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    Crop Genetics · Breeding · Germplasm Resources
    Proteomic Analysis of Wheat Seed in Response to Drought Stress
    ZHANG Yu-feng, HUANG Xiu-wen, WANG Li-li, WEI Liu, WU Zhi-hui, YOU Ming-shan , LI Bao-yun
    2014, 13(5): 919-925.  DOI: 10.1016/S2095-3119(13)60601-2
    Abstract ( )   PDF in ScienceDirect  
    Drought stress is one of the major factors affecting in wheat yield and grain quality. In order to investigate how drought stress might influence wheat quality during grain filling, three wheat cultivars Gaocheng 8901, Jagger and Nongda 3406 were subjected to drought stress during the grain filling stage. Neither globulin and glutenin, nor the relative percentage of amylose significantly changed following drought treatments, whereas albumin and gliadin concentrations did. The SDS-sedimentation, which has a strong linear correlation with wheat baking quality was markedly decreased following drought stress. These results indicated that drought had an adverse effect on wheat quality. In order to investigate the protein complexes in the wheat flour, the data from native PAGE and SDS-PAGE were combined and a total of 14 spots were successfully identified, and of these eight protein types were determined to be potential complex forming proteins.
    Expression Comparisons of Pathogenesis-Related (PR) Genes in Wheat in Response to Infection/Infestation by Fusarium, Yellow dwarf virus (YDV) Aphid-Transmitted and Hessian Fly
    WU Shi-wen, WANG Hong-wei, YANG Zai-dong , KONG Ling-rang
    2014, 13(5): 926-936.  DOI: 10.1016/S2095-3119(13)60570-5
    Abstract ( )   PDF in ScienceDirect  
    Expression profiles of ten pathogenesis-related (PR) genes during plant defense against Fusarium, Yellow dwarf virus (YDV) aphid-transmitted and Hessian fly (Hf) were compared temporally in both resistant and susceptible genotypes following pathogen infection or insect infestation. Quantitative real-time PCR (qRT-PCR) revealed that PR1, PR2, PR3, PR5, PR6, PR8, PR9, and PR15 appeared to be induced or suppressed independently in response to Fusarium, YDV aphid-transmitted or Hf during the interactions. The PR gene(s) essential to defense against one organism may play little or no role in defense against another pathogen or pest, suggesting the alternative mechanisms may be involved in different interactions of wheat- Fusarium, wheat-YDV aphid-transmitted and wheat-Hf. However, strong up- or down-regulation of PR12 and PR14 encoding low molecular membrane acting protein, defensin and lipid transfer protein (LTP), respectively, had been detected after either pathogen infection or insect infestation, therefore showed broad responses to pathogens and insects. It was postulated that low molecular proteins such as defensins and LTPs might play a role in the early stages of pathogenesis in the signaling process that informs plants about the attack from biotic stresses. In addition, a synergistic action between different PR genes might exist in plants to defense certain pathogens and insects on the basis of comprehensive expression profiling of various pathogenesis-related genes revealed by qRT-PCR in this study.
    Acquisition of Insect-Resistant Transgenic Maize Harboring a Truncated cry1Ah Gene via Agrobacterium-Mediated Transformation
    LI Xiu-ying, LANG Zhi-hong, ZHANG Jie, HE Kang-lai, ZHU Li , HUANG Da-fang
    2014, 13(5): 937-944.  DOI: 10.1016/S2095-3119(13)60531-6
    Abstract ( )   PDF in ScienceDirect  
    A novel insecticidal gene cry1Ah was cloned from Bacillus thuringiensis isolate BT8 previously for plant genetic engineering improvement. Truncated active Cry1Ah toxin has a toxicity level similar to that of the full-length Cry1Ah toxin. In this study, plant expression vector pMhGM harboring truncated cry1Ah gene was transformed into maize (Zea mays L.) immature embryos by Agrobacterium tumefaciens-mediated transformation at which maize alcohol dehydrogenase matrix attachment regions (madMARs) were incorporated on both sides of the gene expression cassette to improve gene expression. A total of 23 PCR positive events were obtained with a transformation efficiency of 5% around. Bioassay results showed that events 1-4 and 1-5 exhibited enhanced resistance to the Asian corn borer (Ostrinia furnacalis). These two events were further confirmed by molecular analysis. Southern blot suggested that a single copy of the cry1Ah gene was successfully integrated into the maize genome. Western blot and ELISA showed that the foreign gene cry1Ah was expressed stably at high level in maize and could be inherited stably over generations. The results of a bioassay of T1-T4 transgenic maize plants indicated that the transgenic plants were highly toxic to the Asian corn borer and their resistance could be inherited stably from generation to generation. Thus, events 1-4 and 1-5 are good candidates for the breeding of insect-resistant maize.
