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Table of Content

    16 September 2014, Volume 47 Issue 18
    CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS
    Advances in Research of Transcriptional Regulatory Network in Response to Cold Stress in Plants
    LIU Hui, LI De-jun, DENG Zhi
    Scientia Agricultura Sinica. 2014, 47(18):  3523-3533.  doi:10.3864/j.issn.0578-1752.2014.18.001
    Abstract ( 695 )   HTML ( 9 )   PDF (864KB) ( 3456 )   Save
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    Cold stress seriously influences plant growth, development, and crop yield. In order to survive, plants have evolved complex and high-efficiency regulatory networks to respond and adapt to cold stress. Among these regulatory networks, transcriptional regulation plays crucial roles. Transcription factors can regulate a set of genes by binding to cis-acting regulatory elements in the promoter regions, and play crucial roles in abiotic stress-responsive transcriptional regulatory network in plants. In this review, the authors comprehensively summarized the transcription factors involved in regulating plant response to cold stress, including AP2/ERF (APETALA2/ethylene responsive factor), MYB (myeloblastosis), bHLH (basic helix-loop-helix), NAC (NAM, ATAF1, ATAF2 and CUC2), ZFP (zinc finger protein), WRKY, VOZ (vascular plant one zinc-finger protein), CAMTA (calmodulin-binding transcription activator), and EIN3 (ethylene-insensitive 3). Their structure characteristics were simply summarized, while their functions and regulatory mechanisms were emphatically introduced. Based on the relationships among transcription factors, a transcriptional regulatory network diagram of plant response to cold stress was draw. In this network, CBF (C-repeat binding factor) transcription factors are considered as master molecular switches. CBFs specifically bind to the DRE/CRT (dehydration-responsive element/C-repeat element, A/GCCGAC) cis-acting regulatory element of the promoter region of the cold-responsive genes, such as COR (cold regulated), LTI (low-temperature induced), DHN (dehydrin), RD (responsive to dehydration), etc, and activate their expression. The expression of CBFs are positively regulated by ICE1/2 (inducer of CBF expression 1/2), CAA1 (circadian clock-associated 1), LHY (late elongated hypocotyl), MYB56, ZFP1/182, and CAMTA1/2/3, whereas negatively regulated by MYB15, MYBS3, ZAT12, PIF4/7, WRKY34, and EIN3. The transcriptional activity of ICE1 protein is strictly regulated by post-translational modification, including SIZ1 (SAP and Miz 1)-mediated SUMO (small ubiquitin-related modifier) modification and HOS1 (high expression of osmotically responsive gene 1)-mediated ubiquitination. The HOS1–SIZ1 system strictly regulates and perfectly fine-tunes the expressions of ICE1–CBFs and their targets to cope with temperature change. Moreover, the expression of ICE1 is negatively regulated by JAZ1/4 and positively regulated by ERF2. Except the CBF-dependent cold-response pathway, some transcription factors regulate responses of plant to cold stress through CBF-independent pathways, such as JERF3, MYB2/4/96/3R-2, WRKY19/21/76, NAC1/2, SAP1/8, etc. Uncovering transcriptional regulatory network in response to cold stress in plants provide a theoretical basis for improving the cold tolerance of crops through transgenic technology. Transgenic studies indicate that some transcription factors possess potential usefulness in genetic improvement of cold stress tolerance in plants, such as AtCBF1AtCBF3, AtICE1, AtCCA1α, TaCBF14/15, TaNAC2, TaWRKY19, VrCBF1/4, MdCIbHLH1, PtrbHLH, OsMYB2, GmNAC20, JERF3, and ZFP182. Furthermore, the main problems in current researches and the key points in future studies were also proposed, in hope of providing reference for further revealing the molecular mechanisms underlying plant adaptation to cold stress.
    Characteristics and Function of a GmDREB5-Interacting Protein GmUBC13 in Soybean
    XU Dong-bei, YU Yue-hua, HAN Qiao-ling, MA Ya-nan, GAO Shi-qing, TIAN Ye, XU Zhao-shi, LI Lian-cheng, QU Yan-ying, MA You-zhi, CHEN Ming, CHEN Yao-feng
    Scientia Agricultura Sinica. 2014, 47(18):  3534-3544.  doi:10.3864/j.issn.0578-1752.2014.18.002
    Abstract ( 490 )   HTML ( 2 )   PDF (3782KB) ( 673 )   Save
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    【Objective】The objectives of this research are to identify the interacting proteins of stress related DREB like transcription factor, GmDREB5 in soybean (Glycine max), analyze the characteristics and biological function of its interaction protein, GmUBC13, and study the regulation mechanism that GmDREB5 enhanced the stress tolerance in plants. 【Method】 By yeast two hybrid system, AP2 domain of GmDREB5 was used as a bait to screen the interacting proteins of GmDREB5 from the drought-treated soybean cDNA library, and further interaction between GmDREB5 and its candidate interacting protein was confirmed through interacting testing in yeast and pull-down assay in vitro, then, the properties of interacting protein, GmUBC13, such as phylogenetic tree, protein structure, subcellular localization were analyzed; expression profiles of GmUBC13 were tested under drought, salt, cold and ABA treatment by RT-PCR, and biological functions of GmUBC13 were also identified through transformation of tobacco in this research. 【Result】A GmDREB5-interacting protein, GmUBC13, belonging to the ubiquitin conjugating enzyme protein family, was obtained via screening drought-treated soybean cDNA library, and GmUBC13 contained UBCc (Ubiquitin-conjugating enzyme catalytic domain) domain, highly conserved E3 interaction residues and cysteine catalytic sites. Phylogenetic analyses showed that GmUBC13 shared the high homology with two members of the XV subgroup (containing total sixteen members) of ubiquitin conjugating enzyme like protein family, AtUBC13A (99%) and AtUBC13B (97%) in Arabidopsis and a ubiquitin conjugating enzyme like protein, Os01g0673600 (97%) in rice (Oryza sativa). Interaction between GmDREB5 and GmUBC13 was confirmed by interaction test in yeast and pull-down assay in vitro. The expression of GmUBC13 gene in soybean was analyzed by drought, high salt, low temperature and ABA treatment. After ABA application, the transcript of GmUBC13 was induced after 1 h and reached the peak at 10 h, and slight down-regulated at 24 h. Under drought- and salt-treatment, the transcript of GmUBC13 was induced after 1 h and gradually increased along with the treatment time, and reached the peak at 24 h; under the low temperature, the expression of GmUBC13 induced rapidly and reached the peak at 5 h, and there was no expression at 10 h and 24 h. Further subcellular localization analysis showed that GmDREB5 was located on the nuclear and cytoplasm, and GmUBC13 was located on the nuclear. The analysis result of gene function showed that there was no difference among transgenic tobacco lines GmUBC13-1, GmUBC13-2 and wild type (W38) under normal MS medium, whereas exposed to different concentrations of PEG, the chlorophyll contents decreased, and the length of root and segment above ground were inhibited in all plants. However, the chlorophyll contents were higher, and the length of root and segment above ground was longer in GmDREB13 transgenic plants compared with W38, and there was a significant difference between transgenic lines GmUBC13-2 and W38 under treatment of 8% PEG. For the drought tolerance assay, two-week old transgenic plants and W38 were transferred into soil and withheld water for 21 days, and then re-watered for 6 days. Final results showed that transgenic lines grew healthier than W38, and the survival rates of transgenic lines were significantly higher than W38, suggesting that overexpression of GmUBC13 enhanced drought tolerance in transgenic plant.【Conclusion】 Soybean ubiquitin conjugating enzyme, GmUBC13 interacted with stress related transcription factor GmDREB5, and GmUBC13 was induced by various stress treatments, and overexpression of GmUBC13 enhanced drought tolerance in transgenic tobacco plants.
