Table of Content

    16 February 2016, Volume 49 Issue 4
    Rice OsABC1K3 Gene Mutant and Its Response to High Light Stress
    GAO Qing-song, XU Meng-bin, YUAN Cai-yong, JI Jian-hui, ZHOU Yong, LIANG Guo-hua
    Scientia Agricultura Sinica. 2016, 49(4):  609-620.  doi:10.3864/j.issn.0578-1752.2016.04.001
    Abstract ( 568 )   HTML ( 6 )   PDF (5288KB) ( 699 )   Save
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    【Objective】 High light (HL) stress inhibits plant photosynthesis. Severe HL stress will destroy photosynthetic potential, affecting crop growth and production. The objective of this study was to investigate the physiological function of rice ABC1 (activity of bc1 complex) kinase gene OsABC1K3 in HL stress response pathway. 【Method】 To verify the alternative splicing forms of OsABC1K3, 3′ RACE was performed using primers designed from common sequences of different OsABC1K3 transcripts predicted in the Rice Genome Annotation Project Database. Subsequently, a T-DNA insertion mutant of the gene, osabc1k3, was obtained from the Rice T-DNA Insertion Sequence Database. The homozygous mutants were identified by adding-generation propagation and PCR. Then, the mutant agronomic traits such as plant height, seed setting rate and seed size were examined. The leaf pigment contents were determined according to the method of Arnon. The leaf photosynthetic rate and chloroplast ultrastructure were analyzed using a LI-6400 portable photosynthesis measurement system and transmission electron microscopy, respectively. To perform HL treatment, wild type and mutant seedlings grown at 300 μmol·m-2·s-1 light intensity were transferred into 800 μmol·m-2·s-1. The plant phenotype was photographed, and the variation of chloroplast ultrastructure and chlorophyll content after treatment was analyzed. To perform methyl viologen (MV) treatment, wild type and mutant seedlings were sprayed with 20 μmol·L-1 MV in 0.1% Tween 20 (V/V). Control plants were sprayed with water and 0.1% Tween 20. The leaf phenotype after treatment was photographed, and the superoxide dismutase (SOD) activity and malondialdehyde (MDA) content were measured. Finally, the expression of starch biosynthesis, chloroplast prenyllipid biosynthesis and chloroplast ABC1 kinase genes in mutants was analyzed using quantitative real-time PCR. 【Result】 Only one transcript, LOC_Os05g25840.3, was detected for the OsABC1K3 gene. The plant height, seed setting rate and seed size of osabc1k3 mutants were reduced compared with wild type. The grain width, grain thickness and 1 000-grain weight were reduced by 13.4%, 6.8% and 18.2%, respectively. However, the grain length was not significantly different from that of wild type. The chlorophyll content of mutant leaves was lower than wild type, while the chlorophyll a/b ratio and carotenoid content were higher. In addition, the mutant chloroplasts were morphologically normal, but lacking starch granules. Photosynthetic activity analysis revealed that the photosynthetic rate of mutants did not differ significantly from that of wild type. After 7 days of HL stress, the mutant leaves etiolated. After 9 days, some of the plants died. The chloroplast thylakoid structure of mutants was disrupted after treatment, presenting extensive vacuolation. Further determination of chlorophyll content revealed that the chlorophyll attenuation of mutants was more severe than wild type. However, the necrosis degree, SOD activity and MDA content of mutant leaves were not significantly different from wild type after MV treatment. Furthermore, OsABC1K3 mutation affected the expression of leaf starch biosynthesis, chloroplast prenyllipid biosynthesis and chloroplast ABC1 kinase genes. 【Conclusion】 OsABC1K3 might participate in HL stress response through genetically controlled signaling pathways, rather than direct oxidative stress defense.
    Identification of Heterotic Loci for Kernel Related Traits Using a Maize Introgression Lines Test Population
    GUO Zhan-yong, Lü Pan-qing, ZHANG Xiang-ge, SUN Gao-yang, WANG Hong-qiu, LI Wei-hua, FU Zhi-yuan, TANG Ji-hua
    Scientia Agricultura Sinica. 2016, 49(4):  621-631.  doi:10.3864/j.issn.0578-1752.2016.04.002
    Abstract ( 457 )   HTML ( 1 )   PDF (650KB) ( 551 )   Save
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    【Objective】Kernel shape traits are important components of 100-kernel weight and show high heterosis. Identifying the heterotic loci for kernel related traits and subsequently dissecting the genetic basis architecture of heterosis for kernel related traits can provide useful information for cloning heterotic QTL and the development of elite hybrids by molecular breeding.【Method】A set of chromosome segment substitution lines (CSSLs) population was constructed using the inbred line lx9801 as the receptor parent and the inbred line Chang7-2 as the donor parent, which was used as the martial and test with the inbred line T7296 to construct a set of 184 test hybrid population. The test population, CSSLs population as well as other materials were evaluated in the field by following three replicates in randomized complete blocks with at Changge and Xunxian locations in Henan province in 2013. Ten plants were harvested after the physiological maturity period. Then, four kernel-related traits were investigated as follows: Including kernel length (KL, mm), kernel width (KD, Mm), kernel thickness (KT, mm) and 100-kernel weight (KW, g). The heterotic loci for kernel related traits were identified through significant analysis comparing to each test hybrid and the CK T7296×lx9801 by mean of variance analysis and t test.【Result】The kernel shape traits of the two parents in the CSSLs population had obvious differences; the kernel length and 100-kernel weight of the parent Chang7-2 was higher than lx9801, and the kernel thickness was lower lx9801. The four kernel shape traits in the test population had certain heterosis, the mid-parent heterosis of kernel length were 19.32% and 15.30% in Changge and Xunxian locations respectively, the mid-parent heterosis of kernel width were 10.86% and 10.07%, the mid-parent heterosis of kernel thickness were 6.23% and 4.78%, and the mid-parent heterosis of 100-kernel weight were 20.97% and 25.09%. The results of correlation analysis showed that kernel length expressed a significant positive relationship with kernel width and 100-kernel weight, and kernel width and kernel thickness also showed a significant positive relationship with 100-kernel weight;. kernel width, however, had a negative relationship with kernel thickness. A total of 13 heterotic loci for kernel length were identified for two environments, and one heterotic locus was detected for two environments simultaneously. For kernel width, fourteen heterotic loci were detected, including one main heterotic loci identified at Changge and Xunxian locations respectively. There were twenty-five heterotic loci detected for kernel thickness, and the common heterotic loci on chromosome 2, 3, 7 and 9 were identified for two environments simultaneously. Twenty-four heterotic loci for 100-kernel weight were detected, above of them, two heterotic loci hKW1a and hKW1b were identified on chromosome 1 at the two locations simultaneously. On the chromosome 7, one main heterotic locus hKW7a was identified, which had 20.12% and 11.03% contributions at Changge and Xunxian locations. Additionally, the heterotic locus hKW8a on chromosome 8 had 18.64% and 8.76% contributions at the two environments respectively.【Conclusion】The heterosis of kernel shape traits followed the order: kernel length > kernel width > kernel thickness, and a total of 75 heterotic loci (HL) were identified for three kernel related traits and 100-kernel weight, and 11 HL were identified at the two environments simultaneously. Owing to kernel length having a high correlation with high seed removal rate, and that it is difficult to predict the heterosis of kernel related traits, so the molecular markers linked with the heterotic loci of kernel length could be used for select elite hybrid with high seed removal rate in maize breeding.
