then the pure His-HaPEBP (183.3 ng·μl-1) was obtained through Ni-NTA column and imidazole gradient buffers. HaPEBP was expressed at all larval stages (1st-6th instar larvae and prepupa), and the highest expression level was observed in the 6th instar larvae. It was also expressed in the fat body, midgut, head and integument of the 6th instar larvae and the highest expression was found in the fat body. The expression of HaPEBP in the midgut of 6th instar larvae decreased after treatment with different concentrations of 2-tridecanone. In the low concentration groups (2.5 and 5 mg·g-1), the expression of HaPEBP significantly decreased at 6 h and gradually increased over time. However, the expression of HaPEBP decreased at 12 h in the high concentrations (10 and 15 mg·g-1), the mRNA content decreased firstly and then increased over time. 【Conclusion】The HaPEBP cDNA was cloned and analyzed. The fusion protein His-HaPEBP was synthesized and purified using the prokaryotic expression system. The results of temporal and spatial expression showed that the HaPEBP was highly expressed in 6th instar larvae and fat body. The expression of HaPEBP significantly decreased after treatment with 2-tridecanone in the midgut of 6th instar larvae.
    SOIL & FERTILIZER·WATER-SAVING IRRIGATION·AGROECOLOGY & ENVIRONMENT
    Effects of Film Mulching, Nitrogen Fertilizer, Plant Density and Its Interaction on Nitrogen Accumulation, Translocation and Production Efficiency of Spring Maize on Dryland of Loess Plateau
    LI Ting, LI ShiQing, ZHAN Ai, LIU JianLiang
    Scientia Agricultura Sinica. 2018, 51(8):  1504-1517.  doi:10.3864/j.issn.0578-1752.2018.08.008
    Abstract ( 305 )   HTML ( 8 )   PDF (655KB) ( 376 )   Save
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    【Objective】A field study was to investigate the effects of film mulching, nitrogen fertilizer and plant density on nitrogen accumulation, translocation and production efficiency of spring maize. This research could provide theoretical basis for optimizing high yield and high efficiency cultivation of dryland maize in semi-arid regions.【Method】Treatments included two planting methods (mulching and non-mulching), four nitrogen rates (0, 170, 200 and 230 kgN·hm-2 in 2013 and 0, 170, 225 and 280 kgN·hm-2 in 2014), and three plant densities (5.0×104, 6.5×104 and 8.0×104 plants·hm-2) during 2013 and 2014 growing seasons. The nitrogen accumulation, translocation, grain yield and nitrogen partial factor productivity (PFPN) of different treatments were analyzed.【Result】Film mulching significantly increased N accumulation pre-silking, promoted N accumulation post-silking and N translocation during vegetative stage, and thus significantly increased grain N accumulation and yield. Mulching models×N fertilizer and Mulching models×plant density significantly affected N uptake, accumulation and translocation in spring maize. Under the film mulching, more N fertilizer amounts (200-230 kgN·hm-2) or plant density (6.5×104-8.0×104 plants·hm-2) improved N translocation during vegetative stage, N assimilation post-silking and its contribution to grain, so it significantly increased grain N accumulation. However, under non-mulching, above amount of 170 kgN·hm-2 or plant density of 5.0×104 plants·hm-2 decreased N accumulation post-silking and its contribution to grain, which failed to increase grain N accumulation. N fertilizer×plant density had a significant effect on the N uptake, accumulation and translocation. PFPN and N harvest index (NHI) were positively related to N accumulation pre- and post- silking, translocated N and grain yield, which reached at a significant level. Comprehensive considering grain yield and PFPN, the N application of 200-230 kgN·hm-2 and plant density of 8.0×104 plants·hm-2 with film mulching system could achieve high yield of 13.7-14.6 t·hm-2 and high PFPN of 64.8-68.7 kg·kg-1 in spring maize on the Loess Plateau in northwest China.【Conclusion】The integrated management practice with film mulching, optimal nitrogen rate and plant density could promote the synergistic increase of N accumulation post-silking and N translocation from vegetative organs during grain filling, which was the fundamental reason for increasing grain yield and N fertilizer production efficiency.