    Study on the Mitochondrial Genome of Sea Island Cotton (Gossypium barbadense) by BAC Library Screening
    SU Ai-guo, LI Shuang-shuang, LIU Guo-zheng, LEI Bin-bin, KANG Ding-ming, LI Zhao-hu, MA
    2014, 13(5): 945-953.  DOI: 10.1016/S2095-3119(13)60595-X
    Abstract ( )   PDF in ScienceDirect  
    The plant mitochondrial genome displays complex features, particularly in terms of cytoplasmic male sterility (CMS). Therefore, research on the cotton mitochondrial genome may provide important information for analyzing genome evolution and exploring the molecular mechanism of CMS. In this paper, we present a preliminary study on the mitochondrial genome of sea island cotton (Gossypium barbadense) based on positive clones from the bacterial artificial chromosome (BAC) library. Thirty-five primers designed with the conserved sequences of functional genes and exons of mitochondria were used to screen positive clones in the genome library of the sea island cotton variety called Pima 90-53. Ten BAC clones were obtained and verified for further study. A contig was obtained based on six overlapping clones and subsequently laid out primarily on the mitochondrial genome. One BAC clone, clone 6 harbored with the inserter of approximate 115 kb mtDNA sequence, in which more than 10 primers fragments could be amplified, was sequenced and assembled using the Solexa strategy. Fifteen mitochondrial functional genes were revealed in clone 6 by gene annotation. The characteristics of the syntenic gene/exon of the sequences and RNA editing were preliminarily predicted.
    Decreased Pollen Viability and Thicken Pollen Intine in Antisense Silenced Brassica campestris Mutant of BcMF19
    LIU Jin-long, GAO Ming-hui, LIU Ying , CAO Jia-shu
    2014, 13(5): 954-962.  DOI: 10.1016/S2095-3119(13)60581-X
    Abstract ( )   PDF in ScienceDirect  
    Brassica campestris male fertility 19 (BcMF19; GenBank accession number GQ902048.1), a gene that is specially expressed in tapetum cells and microspores during anther development in B. campestris ssp. chinensis, which is learned from the previous in situ hybridization study. In the present study, we constructed antisense-silenced plants of BcMF19 using B. campestris ssp. chinensis to validate this prediction. The morphology of the pistils, long anthers, and short anthers was significantly affected in 35sbcmf19 compared with the control samples. 4´-6-Diamidino-2-phenylindole staining revealed that two generative nuclei and one large vegetative nucleus were not affected in the mutant compared with control. Statistical analysis of Alexander’s staining results showed that 96% of the control pollen grains had vitality, whereas only 86% of the mutant pollen grains did. Under scanning electron microscopy, the mutant demonstrated numerous abnormal pollen grains and resembled dried persimmon. The frequency of normal pollen grains was approximately 18%. Under transmission electron microscopy, the pollen intine during the binucleate and mature pollen stages in 35sbcmf19 exhibited abnormal thickening, especially at the germinal furrows, compared with control. In vitro pollen germination test showed that the tips of the mutant pollen tubes transformed into globular alveoli and stopped growing compared with control. On the other hand, in vivo pollen germination test suggested that BcMF19 affected the pollen tube extension in the pistil. These findings indicate that BcMF19 is essential to the pollen development and pollen tube extension of B. campestris ssp. chinensis.
    Proteomic Analysis of Fruit Bending in Cucumber (Cucumis sativus L.)
    WANG Li-li, ZHANG Peng, QIN Zhi-wei , ZHOU Xiu-yan
    2014, 13(5): 963-974.  DOI: 10.1016/S2095-3119(13)60406-2
    Abstract ( )   PDF in ScienceDirect  
    In cucumber, fruit shape is an important quality criterion, and fruit bending is known to limit growth, yield, and taste. To investigate the post-transcriptional changes that regulate fruit bending and to better understand the underlying molecular mechanisms, we generated a proteomic profile of the abdomen and back of cucumber bending fruit. Two-dimensional gel electrophoresis (2-DE) allowed the detection of approximately 900 distinct protein spots in each gel, 32 of which were differentially expressed in the abdomen and back of bending cucumber fruit. Ten of the differentially expressed proteins were analyzed using matrix-assisted laser ionization time of flight mass spectrometry (MALDI-TOF/MS). A search of primary databases showed that the identified proteins are involved in various metabolic processes and cellular responses, including photosynthesis metabolism, energy metabolism, defense and stress response, and regulation. The identified proteins included large subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase, which are involved in photosynthesis and photorespiratory metabolism, and isocitrate dehydrogenase, which is involved in the tricarboxylic acid cycle. It is possible that imbalances in catabolic and anabolic processes directly affect the bending of cucumber fruit. The predicted function of the cobalamin- independent methionine synthase isozyme is closely related to ethylene biosynthesis; fruit bending may be regulated by ethylene, or by ethylene signaling crosstalk during fruit development. The 14-3-3 protein is usually considered to be a regulation-related protein, which plays a role in regulating cell hyperplasia, cell differentiation during growth, and apoptosis during senescence. Involvement of guanosine triphosphate (GTP)-binding proteins in signal transmission is known to regulate the development of cells in cucumber fruits and to play a role in fruit shape variation. Patterns of protein expression showed high repeatability. We hypothesize that these proteins may play an important role in growth and bending of cucumber fruits. The results of our study provide insight into the genetic mechanism underlying fruit bending in cucumber, and may help to promote cultivation of new varieties with superior fruits.
    The Relative Contribution of Non-Foliar Organs of Cotton to Yield and Related Physiological Characteristics Under Water Deficit
    HU Yuan-yuan, ZHANG Ya-li, YI Xiao-ping, ZHAN Dong-xia, LUO Hong-hai, Chow Wah Soon, ZHANG Wang-feng
    2014, 13(5): 975-989.  DOI: 10.1016/S2095-3119(13)60568-7
    Abstract ( )   PDF in ScienceDirect  
    Water deficit is one of the most important causes of decreased yield in cultivated plants. Non-foliar green organs in cotton play an important role in yield formation at the late growth stage. Although better photosynthetic performance was observed in a non-foliar organ (bract) compared with leaves under water deficit. However, the physiological response of each organ in cotton to water deficit has not been comprehensively studied in relation to the water status and photosynthesis characteristics. We studied the maintenance of water status of each organ in cotton by measuring their relative water content, proline content and stomatal characteristics. Water deficit significantly decreased the surface area of each organ, but to a lesser extent in non-foliar organs. Our results showed that the relative contribution of biomass accumulation of non-foliar organs increased under water deficit. Non-foliar organs (bracts and capsule wall) showed less ontogenetic decrease in O2 evolution capacity and in RuBPC activity (per dry weight) as well as better antioxidant systems than leaves at various days after anthesis. We conclude that the photosynthesis from non-foliar organs is important for increasing cotton yield especially under water deficit conditions.