    TILLAGE & CULTIVATION·PHYSIOLOGY & ECOLOGY
    Optimization of Samples Layout in Spatial Sampling Schemes    for Estimating Winter Wheat Planting Acreage
    WANG Di, CHEN Zhong-xin, ZHOU Qing-bo, LIU Jia
    Scientia Agricultura Sinica. 2014, 47(18):  3545-3556.  doi:10.3864/j.issn.0578-1752.2014.18.003
    Abstract ( 539 )   HTML ( 1 )   PDF (2737KB) ( 532 )   Save
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    【Objective】 Sample layout is one of the key factors in spatial sampling schemes for estimating crop planting acreage. Optimization of sample layout plays an important role in improving the representativeness of samples versus population and the accuracy of population extrapolation, decreasing the cost of survey sampling. In this study, focusing on the problem that the formulation of samples layout in the spatial sampling scheme for crop acreage estimation was not reasonable (e.g. samples units are  not all independent of each other, when simple random sampling method was used to formulate samples layout; sampling intervals are not able to be reasonably defined, when systematic sampling method was used in the design of samples layout), the author tried to propose a optimal formulation of samples layout to improve the efficiency of the spatial sampling scheme further.【Method】Mengcheng County in Anhui Province, China was chosen as the study area, winter wheat planting acreage as the study object, and square girds as the shape of sampling units. Geostatistics, “3S” techniques (Remote Sensing, Geographic Information Systems and Global Positioning techniques) and traditional sampling methods were used in this study. Firstly, 8 kinds of sampling unit sizes were formulated, and then the study area was split by the sampling units with these 8 kinds of sizes to construct the sampling frame. The winter wheat acreages in all sampling units were calculated based on the spatial distribution data of winter wheat in 2009 and 2010(derived by ALOS AVNIR-2 and Landsat5 TM image, respectively). Secondly, in order to build the Variogram theoretical model of winter wheat planting acreage proportion within the sampling unit (WPS), simple random sampling method was used to draw the initial samples. Spatial correlation and variability of sampling units were analyzed, and spatial correlation threshold was quantitatively determined by the Variogram theoretical model. Thirdly, the equi-spaced pattern (sampling intervals were identical in vertical and horizontal directions, and spatial correlation threshold of samples was chosen as the sampling interval) was used to reasonably formulate the samples layout, following the principle that samples units were independent of each other in the traditional sampling methods. Finally, the population extrapolation accuracy, stability and sampling cost were estimated according to the samples that the spatial layout were reasonably formulated. In order to evaluate the design efficiency of samples layout, relative error, coefficient of variation (CV) and sampling size were selected as the indices, and simple random sampling method as the control treatment. 【Result】The experimental results demonstrate that, the variability of WPS increased with sampling unit size increasing. CV of WPS varies from 32.75% to 43.46% under 8 kinds of sampling unit size levels; There was an intense spatial correlation among all of WPS within a certain range, and the spatial correlation was mainly dominated by structural factors (climate, topography, soil type). Spatial correlation thresholds of WPS increase with sampling unit size increasing; The relative error and CV of population extrapolation that samples layout was optimized were obviously less than those of simple random sampling method at the same sample size, while sampling unit size was small (500m×500m-2 000m×2 000m); Compared those of simple random sampling method, although the relative error and CV were not decreased after optimized design of sample layout, there was an obvious decreasing in sample size, when sampling unit size was larger (2 500m×2 500m-4 000m×4 000m).【Conclusion】In this way, this research can provide a solution for improving the efficiency of spatial sampling scheme to estimate crop planting acreage.
    TILLAGE & CULTIVATION·PHYSIOLOGY & BIOCHEMISTRY·AGRICULTURE INFORMATION TECHNOLOGY
    Grain Filling Characteristics of Maize Hybrids Differing in Maturities
    WANG Xiao-hui, ZHANG Lei, LIU Shuang-li, CAO Yu-jun, WEI Wen-wen, LIU Chun-guang, WANG Yong-jun, BIAN Shao-feng, WANG Li-chun
    Scientia Agricultura Sinica. 2014, 47(18):  3557-3565.  doi:10.3864/j.issn.0578-1752.2014.18.004
    Abstract ( 654 )   HTML ( 7 )   PDF (361KB) ( 989 )   Save
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    【Objective】 A field experiment was conducted to illustrate grain-filling characteristics of maize hybrids differing in maturities in northeast China, aimed to provide scientific information for the regulation of grain weight and selection of maturity.【Method】 Forty high-yield hybrids differing in maturities were used as experimental materials, and they were planted according to 60 000 plants/hm2 with the same environmental conditions. The days of seedling emergence to maturity were investigated, and the whole growing days were applied to divide the maturity of different maize hybrids. The difference of yield and 100-kernel weight of maize hybrids differing in maturities was studied, and the grain-filling processes of maize hybrids differing in maturities were analyzed by Logistic model. The grain-filling parameters of initial grain-filling potential, grain-filling rate and grain-filling duration and their relation to yield were calculated. 【Result】 The results indicated that forty maize hybrids were divided into 4-type maturities according to the whole growing days on the basis of the criterion of Food and Agriculture Organization of the United Nations, and the type I is medium maturity, the type II is mid-late maturity, the type III is late maturity, the type IV is supper-late maturity, respectively. The ratio of type III was 50% and 121-130 d with the whole growing days, the highest among these 4 types, the type I and II were the lowest (12.5%), 101-110 d and 111-120 d with the whole growing days, the ratio of type IV was 25% and 131-140 d with the whole growing days. The yield of late maturity was all the highest among these 4 types, the second of supper-late maturity and the lowest in medium maturity, lower 45%, 44% and 35% than the late maturity, supper-late maturity and mid-late maturity. The yield of mid-late maturity, late maturity and supper-late maturity were not different significantly (P>0.05), but remarkably higher than medium maturity under trial density (P<0.05). Initial stage of grain-filling, the 100-kernel weight of medium maturity was the lowest, and highest in the supper-late maturity. After 30 days of grain-filling, the 100-kernel growth rate of mid-late maturity was the fastest among these 4 types, and the slowest in the medium maturity. The tendency of grain-filling rate of these 4 types was expressed in single peak curve; the peak value of medium maturity was the highest, and the lowest in the supper-late maturity. After the peak of grain-filling, the grain-filling rate of supper-late maturity decreased the slowest, and fell the fastest in the medium maturity. The grain-filling processes of maize hybrids differing in maturities were analyzed by Logistic model, and the total filling period could be divided into early stage, middle stage and late stage. The initial grain-filling potential (R0), maximum grain-filling rate (Rmax), grain-filling duration of early stage (T1), grain-filling rate of middle stage and late stage (v2, v3 ) of medium maturity were significantly higher than other three types in this trial, but the active grain-filling period (P), grain-filling duration of middle stage and late stage (T2, T3), grain-filling rate of early stage (v1) were remarkably lower than the other three types. Furthermore, correlation analysis showed that active grain-filling period, effective grain-filling time (t3), grain-filling duration of early stage, grain-filling rate of middle stage and late stage were significantly and positively correlated to yield, and initial grain-filling potential, maximum grain-filling rate, grain-filling duration of early stage, grain-filling rate of middle stage and late stage were significantly and negatively correlated to yield. 【Conclusion】 Medium maturity varieties were quickly in launch of grain-filling, short time of active grain-filling period and effective grain-filling time, but the mid-late maturity, late maturity and supper-late maturity varieties were slowly in launch of grain-filling, long time of active grain-filling period and effective grain-filling time. Prolonging the active grain-filling period, effective grain-filling time, grain-filling duration of middle stage and late stage and enhancing the mean grain-filling rate of early stage can improve grain yield of maize hybrids differing in maturities.
    Influence of Drought Stress on Physiological Characteristics and Agronomic Traits at Bud Stage of Rapeseed (Brassica napus L.)