    Effects of Phosphorus Fertilization Depth on Yield and Root Distribution of Summer Maize
    YANG YunMa, SUN YanMing, JIA LiangLiang, JIA ShuLong, MENG ChunXiang
    Scientia Agricultura Sinica. 2018, 51(8):  1518-1526.  doi:10.3864/j.issn.0578-1752.2018.08.009
    Abstract ( 347 )   HTML ( 9 )   PDF (386KB) ( 324 )   Save
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    【Objective】This study focused on the effects of phosphorus fertilization depth on yield and root distribution of summer maize in the North China Plain. 【Method】 The research included a field experiment and a soil column experiment. The field experiment was carried out to determine the effects of different phosphorous fertilization positions on grain yield and nutrient uptake. The treatments were CK (no P), T-side (P fertilized at seed side), T-8 (P fertilized at 8 cm depth in the seed row), T-16 (P fertilized at 16 cm depth in the seed row), T-24 (P fertilized at 24 cm depth in the seed row), and T-all (P fertilized at 8 cm, 16 cm and 24 cm depth equally in the seed row). The soil column experiment was carried out to determine the effects of different phosphorous fertilization depths (P 8(8 cm); P 16(16 cm); P 24(24 cm); P-all, (8/16/24 cm)) on summer maize root distribution. 【Result】 Field experiment showed that the phosphorous fertilization depth significantly affected the summer maize grain yield. The maize yield decreased in the order of T-24, T-all, T-16, T-side, T-8 and CK, and the T-24 treatment significantly increased yield by 10% than the T-side treatment. The highest above ground plant P accumulation at the eight-leaf stage was the T-side treatment, and that in silking and harvest stage were T-8 and T-all treatment, respectively. The above ground N uptake at harvest stage showed a significantly increasing trend with the P increased application depth. The soil column experiment showed that the total root length for the P 24 treatment was the highest in all application depths. Compared with CK, P-all and P 8 treatments, the root length of P 24 significant increased by 68%, 18% and 17%, respectively. The maize roots were distributed around the application point of P fertilizer, and the total maize root length increased with the P fertilization depth increasing. 【Conclusion】 Phosphorus fertilizer deep application could induced the maize root grow to deep soil layer and increasing the root length, significantly increased the grain yield. The suitable P fertilizer application depth was 24 cm in this research.
    Effects of Different Slow-Released Nitrogen Fertilizers on Summer Maize and Nitrogen Fate in the Field
    ZHOU LiPing, YANG LiPing, BAI YouLu, LU YanLi, WANG Lei
    Scientia Agricultura Sinica. 2018, 51(8):  1527-1536.  doi:10.3864/j.issn.0578-1752.2018.08.010
    Abstract ( 368 )   HTML ( 13 )   PDF (423KB) ( 572 )   Save
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    【Objective】 The effects of different slow-released nitrogen (N) fertilizer on yield, N fate and N balance of summer maize were studied for providing a theoretical basis in improving N use efficiency and reducing the environmental impact. 【Method】A field in-situ experiment of two years was conducted in a sandy soil in North China Plain and maize variety “Zhengdan958” was used as materials. There were six N fertilizer treatments, including no N fertilizer (CK), urea (CU), polymer-coated urea (CRF), release-controlled urea (LCU), polymer gel urea (CLP) and urea formaldehyde (UF). Plant and soil samples were collected at the maturing stage of maize, and then the contents of N in plant and soil inorganic N were determined. Thereafter, the N uptake, N use efficiency, soil inorganic N accumulation and N loss were calculated. 【Result】(1)The N fertilizer slow-release treatments could obviously improve the yield of summer maize and promote N absorption. Compared with the urea, the average yield under UF, CLP, CRF and LCU increased by 18.9%, 16.8%, 13.7% and 13.6%, respectively, and the average N agronomic efficiency increased by 6.5 kg·kg-1, 4.8 kg·kg-1, 4.0 kg·kg-1 and 3.7 kg·kg-1, respectively. (2)There was a significant difference in the soil 0-100 cm N fertilizer residues between different N fertilizer treatments. The average N recovery efficiency of UF, CLP, CRF, LCU and CU was 54.9%, 42.4%, 38.3%, 38.3% and 22.0%, respectively, and the N fertilizer residue in 0-100 cm soil profile of them accounted for 28.3%, 43.8%, 39.2%, 46.2% and 46.6% of the applied N rate, respectively. Compared with urea, the loss of N fertilizer of CLP, LCU, UF and CRF decreased by 47.6%, 43.1%, 40.8% and 26.7%, respectively. (3) When analyzing the field N balance of different N fertilizer treatments comprehensively, the average N uptake of UF was the highest (245.0 kg·hm-2), followed by CLP which was 222.5 kg·hm-2. The average soil 0-100 cm N residues of UF was the lowest among the slow-released N fertilizers which was only 153.4 kg·hm-2, and the average soil 0-100 cm N residues of CRF, CLP and LCU was 173.1 kg·hm-2, 181.5 kg·hm-2 and 185.7 kg·hm-2, respectively. The average N apparent loss of CLP was the lowest (35.6 kg·hm-2) and that of LCU, UF and CRF was 38.8 kg·hm-2, 41.2 kg·hm-2 and 51.3 kg·hm-2, respectively. 【Conclusion】The N fertilizer slow-release treatments could significantly improve the uptake of N in summer maize and reduce the loss of N in the north of North China Plain, and urea formaldehyde and polymer gel urea performed better for the high efficiency and less N loss.