    Seed Priming Influence on Early Crop Growth, Phenological Development and Yield Performance of Linola (Linum usitatissimum L.)
    Hafeez ur Rehman, Muhammad Qaiser Nawaz, Shahzad Maqsood Ahmed Basra, Irfan Afzal, Azra Yasmeen , Fayyaz ul-Hassan
    2014, 13(5): 990-996.  DOI: 10.1016/S2095-3119(13)60521-3
    Abstract ( )   PDF in ScienceDirect  
    Reduced early crop growth and limited branching are amongst yield limiting factors of linola. Field response of seed priming treatments viz. 50 mmol L-1 salicylic acid (SA), 2.2% CaCl2 and 3.3% moringa leaf extract (MLE) including untreated dry and hydropriming controls was evaluated on early crop growth and yield performance of linola. Osmopriming with CaCl2 reduced emergence time and produced the highest seedling fresh and dry weights including Chl. a contents. Osmopriming with CaCl2 reduced crop branching and flowering and maturity times and had the maximum plant height, number of branches, tillers, pods and seeds per pod followed by MLE. Increase in seed weight, biological and seed yields was 9.30, 34.16 and 39.49%, harvest index (4.12%) and oil contents (13.39%) for CaCl2 osmopriming. Positive relationship between emergence and seedling vigor traits, 100-seed weight, seed yield with maturity time, 100-seed weight and seed yield were found. The study concludes that seed osmopriming with CaCl2 or MLE can play significant role to improve early crop growth and seed yields of linola.
    Low Light Stress Down-Regulated Rubisco Gene Expression and Photosynthetic Capacity During Cucumber (Cucumis sativus L.) Leaf Development
    SUN Jian-lei, SUI Xiao-lei, HUANG Hong-yu, WANG Shao-hui, WEI Yu-xia , ZHANG Zhenxian
    2014, 13(5): 997-1007.  DOI: 10.1016/S2095-3119(13)60670-X
    Abstract ( )   PDF in ScienceDirect  
    Low light stress is one of the most important factors affecting photosynthesis and growth in winter production of cucumber (Cucumis sativus L.) in solar greenhouses in northern China. Here, two genotypes of cucumber (Deltastar and Jinyan 2) are used to determine the effect of low light stress on Rubisco expression and photosynthesis of leaves from emergence to senescence. During leaf development, the net photosynthetic rate (PN), stomatal conductance (gs), Rubisco initial activity and activation state, transcript levels of rbcL and rbcS, and the abundance of rbcL and rbcS DNA in these two genotypes increase rapidly to reach maximum in 10-20 d, and then decrease gradually. Meanwhile, the actual photosystem II efficiency (ФPSII) of cucumber leaves slowly increased in the early leaf developing stages, but it declined quickly in leaf senescent stages, accompanied by an increased non-photochemical quenching (NPQ). Moreover, PN, gs, initial Rubisco activity, and abundance of protein, mRNA and DNA of Rubisco subunits of leaves grown under 100 μmol m-2 s-1 are lower, and require more time to reach their maxima than those grown under 600 μmol m-2 s-1 during leaf development. All these results suggest that lower photosynthetic capacity of cucumber leaves from emergence to senescence under low light stress is probably due to down-regulated Rubisco gene expression in transcript and protein levels, and decreased initial and total activity as well as activation state of Rubisco. Deltastar performs better than Jinyan 2 under low light stress.
    Nitrogen Nutrition Index and Its Relationship with N Use Efficiency, Tuber Yield, Radiation Use Efficiency, and Leaf Parameters in Potatoes
    HU Da-wei, SUN Zhou-ping, LI Tian-lai, YAN Hong-zhi , ZHANG Hua
    2014, 13(5): 1008-1016.  DOI: 10.1016/S2095-3119(13)60408-6
    Abstract ( )   PDF in ScienceDirect  
    Knowledge about crop growth processes in relation to N limitation is necessary to optimize N management in farming system. Plant-based diagnostic method, for instance nitrogen nutrition index (NNI) were used to determine the crop nitrogen status. This study determines the relationship of NNI with agronomic nitrogen use efficiency (AEN), tuber yield, radiation use efficiency (RUE) and leaf parameters including leaf area index (LAI), areal leaf N content (NAL) and leaf N concentration (NL). Potatoes were grown in field at three N levels: no N (N1), 150 kg N ha-1 (N2), 300 kg N ha-1 (N3). N deficiency was quantified by NNI and RUE was generally calculated by estimating of the light absorbance on leaf area. NNI was used to evaluate the N effect on tuber yield, RUE, LAI, NAL, and NL. The results showed that NNI was negatively correlated with AEN, N deficiencies (NNI<1) which occurred for N1 and N2 significantly reduced LAI, NL and tuber yield; whereas the N deficiencies had a relative small effect on NAL and RUE. To remove any effect other than N on these parameters, the actual ratio to maximum values were calculated for each developmental stage of potatoes. When the NNI ranged from 0.4 to 1, positive linear relationships were obtained between NNI and tuber yield, LAI, NL, while a nonlinear regression fitted the response of RUE to NNI.
