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Table of Content

    15 January 2014, Volume 47 Issue 2
    CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS
    Recent Advances in Research of Transcription Factor NAP Subfamily in Plants
    FAN Kai, WANG Xue-De, YUAN Shu-Na, WANG Ming
    Scientia Agricultura Sinica. 2014, 47(2):  209-220.  doi:10.3864/j.issn.0578-1752.2014.02.001
    Abstract ( 615 )   HTML ( 3 )   PDF (523KB) ( 921 )   Save
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    NAP (NAC-Like,Activated by AP3/PI) is a plant-specific transcription factor, which plays an important role in regulation of plant growth and development, leaf senescence and responses to various kinds of stresses. NAP belongs to NAC (NAM, ATAF1/2 and CUC2) transcription factor, which contains a conserved N-terminal NAC domain and a highly divergent transcription activation region (TAR) in C-terminal region. Furthermore, the NAP protein is localized in the nucleus, and the NAP gene structure contains three exons and two introns. Since the first NAP was found to be closely associated with Arabidopsis flower growth and development in 1998, NAP subfamily has been discovered in rice, wheat, cotton, bean, bamboo, grape and so on, which further confirms that NAP subfamily is one of plant-specific transcription factors. Moreover, NAP subfamily has various biological functions. Firstly, NAP participates in the plant growth and development including seed, root and flower. Then NAP subfamily also takes an essential part in the leaf senescence, which indirectly regulates the macromolecules degradation and nutrient-recycling processes. NAP subfamily also responds to stresses such as drought, salt and cold. And then, NAP subfamily can contribute to the improvement of crop quality, which provides a novel method for crop breeding. In the recent researches, it was supposed that NAP is mainly regulated by abscisic acid (ABA) and ethylene, and a Golgi-localized protein phosphatase 2C SAG113 is its direct target gene. Subsequently, as a negative regulator of ABA signal transduction, SAG113 is closely related to the control of water loss especially during leaf senescence. The results indicate that NAP is regulated by ABA and specifically interacts with its target gene (SAG113). Through ABA-NAP-SAG113 PP2C regulatory chain, NAP will promote the expression of its target gene (SAG113). Then the SAG113 expression inhibits the stomatal closure, which leads to water loss and enough oxygen diffusion for ethylene-stimulated fast respiration during leaf senescence. All the results in turn trigger leaf senescence. The study on NAP subfamily will have an effect on both theoretical and practical researches.
    Genetic Identification and Candidate Gene Analysis of Yellow-Green Leaf Mutant 507ys in Rice
    LI Yan-Qun-1, PU Xiang-1, LI Chun-Mei-1, ZHONG Ping-1, SUN Chang-Hui-1, LI Xiu-Lan-2, DENG Xiao-Jian-1, WANG Ping-Rong-1
    Scientia Agricultura Sinica. 2014, 47(2):  221-229.  doi:10.3864/j.issn.0578-1752.2014.02.002
    Abstract ( 429 )   HTML ( 3 )   PDF (758KB) ( 728 )   Save
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    【Objective】 The present study was conducted aiming at genetic identification and candidate gene analysis of yellow-green leaf mutant 507ys. 【Method】 A yellow-green leaf mutant, designated as 507ys, was isolated from the progeny of a japonica rice cv. Nipponbare treated with ethyl methanesulfonate. Phenotypes of the 507ys mutant were observed and its main agronomic traits were analyzed under field conditions in Chengdu, Sichuan. After the 507ys mutant was crossed with normal green varieties, leaf colour phenotypes of the F1 progenies and the segregation ratio of yellow-green and green leaf plants in the F2 populations were investigated. Genetic mapping of the mutant gene was conducted using 360 yellow-green leaf individuals from the F2 mapping population of 507ys/Minghui63. Putative genes in the fine mapped region were analyzed, and the candidate genes in the mutant and its wild-type were sequenced, respectively. Alignment of the deduced amino acid sequences of homologous OsCAO1 proteins was conducted. In addition, contents of photosynthetic pigments in the 507ys mutant and its wild-type were determined by spectrophotometer, and their chlorophyll compositions were well-examined by high-performance liquid chromatography (HPLC) so that the candidate gene resulting in the 507ys mutant phenotype could be further identified.【Result】 The whole plant of the mutant exhibited yellow-green leaf trait throughout the growing period. Compared with its wild-type parent Nipponbare, the contents of chlorophyll and carotenoid decreased by 52.1% and 58.1%, and plant height, the number of productive panicles per plant, the number of spikelets per panicle and seed setting rate reduced by 8.3%, 51.0%, 7.4% and 11.6%, respectively. All F1 plants generated by crossing yellow-green leaf mutant 507ys with normal green varieties Nipponbare and Minghui 63 showed normal green leaf. The segregation ratios of normal leaf plants and yellow-green leaf plants in two F2 populations both 3:1, indicating the 507ys mutant phenotype is controlled by a single recessive nuclear gene. The mutant gene was finally mapped to a region of 60.2 kb between SSR marker RM333 and InDel marker L3 on the long arm near the end of chromosome 10, in which thirteen predicted genes were annotated in the Rice Genome Annotation Project. Sequencing analysis of these candidate genes between the mutant and its wild-type revealed the single base change (G2198A) in the coding region of the OsCAO1 (LOC_Os10g41780) gene for chlorophyllide a oxygenase resulted in a missense mutation (E353K) in the encoded product. HPLC analysis of chlorophyll composition indicated the 507ys mutant accumulated only chlorophyll a and no chlorophyll b.【Conclusion】 The 507ys mutant gene was allelic to OsCAO1 gene. A point mutation in exons of OsCAO1 gene in the 507ys mutant makes chlorophyllide a oxygenase inactive, resulting in the block in chlorophyll b biosynthesis.
    SSR Markers Linked to High and Low Protein Content Strains Derived from 3 Backcross Combinations Under Jidou 12 Genetic Background
    CHEN Qiang-1, 2 , YAN Long-1, YANG Chun-Yan-1, ZHANG Jia-Nan-1, SHI Xiao-Lei-1, DONG Fang-Yang-2, DENG Ying-Ying-1, 2 , HOU Wen-Huan-1, ZHANG Meng-Chen-1, 2
    Scientia Agricultura Sinica. 2014, 47(2):  230-239.  doi:10.3864/j.issn.0578-1752.2014.02.003
    Abstract ( 444 )   HTML ( 1 )   PDF (620KB) ( 490 )   Save
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    【Objective】 The objective of this study is to investigate the QTL and their chromosomal segments that determine the seed protein content of progeny strains of high-protein and low protein content, which derived from the crosses between high combing-ability cultiver Jidou 12 and three other cultrivers. By accomplishment of these analyses, the results will provide a basis for the use of high-protein breeding. 【Method】 High protein germplasm materials with protein content more than 50% were created by backcrossing strategy using Jidou 12 as recurrent parent varieties and three different varieties as donor parents. Four high-protein (50%-53%) strains and three low protein (38%-41%) strains were selected from propeny strains of three backcrosses (Hongfeng 11, Chamoshidou and Suinong 14 as the donor parent of three backcrosses). These strains had the same agronomic traits and had no significant difference with Jidou 12. Differences in QTL loci between progeny strains and Jidou12 were analyzed by using 209, 201, and 199 polymorphic SSR markers, respectively, in strains from three backcrosses. Degrees in QTL expression and sharing in terms of protein contents among different strains and crosses were investigated. 【Result】 The high-protein strains had better genetic recovery than that of low protein strains in the same backcross progeny. The similarity coefficient between two high protein strains, H116 and H117 from Jidou12 × Chamoshidou, and Jidou12, were 83.08% and 84.04%, respectively, which was 71.14% higher than that of the low protein strains. The average of similarity coefficient between four high protain strains and Jidou12 was 79.58%, significantly higher than that of three low protein strains and Jidou12. Linkage analyses using SSR markers indicated that high-protein lines and low-protein lines had remarkable differences in genetic locations. The major difference was inherited from Jidou 12 , which is 27 % (23 loci) on average. SSR markers were initially linked to 22 genomic regions in the three crosses, including 18 for increasing the protein content with allelic variation from Jidou 12. Further analysis identified four different loci in the three backcross population for high-and-low protein chromosome segments: D2 linkage group between 67.71 - 84.18 cM, G chain group between 80.38 - 96.57 cM, C1 linkage group between 75.52 - 80.62 cM, and I chain group bewteen 46.22 - 50.11 cM. 【Conculusion】 Jidou 12 contains a large number of high-protein QTL genetic loci and provides an essential genetic background in high-protein breeding. High-protein genetic loci from donor parents were detected by SSR markers in the progeny populations with Jidou12 as the recurrent parent. This study lays a foundation for excavating excellent genes and high-protein breeding.