    Progress on TGA Transcription Factors in Plant
    TIAN Yi, ZHANG Cai-xia, KANG Guo-dong, LI Wu-xing, ZHANG Li-yi, CONG Pei-hua
    Scientia Agricultura Sinica. 2016, 49(4):  632-642.  doi:10.3864/j.issn.0578-1752.2016.04.003
    Abstract ( 1518 )   HTML ( 32 )   PDF (451KB) ( 2039 )   Save
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    TGA family is an important group of bZIP transcription factors in plant, which regulates the downstream target genes by binding to the as-1 cis-elements in the promoter region and subsequently activating or inhibiting their expression, ?nally regulating the resistance or development of floral organ. The first TGA gene was identified in tobacco. Subsequently, TGA members were widely characterized in arabidopsis, rice and apple. In arabidopsis, they constitute a multigene family comprising 10 members based on genome sequence analyses. The members of TGA family can be divided into five subgroups (Ⅰ: TGA1 and TGA4; Ⅱ: TGA2, TGA5 and TGA6; Ⅲ: TGA3 and TGA7; Ⅳ: TGA9 and TGA10; Ⅴ: PAN) according to their sequence similarity. The members of Ⅰ, Ⅱ and Ⅲ subgroups are mainly involved in disease resistance. Yeast two-hybrid and pull-down results showed that all members of subgroupⅠ, Ⅱ, and Ⅲ could interact with NPR1 protein which is the key positive regulator in SA signaling pathway. EMSA results indicated that the interaction between TGA and NPR1 could enhance the expression of pathogenesis-related (PR) genes and disease resistance. However, their roles were different in basal and systemic acquired resistance (SAR): tga3 mutants showed a defect in basal resistance, whereas the induced resistance was unaffected. TGA1 and TGA4 were involved in regulation of basal resistance and SAR. The tga2/5/6 triple mutants but not tga6 or tga2/5 double mutants were defective in SAR, a phenotype similar to that of the npr1 mutants, indicating functional redundancy among TGA2, TGA5 and TGA6. Yeast two-hybrid screen revealed that TGA transcription factors of subgroup Ⅱ could interact with glutaredoxin GRX480 to mediate the repression of the marker genes of JA signaling by SA, and they also could interact with GRAS protein SCL14 to increase the expression of CYP81D11 and GSTU7 in an NPR1-independent manner and enhance the tolerance to xenobiotics. In addition, the primary and lateral root growth was affected in tga1/4 and nrt2.1/2.2 mutants. ChIP and yeast one-hybrid assay results showed that TGA1 and TGA4 regulated the nitrate response via binding to the promoters of NRT2.1 and NRT2.2 to regulate their expression. TGA3 was also involved in the long-distance transport of cadmium. The members of subgroup Ⅳ and Ⅴ played an importance role in the regulation of floral organ development. tga9/10 mutants have defects in male gametogenesis that were similar to those in roxy1/2 mutants. PAN could interact with NPR1-like proteins BOP1 and BOP2. Both pan and bop1/2 double mutants revealed a pentamerous arrangement of sepals, which suggested that a similar signaling mechanism might be used between floral organ development and disease resistance. Finally, the research directions of TGA transcription factors was discussed, which will provide a reference for researchers in this field.
    Effect of Source-Sink Manipulation on Translocation of Carbohydrate and Nitrogen, Phosphors, Potassium in Vegetative Organs of Conventional Japonica Rice After Anthesis
    XU Bei-bei, YOU Cui-cui, DING Yan-feng, WANG Shao-hua
    Scientia Agricultura Sinica. 2016, 49(4):  643-656.  doi:10.3864/j.issn.0578-1752.2016.04.004
    Abstract ( 493 )   HTML ( 6 )   PDF (559KB) ( 609 )   Save
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    【Objective】This study investigated how source-sink relationship manipulation could affect dry matter, non-structural carbohydrate, nitrogen (N), phosphorus(P) and potassium(K) nutrient translocation of rice after anthesis and clarify the SPP/LAPC that promotes nutrient efficient recycling in rice.【Method】Two japonica rice cultivars, Huaidao 5 and Ningjing 3 were field grown in 2013 and 2014. Leaf-cutting and spikelet-thinning treatments were made on single stems with 6 leaves and consistent flowering process to regulate source-sink relationship at heading stage, define the ratio of spikelets per panicle and leaf area per culm as SPP/LAPC, dry matter, non-structural carbohydrate(NSC), nitrogen, phosphorus and potassium translocation rate (including leaf and stem-sheath) of two cultivars were measured in heading to harvesting period, so as to study the relationship between dry matter/NSC and nutrient translocation, and the relationship between SPP/LAPC and nutrient translocation of leaf and stem-sheath, respectively.【Result】Compared to the control(L0S0), leaf-cutting treatment significantly decreased filled-grain percentage and 1000-grain weight as SPP/LAPC increased significantly, Huaidao5 reduced 8.6%-10.5% and 19.0%-8.0%, respectively, Ningjing3 reduced 9.7%-20.4% and 5.7%-12.6%, respectively; whereas spikelet-thinning treatment significantly increased filled-grain percentage and 1000-grain weight as SPP/LAPC decreased significantly, Huaidao5 average increased 3.4%-6.7% and 1.2%-18.7% by an average, Ningjing3 increased 2.0%-4.3% and 6.9%-17.3% by an average. Percentage of ripened grains and 1000-ripened grain weight showed no significant difference between different spikelet-thinning treatments of the same variety, but differed in years. The 2014 rice season weather was more beneficial to grain filling, filled-grain rate, 1000-grain weight and their increasing range when compared with 2013. Leaf-cutting treatment increased dry matter, NSC and nutrient translocation rate of leaf and stem-sheath from heading to maturity, there were no significant difference between variety and year; whereas performance of indexes mentioned above in spikelets thinning treatments were just on the contrary, different varieties and different years showed no substantive differences, either. There were fundamental differences between leaves and stem-sheath in translocation of dry matter and NSC, transport rate of dry matter and NSC in leaves declined as spikelet thinning proportion increased, while matters mentioned above detained in stem-sheath on apparent. There was a curvilinear correlation(y=(a+bx)/x) between SPP/LAPC at heading stage(x) and dry matter, NSC and nutrient transport rate in leaves and stem-sheath after anthesis (y) with further analysis. Nutrient transport rate of leaves and stem-sheath increased significantly with the increase of SPP/LAPC. While the SPP/LAPC increased around 1.5, transport rate of dry matter, NSC and nutrient approximated to maximum, there were no obvious differences between Huaidao5 and Ningjing3.【Conclusion】A relatively high SPP/LAPC was beneficial to translocation of dry matter, NSC and nutrient in vegetative organs in rice after anthesis, there was a close curvilinear correlation between SPP/LAPC and matters mentioned above in vegetative organs in rice after anthesis, translocation rate reached a maximum when SPP/LAPC was 1.5. The critical SPP/LAPC around 1.5 can be used as the basis for variety breeding and cultivation regulation in mineral nutrients’ efficient reutilization of conventional japonica rice.