    Plant Protection
    High-Level Accumulation of Exogenous Small RNAs Not Affecting Endogenous Small RNA Biogenesis and Function in Plants
    SHEN Wan-xia, Neil A Smith, ZHOU Chang-yong, WANG Ming-bo
    2014, 13(5): 1017-1023.  DOI: 10.1016/S2095-3119(13)60525-0
    Abstract ( )   PDF in ScienceDirect  
    RNA silencing is a fundamental plant defence and gene control mechanism in plants that are directed by 20-24 nucleotide (nt) small interfering RNA (siRNA) and microRNA (miRNA). Infection of plants with viral pathogens or transformation of plants with RNA interference (RNAi) constructs is usually associated with high levels of exogenous siRNAs, but it is unclear if these siRNAs interfere with endogenous small RNA pathways and hence affect plant development. Here we provide evidence that viral satellite RNA (satRNA) infection does not affect siRNA and miRNA biogenesis or plant growth despite the extremely high level of satRNA-derived siRNAs. We generated transgenic Nicotiana benthamiana plants that no longer develop the specific yellowing symptoms generally associated with infection by Cucumber mosaic virus (CMV) Y-satellite RNA (Y-Sat). We then used these plants to show that CMV Y-Sat infection did not cause any visible phenotypic changes in comparison to uninfected plants, despite the presence of high-level Y-Sat siRNAs. Furthermore, we showed that the accumulation of hairpin RNA (hpRNA)-derived siRNAs or miRNAs, and the level of siRNA-directed transgene silencing, are not significantly affected by CMV Y-Sat infection. Taken together, our results suggest that the high levels of exogenous siRNAs associated with viral infection or RNAi-inducing transgenes do not saturate the endogenous RNA silencing machineries and have no significant impact on normal plant development.
    EaIspF1, Essential Enzyme in Isoprenoid Biosynthesis from Eupatorium adenophorum, Reveals a Novel Role in Light Acclimation
    ZHANG Sheng-rui, JIANG Xue, WANG Ping, WU Di, WANG Qing-hua , HOU Yu-xia
    2014, 13(5): 1024-1035.  DOI: 10.1016/S2095-3119(13)60519-5
    Abstract ( )   PDF in ScienceDirect  
    Isoprenoids are a functionally and structurally diverse class of natural organic chemicals. The universal precursors of all isoprenoids, isopentenyl diphosphate and dimethylallyl diphosphate are synthesized through the mevalonate and 2C-methyl- D-erythritol 4-phosphate (MEP) pathways, respectively. Many isoprenoids produced through the MEP pathway play an important role in plant acclimation to different light environments. Eupatorium adenophorum, an invasive weed in China, presents a remarkable capacity to acclimate to various light environments, which constitutes its solid foundation of being a successful invasive species. Thus we aimed at gaining a deeper insight into the regulation of MEP pathway in E. adenophorum to further understand the invasive mechanism. 2C-Methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF or MCS) is an essential enzyme in the MEP pathway. In this paper, a novel IspF gene was cloned and characterized from E. adenophorum. Tissue-specific expression assays revealed a higher expression of EaIspF1 in leaves than in stems and roots. The expression of EaIspF1 was responsive to different light conditions. Some up-regulation of EaIspF1 expression was also found after the treatments with signal compounds and after wounding stress. Interestingly, the over-expression of EaIspF1 in Arabidopsis led to increase carotenoids contents, resulting in an enhanced tolerance to high light. Taken together, these results indicate that the EaIspF1-derived enzyme participates in isoprenoid metabolism and among others, the expression of this gene in E. adenophorum is involved in the regulation of plastidial isoprenoids, which play an important role in acclimation to various light environments.
    The Minimal Active Fragment of the Cry1Ai Toxin is Located Between 36I and 605I
    ZHOU Zi-shan, LIN Hui-yan, LI Ying, SHU Chang-long, SONG Fu-ping , ZHANG Jie
    2014, 13(5): 1036-1042.  DOI: 10.1016/S2095-3119(13)60532-8
    Abstract ( )   PDF in ScienceDirect  
    The novel cry1Ai gene that cloned from Bacillus thuringiensis strain SC6H8 encoded a protein exhibiting strong toxicity against Plutella xylostella and Chilo suppressalis in our previous study. Using the available information for the active fragments of other Cry toxins, eight truncated fragments were constructed to identify the minimal active fragment of Cry1Ai. All truncated fragments were expressed in Escherichia coli strain BL21 (DE3), and the insecticidal activity against 2nd- instar P. xylostella larvae was assessed using full-length Cry1Ai as a positive control. The results indicate that the minimal active fragment of the Cry1Ai toxin against P. xylostella is located between amino acid residues 36I and 605I, which is smaller than the regions previously reported for Cry1A. The first two amino acids (34T and 35P) on helix α-1 and whole helix α-2 of domain I and sheet β-32 of domain III are necessary for Cry1Ai toxin to keep its toxicity against P. xylostella.