    TILLAGE & CULTIVATION·PHYSIOLOGY & BIOCHEMISTRY·AGRICULTURE INFORMATION TECHNOLOGY
    Physiological Mechanism of Silicon-Enhanced Rice Blast Resistance
    GE Shao-Bin, LIU Min, CAI Kun-Zheng, CAI Yi-Xia, LUO Shi-Ming
    Scientia Agricultura Sinica. 2014, 47(2):  240-251.  doi:10.3864/j.issn.0578-1752.2014.02.004
    Abstract ( 506 )   HTML ( 3 )   PDF (561KB) ( 777 )   Save
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    【Objective】 Rice blast is one of the major diseases in rice production and cause serious yield losses every year. As a kind of environment friendly disease control measures, silicon fertilizer application plays an important role in enhancing plant disease resistance, but the mechanism is not totally clear. The purpose of this study was to investigate the physiological mechanism of silicon-enhanced resistance of rice to blast,and to provide a theoretical foundation and methods of controlling rice blast. 【Method】 Two rice near-isogenic lines(NILs) with different resistances to blast, i.e. CO39 (susceptible) and C101LAC (Pi-1) (resistant) were used in the experiments. There were four treatments: no silicon added and no inoculation with M. oryzae(Si-M-); silicon added but no inoculation with M. oryzae(Si+M-); no silicon added but inoculation with M. oryzae(Si-M+); silicon added and inoculation with M. oryzae(Si+M+). Using a grown chamber to control the growing conditions, the hydroponic culture experiments with Hoagland’s nutrient solution were conducted to clarify the effects of silicon on disease incidence, content of silicon, phenolics, SA, ethylene and H2O2 in roots and leaves of rice. 【Result】 Si application significantly reduced the blast incidence and disease index for both two rice lines. And the blast incidence and disease index of C101LAC (Pi-1) were obviously lower than CO39. Si concentration increased significantly in all leaf, stem and root tissues of both two rice lines by Si treatment regardless of inoculation. While Si concentrations in leaves and stems were obviously higher than that in roots. After inoculation with M. oryzae, Si treatment significantly reduced the total phenolic content in leaves of CO39 and C101LAC (Pi-1). In roots of two rice lines, Si application had no significant effect on the total phenolic content. Without inoculation, Si application had no significant impact on the total phenol content in leaves 3 d after inoculation, but reduced it 7 d after inoculation for both rice lines. The content of total phenol was obviously lower in roots than in leaves. After inoculation with M. oryzae, Si treatment significantly reduced the lignin content in leaves of CO39 and C101LAC (Pi-1), but there were no significant effect in roots. Without inoculation, Si application significantly increaced SA content in leaves 3 d and 7 d after inoculation for CO39, but only 7 d after inoculation for C101LAC (Pi-1). Si application also significantly increased SA content in leaves of CO39 3 d after inoculation, but not in C101LAC (Pi-1). SA was not detected in roots for both CO39 and C101LAC (Pi-1). Under the condition of inoculation, Si supply significantly reduced ethylene content in leaves and roots for both CO39 and C101LAC (Pi-1). Without inoculation, Si application significantly reduced ethylene content in roots on 3 d after inoculation for CO39, and 3 d and 7 d for C101LAC (Pi-1). After inoculation with M. oryzae, Si treatment significantly reduced the H2O2 content in leaves, but significantly increased it in roots, for both CO39 and C101LAC (Pi-1). H2O2 content was obviously lower in roots than in leaves for both rice lines. 【Conclusion】 These results indicate that Si can significantly increase the blast resistance of rice. Si addition can adjust biochemical metabolism of phenolic compounds in plants, and leads to variation of the signal compounds such as SA, ethylene, H2O2 in the rice to enhance the resistance against blast.
    Investigation on Response Mechanism of Epicuticular Wax on Arabidopsis thaliana Under Cold Stress
    NI Yu, SONG Chao, WANG Xiao-Qing
    Scientia Agricultura Sinica. 2014, 47(2):  252-261.  doi:10.3864/j.issn.0578-1752.2014.02.005
    Abstract ( 523 )   HTML ( 6 )   PDF (864KB) ( 878 )   Save
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    【Objective】Cold stress is one of the factors influencing the quality and quantity of crops while the epicuticular wax is believed to be the first protective barrier of the plant to external stresses. In the current study, the effects of cold stress on epicuticular wax morphology and constituents and expression of wax related genes in Arabidopsis stems were examined, aiming to understand the interactive mechanism between plant epicuticular wax and cold stress, and to provide suggestions on researches related to crop resistance to abiotic stresses. 【Method】The materials used in current experiment included wild type Arabidopsis (WT) and wax mutants (cer1, cer3, cer4, cer6, cer10, cer20 and kcs1). The seedlings were grown for 5-6 weeks after germination, and then were placed in a temperature controlled cabinet at 4℃ for ten days and eighteen days, seperately. Scanning electron microscope technology was used to investigate the changes of crystal structure. Gas chromatography and mass spectrometry technology were used to measure the contents of total wax and wax constituents. Real-time Q-PCR was used to analyze the transcripts of wax related genes.【Result】Under cold stress, the density, shape and size of wax crystalloids on stem of mutants and WT changed. Wax crystal structures on WT fused to big horizontal plates, greatly covering the surface of stems, which might help protect plant from cold hardness and decrease water loss. The pine-needle crystals on cer1 reduced significantly and some small dendrites appeared. The rod crystals on cer3 and cer10 reduced significantly while the crystals on cer6 and cer20 increased. The vertical rods on kcs1 surface reduced while the horizontal rods appeared with the fusion of crystals. The cold stress had no effect on the crystal morphology on cer4. The GC-MS analysis showed that the contents of wax constituents and total wax also changed under cold stress. The contents of aldehydes and ketones in Arabidopsis wild type reduced significantly while the content of primary alcohols increased. For the mutants, the contents of acids, aldehydes and ketones increased or changed insignificantly while the contents of primary alcohols reduced or changed insignificantly. For cer3 and cer10 with severe cold harm symptoms, the contents of primary alcohols decreased significantly. The contents of wax accumulated significantly under cold stress except cer3 and cer10, mainly attributing to significant increase in alkanes and secondary alcohols. The content of total wax in cer3 changed insignificantly while that in cer10 reduced. For A. thaliana wild type, the expression of CER1 was induced strongly by the cold stress, suggesting that plant can up-regulate the expression of CER1 to promote the alkane synthesis under cold stress. The up-regulated expression of CER4 promoted the synthesis of primary alcohol. The down-regulated expression of KCS1, CER3 and WIN1 under cold stress indicated that the increase of total wax was due to the promotion of the alkane-synthesizing branch. 【Conclusion】 The crystal structure and constituents of epicuticular wax can be altered by cold stress, and the increase of alkanes and secondary alcohols in epiculticular wax might be the main response way to cold stress. The increase of alkanes contributes to the increase of total wax and cer1 might be the main response wax-related gene to cold stress for A. thaliana.