    BAI Peng, RAN Chun-yan, XIE Xiao-yu
    Scientia Agricultura Sinica. 2014, 47(18):  3566-3576.  doi:10.3864/j.issn.0578-1752.2014.18.005
    Abstract ( 593 )   HTML ( 21 )   PDF (466KB) ( 939 )   Save
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    【Objective】Against the characteristics of less precipitation which easily lead to drought in the Yangtze River Basin,especially in the upper reaches of the Yangtze River in the spring in recent years, the effects of water stress on physiological characters and agronomic traits at bud stage of rapeseed were studied, the aims were to reveal the drought resistance mechanism of rapeseed at bud stage, and to provide a theoretical basis for drought-resistant breeding. 【Method】The changes of associated physiological indicators and agronomic traits of rapeseed were studied by drought resistance coefficients of each character using two different drought resistance rapeseed varieties at bud stage (zhongshuang10 with low drought resistance and 94005 with high drought resistance) with drought stress experiment in pot. And filter out some indicators that apply to the local area, which can be used to identify drought resistance at bud stage of rapeseed.【Result】The results showed that the drought resistance coefficients ofnet photosynthetic rate, stomatal conductance, transpiration rate, intercellular CO2 concentration, leaf relative water content, content of chlorophyll, RuBP carboxylase activity, height per plant, stem diameter, primary branches number and seed yield per plant tended to decrease with the extension of drought stress time, and the decrease range was positively related to the time of drought stress, the decrease range of the drought resistance coefficients of stomata conductance and transpiration rate was positively related to the drought resistant ability of rapeseed, the rest of the indicators were negatively related to the drought resistance coefficients of materials. The drought resistance coefficients ofstomata limitation value and water use efficiency of low drought resistance variety presented an upward to downward and downward trend, respectively, but both of that showed an increase trend in high drought resistance variety. With the lasting of drought stress, the relative values of POD, SOD, CAT activities and soluble sugar content and soluble protein content increased firstly and then decreased, and the increase range of the high drought resistance variety was higher than that of the low drought resistance variety. The relative values of cell membrane permeability, malondialdehyde (MDA) content and proline showed an upward trend with the prolonging of drought stress. The correlation analysis showed that the yield was significantly positively correlated with the drought resistance coefficients of net photosynthetic rate, leaf relative water content, chlorophyll content, RuBP carboxylase activity, height per plant, stem diameter and primary branches number, whereas it was significantly negatively correlated with the drought resistance coefficients of cell membrane permeability and MDA content. The principal component analysis divided the yield per plant, net photosynthetic rate, intercellular CO2 concentration, stomata limitation value, water use efficiency, relative water content, chlorophyll content, RuBP carboxylase, POD, SOD, CAT, cell membrane permeability, MDA, soluble sugar, height per plant, stem diameter and primary branches number into the first component and stomata conductance, transpiration rate, proline, and soluble protein were divided into the second component. 【Conclusion】After analyzing the relationship at related indicators of bud stage under drought stress and mature stage after rehydration, showed that, to improve the yield of rapeseed under drought stress, it is need to increase net photosynthetic rate, chlorophyll content, relative water content of rapeseed leaf in bud stage and primary branches number.The variation of each indicator in the first component correlated significantly to drought resistance. These indicators can be used as the principal indicators to identify the drought resistance of rapeseed at bud stage when breeding drought-tolerant rapeseed varieties, and the secondary indicators are in the second component.
    PLANT PROTECTION
    Impact on Strain Growth and Spinosad Biosynthesis by Overexpression of Cyclic AMP Receptor Protein Gene in Saccharopolyspora spinosa
    XU Miao, WU Yang, YANG Yan, XIA Li-qiu
    Scientia Agricultura Sinica. 2014, 47(18):  3577-3587.  doi:10.3864/j.issn.0578-1752.2014.18.006
    Abstract ( 459 )   HTML ( 6 )   PDF (2364KB) ( 672 )   Save
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    【Objective】The objective of this study is to promote spinosad biosynthesis in Saccharopolyspora spinosa and to study the effects on aspects of strain growth and morphological development through overexpression of Streptomyces global regulatory factor cyclic AMP receptor protein gene (crp). 【Method】 crp coli-Streptomyces shuttle vector pUC-spn stored in authors’ lab by Red/ET homologous recombination technology, generating Crp expression vector pUC-spn-PermE-crp. Then, The vector pUC-spn-PermE-crp was introduced into S. spinosa by conjugal transfer, and integrated into the chromosome via single-cross homologous recombination, and the strain whose chromosome was integrated by original plasmid pUC-spn was used as control strain in this study. The apramycin resistant gene and the target gene were amplified by PCR to confirme positive transconjugants. The morphological comparison of the engineering strain S. spinosa-Crp and the control strain S. spinosa-1 on different media were observed. Growth curves were compared between S. spinosa-Crp and S. spinosa-1 in liquid medium. The mycelial morphologies between S. spinosa-Crp and S. spinosa-1 were observed by scanning electron microscopy and the spinosad production of S. spinosa was detected by high performance liquid chromatography.【Result】A vector pUC-spn-PermE-crp expressing Crp was constructed successfully by molecular biology method, and was transferred into S. spinosa by conjugation. PCR detection results exhibited that the 2 kb long target band cm-PermE-crp could be amplified in engineering strain S. spinosa-Crp, suggesting that crp was integrated into chromosome of S. spinosa successfully. The recombinant engineering strain S. spinosa-Crp abtained was genetically stable. On BHI and ISP-2 media, the spore germination and formation rate of engineering strain S. spinosa-Crp delayed compared to the control strain S. spinosa-1, however, the spore germination and formation rate didn’t show significant difference between S. spinosa-Crp and S. spinosa-1 in R6 medium. Compared to the control strain of S. spinosa-1, the secondary growth phenomenon of engineering strains S. spinosa-Crp disappeared cultured in liquid medium, and its biomass became higher. Under the scanning electron microscopy it was showed that the engneering strain had a higher degree of mycelial fragments and less branches, which can improve the level of dissolved oxygen in S. spinosa-Crp in the process of fermentation. Shaking flask fermentation results revealed that spinosad yield of the engineering strain increased by 128% than that of the control strain.【Conclusion】Overexpression of crp had a certain effect on the mycelial morphology and growth of S. spinosa, andeffectively promoted the biosynthesis of spinosad, which lay an important foundation for improvming spinosad production by overexpression of other positive regulatory genes. was amplified by PCR, and an intermediate vector pOJ260-cm-PermE-crp was constructed by restriction enzymes digestion and ligation, in which crp was placed under the control of erythromycin enhanced promoter PermE. The PermE-crp frangment was amplified from pOJ260-cm-PermE-crp and subcloned into Escherichia
    Sublethal Effects of Beauveria bassiana Balsamo on Life Table Parameters of Subsequent Generations of Bemisia tabaci Gennadius
    WANG Deng-jie, ZANG Lian-sheng, ZHANG Ye, WANG Hai-hong, LEI Zhong-ren
    Scientia Agricultura Sinica. 2014, 47(18):  3588-3595.  doi:10.3864/j.issn.0578-1752.2014.18.007
    Abstract ( 494 )   HTML ( 1 )   PDF (497KB) ( 489 )   Save
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    【Objective】 Beauveria bassiana (Bals.) Vuill., an important entomopathogenic fungus, has been widely used to control Bemisia tabaci Gennadius. No much research has been conducted to study the sublethal effects of fungus on life table parameters generations of insects, while sublethal effects of fungal infection can have important implications for the population dynamics of the host offsprings, which ultimately contributes to the status of the target insect as a pest. The objective of this study is to compare life tables of subsequent generations of B. tabaci arising from parental generations exposed in the third nymphal stages to B. bassiana and controls, and to investigate the sublethal effects of fungus on life table parameters of B. tabaci offsprings. 【Method】 Developmental, survival, and fecundity rate data were analyzed by using the age-stage, two-sex life table in fungus-treated B. tabaci. Those exposed to distilled water were considered as control. Means and standard errors of population growth parameters were calculated by using the bootstrap method. The t-test was used to evaluate the differences in the population parameters, development times, and fecundities between treated B. tabaci and control. 【Result】 There were significant differences in pre-adult developmental time, total preoviposition period, 1st-4th instars survival and life table parameters between fungus-treated whiteflies and control. In comparison to controls (20.59 d), parental generations exposed to fungus caused longer pre-adult development times (26.58 d). Fungus exposure caused lower survival rate (0.195) for subsequent generations individuals than control (0.76). The intrinsic rate of increase (r), finite rate (λ), net reproductive rate (R0), mean generation time (T) and gross reproductive rate (GRR) were 0.063 d-1,1.065 d-1, 6.85,30.613 d and 41.883, respectively, for treated whiteflies and 0.137 d-1,1.147 d-1,33.443,25.575 d and 51.44, respectively, for control. The population growth of fungus-treated B. tabaci was slower than control. 【Conclusion】 Offsprings of fungus-treated B. tabaci compared to control increased their population slower, had higher mortality. However, fungal treatment didn’t impose significant effect on fecundity of B. tabaci. This information can provide insights into the long-term effects of applied entomopathgenic fungi on B. tabaci populations.