    Penetration of a Single Domain of Bacillus thuringiensis Cry1Ie-Domain I to a Lipid Membrane In vitro
    GUO Shu-yuan, LI Jie, CHEN Zhen , HE Kang-lai
    2014, 13(5): 1043-1050.  DOI: 10.1016/S2095-3119(13)60589-4
    Abstract ( )   PDF in ScienceDirect  
    Domain I of the activated Crystal protein from Bacillus thuringiensis has a seven α-helix bundle structure, which is responsible for membrane channel formation in its insecticidal mechanism. Cry1Ie is toxic to Asian corn borer, Ostrinia furnacalis (Guenée), and plays important roles in insect biological control. The domain I from Cry1Ie has been expressed and purified in its normal conformation, as embedded in the full length homologous toxin structure. The membrane insertion ability of this single domain was compared with the full length homologous toxin using a monolayer insertion experiment. The results indicated that the Cry1Ie-domain I had the ability to insert into the lipid monolayer, and this ability is greater than that of the IE648 toxin. However, the state of insertion is not stable and remains for only a short period of time. The Cry1Ie-domain I plays no role in receptor binding as it had a nonspecific binding with the brush border membrane vesicles of the Asian corn borer.
    Animal Science · Veterinary Science
    Chromosome Mapping, Expression and Polymorphism Analysis of CRABP1 Gene in Pigs
    ZHAO Shuan-ping1, 4 , TANG Zhong-lin1, ZHOU Rong1, QU Chang-qing3, ZHENG Jian-wei2 and LI Kui1
    2014, 13(5): 1051-1057.  DOI: 10.1016/S2095-3119(13)60431-1
    Abstract ( )   PDF in ScienceDirect  
    Cellular retinoic acid-binding protein 1 (CRABP1) is a well-conserved member of cytosolic lipid-binding protein family. It is an important modulator of retinoic acid signaling. Long serial analysis of gene expression (LongSAGE) analysis suggested that CRABP1 gene was differentially expressed during prenatal skeletal muscle development in porcine. Here, we obtained the full-length coding region sequence and genomic sequence of the porcine CRABP1 gene and analyzed its genomic structures. Subsequently, we examined CRABP1 chromosome assignment using INRA-University of Minnesota 7 000 porcine radiation hybrid panel (IMpRH) and explored its tissue distribution in adult Tongcheng pigs and dynamical expression profiles in prenatal skeletal muscle (33, 65 and 90 days post coitus, dpc) from Landrace (lean-type) (described as L33, L65 and L90) and Tongcheng pigs (obese-type) (described as T33, T65 and T90). The CRABP1 gene was mapped to chromosome 7q11-q23 and closely linked to the microsatellite marker SWR1928. Quantitative real-time PCR showed that CRABP1 mRNA was highly expressed in lung and stomach, moderately expressed in placenta and uterus, and weakly expressed in other tissues. Moreover, CRABP1 gene was down-regulated during prenatal skeletal muscle development in both Landrace and Tongcheng pigs and it was expressed much higher in T33 than L33. Two single-nucleotide polymorphisms (SNPs) were detected by sequencing and mass spectrometry methods, allele frequency analysis indicated that g. 281 (G>A) and g. 2992 (G>A) were deviated from Hardy-Weinberg equilibrium in the Landrace and DLY (Duroc×(Landrace×Yorkshire)) pig breeds.
    Morphological and Hormonal Identification of Porcine Atretic Follicles and Relationship Analysis of Hormone Receptor Levels During Granulosa Cell Apoptosis In vivo
    YU De-bing, YU Min-li, LIN Fei, JIANG Bao-chun, YANG Li-na, WANG Si-yu, ZHAO Ying , WNAG Zheng-chao
    2014, 13(5): 1058-1064.  DOI: 10.1016/S2095-3119(13)60448-7
    Abstract ( )   PDF in ScienceDirect  
    Recent reports have demonstrated that follicular atresia is initiated or caused by granulosa cell apoptosis followed by theca cell degeneration in mammalian ovaries, but the mechanism of follicular atresia is still to be elucidated. Therefore, our present study was designed to examine our hypothesis that the changes of follicular microenvironment induce the granulosa cell apoptosis during pocrine follicular atresia in vivo. We firstly isolated intact porcine antral follicles and identified them into three groups, healthy follicles (HF), early atretic follicles (EAF) and progressed atretic follicles (PAF) through morphology and histology. To further confirm their status, we detected hormone levels in follicular fluids and the expression level of apoptosis gene Bax in granulosa cells. The rate of progesterone (P) and estradiol (E2) was increased with the expression of Bax, indicating hormone can be used as a marker of granulosa cell apoptosis or follicular atresia. Finally, we analyzed the expression level of hormone receptor genes in granulosa cells and their relationship with follicular atresia. In PAF, the expression of Progesterone receptor (PGR) was increased significantly while estradiol receptor (ER) had no notable changes, which suggesting the increased-PGR accelerated the effect of P-stimulated granulosa cell apoptosis. The dramatic increasing of androgen receptor (AR) expression in PAF and the obvious increase of tumor necrosis factor-α receptor (TNFR) in EAF indicated that there are different pathways regulating granulosa cell apoptosis during follicular atresia. Together, our results suggested that different pathways of granulosa cell apoptosis was induced by changing the follicular microenvironment during follicular atresia.