    PLANT PROTECTION
    Relationship Between β-1, 4-Endoglucanase Gene in Endophytic Bacillus Strain BS2 and Its Colonization in Plant
    FAN Xiao-Jing-1, YANG Rui-Xian-1, 2 , QIU Si-Xin-3, HU Fang-Ping-1
    Scientia Agricultura Sinica. 2014, 47(2):  262-272.  doi:10.3864/j.issn.0578-1752.2014.02.006
    Abstract ( 470 )   HTML ( 5 )   PDF (743KB) ( 1021 )   Save
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    【Objective】 The objective of this study is to clone β-1,4-endoglucanase gene (eglS) of endophytic Bacillus amyloliquefaciens strain BS2, investigate the interactive relationship between the gene and plant in colonization. 【Method】The constitutive promoter rpsD sequence was cloned from genomic DNA of B. subtilis 168 by PCR. Homology primers were used to clone eglS from genomic DNA of strain BS2. Then the fragments of rpsD and eglS were ligated and amplified by PCR, and an expression cassette was obtained. The expression cassette fragment was inserted into the shuttle vector pGFP4412 for constructing over-expressive plasmid. pGFP4412 was digested by EcoR I and Xba I. PCR was also used to obtain homologous recombination fragment of eglS. The homologous recombination fragment was inserted into the integration vector pMUTIN-GFP+, which could integrate into chromosome by a single recombination event, to construct knockout plasmid. The over-expressive mutants and knockout mutants were obtained by competent-cell transformation of over-expression plasmids and knockout plasmids, respectively. Complementary mutants were generated by transforming over-expressive plasmids into knockout mutants. The mutants were confirmed by a fluorescence microscope, PCR, enzymatic assays on agar plates and real-time PCR. Enzyme activity of β-1,4-endoglucanase was determined by 3,5-dinitrosalicylic acid colorimetric method (DNS method). The colonizing bacteria amounts of mutants and the wild strain in the tissues of cabbage plants within 30 d were measured after inoculation by root irrigation. 【Result】The over-expressive mutants and complementary mutants were obtained, which showed green fluorescence under a fluorescent microscope. Knockout mutants were identified by PCR and the results showed that the DNA fragment of about 675 bp was amplified from the knockout mutants. However, there were no corresponding bands from the wild-type strain and the integration vector. β-1,4-endoglucanase assays on agar plates showed that the over-expression mutants and complementary mutants presented large and dual hydrolytic circles compared with the wild-type strain. In addition, the clarity of the outer circle was less than that of the inner one. The knockout mutants showed a small and very weak hydrolytic circle around the edge of a colony. The real-time quantitative PCR results confirmed the differentially expression of β-1,4-endoglucanase between the wild-type strain and the mutants. The relative mRNA transcription levels of β-1,4-endoglucanase gene in the over-expressive mutants and complementary mutants were higher than that in the wild-type strain. They were 111 and 82 times as much as that of the wild-type strain, respectively. In contrast, the relative expression level of β-1,4-endoglucanase mRNA in knockout mutants was approximately 0 compared with the wild-type strain. The results of quantity of mutants and the wild strain in the tissues of cabbage plants and enzyme activity assay showed that knockout mutant bacteria had the least amount of number in the same cabbage plant tissues and the same isolation time, and it had significant difference compared with other strains (P<0.05). In addition, the β-1,4-endoglucanase activity of knockout mutants was also the lowest, and it was significantly lower than that in the other strains (P<0.05). The over-expression mutants were significantly higher than that of the knockout mutants and wild strains both in enzyme activity and colonization population in the cabbage plants (P<0.05). The enzyme activity and colonizing bacteria density in the cabbage plants of complementary mutants were basically consistent with the over-expression mutants.【Conclusion】There was a positive correlation between the level of β-1,4-endoglucanase activity and its colonization quantity in the cabbage plants of the endophytic B. amyloliquefaciens BS2. The β-1,4-endoglucanase of strain BS2 played a role in the process of colonization in plants.
    cDNA Cloning, Sequence Analysis and Prokaryotic Expression of Farnesyl Pyrophosphate Synthase from Epicauta gorhami Marseul (Coleoptera: Meloidae)
    FU Nan-Xia, DI Feng, JIANG Ming, 吕Shu-Min , ZHANG Ya-Lin
    Scientia Agricultura Sinica. 2014, 47(2):  273-283.  doi:10.3864/j.issn.0578-1752.2014.02.007
    Abstract ( 480 )   HTML ( 2 )   PDF (1846KB) ( 553 )   Save
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    【Objective】 Farnesyl pyrophosphate synthase (FPPS) plays an important role in deciding the biosynthetic pathway of monoterpene and sesquiterpene, obtaining FPPS from Meloidae insects is the basis for studying the relationship between FPPS and the biosynthetic pathway of cantharidin. The main purpose of this study is to clone cDNA sequence of FPPS from Epicauta gorhami Marseul, to predict the structures, properties and functions of this gene and its encoding protein, and to successfully prokaryotic express its encoding protein. 【Method】 First, according to the conservative sequences from those known insects FPPS, degenerate primers were designed by using CODEHOP software and RT-PCR to gain cDNA template, nested PCR was used to amplificate E. gorhami FPPS conserved region. Next, specific primers were designed according to the conserved fragment of E. gorhami FPPS, RACE technique was used to clone E. gorhami FPPS 3′ and 5′ sequences, then the open reading frame (ORF) region specific primers were designed according to the prediction of the ORF and nested PCR to amplificate encoding area. Finally, splicing different fragments by DNAMAN to obtain the full-length cDNA of FPPS from E. gorham. The sequences of E. gorham FPPS, phylogenetic relationship and properties of Fps, FPPS protein subcellular localization were analyzed by a variety of bioinformatics softwares such as DNAMAN, MEGA 5.05, ProtParam and TargetP 1.1 Server et al. Using pET-28a (+) as a fused expression vector, a recombinant plasmid pET-28a-EgFPPS containing the coding sequence of E. gorhami was constructed. Then inducing its expression in Escherichia coli BL21 (DE3) with IPTG. Next, samples induced at different times were collected and SDS-PAGE was used to analyze the protein expressed by E. coli BL21 (DE3). Using ultrasonic wave to broke the efficiently expressed bacterium liquid, then the supernatant and precipitate were centrifuged and boiled respectively, finally SDS-PAGE was used to analyze the solubility of EgFPPS. Western Blot confirmed the successful expression of EgFPPS in E. coli BL21 (DE3). 【Result】 The full-length cDNA of FPPS from E. gorhami was obtained. It was 1 857 bp in length, containing a 5′-untranslated region (5′-UTR) of 146 bp and a 3′-untranslated region (3′-UTR) of 436 bp. The ORF of 1 275 bp encodes 424 amino acid residues with a predicted molecular weight of 49.2 kD and an isoeletric point value of 8.89, its predicted molecular formula was C2192H3457N605O632S25. Bionformatical analysis showed that its instability index was 38.62 and the GRAVY was -0.429, suggesting that EgFPPS was a stable hydrophobic protein. Homology comparison found that FPPS from E. gorhami and other six species insects had 72.56% homology at amino acid level and all of them had one R**S tetrapeptide which was a consensus cleavage motif in mitochondrial targeting peptide. Besides, seven conserved regions and two aspartate-rich regions (DD**D) could also be identified in EgFPPS. Phylogenetic tree showed that E. gorhami had the closest evolutionary relationship with Tribolium castaneum which belongs to Coleoptera, Tenebrionidae. A mitochondrial targeting peptide was found in the N-terminal of EgFPPS by subcellular localization software, TargetP 1.1 Server. Prokaryotic expression results showed that efficient expression of FPPS protein could be realized after induced with 1 mmol•L-1 IPTG in E. coil BL21 (DE3) for 4 h at 37℃. Solubility analysis showed that the fused protein mainly existed as inclusion bodies. Western Blot confirmed that the molecular weight of the recombinant EgFPPS was about 49 kD, consistent with the predicted result.【Conclusion】The cDNA sequence of FPPS was successfully cloned from E. gorhami, and the prokaryotic expression of its encoding protein was achieved, which lay a foundation for further functional research on the role of FPPS in the biosynthetic pathway of cantharidin in blister beetles.