    SOIL & FERTILIZER·WATER-SAVING IRRIGATION·AGROECOLOGY & ENVIRONMENT
    Distribution of Fertilizer Application and Its Environmental Risk in Different Provinces of China
    LIU Qin-pu
    Scientia Agricultura Sinica. 2014, 47(18):  3596-3605.  doi:10.3864/j.issn.0578-1752.2014.18.008
    Abstract ( 573 )   HTML ( 6 )   PDF (874KB) ( 1443 )   Save
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    【Objective】 Studies on distribution and environmental risk of fertilization in China were made in order to fully recognize the differences in provincial agricultural fertilizer use, its potential threats to the ecological environment and the degrees of environmental risk, and realize the sustainable development of Chinese agriculture. 【Method】 Considering the different impacts of N, P, K fertilizers on environment, a model of fertilization environmental risk assessment was established with weight coefficients to assess the fertilization environmental risk of China. Analystic hierarchy process (AHP) was used to determine the weithts of N, P, K. The environmental safety thresholds of N, P, K was made according to the standard of 250 kg·hm-2 for the construction of ecological counties sponsered by Chinese government and the proportion of 1﹕0.5﹕0.5 for N:P:K surposed by some developed counties. 【Result】Results showed that, at present the rate of farmland fertilizer in China reached 480 kg·hm-2, farmland fertilizer input in most provinces was overloaded. The basic trend was that the southeast part of China puts much more fertilizers onto the farmland than the part of northwest. The first five provinces, whose fertilization intensities were greater than 690 kg·hm-2 in 2012, were Fujian, Guangdong, Henan, Hubei and Jiangsu, while the last seven provinces, whose fertilization intensities were less than 200 kg·hm-2, were Tibet, Qinghai, Gansu, Heilongjiang, Guizhou, Inner Mogolia and Shanxi. The greatest amount of fertilization, 908.7 kg·hm-2, was in Fujian, 6.6 times greater than that of Tibet (137.99 kg·hm-2). Unbalanced proportion of NPK use was obvious in China. The intensity of nitrogen fertilizer use in Guangdong province was 448.5 kg·hm-2, 7.2 times greater than that of Tibet (62.59 kg·hm-2); Phosphate fertilizer input in Henan was as high as 259.8 kg·hm-2, 6 times greater than the lowest one of Guizhou (44.04 kg·hm-2); Potash input in Fujian was up to 264.73 kg·hm-2, 8.6 times greater than the lowest one of Qinghai (30.83 kg·hm-2). China's agricultural fertilizer intensity resulted in the potential risk of environmental pollution. Except for five provinces of Tibet, Qinghai, Guizhou, Gansu and Heilongjiang, which were at safety status, three provinces of Henan, Fujian, Guanddong and Hubei were classified as being severe risk from fertilization, while the rest of provinces were classified as light risks or medium risks. 【Conclusion】 Overuse of fertilizer is significant in China and resulted in severe environmental risk. Several reasons were concluded. Soil condition, fertilization technology and mismanagement were the direct causes for the overuse, while agricultural policies and management system were the fundamental ones.
    Effects of Different Planting Patterns on Water Use of Soil and Crops Annual Productivity in Southern Hilly Dryland
    TANG Wen-guang, XIAO Xiao-ping, TANG Hai-ming, YANG Guang-li
    Scientia Agricultura Sinica. 2014, 47(18):  3606-3617.  doi:10.3864/j.issn.0578-1752.2014.18.009
    Abstract ( 479 )   HTML ( 2 )   PDF (774KB) ( 459 )   Save
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    【Objective】 In order to obtain the best planting patterns, the difference of water use and crops annual productivity in southern hilly dryland were analyzed in the present paper. 【Method】 In southern hilly dryland area, nine types of experimental planting patterns including spring maize-forage grass on the hillside (SPD1), sweet potato-forage grass on the hillside (SPD2), watermelon // spring maize-forage grass on the hillside (SPD3), spring maize-potato on the slope (ZPD1), soybean-potato on the slope (ZPD2), soybean // spring maize-potato on the slope (ZPD3), spring maize-autumn maize-potato under the slope (XPD1), watermelon-autumn maize-potato under the slope (XPD2), watermelon // spring maize-autumn maize-potato under the slope (XPD3) on the hillside, on the slope and under the slope were used to study the effects of different planting patterns on soil water content, crop water consumption (WC), water use efficiency (WUE), crops annual productivity in southern hilly dryland, and land equivalent ratio (LER) between interplanting modes. 【Result】 Compared with the SPD1, soil water content were increased by 22.8%, 21.6% and 24.1% with SPD3, WUE were increased by 9.7%, 17.8% and 15.6% with SPD3, and the crops annual productivity were increased by 8.3%, 16.1% and 14.8% with SPD3, respectively. Meanwhile, compared with the SPD2, soil water content increased by 16.1%, 11.0% and 14.5% with SPD3, WUE increased by 61.9%, 60.6% and 62.5% with SPD3, and the crops annual productivity increased by 62.4%, 61.6% and 64.8% with SPD3, respectively. Compared with the ZPD1, soil water content increased by 12.9%, 13.3% and 20.9% with SPD3, WUE increased by 17.9%, 22.5% and 20.3%, and the crops annual productivity increased by 17.2%, 21.4% and 19.6% with ZPD3, respectively. Compared with the XPD1, soil water content increased by 15.1%, 17.6% and 12.9% with XPD3, WUE increased by 47.5%, 44.6% and 38.5%, and the crops annual productivity increased by 41.5%, 43.3% and 37.6% with XPD3, respectively. Compared with the XPD2, soil water content increased by 6.5%, 4.4% and 5.1% with XPD3, WUE increased by 18.6%, 21.5% and 20.0%, and the crops annual productivity increased by 20.0%, 21.5% and 21.0% with XPD3, respectively. There were no difference in water content among different planting patterns in the same year, but there was a difference in water content among different planting patterns between 2009, 2010 and 2008. In the three planting patterns, the LER of XPD3 was the highest with 1.518, 1.517 and 1.496 in 2008, 2009 and 2010. Compared with the ZPD3, the LER of XPD3 increased by 15.70%, 12.45% and 13.94%, respectively. Meanwhile, compared with the SPD3, the LER of XPD3 increased by 10.32%, 6.31% and 5.65%, respectively. In the three slope types, compared with the ZPD1, the soil water content increased by 5.5%, 27.3% and 17.3% with XPD1 in 2008, 2009 and 2010. And the soil water content increased by 68.0%, 23.3% and 28.7% with ZPD1 in 2008, 2009 and 2010. Compared with the ZPD3, the WUE increased by 105.4%, 101.2% and 102.3% with XPD3. Compared with the SPD3, the WUE increased by 238.2%, 230.2% and 246.2% with XPD3. Compared with the ZPD3, the economic output in a year increased by 195.9%, 104.1% and 101.2% with XPD3. Compared with the SPD3, the economic output in a year increased by 230.6%, 224.4% and 236.3% with XPD3. Meanwhile, Compared with the SPD3, the economic output in a year increased by 62.0%, 62.8% and 67.2% with ZPD3, respectively. 【Conclusion】As a result, the planting patterns were of benefit to enhancement of soil water retention, water use efficiency, and crops annual productivity, such as the watermelon // spring maize-forage grass, soybean // spring maize-potato, watermelon // spring maize-autumn maize-potato. Under the different slope type conditions, the WUE and crops annual productivity with different planting patterns were in the order: under the slope>slope>hillside. Therefore, the reasonable planting patterns should be chosen according to different slope conditions in the southern hilly dryland to achieve water-saving drought avoidance and increase efficiency.