    The Relationship of NO3--N Leaching and Rainfall Types During Summer Fallow in the Loess Plateau Dryland
    XIA MengJie, MA LeLe, SHI QianYun, CHEN ZhuJun, ZHOU JianBin
    Scientia Agricultura Sinica. 2018, 51(8):  1537-1546.  doi:10.3864/j.issn.0578-1752.2018.08.011
    Abstract ( 284 )   HTML ( 7 )   PDF (448KB) ( 549 )   Save
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    Objective Summer fallow after harvesting winter wheat is very common in dryland of the Loess Plateau. The residual nitrate in soil profile after wheat harvest was increased with the application of nitrogen (N) fertilizer. Whether the intensive high rainfall during summer fallow will increase NO3--N leaching loss is an important issue deserving study.Method】Soil profile samples (0-200 cm) were taken before and after summer fallow in three consecutive years (2013 to 2015) in Changwu and Yangling; NO3--N content in soil was analyzed to evaluate the impact of different precipitations and N application rates on NO3--N leaching during summer fallow. ResultThe NO3--N in soil profiles of Changwu was in range of 97 to 328 kg·hm-2, averaged 193 kg·hm-2. The average NO3--N content in soil profiles of 120 and 240 kg N·hm-2 treatments in Yangling was 156 and 366 kg·hm-2, respectively, indicating that NO3--N accumulation in soil was increased with N fertilizer rate. There was significant relationship between NO3--N leaching and rainfall during summer fallow. When rainfall was high (296 mm, abundant rainfall) in 2013 in Changwu, NO3--N accumulation peak leached below 80 cm soil depth from 40-60 cm after summer fallow, indicating strong nitrate leaching. But NO3--N peak didn’t change after summer fallow in 2014 due to the low rainfall (157 mm, deficit rainfall). The rainfall was normal in 2015 (200 mm), so slight nitrate leaching occurred in 0-100 cm soil profile after summer fallow. Soil nitrate has already leached to 100-200 cm soil profile after summer fallow in 2013 and the accumulation increment was 2.5 times than 0-100 cm soil profile due to high rainfall; but in 2014 soil nitrate accumulation increment was mainly in 0-100 cm soil profile. When rainfall in 2013 was low (only 220 mm, deficit rainfall) in Yangling, NO3--N was found slightly move upward of two N applied treatments. It was deficit rainfall but the rainfall increased to 288 mm in 2015, then nitrate leaching occurred through a 20-40 cm thick soil layer in 0-100 cm soil profile after summer fallow. While rainfall was 346 mm (normal rainfall) in 2014, nitrate peaks of 120 kg N·hm-2 and 240 kg N·hm-2 treatments was leached down to 140-160 cm in depth, through a 60-80 cm thick soil layer. Compared with the beginning of summer fallow, soil nitrate accumulation increment was mainly in 0-100 cm soil layer in 2013 due to deficit rainfall. While in 2014, a massive of soil nitrate of N applied treatments leached down to 100-200 cm due to high rainfall, especially under 240 kg·hm-2 N applied treatment. Conclusion NO3--N accumulation content in 0-200 cm soil profile of Loess Plateau dryland after wheat harvest was high. Rainfall was the key factor affects NO3--N leaching; and high rainfall during summer fallow increased NO3--N leaching. When abundant rainfall occured during summer fallow in Changwu, there was high leaching risk; while in Yangling high leaching risk would occur under normal rainfall.