    Nested-PCR Rapidly Detecte Acidovorax avenae subsp. citrulli from Watermelon Seeds
    WANG Jing, BI Yang, ZHU Yan, HAN Shun-Yu, ZHU Xia, SHENG Wen-Jun, LI Min
    Scientia Agricultura Sinica. 2014, 47(2):  284-291.  doi:10.3864/j.issn.0578-1752.2014.02.008
    Abstract ( 450 )   HTML ( 2 )   PDF (579KB) ( 736 )   Save
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    【Objective】The objective of this study is to develop a nested-PCR method to rapidly and accurately detect Aac (Acidovorax avenae subsp. citrulli) in watermelon seeds, and to provide technique support for prevention and control bacterial fruit blotch of watermelon (WFB).【Method】Two pairs of specific primer sets (BX-L1/BX-R5 and BX-L1/BX-S-R2) derived from BOX short repeated sequence of Aac were selected to establish the nested-PCR. 5 µL of suspension were added in the tubes, boiled at 99℃ for 10 min, and then chilled on ice for 5 min, the released pathogen DNA was used as template for PCR reaction. PCR reaction system was performed with 5 μL of 10× reaction buffer (25 mmol?L-1 MgCl2), 0.4 μL of Taq DNA polymerase (DR001B, 5 U?μL-1), 3 μL of each primer (5 μmol?L-1), 4 μL of dNTP(D4030RA, 2.5 mmol?L-1), ddH2O was added to the final reaction volume of 50 μL. DNA amplification was performed with 2 min at 95℃, followed by 35 cycles consisting of denaturation (30 s at 95℃), annealing (45 s at 65℃) extension (1 min at 72℃), and a final extension at 72℃ for 7 min. The nested-PCR was performed using (BX-L1/BX-R5 primers for the first run and the BX-L1/BX-S-R2 sets for the second run by using 1 μL of the first PCR product as the template applying the same thermal profile and number of cycles and annealing (45 s at 66℃). Analytical sensitivity, specificity and reproducibility were assessed, respectively. The nested-PCR method was used to detect a series of dilution of Aac suspension and different bacteria-carrying rates of seeds. 【Result】Aac in different strains was amplified by the specific primer sets BX-L1/ BX-R5 and BX-L1/BX-S-R2, respectively. A target band was amplified from Aac strains but not from A. avenae subsp. cattleyae, A. avenae subsp. konjaci and other bacteria. The nested-PCR assay had a lowest detection limit of 4.7×101 cfu/mL, the sensitivity was 1 000 times higher than the conventional direct-PCR. When the carrier rate of infected seed was 0.1%-0.5% , the positive detection rate was 66.7%. When the carrier rate of infected seed was 1%-10% , the positive detection rate was 83%-100%.【Conclusion】The present study demonstrated that the procedure of nested-PCR is a sensitive, specific, rapid, reproducible method to detect Aac in watermelon seeds.
    SOIL & FERTILIZER·WATER-SAVING IRRIGATION·AGROECOLOGY & ENVIRONMENT
    Effect of Straw Incorporation Substitute for K-Fertilizer Under Different Paddy Soil K Supply Capacities
    LI Ji-Fu-1, LU Jian-Wei-1, REN Tao-1, CONG Ri-Huan-1, LI Xiao-Kun-1, ZHOU Li-1, YANG Wen-Bing-2, DAI Zhi-Gang-2
    Scientia Agricultura Sinica. 2014, 47(2):  292-302.  doi:10.3864/j.issn.0578-1752.2014.02.009
    Abstract ( 466 )   HTML ( 4 )   PDF (643KB) ( 677 )   Save
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    【Objective】In order to provide a scientific basis for crop residue management and K fertilization, the effect of straw incorporation plus K-fertilizer on yield of rice, potassium (K2O) accumulation amount of shoots and potassium efficiency under different paddy soil potassium levels was studied. 【Method】A series of field trials were carried out to study the effect of recommended rate of potassium (K2O 75 kg•hm-2) on rice production and whether straw potassium could replace chemical potassium under wheat-rice/rape-rice cropping systems in selected 10 counties in Ezhong, Jianghan plain and Edong regions during 2011-2012. According to the second national soil census in Hubei province, trial sites (available K content >150 mg•kg-1), Zhongxiang (ZX) and Yicheng (YC) were classified as high potassium supply capacity soils, marked as high-K; the test points (available K content 100-150 mg•kg-1), Tuanfeng (TF), Xiantao (XT), Honghu (HH) and Zhijiang (ZJ) were classified as middle potassium supply capacity soils, labeled as Middle-K; the test points (available K content <100 mg•kg-1), Macheng (MC), Guangshui (GS), Ezhou (EZ) and Qichun (QC) were normalized to low potassium supply capacity soil, labeled as Low-K. The field experiment was carried out in a randomized block design, including six treatments with three replications. The designed treatments were CK (-K), +K, +S, S+1/3K, S+2/3K, and S+K, where K and S denoted K fertilizer and straw, respectively. 【Result】The results showed that the yield of CK treatment for high-K, middle-K and low-K soil were 8372, 8710 and 7767 kg•hm-2, respectively. However, the K-fertilizer and straw returning to field could increase yield of rice and K accumulation amount of shoots at varying degrees. The straw incorporation with K-fertilizer treatment got the best effect compared with CK treatment. The increased yields were 633, 1098 and 814 kg•hm-2, respectively and the yield increase rates were 7.7%, 12.6% and 12.5%, respectively for high-K, middle-K and low-K soils. The increased K accumulations were 40.2, 56.5 and 49.3 kg?hm-2 and the K increase rates were 15.9%, 21.3% and 36.8%, respectively. Straw incorporation could reduce the KfRE (K fertilizer recovery efficiency) of high-K soil, but increase the KfRE of middle-K and low-K soils, significantly. Meanwhile, straw returning to field also lowered the KfAE (K fertilizer agronomic efficiency) of high-K soil, but enhanced the KfAE of others. Compared with KfRE and KfAE, the KRE and KAE (K efficiency of straw-K with chemical-K) were reduced, but the KRE and KAE of middle-K and low-K soils were obviously higher than those of high-K soil. Under the straw returning to field condition, the correlation analysis between increase of yield, K accumulation and K-fertilizer amount indicated that the recommended rate of potassium was more than the actual demand of rice growth in high-K soil and middle-K soil. Using the fertilizer efficiency model and considering straw returning to field, the optimal amount of K-fertilizer was 38.2 kg•hm-2 for high-K soil (available potassium content>150 mg•kg-1), which was 49.1% less than the recommended amount in Hubei province. In the same way, the optimal amount of K-fertilizer was 60.0 kg•hm-2 for middle-K soil (available potassium content 100-150 mg•kg-1), which was 20.0% less than the recommended amount. Whereas for low-K soil (available potassium content<100 mg•kg-1), the current recommended amount of K-fertilizer was deficient. 【Conclusion】 It was concluded that straw potassium could replace partial chemical potassium for higher soil K supply, but insufficient for lower soil K supply if more grain wants to be produced in short-term of straw incorporation.
    Ecological Benefits and Environmental Carrying Capacities of Red Paddy Field Subjected to Long-Term Pig Manure Amendments
    LIU Kai-Lou, LI Da-Ming, HUANG Qing-Hai, YU Xi-Chu, YE Hui-Cai, XU Xiao-Lin, HU Hui-Wen, WANG Sai-Lian
    Scientia Agricultura Sinica. 2014, 47(2):  303-313.  doi:10.3864/j.issn.0578-1752.2014.02.010
    Abstract ( 578 )   HTML ( 3 )   PDF (594KB) ( 741 )   Save
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    【Objective】Returning organic manure into field soil is one of the important ways to achieve cyclic utilization of waste resources. A large number of studies proved that pig manure could improve soil fertility and crop yield. However, long-term pig manure amendments also would lead heavy metal elements such as Cu, Zn and As accumulation in soil, due to the feed additive are widely used which contain high content of heavy metals, and then threat the soil quality and food safety. Therefore, the effect of long-term amendments of pig manure on environment carrying capacity was conducted in red paddy field. 【Method】The changes of rice grain yield and the contents of soil organic carbon and heavy metals such as Cu, Zn, Cr and As were investigated based on samples from the long-term field experiment with continuously pig manure amended since 1981 (it contained samples of initial soil, 5, 16, 22, and 30 years fertilization), in order to assess the environment carrying capacities of red paddy field regarding to the side effects of pig manure amendments based on the Grade Ⅱ thresholds according to the national soil environmental quality standard (GB15618-1995). 【Result】The results showed that long-term amendments of pig manure significantly increased both rice yield and soil organic carbon content by 10.3%-12.0% and 18.8%-23.7%, respectively, compared with long-term chemical fertilizer (NPK) amendment. Soil pH value for both OM1 and OM2 treatments (i.e. pig manure amendments in the early and late rice season) after three decades compared to the initial soil pH before the experiment set up. This indicated that long-term pig manure could not alleviate soil acid status. Moreover, pig manure amendments also significantly increased the contents of heavy metals such as Cu, Zn, Cr and As in soil. After continuous amendments for three decades, the contents of Cu, Zn, Cr and As were increased by 72.0%-82.6%, 29.1%-55.2%, 194.8%-262.6% and 90.5%-192.7%, respectively, compared with the chemical fertilizer (NPK) treatment, but still below the Grade Ⅱ thresholds according to the national soil environmental quality standard (GB15618—1995). Based on the present amendment rate of pig manure, the contents of heavy metal measured in soil would exceed the Grade Ⅱ thresholds if continuously amended pig manure for 22, 67, 44 and 14 years, respectively. From a different perspective, considering the 50-year continuously amended pig manure as an essential prerequisite, the amendment rate of pig manure should not exceed 6.40 t•hm-2•a-1 to ensure the soil heavy metal contents of Cu, Zn, Cr and As is still below the Grade Ⅱ thresholds. 【Conclusion】Long-term amendments of pig manure unambiguously increased both the rice grain production and soil fertility development. Meanwhile, it should not be neglected that the accumulation of heavy metal would lead to environmental pollution. Taking together, the results highlight that tradeoffs between crop production, soil fertility and ecological benefits and environmental carrying capacities subjected to long-term pig manure amendments could help the sustainable development of agriculture and the improvement of the ecological environment in red paddy field.