    SPECIAL FOUCUS: AGRO-PRODUCTS SAFETY
    A Review of Studies on Quality and Safety of Edible Vegetable Agro-Products
    LI Pei-wu, ZHANG Qi, DING Xiao-xia, BAI Yi-zhen
    Scientia Agricultura Sinica. 2014, 47(18):  3618-3632.  doi:10.3864/j.issn.0578-1752.2014.18.010
    Abstract ( 546 )   HTML ( 2 )   PDF (552KB) ( 1062 )   Save
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    Contaminants threating the quality and safety of edible vegetable agri-food are mainly pesticide residues, biotoxins and heavy metal ions. In field of the detection technologies for the pollutants in agri-foods, some obvious progresses in sample pretreatment techniques, confirmed detection technologies and rapid detection technologies have been made. Sample pretreatment techniques have been developed and improved, including solid phase extraction, solid phase micro extraction, supercritical fluid extraction, matrix solid phase dispersion extraction and microwave assisted extraction technology and so on. Both technologies of immunoaffinity adsorption and QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and, Safe) have been quickly developed and widely applied. Confirmatory testing technologies, particularly chromatography-mass spectrometry, have been modified to improve the standard methods for determination of pesticide residues, biotoxin contamination in agricultural products in China, and the chromatographic mass spectrometry detection technology has been in rapid development and application, because it is sensitive, accurate, and reliable, especially suitable for being used as a laboratory arbitration detection. Rapid detection technologies have also been gradually set up from original innovation to terminal product creations. And a series of techniques and products such as various kits, immunoaffinity column, immunochromatographic strips and so on based on enzyme inhibition, immunoassay and non-destructive methods have been developed. Therefore, a multi-level technological options were provided for the determination of qualitative or quantitative analysis, as complements to advanced instrument method. In the field of risk assessment of contaminants in agri-foods, remarkable progress has been made in the construction of agricultural product quality and safety risk assessment system in the last three years. The system comprises of National Agri-food Safety Risk Assessment Agency as the leading organization, professional RAL (risk assessment laboratory) for the specific agri-foods and regional RAL for local agri-foods as the main body, experimental stations as the basis and observation spots in the main production area as the extension has been established. Four types of risk assessment have been proposed for six kinds of hazard with definite characteristics and procedure of each type. The technical system such as hazards identification technologies and data processing, has been preliminarily built for pesticide residues, biotoxins, heavy metal ions, exogenous additives and so on. Seeing from the studies on the control of contaminants in agri-foods, some rules or mechanism of contamination have been being gradually discovered and prevention and control techniques such as processing control technology, reducing and cutting technology have been proposed. The current main problems conclude: 1) the original innovation on the detection of agri-foods quality and safety needs to be strengthened further, especially the simultaneous determination technology for different pollutants and even different classes of contaminants; 2) late starting risk assessment researches and the urgent demand with lack of basic data; 3) weakness in basic research, lack of awareness of pollution mechanism, unclear of the critical control points and shortage of early warning technology. With the researches on edible agri-food quality and safety furtherer and furtherer, self-reliance innovation of hazard detection technology will continuously be strengthened, and the technical methods and series of corresponding analysis products will gradually meet the various testing demands. Through the research on the occurrence of hazard, the formation of nutritional quality and control mechanism, there will be a rapid development in early risk warning and contamination control techniques. Agricultural products quality and safety risk assessment will be an indispensable technical and basic work in agricultural products quality and safety supervision, and the related research will be a hot issue. The risk assessment system will play more and more important roles in the whole process control, management and official supervision of multiple contaminants influencing on agricultural products quality and safety from field to table.
    Advances in Research of Cadmium Metabolism and Control in Rice Plants
    ZHU Zhi-wei, CHEN Ming-xue, MOU Ren-xiang, CAO Zhao-yun, ZHANG Wei-xing, LIN Xiao-yan
    Scientia Agricultura Sinica. 2014, 47(18):  3633-3640.  doi:10.3864/j.issn.0578-1752.2014.18.011
    Abstract ( 569 )   HTML ( 5 )   PDF (357KB) ( 1368 )   Save
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    Cadmium (Cd) is one of the major pollutants in rice grains, for rice plants can absorb Cd easily. Thus, it becomes the potential important pollutant for the food supply and food safety in China. In this review, the authors attempt to show three important steps in the bio-accumulation of Cd in rice plants, including the activation and absorption of Cd by rice roots, the loading and transportation of Cd by rice xylem, and the enrichment of Cd in rice grains by the phloem from rice internodes. The metabolic process of Cd at different rice growing stages, the control mechanism of Cd through transport protein and key genes, and the influence of environmental conditions and inputs to Cd accumulation in rice plants were discussed. The future research of Cd control mechanism in rice was also highlighted from the viewpoint of the sustainable agricultural development and the management of rice safety.
    Deoxynivalenol Contamination in Wheat and Its Management
    SHI Jian-rong, LIU Xin, QIU Jian-bo, JI Fang, XU Jian-hong, DONG Fei, YIN Xian-chao, RAN Jun-jian
    Scientia Agricultura Sinica. 2014, 47(18):  3641-3654.  doi:10.3864/j.issn.0578-1752.2014.18.012
    Abstract ( 600 )   HTML ( 6 )   PDF (733KB) ( 2073 )   Save
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    Deoxynivalenol (DON), one of the type B trichothecene mycotoxins, also known as vomitoxin, can cause sickness such as vomiting, feed refusal, bloody diarrhea, dermatitis, hemorrhaging, and even death because of inhibition of DNA, RNA and protein synthesis at the ribosomal level in nonruminant animals, especially swine. DON is the metabolite of Fusarium graminearum species complex(FGSC) which causes wheat head blight around the world. In the last 10 years, FHB has become a severely epidemic for many times in China (especially in Jiangsu, Anhui, Hubei, Henan and Shandong), north America and European countries, causing that DON becomes severe food safety issue for wheat grains. This paper presents the DON contamination of wheat, distribution and chemotype of DON-produced FGSC, the factors which influence the DON production, and the management of DON contaminated grains to facilitate the risk evaluation and surveillance of DON-contaminated wheat in this country.