    HORTICULTURE
    Physiological Regulatory Effects of Fulvic Acid on Stress Tolerance of Tomato Seedlings Against Phosphate Deficiency
    ZHANG LiLi, LIU DeXing, SHI QingHua, GONG Biao
    Scientia Agricultura Sinica. 2018, 51(8):  1547-1555.  doi:10.3864/j.issn.0578-1752.2018.08.012
    Abstract ( 427 )   HTML ( 5 )   PDF (652KB) ( 588 )   Save
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    【Objective】Phosphorus is one of the key elements of plant growth and development. Phosphorus deficiency can seriously affect crop yield and quality. Fulvic acid, as a natural organic compound, can promote plant growth and improve plant stress resistance. In order to ascertain the physiological regulatory mechanisms of fulvic acid on stress tolerance of tomato seedlings against phosphate deficiency, we will execute this experiment to explain the mitigative mechanisms of “genetic phosphate deficiency” by fulvic acid in tomato seedlings. It has launched the experiment to study on physiological control of tomato seedlings adapting to low phosphorus stress by fulvic acid. 【Method】 Tomato cultivar of ‘JinPeng No.1’ was used as the tested cultivar, and Hogland nutrient solution hydroponics was used in this experiment. When the tomato seedlings grew to three leaves, they were moved to the hydroponic basin for seven days. We studied the roles of applying different concentrations of exogenous fulvic acid (0, 0.04, 0.08, 0.12, 0.16, and 0.20 g·L-1; T0-T5 ) on the growth of tomato seedlings, root development, ability of photosynthesis, uptake and distribution of phosphate, accumulation and secretion of organic acid, and so on under hydroponic environment of phosphate deficiency.【Result】With the increase of the amount of fulvic acid (T1-T5), the physiological indexes of tomato seedlings showed a trend of rising first and then decreasing under low-phosphorus stress. Application of fulvic acid to 0.08 g·L-1 had a significant effect on increasing ratio of root to shoot; improving chlorophyll content, photosynthetic performance of leaves, enhancing phosphorus accumulation, distribution and transport in different tissues of tomato,phosphorus transporter-related gene's expression (PT1 and PHO1). PT1 can promote the roots of tomato seedlings to the environment in the uptake of phosphate alleviate low phosphorus stress and PHO1 can promote phosphate from root to stem and leaf distribution in order to effectively alleviate the shoot phosphorus deficiency. It can also improve the accumulation and secretion of organic acid (oxalate, malate, citrate, succinate, and tartrate), reducing the roots of anaerobic respiration products of lactic acid and acetic acid, promoting the proton pump gene (HA1) expression that can effectively change the environment of insoluble phosphorus to soluble phosphate and increased soluble phosphate content in the environment to promote plant root phosphorus content, improving growth- and development-related gene's expression (GRAS1) as well as decreasing the anthocyanin accumulation. 【Conclusion】Adding fulvic acid at a certain concentration under low-phosphorus stress can significantly improve shoot growth and root development of tomato seedlings. It indicates that the addition of fulvic acid can alleviate the phosphorus deficiency symptoms of plants to a certain extent and reveal the regulation of fulvic acid physiological mechanism of tomato actively against phosphorus deficiency.