    Effect and Assessment of Controlled Release Fertilizer and Additive Treatments on Greenhouse Gases Emission from a Double Rice Field
    WANG Bin, LI Yu-E, WAN Yun-Fan, QIN Xiao-Bo, GAO Qing-Zhu
    Scientia Agricultura Sinica. 2014, 47(2):  314-323.  doi:10.3864/j.issn.0578-1752.2014.02.011
    Abstract ( 527 )   HTML ( 3 )   PDF (726KB) ( 661 )   Save
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    【Objective】It is well known that the issue of greenhouse gases (GHGs) emission from rice ecosystem has been concerned within the scope of climate change research over years. The effect of controlled release fertilizer and additive treatments on GHGs emission and rice yield in a double rice (Oryza sative L) field was investigated to evaluate their potential of GHGs reduction and yield promotion, this study is also very important for the development of low-carbon agriculture and the mitigation research of global warming.【Method】Taking the double rice in Jianghan Plain, Hubei province, Central China as the object, a continuous observation of greenhouse gas emission from six different controlled release fertilizer or additive treatments (CK: conventional urea, CRU1: sulfur-coated urea, CRU2: polymer-coated urea, CU: nitrapyrin crystal urea, DMPP: nitrification inhibitor, EM: effective microorganisms) was conducted by using the automatic static chamber-GC (gas chromatography) method, the rice yield and soil properties were also monitored simultaneously. Variation and characterization of GHGs (CH4 and N2O) emission, greenhouse effect (CO2-e) and greenhouse gas intensity of each treatment were analyzed comprehensively.【Result】The results indicated that CH4 and N2O emission in different fertilizer treatments had an obvious daily and seasonal variation law in double rice ecosystem. Controlled release urea (polymer-coated) caused the lowest CH4 emission during the early rice, while the nitrapyrin crystal urea had the lowest CH4 emission during the late rice growing season. In consideration of N2O, the DMPP had the lowest emission during the two rice growing season compared to the other field applications. Pronounced differences were discovered among 6 treatments on global greenhouse effect (CO2-e,on 100 a horizon) during the whole rice growing season (P<0.05). Among the field applications, CRU1 had the lowest global greenhouse effect, followed by CU, EM, DMPP, CRU2, and CK, respectively. Furthermore, significant greenhouse effect reduction potential was also employed; the polymer-coated urea dominated the fashion with the highest reduction potential of 56.2% compared to traditional fertilization, followed by nitrapyrin crystal urea (45.6%). In the view of rice yield, five other treatments were significantly higher than CK during late rice (stimulated rice yield by 13.5%-16.2%) while no statistical differences were found during early rice. Additionally, GHGI of polymer-coated urea was statistically lower than the other applications including the conventional fertilization (P<0.01). 【Conclusion】Various reduction potential and yield promotion effects existed among different field applications from the double rice cropping system, this influence was significant during the late rice growing season but not remarkable in the early rice,while synthetically consideration of their economic earnings and environmental effects, the application of controlled release urea benefitted the most to the rice production, followed by nitrification inhibitor and biopreparate under the current field management conditions.
    Screening, Identification of Lignin-Degradating Bacillus MN-8 and Its Characteristics in Degradation of Maize Straw Lignin
    LI Hong-Ya, LI Shu-Na, WANG Shu-Xiang, WANG Quan, ZHU Bao-Cheng
    Scientia Agricultura Sinica. 2014, 47(2):  324-333.  doi:10.3864/j.issn.0578-1752.2014.02.012
    Abstract ( 515 )   HTML ( 2 )   PDF (663KB) ( 792 )   Save
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    【Objective】The objective of this study is to obtain the Bacillus with the high lignin-degradation ability and investigate its characteristics in degradation of maize straw lignin, and to provide a basis for microbiological degradation of lignin. 【Method】Bacillus with high ability for degrading lignin was isolated and screened from cattle manure by the decolorization of aniline blue in plate cultivation and the lignin degradation enzyme activity was determined. Among of the obtained Bacillus, the strain with higher ability for degrading lignin was identified through morphology, physiological and biochemical tests, 16S rDNA and gyrB sequence. Then the straw heap fermentation experiment was performed with the inoculation of the bacterial strain obtained above. The changes of lignin peroxidase (LiP), manganese peroxidase (MnP) and the content of lignin in maize straw in the fermentaiton process were monitored to study the lignin-degradation activity of the strain. Its mechanism for degrading lignin was clarified by the GC/MS analysis results of lignin-degradation products. 【Result】 A strain of spore bacterium MN-8 with high ability of lignin-degradation was isolated and screened. Based on the morphology, physiological and biochemical experiments and 16S rDNA sequence analysis, strain MN-8 was identified as Bacillus. The result of 16S rDNA sequence analysis showed that MN-8 was more than 99% similar with Bacillus licheniformis and B. amyloliquefaciens. But the phylogenetic tree based on gyrB sequences showed that strain MN-8 shared 99% identity with B. amyloliquefaciens. Combined with 16S rDNA and gyrB sequence analysis, the strain MN-8 was confirmed as B. amyloliquefaciens. After 16 d incubation in the process of straw heap fermentation, lignin degradation rate was up to 24%. In the two stages of 6-8 d and 10-12 d in the stacking fermentation, the maximum activities of MnP and LiP were appeared. Lignin was degradated apparently in the same stage. And the analysis results of degradation products by GC/MS showed that strain MN-8 could degradate lignin efficiently into some low-molecular-weight matter, such as aromatic compounds 4-hydroxy-3,5-dimethoxy-acetophenone and short chain fatty acids, etc. 【Conclusion】B. amyloliquefaciens MN-8 with high lignin-degradation ability was obtained. It could degradate straw lignin into small molecular substances such as aromatic compounds by the rupture of β-O-4 bond connected between lignin monomer. And its degradation capacity depends on the LiP and MnP.
    HORTICULTURE
    Morphological and Molecular Studies on a Wild Citrus ‘Longmen Xiangcheng’
    ZENG Ji-Wu, JIANG Bo, WU Bo, ZHONG Yun, CHENG Chun-Zhen, MU Hong-Na, GAN Lian-Sheng, PENG Cheng-Ji, ZHONG Guang-Yan, YI Gan-Jun
    Scientia Agricultura Sinica. 2014, 47(2):  334-343.  doi:10.3864/j.issn.0578-1752.2014.02.013
    Abstract ( 559 )   HTML ( 1 )   PDF (894KB) ( 1053 )   Save
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    【Objective】 Wild citrus plants are growing in the Nankun mountain, Longmen county, Guangdong Province, which is named ‘Longmeng Xiangcheng’ by the locals. This study aimed to reveal the genetic differences between ‘Longmeng Xiangcheng’ and other citrus species by using morphological and molecular analysis. 【Method】The habitat environment of ‘Longmen xiangcheng’ was investigated, and the morphology of leaf, flower and fruit was described. Fruit were analyzed for content of total soluble solid, sugar, titratable acid and ascorbic acid. Pollens were observed under scanning electronic microscope. The phylogenetic relationship between ‘Longmen xiangcheng’ and other citrus species was investigated using AFLP and SNP markers. The two alleles of β-carotene hydroxylase gene (CHX) were cloned, sequenced and compared with those of other citrus. 【Result】 The natural habitant environment of ‘Longmeng Xiangcheng’ indicates that it is truly a wild citrus. ‘Longmeng Xiangcheng’ resembles to pummelo in fruit and seed morphology and is also mono-embryonic. But it is smaller than pummelo in size, extremely low content of juice and edible portion, and bitter taste of the fruit indicates that it is more primitive. Its lanceolate leaves, linear leaf wings and united filaments show clearly similarities to those of mandarins. However, its young leaves, young flowers, and filaments are tinged with purple, indicating its primitiveness. The phylogenetic tree based on AFLP profiles show that it is not clustered with any of the other 23 analyzed citrus, though it is placed in between pummelos and mandarins. The genotyping results showed that its genotypes at 3, 1, 4 of the 8 SNP loci are mandarin homozygotes, mandarin-pummelo heterozygote, and pummelo homozygotes, respectively, and no other analyzed citrus accessions contained such a unique combination of genotypes. The CHX gene sequence data revealed that ‘Longmen Xiangcheng’ possesses two very similar alleles carring some very unique SNPs not found in any known citrus CHX gene sequences. Phylogenetic analyses based on CHX gene sequences showed that ‘Longmeng Xiangcheng’ is distant from other citrus, and ‘Longmen Xiangcheng’ can be placed as an independent clade in trees.【Conclusion】‘Longmen Xiangcheng’ is truly a wild citrus. Its morphology is characterized by possessing with both primitive and advanced traits. It is quite unique and different from any known citrus as reveled by molecular DNA data.