    Effects of Plant Growth Regulators S3307 and DTA-6 on Carbohydrate Content and Yield in Soybean
    LIU Chun-juan, FENG Nai-jie, ZHENG Dian-feng, GONG Xiang-wei, SUN Fu-dong, SHI Ying, CUI Hong-qiu, ZHANG Pan-pan, ZHAO Jing-jing
    Scientia Agricultura Sinica. 2016, 49(4):  657-666.  doi:10.3864/j.issn.0578-1752.2016.04.005
    Abstract ( 657 )   HTML ( 1 )   PDF (416KB) ( 730 )   Save
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    【Objective】The aim of this paper was to investigate the effect of plant growth regulators on carbohydrate content, sucrose metabolizing enzyme activities and yield of soybean in the source-sink theory aiming to provide scientific basis for PGRs on agricultural production.【Method】Foliar spray field experiments were conducted successively in 2013 and 2014 in the main soybean producing zone, Heilongjiang Bayi Agricultural University. The experiment was in the early flowering by foliar spray one time. Taking Hefeng50 and Kefeng16 soybean varieties as the main research materials. Two chemical regulators, 2-N,N-diethylamino ethylcaproate (DTA-6, 60 mg·L-1) and uniconazole (S3307, 50 mg·L-1) were tested with water as the control. The first sampling was carried out after sprayed 30 d, and then leaf samples were collected once every five days. The contents of sucrose, starch or fructose in leaves and seeds, and invertase activity,SPS or SS activity in leaves were measured. The grain yield of soybean were investigated.【Result】At the early seed filling stage(30-35 days since spraying of DTA-6 and S3307), the sucrose,fructose and starch contents of leaves showed the downtrends, and that of seeds showed increasing trends, which showed that more carbohydrates were applied to seeds growth and development. At the middle seed filling stage(35-45 days since spraying of DTA-6 and S3307), the sucrose, fructose and starch contents of leaves always showed increasing trends by S3307 .The sucrose and fructose contents were all higher than the control, which was based to provide sufficient materials for the grain filling. At the late seed filling stage(after the 50th day since the spraying by DTA-6 and S3307), the sucrose contents of leaves reached the maximum by DTA-6 and S3307, and reached significant levels to CK. The starch content of leaves sprayed with S3307 were higher than CK, and the fructose contents of leaves sprayed with DTA-6 were higher than CK. The sucrose contents of seeds were significantly increased by both S3307 and DTA-6 treatments. The fructose contents of two varieties seeds were increased by S3307, but that of Hefeng50 seed was decreased by DTA-6. The starch content of Hefeng50 seed but that were of Kefeng16 seed were increased by both S3307 and DTA-6 treatments, which showed that PGRs were different effect for soybean varieties. With sucrose contents were increased, SPS and SS activities were improved by S3307 and DTA-6. In most times, foliar spray of S3307 and DTA-6 could significantly decrease invertase activity of leaves to regulate carbohydrate metabolism in different varieties. Thereby, sucrose was neutral for carbohydrate metabolism. Balance of carbohydrate metabolism in source and sink by S3307 and DTA-6 to significantly increase yield. To controls(H-CK and K-CK),the yield of PGRs (H-S, H-D and K-S, K-D) treatments were significantly increased by 20.07%, 14.57% and 10.54%, 10.95%. By the correlation analysis, The sucrose contents of the leaves were positively correlated with SPS, SS activities and starch contents of leaves and the sucrose, fructose and starch contents of seeds (0.893**, 0.888** and 0.981** or 0.918**, 0.832 and 0.810), or were negatively correlated with activities and invertase activities and fructose contents of leaves (-0.872 and -0.862).【Conclusion】The activity of SS and SPS which are of great importance in sucrose synthesis were increased and the activity of invertase was decreased by PGRs, the source-sink physiological metabolism of carbohydrates was regulated to increase yield by S3307 were higher than DTA-6.
    Multiplex RT-PCR for Simultaneous Detection of Bean pod mottle virus and Soybean mosaic virus in Imported Soybean Seeds
    SHEN Jian-guo, GAO Fang-luan, CAI Wei, JIN Jing, LIAO Fu-rong, WU Zu-jian
    Scientia Agricultura Sinica. 2016, 49(4):  667-676.  doi:10.3864/j.issn.0578-1752.2016.04.006
    Abstract ( 446 )   HTML ( 4 )   PDF (654KB) ( 833 )   Save
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    【Objective】 A multiplex RT-PCR method was developed for simultaneous and rapid detection of Bean pod mottle virus (BPMV) and Soybean mosaic virus (SMV), both of which could cause fairly similar symptom in imported soybean seeds. 【Method】 Two sets of specific primer for the conserved coat protein genes in viral genome regions were designed according to the sequences from GenBank. BPMV and SMV infected healthy soybean seeds were selected as experimental materials with double strand RNA (dsRNA) used as template for multiplex RT-PCR. A series of assays (involving concentrations of primer pairs, Tm value and the number of cycling) were conducted to optimize the multiplex RT-PCR method, of which the specificity was analyzed by detecting BPMV and SMV from the extract of infected and healthy soybean seeds. In order to determine the sensitivity of the established multiplex RT-PCR, a ten-fold serial dilution (from 10-1 to 10-6) of infected soybean seed extract was obtained by adding corresponding volume of healthy soybean extract. For the reliability analysis, the amplified DNA fragments of multiplex RT-PCR were recovered, purified, inserted into pMD18-T vector and sequenced. Suspected virus-infected seeds from the United States, Argentina, China and Brazil were detected using the multiplex RT-PCR and single RT-PCR. In addition, multiplex one-step immunocapture reverse transcription-polymerase chain reaction (IC-RT-PCR) and multiplex one-step tubecapture reverse transcription-polymerase chain reaction (TC-RT-PCR) were conducted by transferring extract of virus-infected soybean seeds to PCR tubes with or without anti-BPMV/SMV antibodies coated, followed by RT-PCR amplification. 【Result】 The optimal parameters for the multiplex RT-PCR were 0.4 μmol·L-1 for each of the primers, 52 for the Tm value and 35 for the number of cycling. Results of specificity analysis showed two amplified DNA fragment bands (542 and 221 bp, respectively) from BPMV/SMV infected soybean seeds, a 542 bp band from BPMV infected soybean seeds and a 221 bp band from soybean seeds infected with SMV only. No amplified DNA bands were observed from the healthy soybean seeds. Results of sensitivity determination revealed that neither multiplex RT-PCR nor single RT-PCR could amplify any DNA fragments from the infected soybean seeds at a concentration of 10-3 dilution of extract of infected soybean seeds, suggesting they shared same sensitivity with a limit of 10-2 dilution of the extract of infected seeds. PCR products, with lengths of 542 bp for BPMV and 221 bp for SMV, respectively, were demonstrated to share high homology with corresponding genes published before by sequencing and analyzing, validating the reliability of the multiplex RT-PCR method. The method was also employed to detect BPMV and SMV from soybean seeds originating from different countries. Three out of four samples of soybean seeds from the US were infected with BPMV and one with SMV, one sample from Argentina and two samples from China with SMV, which was in line with results of single RT-PCR method. Furthermore, two specifically amplified DNA bands were observed from BPMV/SMV infected seeds when performing one-step multiplex IC-RT-PCR and one-step multiplex TC-RT-PCR, whereas no bands showed in the healthy seeds. 【Conclusion】 The multiplex RT-PCR method in this paper could be used for rapid detection of BPMV and SMV in imported soybean seeds.
    The Super Cooling Point Change of Harmonia axyridis Under   Low Temperature Stress and Its Cold-Resistance Genes’   Expression Analysis
    WU Meng-jing, XU Qing-ye, LIU Ya, SHI Xing-rong, SHEN Qi-da, YANG Meng-meng, WANG Shi-gui, TANG Bin
    Scientia Agricultura Sinica. 2016, 49(4):  677-685.  doi:10.3864/j.issn.0578-1752.2016.04.007
    Abstract ( 370 )   HTML ( 1 )   PDF (469KB) ( 580 )   Save
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    【Objective】Harmonia axyridis is an important predatory lady beetle as natural enemies attacking agricultural and forestry pests. H. axyridis has strong cold-hardiness as it can overwinter as disapausing adults in Northeast population of China. The objective of this study is to use the super cooing point and the changes of cold resistance gene mRNA level as the index of cold-resistance ability, and to define the effect of low temperature stress on improving cold resistance of H. axyridis.【Method】The wild H. axyridis adults were collected and treated for 2, 12 and 24 h by three different low temperatures of 0, 5 and 10 to get the best low temperature stress. The changes of super cooling point were detected all the time during the period of low temperature processing. The H. axyridis adults of experimental populations and wild adults were used as the control groups and experimental groups, respectively. All of these adults were treated under the condition of 5 low temperature and stored for 10, 20 and 30 d, and the changes of super cooling point and survival rate were measured after the treatment. Six important cold hardiness genes were sure by transcriptome and digital gene expression tag profiling (DGE). The relative expressions of six important cold hardiness genes at mRNA level were detected by real-time fluorescent quantitative PCR. 【Result】The super cooling point was decreased significantly after acclimation for 12 h at 5. Acclimation for 12 h at 5 could effectively decrease the super cooling point and other indexes of cold resistance in H. axyridis. Setting treatment group which was acclimated for 12 h at 5 and control group deposited for 10 , 20, and 30 d at 5, the survival rate and super cooling point of control group were all higher than the treatment group. In the same time, the 6 genes’ relative expression of mRNA level results showed HSP21.4, trehalase, transketolase, ATP-grasp fold domain protein and dopa decarboxylase were up-regulated when cold acclimation, but erythrocyte binding protein was down-regulated. HSP21.4 was always highly expressed in cryopreservation, and the treatment group was strikingly higher than the control group. Others were on low expression in cryopreservation, and strikingly lower than the control group. 【Conclusion】The low temperature stress can reduce the super cooing point of the H. axyridis, but it can’t improve the survival ability of summer high temperature resistant population. HSP21.4 and erythrocyte binding protein can help H. axyridis to improve the cold-resistance ability respectively by high expression and low expression. Trehalase, transketolase, ATP-grasp fold domain protein and dopa decarboxylase are highly expressed in cold acclimation to resist cold environment.