    Effects of Intercropping Reineckia Carnea on Soil Enzyme Activity and Kiwifruit Fruit Yield, Quality in Kiwifruit Orchard
    ZHANG Cheng, WANG QiuPing, ZHOU KaiTuo, WU XiaoMao, LONG YouHua, LI JiaoHong, YIN XianHui
    Scientia Agricultura Sinica. 2018, 51(8):  1556-1567.  doi:10.3864/j.issn.0578-1752.2018.08.013
    Abstract ( 361 )   HTML ( 12 )   PDF (783KB) ( 318 )   Save
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    【Objective】The objective of this study was to investigate the effects of intercropping Reineckia carnea (R. carnea)on the soil microorganism quantity, enzyme activity, kiwifruit fruit yield and quality in kiwifruit orchard, and their correlations were studied, so as to provide a scientific basis of enriching the ecological theory, planting the high quality of kiwifruit and applying the cultivation model of intercropping R. carnea in kiwifruit orchard.【Method】 R. carnea was intercropped in kiwifruit orchard, and non-intercropping as a control. The investigation about populations of bacteria, actinomycete and fungus, activities of urease, sucrase, phosphatase and catalase, water content in the rhizosphere soil of kiwifruit trees at different growth stages, as well as yield and quality of kiwifruit fruits, were carried out, as then the correlations between these parameters were analyzed. 【Result】The results showed that the rhizosphere soil microorganism numbers, four enzyme activities, water content in kiwifruit orchard were increased significantly, the yield of kiwifruit fruits was increased, and the quality of kiwifruit fruits was improved by intercropping R. carnea. There were the increase trend of the above-mentioned results with the extension of intercropping year. Compared with the control, numbers of bacteria, actinomycete and fungus in the rhizosphere soil intercroppedR. carnea for 4 years were increased by 27.85%-54.34%, 28.57%-96.05% and 16.39%-148.38%, respectively; soil urease, sucrase, phosphatase, catalase activities and water content increased by 0.20-0.61 times, 0.30-0.87 times, 0.41-0.65 times, 0.23-0.32 times and 0.05-0.16 times, respectively. Single fruit volume and yield increased by 3.26% and 4.88%, respectively, when the fruit were edible, its vitamin C, soluble solids, total soluble sugar, dry matter and titratable acid increased by 15.24%, 5.46%, 12.11%, 5.63% and 4.12%, respectively. Based on correlation analysis, soil microbial population showed a highly significant or significant positive correlation with urease and phosphatase activities in the rhizosphere soil of R. carnea, which was negatively correlated with catalase activity. And there was a significant correlation among soil microorganism number, enzyme activities, fruit weight and quality. 【Conclusion】The intercropping R. carnea in kiwifruit orchard could increase the soil microorganism populations, enzyme activities and kiwifruit yield, and improve the kiwifruit quality.
    FOOD SCIENCE AND ENGINEERING
    Browning Inhibition and Energy Metabolism Mechanism of  Agaricus bisporus by Ergothioneine Treatment
    LIN KaiLi, HUANG Qi, HUANG QiHui, JIN ZhenLiang, JIANG TianJia, ZHENG XiaoLin
    Scientia Agricultura Sinica. 2018, 51(8):  1568-1576.  doi:10.3864/j.issn.0578-1752.2018.08.014
    Abstract ( 311 )   HTML ( 7 )   PDF (486KB) ( 484 )   Save
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    Objective】How to control and reduce the degree of browning in the process of transport and storage is the focus of Agaricus bisporus research work. The objectives of this research were to study the effect of ergothioneine on postharvest browning of Agaricus bisporus, and further investigate the relevance between browning and energy metabolism, so as to provide scientific basis and production guidance for browning control and shelf life extension. Method】A representative Agaricus bisporus cultivar AS2796 was chosen as the experimental material. The harvested mushroom was sprayed with 0.12 mmol?L-1 ergothioneine. The changes of whiteness, browning, MDA content, adenosine triphosphate (ATP) content, adenosine diphosphate (ADP) content, adenosine monophosphate (AMP) content, energy charge and the activities of succinate dehydrogenase (SDH), cytochrome oxidase (CCO), H+-ATPase and Ca2+-ATPase of treated samples were determined during storage at 4 for 17 days. Result】Internal organization browning and MDA content of Agaricus bisporus increased gradually, whereas the whiteness decreased during storage. As compared with the control group, ergothioneine treatment delayed the increase of MDA content and browning degree, effectively reduced the bacteria browning and internal organization browning of Agaricus bisporus. Also, whiteness value of cap of treated Agaricus bisporus was higher than the control group over time. The content of ATP was at a high level, and the lowest point (storage day 17) was also above 0.6 mg?g-1. Among them, the ATP content of Agaricus bisporus was increased in the early stage of storage, and decreased in the later stage. During the whole storage period, ADP and AMP content both showed a general increasing trend, the ATP and AMP content of treated Agaricus bisporus were distinctly higher than that of the control (P<0.05), while the ADP content was lower than the control group. The energy charge showed a decreasing trend, and the treated samples were slightly higher than that of the control. Results demonstrated that ergothioneine treatments delayed the decline of ATP content and enhanced the utilization level of ATP in cells of Agaricus bisporus. The activities of SDH, H+-ATPase and Ca2+-ATPase were obviously higher than that of control during the whole storage (P<0.05). The activity of CCO at the end (day 17) was higher than that of the control, and was lower than the control in the middle of storage (day 9, day 13). Compared with the control group, ergothioneine treatments inhibited the decrease of the activities of SDH, H+-ATPase, and Ca2+-ATPase significantly during the storage, which maintained better mitochondrial function. Conclusion】All the results indicated that Ergothioneine treatments effectively reduced the postharvest Agaricus bisporus browning, it had a closely relationship with energy metabolism during storage.