    Sequence Analysis of NBS-Type RGAs and Their Relationship with Anthracnose Resistance in Walnut
    AN Hai-Shan-1, YANG Ke-Qiang-1, 2
    Scientia Agricultura Sinica. 2014, 47(2):  344-356.  doi:10.3864/j.issn.0578-1752.2014.02.014
    Abstract ( 420 )   HTML ( 4 )   PDF (848KB) ( 576 )   Save
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    【Objective】Isolating NBS-type (Nucleotide binding site) resistance gene analogs (RGAs) from walnut (Juglans regia L.) using homology-based method would provide a foundation for molecular-assisted selection and for cloning R gene during walnut breeding. 【Method】Thirty-five walnut superiors were used as plant materials in the study and their resistance to anthracnose was identified with inoculation. NBS-type RGAs were isolated by a PCR strategy using degenerate primers specific to P-loop and GLPL, conserved motifs of NBS domain in plant R gene. The relationship between NBSs and walnut anthracnose resistance was analyzed. Sequence identification of obtained sequences was performed against known RGAs deposited in GenBank using BLASTN/X algorithms. Similar and phylogenetic analysis were elaborated using MEGA 5.2 and DNAman 7.0 software.【Result】Out of 35 tested walnut superiors, 20 superiors were identified as resistant (R) with a relative resistance index (RRI) ranging from 0.63 to 0.82; 5 of them were medium resistant (M), whose RRI ranged 0.27-0.56; the rest 10 superiors as susceptible (S) with the RRI ranging 0.00-0.21. NBSs were amplified only in 20 resistant superiors and no bands were found in the remaining 15 superiors (5 medium resistant and 10 susceptible), indicating that the NBS-RGAs were associated with the resistance to walnut anthracnose (Colletotrichum gloeosporioides). BLASTN showed the obtained NBSs shared high similarities to cloned jrRGAPGs with a identity ranging 89%-100%; and they shared than 69% homology to other species from GenBank; BLASTX revealed 77%-99% and 49%-66% similarity to the NBS proteins from jrRGAPGs and other species, respectively. Multiple alignment analysis revealed that these NBS-type RGAs contained some well-characteristics motifs of NBS genes, including P-loop, kinase-2, kinase-3 and GLPL. The nucleotide polymorphism and diversity (Pi) were highly conserved at each motif than non-conservative fragments, indicating their conservative structures. Phylogenetic analysis revealed that the NBSs were clustered into seven subgroups at nucleotide level. They were grouped into two clades (TIR and non-TIR) and were subdivided into 7 groups based on their amino acid sequence similarity. Ratio of non-synonymous to synonymous nucleotide substitution (dN/dS) among NBS-RGAs varied from 0.00 to 0.95 (lower than one) for different classes, suggesting a purifying selection. Similarity percentages of deduced amino acid among these 7 NBS subgroups ranged from 28.3% to 63.5% with identifies to R genes ranging from 22.0% to 48.5%. 【Conclusion】 NBS-type RGAs isolated in the study were associated with anthracnose resistance, they were highly similar to cloned R genes and contained some conserved motifs. Walnut NBS-type RGAs undergo a purifying selection.
    A Study on Comprehensive Evaluation of the Processing Tomato Varieties Multiple Traits
    HAN Ze-Qun, JIANG Bo
    Scientia Agricultura Sinica. 2014, 47(2):  357-365.  doi:10.3864/j.issn.0578-1752.2014.02.015
    Abstract ( 534 )   HTML ( 2 )   PDF (673KB) ( 560 )   Save
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    【Objective】In order to determine the processing tomato varieties trait evaluation, a comprehensive evaluation system for processing tomato agronomic traits was established. The systern can be used for providing decision support for identification and screening of its excellent resources.【Method】Through a field experiment in Xinjiang in 2012, 22 processing tomato varieties (lines) were tested and analyzed, 17 agronomic traits of the 22 processing tomato varieties were measured. These 17 agronomic traits include fruit weight (FW), average yield (AY), lycopene (L), soluble solids (TSS), total acid (TA), total sugar (TS), sugar-acid ratio (SAR) , virus disease resistance (VDR), longitudinal diameter (VD), transverse diameter (TD), fruit shape index (FSI), color (CR), single fruit resistant to pressure (SFRP), the average spacing width (ASW), average height (APH), the average number of branches (ANB), and the growth period (GP). The principal component analysis and cluster analysis (PCA-CA) were also used to conduct a comprehensive analysis of the main agronomic traits of processing tomato after Matlab.2012a software programming for the sample data processing.【Result】The average coefficient of variation of 17 agronomic traits was 18.54%, the virus disease resistance was the largest and the coefficient of variation was 88.42%, and the smallest coefficient of variation of the color difference was 7.33%. The processing tomato evaluation variables were relatively independent but closely related. The 17 evaluation variables could be compressed into six indicators (the first six principal components cumulative contribution rate is 86.0369%, which reflected most of the 17 traits information) ,after analysis of 17 agronomic traits coefficient of the six main functional components corresponding to 17 agronomic traits could be summarized as fruit traits factor, quality factor inherent fruit, fruit appearance quality factor, yield factors, disease factors and the five major indicators could be used to analylize the quality of tomato, fruit resistance to pressure, weight, total acid, lycopene, longitudinal and transverse diameters, the average yield, disease-resistant viruses are the main characters of 8 traits. By using the class average of 22 different tomato varieties Q-type cluster analysis, all tomato varieties could be divided into three groups when Euclidean distance was 6.40.【Conclusion】The principal component analysis and cluster analysis were used to analyze 17 agronomic traits of the 22 processing tomato varieties (lines) to establish a comprehensive evaluation system for processing tomato. It can give a more comprehensive and objective evaluation and analysis from different perspectives and provide a reference for high-quality processing tomato cultivar selection.