    Effects of Long-Term Application of Chloride Containing Fertilizers on the Biological Fertility of Purple Soil Under a Rice-Wheat Rotation System
    YANG Lin-sheng, ZHANG Yu-ting, HUANG Xing-cheng, ZHANG Yue-qiang, ZHAO Ya-nan, SHI Xiao-jun
    Scientia Agricultura Sinica. 2016, 49(4):  686-694.  doi:10.3864/j.issn.0578-1752.2016.04.008
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    【Objective】 Chlorine (Cl) is an essential mineral element for plant growth, although Cl-containing fertilizers (such as ammonia chloride and potassium chloride) have a high concentration of Cl? and a salt index that may be toxic to plants. Little information was currently known about the effect of long-term application of Cl-containing fertilizer on crop yield, soil fertility, and especially on soil biological fertility. Therefore, it was meaningful to explore the mechanism of the effect of the soil fertility on the soil biological fertility and the quantitative application of Cl-containing fertilizer’s impact on crop yield as well as improving soil fertility. 【Method】 Based on a 23-year field experiment in national monitoring station of soil fertility and fertilizer efficiency on purple soils, soil samples from treatments with no fertilizer (CK), applications of nitrogen, phosphorous and potassium fertilizer (NPK), applications of NPK together with rice straw return (NPK+S), and applications of Cl-containing NPK fertilizer with rice straw return ((NPK)Cl+S) were collected to study the soil microbiological community structure by the PLFA method, soil enzyme activities, and crop yield. 【Result】 Results showed that the long-term application of Cl-containing fertilizer ((NPK)Cl+S) reduced activities of urease, alkaline phosphatase, and catalase by 35.7%, 18.0%, and 69.8% the in rice cropping season and by 31.6%, 24.5%, and 75.6% in the wheat cropping season, respectively. Principal component analysis revealed that urease, alkaline phosphatase, and catalase were more representative than acidic, neutral phosphatase, and nitrate reductase as a comprehensive evaluation index. PLFA analysis showed that both the microbiological number and the abundance of (NPK)Cl+S treatments was the lowest, The biomass decreased 24.7% with NPK+S treatments relatively and a 43.2% reduction compared with the control. Chlorine fertilizer applications significantly affected the composition and quantity of microorganisms. The major behaviors are gram positive bacteria that may be relative to soil acidification and decline of soil enzyme activities. It’s also noted that (NPK)Cl+S treatments had a tendency to decrease crop yield. For example, rice and annual yield were decreased by 6.8% and 3.3% in average over 22 years. 【Conclusion】 Overuse of chloride containing fertilizer by applying ammonia chloride and potassium chloride together resulted in acidification of neutral purple soil, and then changed the soil microbial community and had the tendency to decrease soil biological fertility together with a declining rice yield. Thus we suggested not using two kinds of Cl-containing fertilizers together, especially for ammonia chloride which already has a high concentration of Cl- and salt index.
    Effects of Reduced Application of Nitrogen and Irrigation on Soil Nitrate Nitrogen Content and Nitrogen Balance in Greenhouse Production
    LI Ruo-nan, WU Xue-ping, ZHANG Yan-cai, WANG Li-ying, CHEN Li-li, ZHAI Feng-zhi
    Scientia Agricultura Sinica. 2016, 49(4):  695-704.  doi:10.3864/j.issn.0578-1752.2016.04.009
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    【Objective】Excessive applications of nitrogen and irrigation water in greenhouse vegetable production lead to lower nitrogen (N) use efficiency and unsustainable use of land. The aim of this study was to investigate the potential of a saving on N and water in vegetable production and the best combination of N and irrigation rates.【Method】An experiment was conducted in a 4-year cultivated greenhouse with winter-spring cucumber and autumn-winter tomato double cropping system. Two irrigation levels as conventional irrigation (W1, >100% field capacity) and reduced irrigation (W2, 75% field capacity) and 4 N levels as conventional N rate (N1), saving 25% N compared to conventional management (N2), saving 50% N compared to conventional management (N3) and no nitrogen control (N0) was designed. The corresponding N rates were 1200, 900, 600 and 0 kg·hm-2 for cucumber season and 900, 675, 450 and 0 kg·hm-2 for tomato season. Five combination treatments were designed as W1N1, W2N2, W2N3, W1N0, and W2N0 in this experiment.【Result】(1) Severe accumulation of NO3--N was observed in the conventional management, and the NO3--N was leached away from the surface soil layer. No significant NO3--N accumulation was observed in W2N3 treatment. The average NO3--N content in W2N3 treatment at 0-60 cm depth was at the proper level, and the average NO3--N content at 0-100 cm depth decreased by 13.9%-31.1% compared to W1N1 treatment. Moreover, the apparent N loss decreased by 56% and the N use efficiency increased by 2.4-3.3 percentage point in W2N3 with relatively higher economic benefit. (2) Based on the significant correlations between the NO3--N content and the yield, the optimal NO3--N contents were 37.4-72.9 mg·kg-1 for cucumber growing, and the corresponding values were lower than 90 mg kg-1 for tomato growing. (3) In order to keep the proper NO3--N value for uptaking by plants at 0-20 cm depth, 600 kg·hm-2of N with 450-550 mm of irrigation were recommended for cucumber with the yield level of 160-180 t hm-2, and 250 kg·hm-2 of N with 170-200 mm of irrigation were recommended for tomato of the yield level of 70-80 t·hm-2. (4) The mechanism for improving N use efficiency by reducing N and irrigation applications is as follows: more NO3--N could distribute around rootzone as saving 20%-30% of water, and less NO3--N could be accumulated in the soil profile as saving 50% of N supply. 20%-30% of water saving combined with 50% of N reducing could keep the NO3--N supply at a proper level at rootzone and decrease the loss amount of N, which become an effective way to improve N use efficiency.【Conclusion】The potential of a saving on water and nitrogen fertilizer is huge in North China Plain. Reasonable irrigation amount is the base to reduce N application and improve N use. Recommending reasonable N rate under the proper irrigation amount is an effective way to improve N use efficiency. Saving 20%-30% irrigation water combined with reducing 50% N fertilizer could effectively decrease the NO3--N accumulation in the soil and the apparent N loss and improve the N use efficiency.
    Effects of the Application of Flue Gas Desulphurized Gypsum and Water Leaching on Sodic Soil Aggregates
    TAN Huang, YANG Pei-ling, REN Shu-mei, YU Hao-liang, WANG Jia-hang
    Scientia Agricultura Sinica. 2016, 49(4):  705-716.  doi:10.3864/j.issn.0578-1752.2016.04.010
    Abstract ( 392 )   HTML ( 1 )   PDF (497KB) ( 421 )   Save
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    【Objective】The characteristics of soil aggregates are important factors that are generally used for soil structure evaluation. The objective of this study was to investigate the influence of the application of flue gas desulphurized (FGD) gypsum and water leaching on sodic soil aggregates, which can help assess the enhancement of soil structure after reclamation.【Method】A field experiment with nine reclamation treatments (three levels of FGD gypsum amount × three levels of leaching water) was conducted in the Hetao irrigation district in northern China. Intact soil samples were collected at 0-10cm, 10-20cm, 20-40cm, and 40-60 cm soil depths after reclamation. The bulk density, saturated hydraulic conductivity (Ksat), and characteristic parameters of soil aggregates, including the mean weight diameter (MWD), geometric mean diameter (GMD), the aggregate fractal dimension D, and the proportion of aggregates with a diameter higher than 0.25mm (DR0.25 and WR0.25, respectively) obtained by the dry and wet sieving methods, were used for analysis.【Result】The results indicated that reclamation treatments with both FGD gypsum and leaching water significantly decreased the bulk density and increased Ksat and the MWD and GMD of mechanic-stable aggregates at a 0-40 cm soil depth. In addition, the combined treatment with a high level of FGD gypsum (14.5 t·hm-2) and a low level of leaching water (1.52×103 t·hm-2) G2W1 showed the highest MWD and GMD of mechanic-stable aggregates. In general, the single reclamation treatments didn’t show a significant impact on the characteristics of soil aggregates, while the single treatments with leaching water alone had a significant higher fractal dimension D than the ones with FGD gypsum alone. The fractal dimension D in all combined treatments with a high level of FGD gypsum was lower than that of other treatments at a 0-60 cm soil depth. The MWD and GMD of the water-stable aggregates were significantly lower than those of the mechanic-stable ones. Most parameters of the sodic soil aggregates showed significant negative relations with the content of exchangeable sodium, exchangeable sodium percentage (ESP), and pH. The Ksat was positively related to the GMD, MWD, and DR0.25 of the mechanic-stable aggregates.【Conclusion】The combined reclamation treatments with both FGD gypsum and leaching water significantly increased sodic soil aggregates’ stability at a 0-40cm soil depth, suggesting that the structure of sodic soil was improved after reclamation. The reclamation with FGD gypsum alone maintained the stability of soil aggregates, while the ones with leaching water alone may have a negative influence on the formation of aggregates.