    ANIMAL SCIENCE·VETERINARY SCIENCERE·SOURCE INSECT
    Transcriptomic Analysis of IVF Embryonic Development in the Yak (Bos grunniens) Via RNA-Seq
    ZI XiangDong, LUO Bin, XIA Wei, ZHENG YuCai, XIONG XianRong, LI Jian, ZHONG JinCheng, ZHU JiangJiang, ZHANG ZhengFan
    Scientia Agricultura Sinica. 2018, 51(8):  1577-1589.  doi:10.3864/j.issn.0578-1752.2018.08.015
    Abstract ( 365 )   HTML ( 14 )   PDF (568KB) ( 443 )   Save
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    【Objective】The objectives of this study were to investigate the transcriptome differences and identify function, classification and metabolic pathways of the differentially expressed genes (DEG) at different developmental stages of yak embryos derived from in vitro fertilization (IVF), which are necessary to better understand the mechanism that regulates embryonic development and provide theoretical basis for improving in vitro embryo production in the yak (Bos grunniens). 【Method】Total RNA was extracted from IVF derived yak embryos at 2-cell, 4-cell, 8-cell, morula and blastocyst stages and amplified via the Smart-seq2 method, and the constructed RNA libraries were sequenced using the HiSeqTM2500 high-throughput sequencing method. 【Result】After IVF, the average cleavage rates and blastocyst rates were 69.3% and 26.2%, respectively. A total of 47 355 570 to 50 855 888 clean reads were obtained from 2-cell, 4-cell, 8-cell, morula and blastocyst stages, respectively, of which, 85.65% to 90.02% were covered in the yak reference genome. In total, the number of transcripts mapped to yak genome was highest for 8-cell (14 893) and lowest for blastocysts (9 827). The transcripts mainly had five patterns of alternative splice, of which, the two largest proportions were transcription start site (TSS) and transcription terminal site (TTS). The SNP numbers of the five stages of yak embryonic transcripts were 116 601, 234 131, 196 420, 70 841 and 94 840, respectively. A total of 1 221, 1 116, 142 and 564 transcripts were first detected at the 4-cell, 8-cell, morula and blastocyst stages, respectively. As embryo development proceeded, maternally derived transcripts such as BMP15, KIT, GDF9, STAT3, ZP3 and ZP4 etc.were decreased, whereas embryonic transcripts such as SARS,IL18, ACO2, TXN2, ATP5B, PCGF4, UBE3A, MAPK13, SNURF and JUP etc. were increased at specific stages. When |log2ratio| ≥1 and Q-value<0.05 were set as thresholds for identifying DEGs, a total of 6 922, 7 601, 8 071 and 10 555 DEGs were identified from 2-cell vs. 4 cell, 4-cell vs. 8-cell, 8-cell vs. morula, and morula vs. blastocyst, respectively. The GO distributions of the DEGs were classified into three categories: biological processes (BP), cellular components (CC), molecular functions (MF) with a total of 62 subcategories of two successive stages. KEGG enrichment analysis of DEGs showed that DEGs of 2-cell vs. 4-cell participated in 308 pathways, and significantly enriched in 11 pathways such as spliceosome, RNA transport and ubiquitin mediated proteolysis etc. DEGs of 4-cell vs. 8-cell participated in 310 pathways, and significantly enriched in 9 pathways such as olfactory transduction, neuroactive ligand-receptor interaction and nucleotide excision repair etc. DEGs of 8-cell vs. morula participated in 316 pathways, and significantly enriched in 10 pathways such as olfactory transduction, ubiquitin mediated proteolysis and neuroactive ligand-receptor interaction etc. DEGs of morula vs. blastocyst participated in 315 pathways, significantly enriched in 2 pathways i.e., spliceosome and RNA transport.ConclusionThis is the first study for analyzing the transcriptomes of IVF derived yak-embryos at different stages using high-throughput sequencing. A number of DEGs and their function, classification and metabolic pathways were discovered, which enriched transcriptome information for yak embryos. In addition, the results provided a foundation and reference to uncover the mechanism that regulates embryonic development and improves in vitro embryo production of the yak species. 