    Effect of Straw and Biochar on Soil Bulk Density, Cation Exchange Capacity and Nitrogen Absorption in Apple Orchard Soil
    GE Shun-Feng, PENG Ling, REN Yi-Hua, JIANG Yuan-Mao
    Scientia Agricultura Sinica. 2014, 47(2):  366-373.  doi:10.3864/j.issn.0578-1752.2014.02.016
    Abstract ( 594 )   HTML ( 5 )   PDF (535KB) ( 950 )   Save
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    【Objective】The soil organic carbon content is low but the nitrogen fertilizer application rate is high in apple orchards in China. This study was conducted in order to provide a theoretical basis for the appropriate application of straw and biochar in apple production. 【Method】Two-year-old ‘Fuji’ apple trees (Malus domestica Borkh. cv Red Fuji/Malus hupehensis) trees were used to study the effect of straw and biochar on soil bulk density, cation exchange capacity (CEC), tree growth, and 15N transformation (tree uptake, ammonia volatilization, N2O emission, and soil residual) using 15N trace technique. There were four treatments: CK (control), N (only nitrogen fertilizer), N + B (nitrogen fertilizer + biochar) and N + S (nitrogen fertilizer + straw). 【Result】 The variation trend of soil bulk density in 0-5 and 5-10 cm soil layers was consistent in the four different treatments. There was no significant difference between CK and N treatment, but both were significantly higher than those in N + B and N + S treatments. For the two added exogenous carbon treatments, soil bulk density in N + B treatment was significantly lower than that of N + S treatment. Compared with the N treatment, soil bulk density in 0-5 and 5-10 cm soil layers in N + S and N + B treatments decreased by 0.06, 0.09 and 0.07, 0.11 g•cm-3, respectively. Compared with CK (18.32 cmol•kg-1) and N treatment (19.61 cmol•kg-1), the CEC of 0-10 cm soil layer increased significantly in N + S treatment (22.27 cmol•kg-1) and N + B treatment (25.35 cmol•kg-1). The highest total weight of apple trees, 15N uptake amount and 15N utilization efficiency existed in N + B treatment, followed by N + S treatment, and the lowest of those values were found in N treatment. Compared with CK, the amounts of ammonia volatilization significantly increased in the three N application treatments (N, N + S and N + B). Compared with N treatment, N + S and N + B treatments significantly reduced the N loss through ammonia volatilization, especially in N + B treatment. Compared with CK, the amounts of N2O emission were significantly increased in the three N application treatments (N, N + S and N + B), and the highest was found in N + B treatment, followed by N + S treatment, and N treatment was the lowest. So, addition of exogenous carbon could increase N2O emission rate, but no significant difference was found among the three nitrogen application treatments. When the CK background value was removed, total N gaseous losses (ammonia volatilization + N2O emissions) in N, N + S and N + B treatments accounted for the proportion of N application rate were 6.54%, 4.33% and 3.04%, respectively. The highest 15N residual rate in 0-50 cm soil layer was found in N + B treatment, followed by N + S and N treatment; while the highest 15N residual rate was found in N treatment, followed by N + S and N + B treatment in 50-100 cm. The highest N recovery rate was found in N + B treatment (42.26%), followed by N + S treatment (37.22%), and N treatment (31.54%) was the lowest; so the highest N loss rate appeared in N treatment (68.46%), followed by N + S treatment (62.78%) and N + B treatment (57.74%). 【Conclusion】Application of straw and biochar into apple orchard soil could decrease the soil bulk density, increase the soil cation exchange capacity, improve plant growth, promote N uptake by plant, increase the N fixed by soil and decrease N gaseous loss. The result will get better when application of biochar.
    STORAGE·FRESH-KEEPING·PROCESSING
    Study of Optimization of Preparation of Se-enriched Rice Bran Protein and Its Nutritive Compound of Mixed Proteins
    HU Qiu-Hui-1, 2 , CHEN Xi-1, FANG Yong-1, 2 , CHEN Yue-1, YANG Wen-Jian-1, MA Ning-1, ZHAO Li-Yan-2
    Scientia Agricultura Sinica. 2014, 47(2):  374-382.  doi:10.3864/j.issn.0578-1752.2014.02.017
    Abstract ( 476 )   HTML ( 1 )   PDF (584KB) ( 536 )   Save
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    【Objective】The conditions of alkali extraction method of Se-enriched rice bran protein (RBP) was optimized. Nutritive compound of mixed proteins was studied and evaluated according to the principle of protein complementarity. 【Method】RBP was extracted using alkali method, and the conditions of extraction were optimized by orthogonal experiment L9 (34). RBP was mixed with soybean protein (SP) in different proportions (60%, 70%, 80%, 90%, and 100%) after extraction. Kjeldahl method, hydride generation-atomic fluorescence spectrometry (HG-FAS) and portable color meter were used to determine the protein contents, selenium contents and color values of RBP, soy protein (SP) and the compound of mixed proteins, respectively. Amino acids contents of different protein products were measured by high-speed amino acid analyzer. Nutritive evaluation for the compound of mixed proteins was assessed by WHO/ FAO and amino acid ratio coefficient method. 【Result】By investigating extraction rate, Se-content and color values of RBP in orthogonal experiment, the optimal conditions were a volume-weight ratio of 20:1, 0.08 mol•L-1 NaOH, an extraction time of 3 h and a temperature of 30℃. A protein product of (80.2±1.1)% protein content, (0.269±0.132)mg•kg-1 Se and 69.7±1.2 color value was obtained. Selenium and protein contents of regular rice bran, Se-enriched rice bran and their protein products using optimum extraction conditions were compared. No significant difference was showed between the protein contents of Se-enriched rice bran and regular rice bran, a similar result was also found in their protein products (P<0.01). However, both selenium content of Se-enriched rice bran (0.401±0.021 mg•kg-1) and RBP (0.269±0.132 mg•kg-1) were significantly higher than that of regular rice bran ((0.023±0.010)mg•kg-1) and its protein ((0.052±0.011)mg•kg-1), respectively (P<0.01). Protein contents and selenium contents of RBP mixed with SP in different proportions were also compared in this study. Results showed that selenium contents of all the compound of mixed proteins were significantly higher than that of SP [(0.013±0.005)mg•kg-1]. With the increase of RBP proportion in different compounds, selenium contents were increased from (0.141±0.014)mg•kg-1 to (0.269±0.032)mg•kg-1. Nutritional value of RBP and the compounds were assessed by WHO/FAO and amino acid ratio coefficient method. According to the score of ratio coefficient (SRC), the nutritional value of each protein products were arranged in descending order as follow: 60% RBP > 70% RBP > 50% RBP > 80% RBP > 90% RBP > RBP. SRC values of all compounds were higher than that of RBP (SRC = 64.1). This result indicated that the nutritional values of the compounds of mixed proteins were all higher than that of RBP, and 60% RBP possess the highest nutritional value, of which selenium content and protein content were (0.167±0.024)mg•kg-1 and (84.1±0.8)%, respectively. 【Conclusion】Nutritional value could be enhanced and a high quality protein rich in trace element selenium could be obtained by mixing SP with RBP.
    Effect of Enzymolysis Conditions on Glucosinolates in Rapeseed Meal and Identification of Their Degradation Products
    DING Yan-1, LI Li-Qian-1, CAO Rong-2, TANG Gen-Sheng-2, GU Zhen-Xin-1, HAN Yong-Bin-1
    Scientia Agricultura Sinica. 2014, 47(2):  383-393.  doi:10.3864/j.issn.0578-1752.2014.02.018
    Abstract ( 445 )   HTML ( 1 )   PDF (640KB) ( 1139 )   Save
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    【Objective】The aim of this study was to figure out the optimal factors for directional conversion of glucosinolates to isothiocyanates in rapeseed meal degraded by endogenous and exogenous myrosinase and explore the impact of exogenous myrosinase on the components and content of glucosinolates degradation products. It will provide a basis for the comprehensive utilization of rapeseed meal. 【Method】 Myrosinase activity was evaluated in 4 kinds of buffer solutions to establish a better reaction system. The effects of different enzymolysis conditions included solid liquid ratio, hydrolysis time, reaction temperature, pH value, and amount of ascorbic acid on the conversion of glucosinolates in rapeseed meal to isothiocyanates by endogenous and exogenous myrosinase were investigated through single factors and orthogonal array design methods. The optimal condition for the conversion was obtained by analyzing the yield of isothiocyanates. Taking methylene chloride as the extraction agent, the degradation products were concentrated in rotary evaporator. The products from endogenous and exogenous myrosinase were detected by gas charomatographic mass spectrometry (GC-MS). The kind and content of compounds from the products were monitored. 【Result】 Results showed that among the four reaction systems, the myrosinase had the highest activity under citric acid-phosphate buffer and under the same conditions, exogenous myrosinase activity was significantly higher than that of the endogenous myrosinase. The content of isothiocyanates had no obvious increase when exogenous myrosinase was more than 20% of rapeseed meal, and it was good to add 20% exogenous myrosinase of rapeseed meal to rapeseed meal. The optimal enzymolysis conditions were as follows: ratio of solid to liquid was 1:15, pH was 6.0, reaction temperature was 50℃, amount of ascorbic acid 0.005 mg•g-1, the degradation time was 1h. Under this condition, the isothiocyanates content was 1.799 mg•g-1 which were 7.64 times than these by the autolysis in rapeseed meal. The isothiocyanates content generated by exogenous myrosinase was significantly higher than that by endogenous myrosinase, but both contents had the same trend with the reaction conditions. Seven compounds from exogenous myrosinase were identified by GC-MS from the enzymatic hydrolysates of glucosinolates and accounted for more than 67% of the extracted degradation products which included 1 kind of nitrile and 2 kinds of oxazolidinones and 4 kinds of isothiocyanates. Allyl isothiocyanate, 1-butene,4-isothiocyanate, cyclopentyl isothiocyanate and benzene, and 2-ethyl- isothiocyanate were obtained among glucosinolate degradation products. Besides, 1-butene,4-isothiocyanate had the highest content compared with the other compounds and accounted for 26.77% in the detected products. The nitrile was benzenepropanenitrile. Goitrin and 2,1-benzisoxazole were the two oxazolidinones. Four compounds were analyzed in degradation products from endogenous myrosinase and had lower content than the seven compounds detected in the products from exogenous myrosinase.【Conclusion】This study indicated the isothiocyanates content increased significantly by exogenous myrosinase. It can also be concluded from the experiments the optimal enzymolysis condition for glucosinolates and the main compounds of enzymatic hydrolysates of glucosinolates from rapeseed meal. 1-Butene,4-isothiocyanate had the highest content in the 4 kinds of isothiocyanates analyzed in the test.