    The Correlation Analysis Between Quality and Creep Property of ‘Fuji’ Apple
    FANG Yuan, ZHAO Wu-qi, ZHANG Qing-an, GUO Yu-rong
    Scientia Agricultura Sinica. 2016, 49(4):  717-726.  doi:10.3864/j.issn.0578-1752.2016.04.011
    Abstract ( 438 )   HTML ( 10 )   PDF (355KB) ( 617 )   Save
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    【Objective】 The aim of this work was to study the feasibility of predicting the nutritional compositions and texture properties of ‘Fuji’ apples by analyzing the correlation between creep property of whole apple and texture, nutritional composition, respectively. 【Method】 The creep property, texture profile analysis and nutrition components of ‘Fuji’ apples in different storage time were measured in order to define the proper creep model and to explore the correlation between the indexes of texture profile analysis, nutrient components and the variables of creep property. Prediction models for ‘Fuji’ apples quality were constructed and verified based on the variables of creep property. 【Result】 The Burger’s model satisfactorily described the creep behavior of intact ‘Fuji’ apples and its coefficient of determination reached 0.982. Soluble solid content (SSC), titratable acidity, vitamin C content, hardness, cohesiveness, chewiness and resilience were significantly positively correlated (r=0.56-0.94) with the delayed elasticity coefficient E2 and the viscosity coefficient η1, respectively. Highly negative significant correlation (r=-0.40, r=-0.45) showed between soluble solid content, titratable acidity and viscosity coefficient η2, and vitamin C content had a significant negative correlation (r=-0.34) with viscosity coefficient η2. But there was no significant correlation between the adhesiveness, springiness and the variables of creep property. The correlation coefficients of models for predicting the soluble solid content, titratable acidity, vitamin C content, hardness, cohesiveness, chewiness and resilience of apple were 0.939, 0.922, 0.881, 0.917, 0.594, 0.857, and 0.584, respectively. Moreover all of the models have statistical significance (P<0.05). There was no significant difference between the predicted values of models and the measured data, and the correlation coefficients were 0.929, 0.917, 0.875, 0.920, 0.628, 0.824, and 0.633, respectively. 【Conclusion】 The variables of creep property showed close correlation with the parameters of texture profile analysis and the data of nutritional ingredients. The measured data were well fitted to the SSC prediction models, titratable acidity, vitamin C content, hardness and chewiness, which suggest that the creep property can be proposed for the reliable prediction of ‘Fuji’ apples quality.
    Extrusion Process of Germinated Brown Rice Compounded of Flammulina velutipes and Extrudant Quality Properties
    FANG Yong,WANG Hong-pan, YANG Wen-jian, PEI Fei, LIU Jun-fei, TANG Xiao-zhi,MA Ning, HU Qiu-hui
    Scientia Agricultura Sinica. 2016, 49(4):  727-738.  doi:10.3864/j.issn.0578-1752.2016.04.012
    Abstract ( 375 )   HTML ( 10 )   PDF (559KB) ( 577 )   Save
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    【Objective】In order to optimize the extrusion process conditions, the effects of extrusion operating parameters on nutritional quality, physicochemical properties and volatile aroma components in final products of germinated brown rice compounded of Flammulina velutipes were investigated. The study will provide theoretical basis for quality evaluation and development of functional snack foods. 【Method】 To analyze the effects of extrusion operating parameters on GABA content, soluble dietary fiber content, soluble protein content and radial expansion ratio, germinated brown rice compounded of Flammulina velutipes were extruded to obtain a composite type of puffed products with a twin-screw extruder at different moisture content, extrusion temperature and screw speed conditions. Changes of the amino acids content and physicochemical properties such as hardness, bulk density, water-absorption index, water-solubility index and color difference of germinated brown rice compounded of Flammulina velutipes extrudates were analyzed compared with non-supplemented germinated brown rice products. In addition, the volatile flavor compounds in the puffed products were also analyzed comparatively using electronic nose. 【Result】The quality properties of germinated brown rice compounded of Flammulina velutipes extrudateschanged with the extrusion operating parameters. The GABA content showed a trend from decline to rise as the moisture content kept increasing, dropped sharply as the extrusion temperature increased, and increased at first, but then decreased. With the moisture content, the extrusion temperature and the screw speed increasing, both the SDF content and the soluble protein content had a decline trend after an initial ascent. The radial expansion ratio decreased with the moisture content and the extrusion temperature increasing, while increased first but then decreased as the screw speed continued increasing. The optimal conditions of the extrusion were moisture content 17%, extrusion temperature 140℃ and screw speed 150 r/min from the single factor test. Under these conditions, the content of GABA, SDF and soluble protein were (210.44 ± 0.39) mg·kg-1, (0.735 ± 0.028) g·100 g-1 and (1.23 ± 0.01) mg·g-1, respectively, and the radial expansion ratio of the extruded products was 2.67 ± 0.02. Compared with the un-extruded compounded powder and germinated brown rice extruded products, the total amount of amino acids of germinated brown rice compounded of Flammulina velutipes extrudatesincreased by 1.7% and 2.9%, respectively, and essential amino acid content increased by 2.8% compared with non-supplemented germinated brown rice extrudates, among which, the arginine and lysine content increased by 6.6% and 5.7% respectively, indicating that the addition of Flammulina velutipes powder could increase the amino acid content significantly. The extruded products from germinated brown rice compounded of Flammulina velutipes exhibited lower radial expansion ratio, lower degree of gelatinization, lower water-solubility index and lower L* value, but higher a*, b*and ΔE values than extruded products of non-supplemented germinated brown rice, which indicated that germinated brown rice compounded of Flammulina velutipes extrudates showed greater browning degree. The analytical results of electronic nose showed that the volatile flavor compounds were significantly different between the extruded products from germinated brown rice compounded of Flammulina velutipes and non-supplemented germinated brown rice, which reflected the overall flavor profile of different samples accurately and quickly. 【Conclusion】 The products of germinated brown rice compounded of Flammulina velutipes had comprehensive nutrition, good taste and unique flavor characteristics after the optimization of extrusion technology. Extrusion technology was an effective method to improve the nutritional quality and taste flavor of germinated brown rice.