    Isolation, Culture and Myogenic Differentiation of Muscle Stem Cells in Goat Fetal
    SUI MengHua, ZHENG Qi, WU Hao, DING JianPing, LIU Yong, LI WenYong, CHU MingXing, ZHANG ZiJun, LING YingHui
    Scientia Agricultura Sinica. 2018, 51(8):  1590-1597.  doi:10.3864/j.issn.0578-1752.2018.08.016
    Abstract ( 295 )   HTML ( 10 )   PDF (1305KB) ( 272 )   Save
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    【Objective】 To establish the method for isolation, culture, identification and myogenic differentiation of fetal muscle stem cells from Anhuai goat in vitro, and to provide experimental materials for further research on the molecular mechanism of goat muscle stem cell proliferation and differentiation. 【Method】 In this study, the goat fetal longissimus muscle tissue was selected and cut into meat emulsion with ophthalmology, digested with 0.1% type I collagenase for 40 min and then digested with 0.25% trypsin for 15 min. The isolated cells were cultured in growth medium (20% FBS + 80% DMEM / F12 + Penicillin) in a 37℃, 5% CO2 incubator. After culturing for 2h, the cells were purified by differential adherent technique. After 2h, the cells were purified again. The cells were subcultured when they reached 70% density. 30min adherent method was used for further purification with each subculture of muscle stem cells until the sixth passages. Muscle stem cell marker genes Pax7 and MyoD1 were detected with the purified cells for identification. When muscle stem cells grew to a density of about 70%, the growth medium was displaced with differentiation medium (2% FBS + 98% DMEM / F12 + Penicillin)for myoblasts induction and the morphology of the cells was monitored . One day after the induction of the cells, the marker protein of muscle stem cells Myog was detected. In addition, total RNAs of cells induced at 0, 1, 3, 5, and 7 days were separately extracted and their relative expression amounts of MyoD1 and Myog genes were measured by qPCR. 【Result】 Isolated cells showed adherent growth, and their morphology tended to be long spindle after stabilization. Pax7 and MyoD1 expression were detected in the 6th passage cells by immunofluorescence. After induction by differentiated medium, the cells started to differentiate and fuse with each other into myotubes with a certain directionality as the induction prolonged. Myog protein was detected by immunofluorescence assay. Differentiation marker genes MyoD1 and Myog were detected by qPCR. Expression of MyoD1 could be detected in the first day of induction and maintained until the 3rd day, and its level began to decline from the 5th day but still significantly higher than the proliferative phase. A similar dynamic was observed with the relative expression level of Myog in the differentiating cells.【Conclusion】In this experiment, the fetal muscle stem cells of Anhuai goat was obtained with high purity, which showed good myogenic potential after induction. The results provide material for further research on the mechanisms of myogenic differentiation of muscle stem cells.
    RESEARCH NOTES
    Molecular Cloning of Leaf Developmental Gene EjGRF5, Its Promoter and Expression Analysis in Different Ploidy Loquat (Eriobotrya japonica (Thunb.) Lindl.)