    Use of the N-alkanes to Estimate Intake, Apparent Digestibility and Diet Composition in Sheep Grazing on Stipa breviflora Desert Steppe
    HU Hong-lian, LIU Yong-zhi, LI Ya-kui, LU De-xun , GAO Min
    2014, 13(5): 1065-1072.  DOI: 10.1016/S2095-3119(13)60502-X
    Abstract ( )   PDF in ScienceDirect  
    The application of n-alkane as markers to estimate herbage intake, apparent digestibility and species composition of diet consumed by grazing sheep was studied. Six local Mongolian sheep were used to determine dry matter (DM) intake, apparent DM digestibility and species composition of diet during summer, autumn and winter. Animals were orally dosed twice daily with n-alkane gelatin capsules containing 60 mg C32-alkane as an external marker. Diet composition was estimated by comparing the odd-chain n-alkanes pattern profile (C27-C31) of the consumed plant species with the n-alkanes fecal concentrations of grazing animals, using a non-negative least squares algorithm called EATWHAT software package. The alkane pair C32:C33 and C33 alkane were used to estimate DM intake and diet apparent DM digestibility, respectively. The results showed that daily dry matter intake of the sheep were 1.77, 1.61 and 1.18 kg d-1 in summer, autumn and winter, respectively. Apparent DM digestibility, crude protein (CP), metabolizable energy (ME) and neutral detergent fiber (NDF) intake of diet consumed by sheep decreased significantly (P<0.01) from summer to winter, with no evident changes in ADF and ADL intake. Diet composition indicated Artemisia frigida Willd was the most dominant diet component, contributed 79.68, 68.12 and 86.26% of sheep’s diets in summer, autumn and winter, respectively. Cleistogenes songorica Ohwi and Convolvulus ammannii Desr were the important components of the diet. Although Stipa breviflora Griseb is one of the main plant species in the study area, the sheep rarely choosed it. The study indicated that CP and ME in diet consumed by sheep were deficient in winter. Therefore, appropriate supplementation strategies should be indispensable during this period.
    Comparative Proteomic Analysis Shows an Elevation of Mdh1 Associated with Hepatotoxicity Induced by Copper Nanoparticle in Rats
    DONG Shu-wei, GAO Zhao-hui, SHEN Xiao-yun, XUE Hui-wen , LI Xia
    2014, 13(5): 1073-1081.  DOI: 10.1016/S2095-3119(13)60389-5
    Abstract ( )   PDF in ScienceDirect  
    Copper nanoparticle is a new material widely used in biological medicine, animal husbandry and industrial areas, but its potential toxicity to human health and environment remains unclear. In order to study the hepatotoxic mechanisms of nanoparticles copper, two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI/TOF MS) of proteomics technology were used to isolate and identify the differentially expressed proteins from liver, which associated with hepatotoxicity induced by copper nanoparticle in rats. In this study, we have screened 15 kinds of proteins related with hepatotoxicity, of which spot8212 was identified as Malate dehydrogenase (Mdh1). The mRNA expression trend of Mdh1 was consistent with the result of 2-DE by RT-PCR validation. Bioinformatics analysis showed that Mdh1 was stable and no signal peptides, subcellular location was in endoplasmic reticulum; it contained many functional sites such as malate dehydrogenase activity signal sites 155LTRLDHNRAKSQI167; α helixes and random coils were the two main elements. Homologous analysis demonstrated high homologous of Mdh1 in rats with mouse and human, and the phylogenetic tree of Mdh1 was constructed. The result indicated that copper nanoparticle could regulate up the Mdh1 protein expression so as to compensate the energy deficit. Energy metabolic disturbance may be a pathway for copper nanoparticle particles to exert the hepatotoxic effects in rats.
    Spectrum-Effect Relationship Between High Performance Liquid Chromatography Fingerprints and Anticoccidial Activities of a Compound Chinese Medicine
    XIAO Sui, FEI Chen-zhong, ZHANG Li-fang, ZHENG Wen-li, ZHANG Ke-yu , XUE Fei-qun
    2014, 13(5): 1082-1089.  DOI: 10.1016/S2095-3119(13)60313-5
    Abstract ( )   PDF in ScienceDirect  
    Quality control and screening of active substances in traditional Chinese medicines have been performed using fingerprint analysis. The spectrum-effect relationship between chromatography fingerprints and efficacy of herbal drugs is considered as a potentially useful method for determining active ingredients in complex mixtures. The study was designed to develop a method for determining the bioactive components of a compound Chinese medicine called Tiefeng based on spectrum-effect relationships between high-performance liquid chromatography (HPLC) fingerprints and anticoccidial activities. Four peaks of the established HPLC fingerprint indicate the main bioactive components of this medicine. In addition, pharmacodynamic atlas was defined and used to assess the anticoccidial activity of Tiefeng from different sources for the first time. We found that the level of anticoccidial activity of Tiefeng was consistent with the degree of similarity between the pharmacodynamic atlas and chromatogram of any sample. Furthermore, effect of this medicine was related with the main active constituents, along with the origin and the harvesting time.