    Genome-Wide Association Study of 25 Hematological Parameters in Western DLY Pigs and Two Chinese Erhualian and Laiwu Populations
    LIU Chen-long, YANG Hui, ZHANG Hui, ZHANG Zhi-yan, DUAN Yan-yu
    Scientia Agricultura Sinica. 2016, 49(4):  739-753.  doi:10.3864/j.issn.0578-1752.2016.04.013
    Abstract ( 383 )   HTML ( 5 )   PDF (4507KB) ( 498 )   Save
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    【Objective】 The genome-wide association of 25 hematological traits in Western DLY pigs and two Chinese Erhualian and Laiwu populations was analyzed in this study. The findings would benefit the final identification of causative genes, and provide insights into the potential molecular basis on hematological traits and provide clues for pig anti-disease breeding and production. 【Method】 A total of 610 DLY ternary hybrid pigs at (180±5) days, 336 Erhualian pigs and 333 Laiwu pigs at (300 ±5) days after birth were slaughtered at Guohong commercial abattoir. Blood samples at 2mL of each pig were immediately collected in anticoagulation tubes at slaughter. Twenty-five hematological traits were determined with commercial kits. Genomic DNA was extracted from ear tissues using a standard phenol/chloroform method, the concentration and quality were determined by NANODROP 1000 analyzer. All individuals were genotyped with Illumina porcineSNP60 Bead-Chip. After the genotype quality control by PLINK software, the GWAS were performed with the mixed linear model using GenABEL package in the R software.【Result】 After quality control of phenotype and genotype, there were 552 DLY, 325 Erhualian and 281 Laiwu left for the study, respectively; and there were 56 216 SNPs left for DLY pigs, 49 343 SNPs for Laiwu pigs, 35 974 SNPs for Erhualian pigs and 32 967 SNPs for Meta analysis. The GWAS identified 610 significant loci for 25 hematological traits, of which 135 SNPs were at the genome significant level and 475 SNPs were at the suggestive level, on all pig chromosomes in three populations. In DLY ternary hybrid pigs, 32 genome significant SNPs were identified in 8 traits (LYM,LYMR,BAS,BASR,MCV,RDW_CV,MCH and PDW) and other 85 suggestive level SNPs were also identified. In Erhualian pigs, 33 genome significant SNPs were identified in 9 traits (LYM, MCH, MCHC, MON, MONR, MPV, NEUR, P_LCC and PCT) and other 139 suggestive level SNPs were also identified. In Laiwu pigs, 54 genome significant SNPs were identified in 6 traits (BASR, HCT, MCH, MCHC, MCV and RBC) and other 169 suggestive level SNPs were also identified. In Meta results, 16 genome significant SNPs were identified in 6 traits (RBC, HCT, MCH, MCHC, MCV and MON) and other 82 suggestive level SNPs were also identified. Five promising candidate genes were found in the neighboring region of the lead SNPs at the genome-wide significant loci, such as F13A1 for BASR, SPTA1 for HCT, DBNL for LYM. SLC25A28 for MCHC and CTSC for NEUR. 【Conclusion】 A total of 610 loci surpassing the suggestive significant level were identified for 25 hematological traits on all pig chromosomes in Western DLY pigs and two Chinese Erhualian and Laiwu populations, revealing the complex genetic architecture of hematological traits. Neighboring the lead SNPs at the 54 genome-wide significant loci, five promising candidate genes were found, such as F13A1 for BASR, SPTA1 for HCT, DBNL for LYM, SLC25A28 for MCHC and CTSC for NEUR, and thus providing clues for analysis of the hematological traits and immune disease of commercial pigs and pure indigenous breeds.
    Identification and Bioinformatics Analysis of Differential Expression MicroRNAs in the Spleen Between Beijing Fatty Chickens and Leghorns Chickens
    GENG Li-ying, PAN Su-min, CHEN Juan, ZHU Wen-jin, GONG Yuan-fang, LIU Zheng-zhu, PENG Yong-dong, ZHAO Shu-yu, ZHANG Chuan-sheng, LI Xiang-long
    Scientia Agricultura Sinica. 2016, 49(4):  754-764.  doi:10.3864/j.issn.0578-1752.2016.04.014
    Abstract ( 410 )   HTML ( 1 )   PDF (1350KB) ( 517 )   Save
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    【Objective】 To investigate the differences of spleen weight, histological structure and miRNA expression profile in Beijing Fatty Chicken and Leghorn Chicken, we screened the candidate genes related with the immune response capacity difference in two kind of chickens, so as to lay a foundation for poultry breeding for disease resistance. 【Method】 45 one-day-age Beijing Fatty Chickens and 45 female Leghorn pullets hatched in the corresponding period were selected as the experimental materials, fed in the same nutritional and environmental condition. The body weight and spleen weight were measured in 42 days. Three individuals were randomly selected for each species (body weight was close to the average value of the group). Half of the spleen was made into the paraffin sections. The spleen histological structure, number of splenic corpuscles and thickness of the periarterial lymphatic sheath were observed using HE staining. The small RNA was extracted from other half of the spleen tissue and composed into 2 RNA pools through balanced mix. The cDNA library was constructed. The high-throughput sequencing was conducted using a Solexa platform. The expressed genes were obtained through comparing the sequencing result and reference genome database. The new miRNAs gene was predicted using Mireap software. The gene expression was calculated using RPKM (reads per kb per million reads) method. The differentially expressed genes were screened according to the criteria of FDR(false discovery rate)<0.001 and |log2 ratio (T/CK)| ≥1. The target genes differentially expressing miRNA were predicted using TargetScan and Pictar softwares, the GO and KEGG database functions were annotated. The enrichment analysis of the target gene protein was conducted using DAVID software. The predicted target genes were intersected with the known QTL region annotation genes related with spleen weight and spleen index. 【Result】The body weight of the Beijing Fatty Chicken and spleen weight were significantly higher than those of Leghorns (P<0.01). Histological observation showed that the tissue structure of the spleen was integrated in the two chicken breeds. The white pulp, red pulp, splenic corpuscle and periarterial lymphatic sheath could be clearly observed. Compared with the Leghorn chicken, the number of splenic corpuscles was more, periaterial lymphatic sheaths were thicker and intrathecal lymphocytes were more intensive in the Beijing Fatty Chicken. 486 known miRNAs, 130 candidate miRNAs and 216 coexpressed miRNAs were identified in the two groups of samples using high-throughput sequencing. There were 35 kinds of miRNAs differentially expressed between two groups. Among them, 8 kinds were up-regulated and 27 kinds were down-regulated in the Beijing Fatty Chicken. Their fold change was 1.002-10.41. The first five expressive abundances of differentially expressed genes were miR-2954, miR-6606-5p, miR-146b-5p, miR-21-3p and miR-21-5p. The bioinformatics analysis of 2767 target genes showed that they were involved in the biological processes of T, B lymphocyte cell differentiation, immune organ development, protein phosphorylation and cell morphogenesis. They were significantly enriched in ubiquitin mediated proteolysis, apoptosis, phosphatidylinositol signaling system etc. 14 pathways. The miR-6606-5p and miR-146b-5p common target genes- BLM and miR-2954 target gene IRF8 were localized in the QTL region related with spleen weight or spleen index. 【Conclusion】(1) There were some differences in spleen weight, histological structure and miRNA expression profile of the 42-day-old Beijing Fatty Chickens and Leghorn Chickens. (2) The differential high abundance miRNA (miR-2954, miR-6606-5p, miR-146b-5p, miR-21-3p and miR-21-5p) might be involved in the regulation of T, B lymphocytes differentiation, proliferation, activation and other processes through regulating target gene, so as to affect the spleen weight, organizational structure and immune response capacity.