    LIU Chao, WANG LingLi, WU Di, DANG JiangBo, SHANG Wei, GUO QiGao, LIANG GuoLu
    Scientia Agricultura Sinica. 2018, 51(8):  1598-1606.  doi:10.3864/j.issn.0578-1752.2018.08.017
    Abstract ( 299 )   HTML ( 9 )   PDF (1038KB) ( 488 )   Save
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    【Objective】 In order to provide more details for further studying the mechanisms of EjGRF5 gene in regulating the growth vigor of different ploidy loquat leaf, the aims of this study are to isolate the code region of EjGRF5 gene which is involved in the regulation of leaf development and its promoter sequence, and illustrate the expression pattern of the EjGRF5 in different ploidy loquat. 【Method】 The primers were designed by using the EjGRF5 reference sequence obtained from the RNA-Seq, and the full length of EjGRF5 was cloned by using the genome DNA of Longquan-1 tetraploid, and then the full length and reference sequences were compared to obtain the targeted sequence. The Bioedit7.2 and SignalP4.1 were used to analyze the structure of the EjGRF5 CDS and the physical and chemical properties of EjGRF5; Mega7.0 was used to construct the EjGRF5 phylogenetic tree. The online software of LocTree3 and SoftBerry ProtComp9.0 was adopted to predict the subcellular location of EjGRF5. The genome walking technique was employed to amplify the promoter sequence, and the online software PlantCARE was adopted to analyze the structure of the promoter. The expression patterns of EjGRF5 in triploid loquat and their parents (4x, 2x) were analyzed preliminarily. 【Result】 When Comparing the sequenced data with the reference sequence of EjGRF5, the results showed that the full length of EjGRF5 is 1368 bp and it contains three extron and two intron sequences. The CDS length of EjGRF5 is 987 bp. The results of phylogenetic analysis revealed that the EjGRF5 protein is highly homologous with some other species in Rosaceae and is closest to Pyrus bretschneideri. The result of subcellular localization prediction showed that EjGRF5 protein is located in the nucleus. The promoter analysis indicated that there were multiple putative cis-acting elements involved in the responsive elements, including abscisic acid (ABA), ethylene, heat, anaerobic inductive, gibberellin (GA) and light. Moreover, the number of light responsiveness element has reached 11. QRT-PCR result showed that the expression level of EjGRF5 exhibit varying degrees of up-regulation in almost all of the triploids except the hybrids A-6 and B-3 compared with the middle-parent value (MPV). Among the hybrids, the expression of A-3 was 20 times higher than that of MPV and A-5 was about 18 times higher than that of MPV. 【Conclusion】 The coding region and CDS sequence of EjGRF5 gene which is related to the development of loquat leaves were isolated, and the qRT-PCR results indicated that the expression of EjGRF5 in triploid loquat exhibited a trend of up-regulation.
    The Cloning of Porcine CBR1 Gene’s Different Copy Forms and Analysis of Its Polymorphism
    QI ChuanXiang, XU KUI, MU YuLian, YANG ShuLin, LI Kui, WU TianWen
    Scientia Agricultura Sinica. 2018, 51(8):  1607-1616.  doi:10.3864/j.issn.0578-1752.2018.08.018
    Abstract ( 279 )   HTML ( 6 )   PDF (1046KB) ( 127 )   Save
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    【Objective】 In order to obtain and analysis the different copy forms of CBR1 gene coding region, and analysis the correlation between polymorphism and reproductive performance of different copy forms of CBR1 gene in pigs.【Method】 Based on the cDNA of the Wuzhishan pig’s testis , the different copy sequences of CBR1 gene were obtained by cloning sequencing technology. The amino acid sequences encoded by different copy forms of CBR1 gene were analyzed by DNASTAR software. Homology between different copy forms of porcine CBR1 gene, and that compared with other mammals were analyzed using DNAMAN software. The physical and chemical properties of different proteins of porcine CBR1 gene were analyzed by Protparam online software on ExPaSy. 136 Yorkshire and 47 Landrace pig ears samples were selected for DNA extraction and PCR-sequencing to explore the relationship between polymorphism of CBR1 gene and porcine reproductive performance. 【Result】 All the three different copies of CBR1 gene encoded 3 exons, and the sequence length of the coding region were 870bp, 870bp and 846bp respectively. Their 12-18th amino acid residues were GlyXXXGlyXGly, which were in accordance with the CBRs family structural characteristics. The results of homology comparison showed that the CDS sequence homology among different copies of CBR1 gene was more than 87%, and more than 82% among different mammals, indicated that the CBR1 gene of mammal was conserved in evolution process. The physical and chemical properties of 3 different copies of CBR1 were similar, all of them were unstable and belong to hydrophilic proteins, which indicated that they may play a similar role in vivo. Polymorphism analysis showed that 5 SNPs were found in copy V1, 4 SNPs were found in copy V2, and 2 SNPs were found in copy V3. In the Yorkshire pigs, the A/T mutation at the promoter upstream region 153 bp and the C/T mutation at exon3 379 bp in V1; AC insertion at the promoter upstream region 10 bp, C/T mutation at exon 1 63 bp and G/T mutation at intron1 76 bp in V2, were significantly correlated with the multiparous alive litter size, while the SNPs in Landrace pigs were not associated with reproductive performance. 【Conclusion】 Coding region sequence of 3 different copies of CBR1 gene were obtained; 5 SNPs were found significantly associated with multiparous alive litter size , suggested that CBR1 gene may be used as a valuable candidate gene for improving breeding performance of Yorkshire pigs.