    Soil & Fertilization · Irrigation · Agro-Ecology & Environment
    Differential Responses of Nitrifier and Denitrifier to Dicyandiamide in Short- and Long-Term Intensive Vegetable Cultivation Soils
    LIU Yi, YANG Yang, QIN Hong-ling, ZHU Yi-jun , WEI Wen-xue
    2014, 13(5): 1090-1098.  DOI: 10.1016/S2095-3119(13)60740-6
    Abstract ( )   PDF in ScienceDirect  
    Nitrification inhibitors, such as dicyandiamide (DCD), have been shown to decrease leaching from urea- and ammonium- based fertilizers in agricultural soils. The effect of nitrification inhibitors on nitrifier and denitrifier in short- and long-term intensive vegetable cultivation soils was poorly understood. In this study, the pot trial was conducted to investigate the differential responses of nitrifier (amoA-containing bacteria) and denitrifier (nirK-containing bacteria) to DCD in short-(soil S) and long-term (soil L) intensive vegetable cultivation soils. Quantitative polymerase chain reaction (qPCR) and terminal restriction fragment length polymorphism (T-RFLP) were employed to detect the abundance and composition of amoA- and nirK-containing communities. The results indicated that application of DCD led to a consistently higher NH4 +-N concentration during the whole incubation in soil L, while it was quickly decreased in soil S after 21 days. Furthermore, DCD induced more severe decrease of the abundance of amoA-containing bacteria in soil L than in soil S. However, the abundance of the nirKcontaining community was not significantly affected by DCD in both soils. Long-term vegetable cultivation resulted in a super-dominant amoA-containing bacteria group and less divergence in soil L compared with soil S, and DCD did not cause obvious shifts of the composition of ammonia-oxidising bacteria (AOB). On the contrary, both amoA- and nirK-containing bacterial compositions were influenced by DCD in soil S. The results suggested that long-term intensive vegetable cultivation with heavy nitrogen fertilization resulted in significant shifts of AOB community, and this community was sensitive to DCD, but denitrifiers were not clearly affected by DCD.
    Reducing Ammonia Volatilization from Maize Fields with Separation of Nitrogen Fertilizer and Water in an Alternating Furrow Irrigation System
    HAN Kun, ZHOU Chun-ju , WANG Lin-quan
    2014, 13(5): 1099-1112.  DOI: 10.1016/S2095-3119(13)60493-1
    Abstract ( )   PDF in ScienceDirect  
    The objective of this 2-yr field trial, with a central composite rotatable design, was to assess and quantify the effects of separation of nitrogen fertilizer and water with alternating furrow irrigation (SNWAFI) practices on soil ammonia (NH3) emission during the summer maize (Zea mays L.) growing season. Ammonia volatilization after N sidedress and irrigation ranged from 4.8 to 17.0 kg N ha-1 and 6.2 to 20.6 kg N ha-1, respectively, in 2008 and 2009. The lower N input contributed to lower NH3 loss but lower yield, whereas the higher N input induced higher yield as well as higher NH3 loss. Ammonia intensity (NH3 volatilization per crop yield) after N sidedress and irrigation was 1.2-3.0 kg NH3-N t-1 yield in 2008 and 1.1-3.2 kg NH3-N t-1 yield in 2009. The predicted minimum NH3 intensity in 2008 was 1.6 kg NH3-N t-1 yield and was obtained with the combined application of 127 kg N ha-1 and 108 mm irrigation water. In 2009, the predicted minimum NH3 intensity was 1.3 kg NH3-N t-1 yield and was obtained with the combined application of 101 kg N ha-1 and 83 mm irrigation water. We conclude that SNWAFI practices with optimum rates of water and fertilizer can significantly reduce soil NH3 intensity and maintain yield. It was more beneficial for sustainable farming strategies to minimize the NH3 intensity rather than reduce absolute NH3 emissions alone.
    Nitrogen Removal Improvement by Adding Peat in Deep Soil of Subsurface Wastewater Infiltration System
    CHEN Pei-zhen, CUI Jian-yu, HU Lin, ZHENG Miao-zhuang, CHENG Shan-ping, HUANG Jie-wen , MU Kang-guo
    2014, 13(5): 1113-1120.  DOI: 10.1016/S2095-3119(13)60401-3
    Abstract ( )   PDF in ScienceDirect  
    In order to enhance the nitrogen removal, a subsurface wastewater infiltration system (SWIS) was improved by adding peat in deep soil as carbon source for denitrification process. The effects of addition of carbon source in the underpart of the SWIS on nitrogen removal at different influents (with the total nitrogen (TN) concentration 40 and 80 mg L-1, respectively) were investigated by soil column simulating experiments. When the relatively light pollution influent with 40 mg L-1 TN was used, the average concentrations of NO3 --N and TN in effluents were (4.69±0.235), (6.18±0.079) mg L-1, respectively, decreased by 32 and 30.8% than the control; the NO3 --N concentration of all effluents was below the maximum contaminant level of 10 mg L-1; as high as 92.67% of the TN removal efficiency was achieved. When relatively heavy pollution influent with 80 mg L-1 TN was used, the average concentrations of NO3 --N and TN in effluents were (10.2±0.265), (12.5±0.148) mg L-1 respectively, decreased by 20 and 21.2% than the control; the NO3 --N concentration of all effluents met the grade III of the national quality standard for ground water of China (GB/T 14848-1993) with the values less than 20 mg L-1; the TN removal efficiency of 94.1% was achieved. In summary, adding peat in the underpart of the SWIS significantly decreased TN and NO3 - -N concentration in effluents and the nitrogen removal efficiency improved significantly.