    Risk Assessment of Pesticide Residues in Apples
    NIE Ji-yun, LI Zhi-xia, LIU Chuan-de, FANG Jin-bao, WANG Cheng, GUO Yong-ze, LEI Shao-rong, LI Hai-fei, XU Guo-feng, YAN Zhen
    Scientia Agricultura Sinica. 2014, 47(18):  3655-3667.  doi:10.3864/j.issn.0578-1752.2014.18.013
    Abstract ( 817 )   HTML ( 14 )   PDF (478KB) ( 1701 )   Save
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    【Objective】Risk assessment of pesticide residues in apples will provide a scientific basis for apple consumption, apple pesticide residue supervision, and establishment of maximum residue limits (MRLs) for pesticides in apple. 【Method】Residues of 102 pesticides in 200 apple samples from main producing areas in China were detected. Chronic dietary intake risk and acute dietary intake risk of pesticide residues in these samples were assessed, respectively, by %ADI and %ARfD. Estimated maximum residue limits (eMRLs) of 26 pesticides with detectable residues were calculated by ADIs, large portion of apple, and body weight of consumer. The Matrix Ranking developed by the Veterinary Residues Committee of the United Kingdom was referred to rank the risk of pesticides and apple samples.【Result】25.5% of pesticides (26/102) and 89.5% of apple samples (179/200) had detectable residues. 0.5% of apple samples contained residue above the MRL (of omethoate). For 26 pesticides with detectable residues, their chronic dietary intake risks expressed as %ADI were 0.00%-1.07% with an average of 0.13%, and their acute dietary intake risks expressed as %ARfD were 0.18%-22.41% with an average of 4.12%. These 26 pesticides were divided into 3 groups by risk score, i.e. high risk group (8 pesticides), medium risk group (10 pesticides), and low risk group (8 pesticides). Apple samples studied were ranked by risk index, 1.5% of which had high pesticide residue risk, and 98.5% of which had medium, low, or lower pesticide residue risk. Among 26 pesticides with detectable residues, it is unnecessary for chlorbenzuron to establish MRL in apple, MRLs of 6 pesticides (e.g. flusilazole) are overly strict, MRLs of 5 pesticides (e.g. dimethoate) are overly loose, and MRLs of 18 pesticides (including chlormequat, difenoconazole, tebufenozide, diazinon, flusilazole, thiophanate-methyl, dimethoate, bifenthrin, cyhalothrin, fenvalerate, propargite, buprofezin, dicofol, methidathion, tebuconazole, diniconazole, phosmet, and imazali) were proposed to be 4, 1, 2, 0.5, 0.6, 7, 0.2, 1, 2, 2, 1, 0.8, 0.2, 0.1, 3, 0.5, 1, and 3 mg•kg-1, respectively.【Conclusion】Pesticide residues in Chinese apples had a relatively high detection rate, but pesticide residues in 99.5% of samples were under MRLs. Both chronic and acute dietary intake risks of pesticide residues in Chinese apples were very low. Residues of 8 pesticides in apples should be focused on, including omethoate, phosphamidon, methidathion, chlorpyrifos, diazinon, bifenthrin, phosmet, and dimethoate. And MRLs for 18 pesticides (e.g. chlormequat) in apples were proposed to be established or revised.
    Study on Immunochromatography for Afaltoxin Determination in Agricultural Product
    ZHANG Zhao-wei, LI Pei-wu, ZHANG Qi, DING Xiao-xia
    Scientia Agricultura Sinica. 2014, 47(18):  3668-3674.  doi:10.3864/j.issn.0578-1752.2014.18.014
    Abstract ( 469 )   HTML ( 3 )   PDF (368KB) ( 840 )   Save
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    【Objective】 Aflatoxin contaminates agricultural product severely, threatens health and life of people and livestocks. It is one of the major focus issues and attracts both governmental and social concerns. Thus, it is required to establish a rapid and sensitive determination method for aflatoxin. High specific and sensitive monoclonal antibody against aflatoxin has been developed. In this study, using the as-prepared monoclonal antibody against aflatoxin, the aim is to establish time-resolved fluorescence immunochromatography for afaltoxin determination in agricultural product, in order to provide technical support for agricultural product quality supervision and risk assessment. 【Method】 Herein, monoclonal antibody against aflatoxin was coupled with emulsion europium for labeling. With the home-made time-resolved fluorescence immunochromatographic strip, aflatoxin could be determined quantitatively, by using the signal value ratio of test line to control line and natural logarithm of concentration in standard aflatoxin solution. Regarding to various agricultural product samples (as peanut, rice and vegetable oil), an integration technology was developed that combined grinding and homogenization in one step. The methodological evaluation was conducted, in which aflatoxin in real agricultural products such as peanut, rice and vegetable oil. Moreover, these results via time-resolved fluorescence immunochromatography were compared with those via HPLC method. 【Result】 Results showed a detecting limit of 0.3 μg·kg-1, a liner range of 0.8-25, 0.8-15, 0.8-30 μg·kg-1, for peanut, rice, plant oil, respectively. The standard curves were recorded as y=0.238x+0.654 (R2=0.992), y=0.321x+0.811 (R2=0.990), and y=0.146x+0.173 (R2=0.993), for peanut, rice, and plant oil, respectively. Recoveries from inner accuracy and precision were between 81.0% and 113.0% with RSD of 7.2%-14.2%, while recoveries from inter accuracy and precision were of 75.8%-114.9% with RSD of 7.7%-15.3%. It was suggested that this method allowed considerable inner- and inter- accuracy and precision, It was found to be the relative error below 10% between results via time-resolved fluorescence immunochromatography and those with HPLC, which suggesting that a high degree between time-resolved fluorescence immunochromatographic method and industry standard method.【Conclusion】Results showed that TRFIA possesses high sensitivity, wide liner range, high repeatability and stability, thus it is suitable for rapid assay in China and poses wide application prospect.
    Identification of Aflatoxin Producing Strains by Using PCR-RFLP Method
    SUN Chang-po, CHANG Xiao-jiao, WU Song-ling, SHEN Han
    Scientia Agricultura Sinica. 2014, 47(18):  3675-3683.  doi:10.3864/j.issn.0578-1752.2014.18.015
    Abstract ( 476 )   HTML ( 2 )   PDF (670KB) ( 907 )   Save
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    【Objective】The objective of this study is to develop an identification method of aflatoxin-producing strains with the advantages of high accuracy, good sensitivity, and convenience, which provide early warning to aflatoxin contamination of stored grain, provide technical support for the safety control in food industry and provide the non-toxic microbial sources for biological control of aflatoxin contamination.【Method】The key gene of aflatoxin synthesis, aflR, which belongs to Aspergillus flavus, Aspergillus parasitieus, Aspergillus oryzae and Aspergillus sojae, was analyzed by bioinformatics method. The potential strains producing aflatoxin were identified by PCR-RFLP technology. To verify these experimental results above, their toxin-producing ability was tested and analyzed by HPLC after these strains were cultured and fermented with soybean medium.【Results】The sequences of their aflR gene were analyzed in this research and the results showed high homology among their sequences. On the other hand, the partial bases of their aflR and promoter mutated regularly to induce variation of restriction sites NheⅠ, PvuⅡ, and HincⅡ. The genomic DNA were extracted from all these strains, the sequences of their aflR and promoter were amplified by PCR which includes 798 bp and 515 bp respectively. These promoters of aflR gene were digested by NheⅠand produced 176 bp and 339 bp two short fragments from toxin-producing strains. However, there was no restriction site of NheⅠin those of the non-toxin-producing strains. The results showed thatthe aflR of A. flavus and A. parasitieus were digested into three fragments (387 bp, 250 bp, 161 bp) and two fragments (548 bp, 250 bp) by HincⅡ, and those ofA. flavus andA. parasitieus were digested into two fragments (652 bp, 146 bp) and three fragments (413 bp, 239 bp, 146 bp) by PvuⅡ, respectively. The analysis of aflatoxin-producing demonstrated that both A. oryzae and A. sojae could not produce aflatoxin under the same cultural condition. It showed the same results that no aflatoxin produced by the strains in PCR-RFLP analysis. Among toxin-producing Aspergillus strains, the differences of their toxin-producing ability were remarkable. Not only their toxin yields rose a great many times in variation but their compositions were different.【Conclusion】This study illustrated the differences existed in the key gene aflR sequence among the strains. The active toxin-producing A. flavus and non-toxin-producing ones as well as toxin-producing A. flavus and A. parasitieus were identified by PCR-RFLP rapidly. The development of the method for identifing toxin-producing Aspergillus strains is probably useful for safety check and quality control not only in productive process of cereal and oil products but during food storage.