    Diagnosis of the Ascosphaera apis by the Loop-Mediated  Isothermal Amplification
    XI Wei-jun, LI Jiang-hong, CHEN Da-fu, LIANG Qin
    Scientia Agricultura Sinica. 2016, 49(4):  765-774.  doi:10.3864/j.issn.0578-1752.2016.04.015
    Abstract ( 424 )   HTML ( 1 )   PDF (1619KB) ( 547 )   Save
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    【Objective】 The objective of this study is to establish a rapid and sensitive method for detecting the Ascosphaera apis in epidemiological investigations by the loop-mediated isothermal amplification (LAMP), and to provide technical supports for monitoring and prevention of chalkbrood in honeybee.【Method】Four specific primers were designed based on the particular sequence of ITS of the A. apis using the software of PrimerExplorer V4.0 online. The primers included A. apis-F3 (5′-ACATTGC GCCCTCTGGTA-3′), A. apis-B3 (5′-TGGTTAGACCGGACAGTCG-3′), A. apis-FIP (5′-TAAGACGGGACGATCGCCCAACC TGTCCGAGCGTCATTG-3′) and A. apis-BIP (5′-GAAAGGCAGTGACGGCGTCGGGCCACTAGAGCGAAAGAC-3′). LAMP were performed under different concentrations such as Mg2+ (0, 2, 4, 6, 8, 10, 12, and 14 mmol·L-1), dNPTs (0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, and 1.8 mmol?L-1), FIB/BIF (0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, and 1.8 mmol·L-1), betaine (0, 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 mol?L-1), reaction temperatures (58, 60, 63, and 65) and reaction times (30, 40, 50, and 60 min), respectively. The LAMP result was determined by both the turbidimeter and agarose gel electrophoresis in order to optimize the reaction conditions. The specificity of LAMP was tested by using genomic DNA of Nosema ceranae, Nosema apis, Deformed wing virus (DWV), Sacbrood virus (SBV), Black queen cell virus (BQCV) and Israel acute paralysis virus (IAPV). In addition, the LAMP amplified products were digested with PvuⅠrestriction enzyme. The sensitivity of LAMP was compared with normal PCR by employing ten-fold serially diluted DNA templates of A. apis (including 0.2231, 0.2231×10-1, 0.2231×10-2, 0.2231×10-3, 0.2231×10-4, 0.2231×10-5, 0.2231×10-6, 0. 2231×10-7, and 0.2231×10-8 μg·μL-1). The practical reliability was achieved in clinical trials.【Result】A rapid and specific method for detction of A. apis was established. The optimum conditions for LAMP reaction were as follows: 4 mmol·L-1 Mg2+, 1.2 mmol·L-1 dNTPs, 1.6 mmol·L-1 FIP/BIP, 0.4 mol·L-1 betaine, and the method was performed at 63 for 60 min. The templates of A. apis, N. ceranae, N. apis, DWV, SBV, BQCV and IAPV were tested, the result showed that only the A. apis DNA template could produce typical ladder-like bands. The sensitivity test showed that the PCR could detect the DNA templates as low as 0.2231×10-5 μg·μL-1. While the LAMP could detect ten times lower DNA templates of 0.2231×10-6 μg·μL-1. The LAMP is effective, simple and time saving for detecting the A. apis infect honeybee. 【Conclusion】The method of LAMP was proved to be a precise, faster, lower cost in diagnosis of chalkbrood in honeybee. It also could be applied in both research institutions and field.
    Expression and Interaction Between ROH1 and EXO70A1 in Reproductive Development
    ZHANG He-cui, LIU Jing, LIAN Xiao-ping, ZENG Jing, YANG Kun, ZHANG Xue-jie, YANG Dan, SHI Song-mei, GAO Qi-guo, ZHU Li-quan
    Scientia Agricultura Sinica. 2016, 49(4):  775-783.  doi:10.3864/j.issn.0578-1752.2016.04.016
    Abstract ( 348 )   HTML ( 7 )   PDF (1373KB) ( 401 )   Save
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    【Objective】The objectives of this study were to clone ROH1 and EXO70A1 from the Brassica oleracea bloom, and establish whether EXO70A1 and ROH1 are involved in the reproductive development of Brassica oleracea. 【Method】The coding sequences of ROH1 were cloned by PCR from Brassica oleracea. In addition, the coding sequences of EXO70A1 was cloned from Brassica oleracea mRNA. RT-PCR was used to analyze expression characteristics of ROH1, while Real Time PCR was used to analyze expression of ROH1 and EXO70A1 in 1 hour post-pollination. A Yeast Two-hybrid System was used to determine the interaction between ROH1 and EXO70A1.The encoding sequences of ROH1 was subcloned into vector pGADT7 and Exo70A1 was subcloned into vector pGBKT7. Then the interaction between ROH1 and EXO70A1 was tested in selection medium by yeast two-hybrid System.【Result】We demonstrated that the ROH1 gene is a single-exon gene encoding a 398-amino-acid protein in Brassica oleracea. With the ROH1 in Arabidopsis, the sequence losses 16 amino acid residues in Brassica oleracea, In addition, ROH1 expressed in stems, styles, anther, young roots and leaves. Notably, expression levels varied in different tissues, with strong expression in styles, anthers and leaves, but with weak expression in stems and roots. After pollination, the expression of ROH1 gene increased between 0-15 min, decreased from 15 min, and peaked at 1 hour post-pollination The expression of EXO70A1 gene tracked ROH expression pattern. The changes in expression of EXO70A1 and ROH1 implied that they were involved in reproductive development. The expression of ROH1 and EXO70A1 appeared in overlapping region at 30 min. This overlap suggested that a potential interaction between ROH1 and EXO70A1 in Brassica olerace. The recombinant expression vectors did not exhibit autoactivation and toxicity. Yeast Two-hybrid system experiments established that the recombinant expression vectors could grow well on nutritional media without transcription activation of the reporter genes AUR1-C, MEL1, HIS3 and ADE2, and it showed an interaction between ROH1 and EXO70A1 in Brassica oleracea.【Conclusion】The strong interaction between ROH1 was detected during reproductive development in Brassica oleracea. It is speculated that ROH1 and EXO70A1 affects the reproductive development by regulating the secretion of the stigma EXO70A1, putting forward a novel insight into the reproductive development process in Brassica oleracea system.
    Identification of Five Viroids Infecting Citrus with Herbaceous Plant Tomato
    DONG Yan-na, ZHENG Yin-ying, XU Wen-xing
    Scientia Agricultura Sinica. 2016, 49(4):  784-790.  doi:10.3864/j.issn.0578-1752.2016.04.017
    Abstract ( 454 )   HTML ( 1 )   PDF (1240KB) ( 457 )   Save
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    【Objective】 A suitable season and a long time are needed for the symptom visualization by grafting onto woody indicator plant to identify the viroids infecting citrus. Moreover, the system is not well developed for genetic transformation and function identification in citrus indicator plants, thus, it is difficult to study the interaction between viroids and their host in such plant. Therefore, it will be important to find suitable herbaceous plants as indicator and experimental host for viroid idenfication and study on the interaction between viroids and their host. In this study, five viroids including Citrus exocortis viroid (CEVd), Citrus bent leaf viroid (CBLVd), Hop stunt viroid (HSVd), Citrus dwarfing viroid (CDVd), and Citrus bark cracking viroid (CBCVd) have been reported to be the major ones infecting citrus trees in China. The objective of this study is to identify whether the five viroids infect tomato (Solanum lycopersicum), and then to clarify whether the tomato can be adopted as the herbraceous and experimental host plant for the five viroids.【Method】 After enzyme-digested with Nde I at 37 into linearized form, the pGEM-T plasmids containing the dimerized cDNA of each wild type of five citrus viroids (CEVd.188, 370 nt in length; CBLVd.032, 328 nt in length; HSVd.cit106, 296 nt in length; CVd-III.072, 295 nt in length; CVd IV Ca, 285 nt in length) were in vitro transcribed with T7 RNA polymerase at 37, the obtained dimeric RNAs were inoculated into tomato leaves of S. lycopersicum vars. Alisa Craig and Rutgers by mechicanlly rubbling manners, and incubated in a greenhouse at 24℃. New developed tomato leaves were collected and extracted their total RNAs after 60 days post inoculation (dpi), and subjected to detection of the inoculated viroids by RT-PCR. The resulted RT-PCR products were analyzed on agarose gel, purified by excising the target bands from the gel, ligated with pMD18-T vector, and transformed into competent cells of Escherichia coli DH5α. After idetification by PCR, positive clones were choicely sequenced and their cDNA sequences were aligned using software DNAMAN version 6.0. 【Result】After transcribed based on the lineralized plasmids, analyses on agarose gel revealed the target RNA bands with expect sizes for all the plasmids. After inoculation, RT-PCR identification of the inoculated viroids revealed that the infectivity significantly varied among the five viroids. Of which, CEVd, CDVd and HSVd infected most inoculated seedlings, with total infectivity of 7/8, 5/8 and 7/8, respectively. Sequencing five to ten clones chosen in random showed that each parental sequence could be found in the progeny sequences, and their progeny sequences had similarities more than 99.7% with the parental sequences, with varibilities less than 0.3% (less than 10 mutations). While CBLVd and CBCVd could not infect the inoculated seedlings or had a vey low infectivity (1/8) for the latter. 【Conclusion】 CEVd, CDVd and HSVd can infect S. lycopersicum vars. Alisa Craig and Rutgers, which could be acted as herbaceous indicator and experimental plants. Whereas it is difficult to infect vars. Rutgers and Alisa Craig for CBLVd and CBCVd. For the inoculated seedlings of var. Alisa Craig, they all were significantly dwarfed than the control seedlings inoculated with buffer for all these five viroids; whereas for the inoculated seedlings of var. Rutgers, only the ones inoculated with CEVd showed the dwarfed effect compared with the controls.