    ANIMAL SCIENCE·VETERINARY SCIENCERE·SOURCE INSECT
    Cloning of Buffalo (Bubalus bubalis) Oct4 and Nanog Genes and Ectopic Expression in Buffalo Fetal Fibroblasts
    DENG Yan-Fei, LIU Qing-You, DENG Hai-Ying, LUO Chan, YANG Su-Fang, SHI De-Shun
    Scientia Agricultura Sinica. 2014, 47(2):  394-402.  doi:10.3864/j.issn.0578-1752.2014.02.019
    Abstract ( 395 )   HTML ( 1 )   PDF (616KB) ( 565 )   Save
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    【Objective】Oct4 and Nanog were the pluripotency-related transcription factors, and play important roles in the differentiation of stem cell and embryo development. In this study, the buffalo Oct4 and Nanog genes were cloned, the gene sequences and protein sequences were analysed, and eukaryotic expression vectors were constructed. This study will provide a good foundation for investigating the functions of Oct4 and Nanog, especially their roles in the embryo development, establishing buffalo embryonic stem cell lines and inducing pluripotent stem cell lines (iPSC). 【Method】The RNA and DNA were extracted from fetus germ ridge and somatic cells, and the RNA was reverse-transcribed into first-strand cDNA. The primers were designed specifically according to the bovine gene sequences (Oct4:NM_174580,Nanog:NM_001025344). The coding sequences (CDS) and DNA fragments of Oct4 and Nanog were cloned by RT-PCR and PCR, respectively, three fragments for Oct4 gene and two fragments for Nanog. The protein structure prediction and homology comparison were analysed by online software. After digestion with enzymes (EcoRⅠ, XholⅠfor Oct4 and XholⅠ, NotⅠfor Nanog), the CDS of Oct4 and Nanog were inserted into the pMX retrovirus vector using T4 ligase, respectively, named pMX-Oct4 and pMX-Nanog. Buffalo fetal fibroblasts (BFFs) were cultured by the method of tissue explants. The retrovirus supernatant was collected from the retroviral system after virus packaging, and infected the BFFs 12-15 h. After culturing for 48 h, the ectopic expression of Oct4 and Nanog in the BFFs were detected by RT-PCR and immunofluorescence assay.【Result】The results showed that the CDS and DNA length of Oct4 was 1083 bp and 4509 bp, respectively, encoding 361 amino acids, and containing 5 exons and 4 introns. The CDS and DNA length of Nanog was 903 bp and 4473 bp, respectively, encoding 361 amino acids, and containing 4 exons and 3 introns. The gene sequences were submitted to GenBank online, and the accession numbers were JN991003 and JN991004, respectively. The amino acids of buffalo Oct4 and Nanog exhibited high homology with bovine, pig, human and mouse, the percentage of conservatism displayed Oct4 (98%, 96%, 91% and 81%) and Nanog (90%, 81%, 69% and 47%). Buffalo Oct4 and Nanog protein structures were similar to that of mice, Oct4 containing POU-domain and Nanog containing HOX-domain. Using the retrovirus vectors pMX-Oct4 and pMX-Nanog, buffalo Oct4 and Nanog genes could be transfected into BFFs, and their mRNA and protein could be expressed in the BFFs. 【Conclusion】 The CDS and DNA fragments of buffalo Oct4 and Nanog were cloned, their base sequence and amino acid sequences were conservation during evolution. Oct4 and Nanog were transfected into BFFs and expressed in BFFs using the retrovirus transgene method. The retrovirus-based target genes transfer could be used in the buffalo genetically modified research and buffalo iPSC generation.
    Recent Advances in Research of the Expression and Role of C-Type Natriuretic Peptide as a Regulator in Control of Animal Ovary Performance
    JIA Zhen-Wei
    Scientia Agricultura Sinica. 2014, 47(2):  403-410.  doi:10.3864/j.issn.0578-1752.2014.02.020
    Abstract ( 413 )   HTML ( 2 )   PDF (432KB) ( 702 )   Save
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    C-type natriuretic peptide (Cnp), a member of the natriuretic peptide family of structurally related peptides, is widely distributed in animal brain, kidney, heart and vascular tissues with important roles in cardiovascular homeostasis, regulation of cell proliferation and skeletal development. It is generally believed that there are three known natriuretic peptide receptors (NPR) in mammals: NPR1, NPR1, and NPR3. Cnp exerts its biological actions by the preferentially activation of NPR2. The components of the Cnp signaling pathway mainly include ligand (Cnp), transmembrane receptor (NPR2) and cGMP. Binding of Cnp to NPR2 results in the change of receptor kinase homology domain conformation, which can further activate downstream pathway through stimulation of cGMP production by the activation of receptor guanylate cyclase. At present, some studies have shown that Cnp is expressed predominantly by mural granulosa cells, which line the inside of the follicular wall, whereas Npr2 is expressed predominantly by cumulus cells, indicating a similar pattern of Cnp and Npr2 expression in female animal ovary. In addition,the expression of Cnp and Npr2 is hormonally regulated during animal follicular growth in vivo. FSH- promoted expression of Cnp and Npr2 is mediated by estradiol. Meanwhile, Oocyte-derived paracrine factors promot expression of Npr2 in cumulus cells. LH- reduced expression of Cnp and Npr2 probably is due to activation of EGF signal pathway. Notably, research showed that Cnp, similar to FSH, can induce the expression of diverse ovarian genes important for follicle maturation and steroidogenesis, which is beneficial to cumulus oophorus formation, thereby promote follicular development, indicating that similar to the known follicle-stimulating and oocyte maturation effects of FSH, and Cnp /FSH exerts their biological actions via cGMP /cAMP signal pathway, respectively. Besides, recent studies have revealed that Cnp, secreted by mural granulosa cells, plays an essential role in maintaining meiotic arrest of oocyte. Cnp exerts its biological action as a local factor by binding to NPR2 in cumulus cells, followed by the production of cGMP via the guanylyl cyclase catalytic domains of NPR2, then cGMP diffuses into the oocyte from companion cumulus cells via gap junctions, and inhibits oocyte PDE activity and cAMP hydrolysis, at the same time, higher level of cAMP inhibits oocyte MPF activity, subsequently preventing meiotic resumption of oocyte; whereas Cnp does not affect meiotic maturation of naked oocytes, suggesting the indirect effect of Cnp on meiotic maturation of oocyte by acting as a regulator to cumulus cells. Additionally, pre-treatment of oocytes with Cnp exerts a beneficial effect on cytoplasmic maturation, thereby enhances the developmental capacity of oocyte following in vitro maturation. To sum up, Cnp produced within the ovary might maintain meiotic arrest of oocyte, as well promote cytoplasmic maturation of oocyte by affecting the physiological function of cumulus cells during animal follicular growth, which ensure the higher capability of development after oocyte maturation and fertilization. In light of the findings above, Cnp may be of special importance in the promotion of oocyte development in vitro as one meiotic inhibitor in domestic animals. Hence, this paper reviewed the structure of Cnp, its signal transduction pathway and expression in ovary as one of functional molecules that modulate ovary function in animals.