    Degradation Dynamics and Residues Analysis of Abamectin in Cucumber and Soil
    JIN Fen, WANG Jing, WEI Shan-shan, DU Xin-wei, SHAO Hua, JIN Mao-jun, WANG Shan-shan, SHE Yong-xin
    Scientia Agricultura Sinica. 2014, 47(18):  3684-3690.  doi:10.3864/j.issn.0578-1752.2014.18.016
    Abstract ( 702 )   HTML ( 4 )   PDF (437KB) ( 712 )   Save
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    【Objective】 The objective of this study is to study the degradation dynamics of abamectin in cucumber and soil, and to formulate a scientific basis for the reasonable dose and the pre-harvest interval on controlling cucumber root-knot nematode with hole application. 【Method】 Degradation dynamics experiments were conducted by application abamectin once on cucumber and soil with hole application at 5.62 g·m-2(1.5 times recommended dosage) after transplanting cucumber seedlings. Degradation dynamics samples were collected and determined at random from each plot at different time intervals i.e, 0 day (2 h after application), 1, 3, 5, 7, 10, 14, 21, 28, and 45 days after the abamectin application. Two doses were set in terminal residue experiment. Abamectin was applied once at 3.75 g·m-2 (low concentration, recommended dosage) and 5.62 g·m-2(high concentration, 1.5 times recommended dosage) after transplanting period of cucumber seedlings. Terminal residue samples were collected and determination of abamectin during the harvesting periods. The dispersive solid phase extraction-high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was used to determine the abamectin residue in cucumber and soil. The homogenized cucumber samples, plant samples or soil samples (10 g) were firstly weight into a polypropylene centrifuge tube. After adding 4 g Na2SO4, and 1 g NaCl, the sample was extracted by 10 mL acetonitrile. Secondly, the extraction solution was sonicated and centrifuged for several minutes. Then 2 mL of the clarified supernatant was transferred into a clean centrifuge tube containing 50 mg of C18 and 50 mg PSA dispersive solid phase extraction (DSPE) sorbents for purification. Finally, the solution filtered through a 0.22 μm membrane prior to LC-MS/MS analysis. 【Result】The established method for determination of abamectin in cucumber and soil with DSPE combined LC-MS/MS was rapidly and easy of operation. The recoveries of abamectin in cucumber and soil were 78%-101% with RSD between 1.9% and 9.4% with spiking at 10 μg·kg-1, 50 μg·kg-1 and 100 μg·kg-1. The limit of quantitation (LOQ) of this method was 10μg·kg-1, which could meet the requirement of max residue limits (MRLs) of abamectin. In degradation dynamics experiments, abamectin was not detected in all degradation plant samples collected from in Beijing and Harbin, which suggested that it is not a systemic pesticide. However, abamectin was detected in all soil samples in degradation experiment. The degradation dynamic equations of abamectin in soil were fitted the first-order reaction dynamic equations, and the half-lives of degradation of abamectin were estimated at 7.9 d and 18.7 d in soil. The residue period of abmectin was longer with hole application compared to that with spray application in soil. Abmectin residue was detected in three soil samples collected from Beijing in 2012 with concentration of 10 μg·kg-1, 30 μg·kg-1 and 170 μg·kg-1, respectively. And similarly, abmectin residues were found only in two soil samples collected from Harbin with concentration of 10 μg·kg-1. 【Conclusion】The abamectin sustained release granules (1%) applied with hole application at 3.75 g·m-2 can be considered safe in cucumber planting.
    Gene Cloning and Shuttle Expression Plasmid Construction of Pig β2 Adrenergic Receptors
    WANG Jian, WANG Jing, WANG Miao, KONG Xiang-ya, ZHU Feng-bin, SHAO Xiao-xue
    Scientia Agricultura Sinica. 2014, 47(18):  3691-3699.  doi:10.3864/j.issn.0578-1752.2014.18.017
    Abstract ( 399 )   HTML ( 1 )   PDF (997KB) ( 458 )   Save
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    【Objective】 Receptor assay is a new method suitble for rapid multianalysis for detection of β-agonists compared to traditional immunoassay. The key problem of this method is acquiring pure recombinant receptor with high affinity and selectivity. To provide the basic material for screening of optimal expression system and expression condition, the specific recombinant shuttle expression plasmid was constructed with pig β2AR gene. 【Method】 Total RNA was extracted from fresh pig liver and a pair of primers was designed and synthesized according to the published pig β2AR gene sequence in Genbank(AF000134). The recovery of RT-PCR product from agarose gel was connected with cloning vector pMD-18T by T4 DNA ligase at 4℃ overnight. The ligation product was then transformed into competent cell DH5α and the plasmid was extracted after blue and white spot selection. And that the plasmid was confirmed by PCR, double enzyme digestion and sequencing analysis. The DNA sequence and deduced amino acid sequence were firstly analyzed by BLAST, and then phylogenetic tree construction and hydrophobicity were performed, respectively. In order to enhance the expression and binding affinity of receptor protein, N-terminal 186 bp of the cloned gene were truncated, and 6×His-tag was added at C-terminal. Finally, the genetically modified genes were respectively cloned to pTriEx-1.1 Hygro vector, and the ligation products were transformed into competent cell NovaBlue. The recombinant plasmids were extracted from single colonies and identified after Amp resistance screening. 【Result】 The purity, concentration and integrity of the extracted total RNA could meet the requirements of successive test by UV spectrophotometer testing and agarose gel electrophoresis. The RT-PCR product was 1 257 bp by sequencing, which encods 418 amino acids. The sequence has been submitted in Genbank as accession number KF023571.1. The deduced protein was predicted to have a computed molecular mass of 46.73 kD by Compute pI/Mw. Compared with the published pig β2AR gene sequence (AF000134), the identity of nucleotide between them was 99.68%, and the rate of deduced amino acid was 99.28%. Furthermore, all of the amino acids at the ligand binding sites were cloned correctly. BLAST analysis indicated that there was a high homology of β2AR between pig and some species. From phylogenetic tree analysis based on β2AR gene, Sus scrofa is more close to Pecan tajacu and not closely related to Tscherskia triton and Microtus ochrogaster. Hydrophobicity analysis illustrated that the N-terminal and C-terminal of the receptor were dominated by hydrophobic amino acids and hydrophilic amino acids, respectively. Recombinant plasmid named pTriEx-1.1Hygro-β2AR1-418 and pTriEx-1.1Hygro- β2AR63-418 were constructed successfully after verification of PCR, double enzyme digestion and sequencing. 【Conclusion】 The β2AR gene and the amino acids at active sites were highly consistent with the published records. Besides, pTriEx-1.1 Hygro vector contains promoters suitble for Escherichia coli, insect cells and mammalian cells, respectively. It is a good material to explore the expression effect of target gene in different expression systems. Consequently, both of the recombinant shuttle expression plasmid pTriEx-1.1Hygro-β2AR1-418 and pTriEx-1.1Hygro-β2AR63-418 can be applied to the further studies on β2AR expression and purification in all three of expression systems.