    Effects of Different Planting Methods on Growth of Alfalfa and Weeds in Gannan Alpine Region
    YU Xiao-jun, CHAI Jin long, XU Chang-lin, SHI Shang-li, XIAO Hong, MA Long-xi, CAO Guo-shun
    Scientia Agricultura Sinica. 2016, 49(4):  791-801.  doi:10.3864/j.issn.0578-1752.2016.04.018
    Abstract ( 402 )   HTML ( 1 )   PDF (409KB) ( 619 )   Save
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    【Objective】Lacking the appropriate cultivation techniques of alfalfa and weed control technology in the Gannan alpine region has seriously affected the development of the alfalfa and animal husbandry. In order to provide a basis for the establishment of alfalfa grassland, the effects of different planting methods on the growth characteristics of alfalfa and weeds in the Gannan alpine region were studied. 【Method】The experiments were conducted in Xiahe alpine meadows, Gansu province. Four planting method treatments included I: black plastic film mulching on ridge and furrow, II: black plastic film mulching parallel to the ground, III: ridge and furrow, and Ⅳ: common planting methods were designed to discover the effects of those four treatments on the growth characteristics of alfalfa and weeds. 【Result】 The alfalfa height of the treatment of III (19.1 cm) and Ⅳ (14.4 cm) were significantly lower than that of the treatment I and II (P<0.05). Under treatment I, the branch number of alfalfa can be up to 7.7 per plant, significantly higher than that of treatment II( 5.2), III(4.8) and Ⅳ(4.5) (P<0.05). There was no significant difference between the treatment I (2.70 mm) and II (2.50 mm) for the main stem diameter (P>0.05), but both of them were significantly higher than III and Ⅳ (P<0.05). The alfalfa crown diameter of treatment I was 6.19 mm, significantly higher than that of the treatment II (5.29 mm) (P<0.05), the treatment II was significantly higher than that of the treatment III (3.99 mm), and the treatment Ⅳ (2.80 mm) was significantly lower than that of the former three (P<0.05). Crown depth of treatment I was 2.73 cm, significantly higher than II (2.24 cm) (P<0.05). Compared with the treatment III and Ⅳ, alfalfa crown depth of I increased by 56.0 % and 29.4 %. Under the condition of I, dry matter of alfalfa forage production was up to 1 503.2 kg·hm-2, significantly higher than that of the treatment II (1 089.6 kg·hm-2), III (317.6 kg·hm-2) and Ⅳ (224.4 kg·hm-2) (P<0.05). The root volume, root surface area, and root biomass of treatment I and II were significantly higher than treatment III and Ⅳ at the depth from 0 to 10, 10 to 20, 20 to 30, 30 to 40 cm and 0 to 40 cm (P<0.05). The root volume, root surface area. The root volume, root surface area, and root biomass at the deep from 0 to 10 cm, 10 to 20 cm, 20 to 30 cm and 0 to 40 cm of the treatment III was significantly higher than that of the treatment Ⅳ except that of the deep from 30 to 40 cm(P<0.05). In the first planting year of artificial alfalfa, 21 kinds of weeds were found. The main weeds included Nepeta cataria, Euphorbia esula, Hypecoum leptocarpum, Chenopodium album and some other annual plants. The number of weed species of treatment III and Ⅳ were 14.3 and 13.3 respectively, and there was no significant difference between III and Ⅳ (P>0.05); while both of them were significantly higher than that of the treatment I (9.3) and II (9.7). The highest total density of weeds was the treatment III and was 272.3 plant/m2, then the treatment Ⅳ (241.7 plant/m2); and the former two were significantly higher than that of the treatment I (86.0 plant/m2) and II (74.0 plant/ m2) (P<0.05). Dry matter of weeds aboveground of treatment I was the highest for 186.8 g·m-2, then was the treatment II (157.7 g·m-2), III (88.5 g·m-2) and Ⅳ (79.0 g·m-2). 【Conclusion】Both using black plastic film mulch on ridge and furrow and film mulch parallel to the ground improved the plant height, main stem diameter, crown diameter, crown depth and the dry matter of alfalfa, significantly increased alfalfa root biomass, roots surface area and root volume. Also, it improved dominant weed biomass and weed plant height, while greatly reduced the number of weed species and density.
    Research on the Quality Evaluation for Peach and Nectarine Chips by Explosion Puffing Drying
    Lü Jian, LIU Xuan, BI Jin-feng, ZHOU Lin-yan, WU Xin-ye
    Scientia Agricultura Sinica. 2016, 49(4):  802-812.  doi:10.3864/j.issn.0578-1752.2016.04.019
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    【Objective】 The aim of the paper was to investigate the variations in the comprehensive quality of peaches and nectarine chips from the different cultivars and establish scientific evaluation models for peach and nectarine chips. 【Method】 Peach and nectarine fruit from 49 varieties grown in the north of China were selected for the testing materials. 17 quality evaluation indexes were measured, including organoleptic quality indexes (e.g. color, hardness, crispness, explosion ratio, and so on), physical and chemical characteristic indexes (e.g., soluble solid content, crude fat content, crude protein content, crude fiber content, and so on) and processing quality indexes (output ratio and rehydration ratio). The method of a variable coefficient was used to investigate the differences in quality evaluation indexes from different peach and nectarine cultivars. The relationship between these indexes and the characteristic indexes were selected by factor analysis (FA). The weights and the levels of the characteristic indexes were calculated by an analytic hierarchy process (AHP) and a range analysis method, respectively. The levels in cultivars effectively were classified by discrimination functions which were obtained by K-means cluster (KC) and discriminate analysis (DA). 70% of the samples were selected as the testing samples, and the others were used as the verification set data, which could verify the fitness of the functions. 【Result】 (1) 17 quality evaluation indexes dispersed with the coefficient of variation ranging from 0.70%-344.02%. (2) Five characteristic indexes were determined based on the method of FA, which explained 74.626% of the total variances. The first principal component (PC1) was related to reducing sugar content and sugar-acid ratio, which were the taste quality factors. The principal component (PC2) was related to the output ratio and rehydration ratio, which were the processing quality factors. The third principle component (PC3) was related to the L value and b value, which were the color quality factors. And the fourth and fifth principal components (PC4 and PC5) were related to the crude protein content and explosion ratio, respectively, which had higher weights than the others. Five quality evaluation indexes were selected as the characteristics indexes, namely, reducing sugar, rehydration ratio, L value, crude protein content, and expansion ratio. (3) Based on the AHP, the weights of the characteristics indexes were 0.0824, 0.1724, 0.2732, 0.0480, and 0.4240, respectively. Also, the scoring standard of the characteristic indexes were established. (4) Discrimination functions of different grades were established, which had satisfactory recognition accuracy up to 100%, and only one sample was discriminated inaccurately. Ruipan19, Delaifulaika, and Dajiubao were the best cultivars used to produce peach and nectarine chips, Ruipan 21, Juhuang, and Yanhong were the good cultivars used to produce peach and nectarine chips, and Rupan 20, Sengelin, and Huangjinxiu were the worst cultivars used to processing peach chips. 【Conclusion】 Peach and nectarine chips’ comprehensive quality can be evaluated by 5 characteristics indexes, namely, reducing sugar content, rehydration ratio, L value, crude protein content, and expansion ratio. The grading standard and the scoring standard of the 5 indexes set up a scientific foundation for evaluating peach and nectarine chips’ quality. The established discrimination functions were effective in discriminating peach and nectarine chips’ quality.