    Food Science
    In vitro Selection of DNA Aptamers and Fluorescence-Based Recognition for Rapid Detection Listeria monocytogenes
    LIU Guo-qing, LIAN Ying-qi, GAO Chao, YU Xiao-feng, ZHU Ming, ZONG Kai, CHEN Xuejiao
    2014, 13(5): 1121-1129.  DOI: 10.1016/S2095-3119(14)60766-8
    Abstract ( )   PDF in ScienceDirect  
    Aptamers are specific nucleic acid sequences that can bind to a wide range of nucleic acid and non-nucleic acid targets with high affinity and specificity. Nucleic acid aptamers are selected in vitro from single stranded DNA or RNA ligands containing random sequences of up to a few hundred nucleotides. Systematic evolution of ligands by exponential enrichment (SELEX) was used to select and PCR amplify DNA sequences (aptamers) capable of binding to and detecting Listeria monocytogenes, one of the major food-borne pathogens. A simplified affinity separation approach was employed, in which L. monocytogenes in exponential (log) phase of growth was used as the separation target. A fluorescently-labeled aptamer assay scheme was devised for detecting L. monocytogenes. This report described a novel approach to the detection of L. monocytogenes using DNA aptamers. Aptamers were developed by nine rounds of SELEX. A high affinity aptamer was successfully selected from the initial random DNA pool, and its secondary structure was also investigated. One of aptamers named e01 with the highest affinity was further tested in aptamer-peroxidase and aptamer-fluorescence staining protocols. This study has proved the principle that the whole-cell SELEX could be a promising technique to design aptamer-based molecular probes for dectection of pathogenic microorganisms without tedious isolation and purification of complex markers or targets.
    Characterization and Comparison of Ochratoxin A-Ovalbumin (OTA-OVA) Conjugation by Three Methods
    WANG Xi-chun, BAO Ming, WU Jin-jie, LUO Ying, MA Liang-you, WANG Ying, ZHANG Ai-hua, HE Cheng-hua , ZHANG Hai-bin
    2014, 13(5): 1130-1136.  DOI: 10.1016/S2095-3119(14)60767-X
    Abstract ( )   PDF in ScienceDirect  
    In order to generate an antibody against a small hapten molecule, the hapten is cross-linked with carrier protein to make it immunogenic. In this study, the hapten (ochratoxin A, OTA) was coupled to ovalbumin (OVA) by an active ester reaction. To develop a technique for detecting the conjugation, the hapten-protein conjugate (OTA-OVA) was characterized thoroughly by immunoarray technology, ultraviolet (UV) spectroscopy and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), respectively. The molecular weight of OTA-OVA was 50 350.141 Da, and the molecular weight of OVA was 44 887.506 Da, which were determined by MALDI-TOF-MS, respectively. In OTA-OVA, the molecular coupling ratio was 13:1 by MALDI-TOF-MS while the molecular coupling ratio was 10:1 by UV. In this experiment, UV and MALDI-TOF-MS were selected as the efficient methods to evaluate the cross-linking effect and calculate the molecular coupling ratio.
    Solvent Effects on the Ultrastructure and Chemical Composition of Cuticular Wax and Its Potential Bioactive Role Against Alternaria alternata in Pingguoli Pear
    CHEN Song-jiang, LI Yong-cai, BI Yang, YIN Yan, GE Yong-hong , WANG Yi
    2014, 13(5): 1137-1145.  DOI: 10.1016/S2095-3119(13)60374-3
    Abstract ( )   PDF in ScienceDirect  
    Effects of different polarity solvents on the ultrastructure and chemical composition of cuticular wax in Pingguoli pear as well as their bioactive role against Alternaria alternate were studied and the results showed that the highest wax content was extracted with chloroform, and its wax content was up to 322.2 μg cm-2. Long-chain fatty acids predominated in menthol extracts and n-alkanes were predominant in wax extracted with ether, chloroform and n-hexane. Pingguoli pear fruit surface was covered by a smooth and amorphous wax layer with small, scattered crystal. The morphology of recrystallized wax in vitro after removal with different solvents was not similar to that of the intact fruit surface. Removal of cuticular wax with various solvents significantly enhanced A. alternata infection, except for wax removed by methanol. The solvent extracts of methanol and chloroform stimulated the spore germination and mycelium growth of A. alternata, but the ether and n-hexane extracts showed antifungal activity.
    Molecular Characteristics of New Wheat Starch and Its Digestion Behaviours
    ZHOU Zhong-kai, HUA Ze-tian, YANG Yan, ZHENG Pai-yun, ZHANG Yan , CHEN Xiao-shan
    2014, 13(5): 1146-1153.  DOI: 10.1016/S2095-3119(13)60621-8
    Abstract ( )   PDF in ScienceDirect  
    In order to understand the effect of starch molecular characteristics on the gel structure, which subsequently influence the gel digestion behaviours, three wheat starches, control (conventional wheat starch), two new wheat cultivars with different genetic backgrounds (by knocking out SBE IIb and SBE IIa, respectively) were used in this study. In comparison with control, slight differences in the morphology of the starch granules of new wheat 1 were observed, whereas the starch granules of new wheat 2 had irregular shapes both for A-type granules and B-type granules. Starch molecular weight size was determined by SE-HPLC, and the results indicate that there was a subtle increase in the amylose content in the starch of new wheat 1 compared to that of control. The starch of new wheat 2 had the highest amylose content, and the molecular weight (MW) of its amylopectin was the lowest among the three starches. Fourier transform infrared spectroscopy (FTIR) was employed to investigate starch gel structure and the results suggest that the molecules of starch gel from new wheat 2 are more likely to re-associate to form an organized conformation. The digestion behaviours of the three starch gels were measured using a mixture of pancreatin α-amylase and amyloglucosidase. The results indicated that the starch gels of control and new wheat 1 had very high digestibility of 91.7 and 91.9%, respectively, whereas the digestibility of wheat 2 starch gel was only 36.2%. In comparison with the digestion curve patterns of control and new wheat 1 starch gels, the new wheat 2 exhibited a much lower initial velocity. These results indicated that the molecules in the starch of new wheat 2 are more readily to re-associate to form an organized structure during gel formation because of its unique molecular characteristics.