    ANIMAL SCIENCE·VETERINARY SCIENCERE·SOURCE INSECT
    Genome-Wide Association Study on Porcine Serum Glucose (GLU) and Glycosylated Serum Proteins (GSP) with High Density SNP Markers
    ZENG Zhi-jun, LIU Chen-long, YANG Hui, YANG Bin, YANG Zhu-qing, CHEN Cong-ying
    Scientia Agricultura Sinica. 2014, 47(18):  3700-3707.  doi:10.3864/j.issn.0578-1752.2014.18.018
    Abstract ( 409 )   HTML ( 1 )   PDF (1820KB) ( 895 )   Save
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    【Objective】 Serum glucose (GLU) and glycosylated serum protein (GSP) contents in a Sutai population and a large-scale White Duroc×Erhualian F2 intercross at the age of 240 days were measured. A genome-wide association study was carried out to identify the SNPs or chromosomal regions associated with GLU and GSP. The aim of the study is to establish a foundation for identification of causative genes influencing the serum GLU and GSP, and provide the clues for genetic analysis of human hypoglycemia and diabetes.【Method】 The experimental pigs used in this study included 435 Sutai pigs that were bought from Sutai Pig Breeding Center in Suzhou city and 760 F2 individuals from White Duroc × Erhualian intercross that was constructed by Key Laboratory for Animal Biotechnology of Jiangxi Agricultural University. All experimental pigs were fed under the same farm conditions and slaughtered at the age of 240 days at Guohong abattoir. The collected blood samples were kept at room temperature for 5 hours, then centrifuged at 4℃, 3 000 r/min for 20 min. The serum GLU and GSP 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 DNA samples were diluted to 20ng•μL-1, and then stored at -20℃ until used. All experimental animals were genotyped with Illumina porcine 60K SNP chip. Quality control of genotyping results was carried out using PLINK software. The genome-wide association studies were performed with the mixed linear model with the SNPs passed the quality control by using GenABEL software in the R packages to identify the significant SNPs associated with GLU and GSP at 240 days in the Sutai and White Duroc × Erhualian F2 intercross. The possible candidate genes were chosen for each of significant region according to gene annotations in Ensembl or NCBI websites. 【Result】A total of 5 SNPs that significantly associated with GLU and GSP were identified. In the White Duroc × Erhualian F2 resource population, one SNP (ALGA0057739, P=1.58×10-5) was identified that significantly associated with GSP at chromosome 10: 24.67Mb, which explained 3.72% of phenotypic variance, but any SNPs that significantly associated with GLU in this population were not detected. In the Sutai population, two SNPs (ALGA0108699 and DRGA0017552, P=1.45×10-5) that significantly associated with serum GSP were identified and explained 3.72% of phenotypic variance, but these two SNPs could not be mapped to pig reference genome by using Sus scrofa 10.2 reference genome assembly. Through comparative genome analysis of human and pig, it was found that these two SNPs locate on SSC8 and map to the 180.0-193.0kb downstream of STPG2 gene 3’ end. Any SNPs that significantly associated with GLU in Sutai pigs were not identified. No significantly associated SNPs were identified for GSP in the Meta analysis. But the authors found two SNPs at SSC1: 250.32Mb (DRGA0002016, P=2.48×10-5) and SSC14: 43.97Mb (ASGA0062984, P=1.29×10-5), respectively, that were significantly associated with GLU. The candidate genes around the top SNPs and its functional annotations were searched, and it was found that ASPM, TRPM3 and KCTD10 were the important candidate genes for GLU and GSP. 【Conclusion】A total of 5 SNP regions that significantly associated with serum GLU and GSP were identified. ASPM, TRPM3, STPG2 and KCTD10 are the important candidate genes influencing GLU and GSP.
    Effects of Zearalenone Contaminated Diets on Serum Metabolite and Histopathology of Liver and Kidney in Weaned Piglets
    JIANG Shu-zhen, SUN Hua, HUANG Li-bo, YANG Zai-bin, WANG Shu-jing, LIU Fa-xiao, F. Chi
    Scientia Agricultura Sinica. 2014, 47(18):  3708-3715.  doi:10.3864/j.issn.0578-1752.2014.18.019
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    【Objective】Zearalenone (ZEA) is one of estrogenic mycotoxins produced mainly by Fusarium fungigrowing on grains and their derived products. The aims of the present study were to investigate the effect of ZEA on serum metabolite, histopathology of liver and kidney and liver ultrastructure in weaned piglets.【Method】A total of twenty healthy weaned gilts (Landrace × Yorkshire × Duroc) with an average body weight of (10.36 ± 1.21) kg (mean ± SD) were used in the study. Gilts were randomly allocated into 4 treatments according to body weight and fed individually in a metabolic cage for 18 days after 7-d adaptation. Piglets were fed a basal diet only (control) or basal diet supplemented with ZEA at a dietary concentration of 1.1, 2.0 or 3.2 mg·kg-1. Piglets were fasted for 12 h at the end of the experimental period. After the collection of blood samples, piglets were immediately killed by electrocution and livers and kidneys were isolated. Samples of livers and kidneys from each pig were quickly collected, one portion was fixed in 10% buffered formalin for histopathology evaluation, the second cutted into 0.5 mm3 was fixed in 2.5% polyoxymethylene-glutaraldehyde for ultrastructure analysis. 【Result】Results showed that piglets fed diets containing 3.2 mg·kg-1 ZEA had increased serum bilirubin and decreased serum levels of globulin and triglyceride compared with the control (P<0.05). Increasing dietary ZEA linearly decreased serum levels of globulin and triglyceride (P<0.05). Liver sections from piglets treated with 1.1, 2.0 and 3.2 mg·kg-1 ZEA showed hepatocyte swelling and granular degeneration compared with control. Histological examination of kidney in piglets fed 2.0 and 3.2 mg·kg-1 ZEA revealed the degeneration and swelling in renal tubular epithelial cells and smaller lumina, and some renal tubules were filled with a large number of transparent bottom or proteinuria. Cytophagosome and leucocyte neutrophil were observed in the 2.0 and 3.2 mg·kg-1 ZEA treatments. 【Conclusion】The above findings indicated that ZEA at level of 2.0 mg·kg-1 was sufficient to induce hepatorenal toxicity of piglets, which may have implications for human health and animals production consuming ZEA-contaminated food or feed.
    Regulation of GnIH on Reproduction of Female Pig
    WANG Yao, LI Xun, LI Min-jie, GUO Meng-jie, LEI Zhi-hai
    Scientia Agricultura Sinica. 2014, 47(18):  3716-3724.  doi:10.3864/j.issn.0578-1752.2014.18.020
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    【Objective】The expression of GnIH mRNA in female pig, the effect of GnIH on steroid hormone secretion and cell proliferation proteins expression in granulosa cells cultured in vitro, and morphological relationship between GPR147 and GnRH were investigated in this study. The purpose of all the studies was to explore the regulatory mechanism of GnIH to pig reproduction in vitro, at molecular and cell levels, and from cell morphology images.【Method】Twenty-four tissue and organ samples were collected from healthy female piglets aged 30 d, expression levels of GnIH mRNA were detected by semi-quantitative RT-PCR. The effect of GnIH on steroid hormone secretion and cell proliferation proteins expression in granulosa cells cultured in vitro was analyzed by the method of radioimmunoassay and Western blotting. Hypothalamus tissues were also collected from healthy female piglets aged 30 d and used for preparation of histological sections. The morphological relationship between GnIH receptor and GnRH was revealed by immunohistochemistry and examined with confocal microscopy.【Result】The result showed that GnIH mRNA mainly expressed in the central nervous system, especially in the diencephalon. After in vitro cultured, the granulosa cells were treated with 10-6, 10-8, 10-10 and 10-12 mol•L-1 GnIH. Compared with the control cells, 10-6 and 10-10 mol•L-1 doses of GnIH significantly reduced (P<0.01) E2 secretion. All concentrations of GnIH could attenuate P4 secretion. GnIH significantly inhibited (P<0.05) PCNA and cyclin B1 expression in cultured granulosa cells. Moreover, immunohistochemistry analysis revealed the close contact between immunoreactive fibers or cell bodies of GPR147 and GnRH in pig hypothalamus tissue. 【Conclusion】These results demonstrated that GnIH not only plays a central regulatory role on GnRH secretion in the hypothalamus, but also peripherally regulates pig reproduction by affecting steroid hormone secretion and cell proliferation proteins expression in the ovary.