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Table of Content

    01 January 2016, Volume 49 Issue 1
    CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS
    Observation, Genetic Analysis and Gene Mapping of an Open Hull Semi-Sterility Mutant in Rice (Oryza sativa)
    CHEN Li-kai, HUANG Ming, LIU Yong-zhu, WANG Hui, CHEN Zhi-qiang, GUO Tao
    Scientia Agricultura Sinica. 2016, 49(1):  1-13.  doi:10.3864/j.issn.0578-1752.2016.01.001
    Abstract ( 771 )   HTML ( 10 )   PDF (7668KB) ( 730 )   Save
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    【Objective】The paper is order to carry out a morphological characteristics investigation and genetic analysis of an open-hull semi-sterility mutant (ohss) of rice (Oryza sativa L.), induced by spaceflight, laid the groundwork for further gene cloning and function analysis by fine mapping and preliminary screening of the candidate gene responsible for the mutated trait.【Method】The ohss mutant was derived from rice variety Hanghui 7, which was induced on Spaceship “Shenzhou 8”. The morphological characteristics of ohss were anatomically observed to analyze the mutagenic features of floral organ development. Subsequently, pollen fertility, natural seed setting rate and bagged seed setting rate were investigated for fertility evaluation. Five plants of ohss mutation and WT were random selected to survey the panicle and grain related traits. SSR markers covering the whole genome were used to detect the mutagenic effect of ohss. Moreover, genetic analysis was conducted using the crosses between ohss and three wild type varieties, Hanghui 7, Francis and 02428 where the phenotypes of the F1 and F2 were surveyed and c2 test was performed. A population from the cross of 02428/ohss was used to map the ohss(t) geneusing SSR markers and newly developed InDel markers. The candidate gene was predicted based on the RAP gene annotation database of the mapping regionand screened through sequences alignment and expression of candidate genes.【Result】Compared with wild-type, panicles of mutant ohss were enclosed and florets showed abnormalities at the reproductive stage, and the palea and lemma were weak, distorted and dehiscent, with organ similar to the palea in the floret, while some of florets had no palea differentiation. Sterility testing showed that pollen grain rate of the abnormal spikelet of ohss was 58.7%, leading to significantly lower seed set, less panicle weight per plant and filled grain number per panicle compared with the wild-type. Mutation survey based on SSR markers revealeda total of 0.0336 of variation frequency was caused between ohss and WT, and variation frequency of different chromosome varied from 0.0143 to 0.0889, except chromosome 7 and 12. Genetic analysis indicated that the mutant phenotype in ohss was controlled by a single recessive nuclear gene, namely ohss(t), which had been fine mapped to a 27.6 kb physical distance between two InDel markers, InDel6043 and InDel6070 on chromosome 3, where three annotated genes were predicted. Based on the result of sequencing, semi-quantitative RT-PCR and real time quantitative PCR, there was no mutation occurring in the coding and promoter sequence of OsMADS1 of ohss,but strong changes on gene expression pattern.【Conclusion】The mutated trait of ohss was controlled by a single recessive nuclear gene ohss(t), which was fine mapped to a 27.6 kb physical distance between InDel6043 and InDel6070 on chromosome 3. No nucleotide sequence mutation was found to occur in the coding sequence or the 5′UTR of OsMADS1,but expression of OsMADS1 was strongly inhibited in ohss(t).
    Identification of Interaction Domain of SRK-ARC1-Exo70A1 and Interaction Strength Analysis in Brassica oleracea var. capitata L.
    SHI Song-mei, GAO Qi-guo, LIAN Xiao-ping, BI Yun-long, LIU Xiao-huan, PU Quan-ming, LIU Gui-xi, LIU Jing, REN Xue-song, YANG Xiao-hong, ZHU Li-quan, WANG Xiao-jia
    Scientia Agricultura Sinica. 2016, 49(1):  14-26.  doi:10.3864/j.issn.0578-1752.2016.01.002
    Abstract ( 351 )   HTML ( 1 )   PDF (1983KB) ( 572 )   Save
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    【Objective】 The study was for further study on the mechanism of mutual recognition among S receptor kinase (SRK), ARM-repeat containing 1 (ARC1), and Exo70A1, which were the key signal elements in self-incompatible response, and to identify the interaction domains and compare their interaction strength.【Method】According to a bioinformatics analysis, we obtained the different functional domains of three proteins. And on this basis, the truncated fragments containing different functional domains were amplified from Brassica oleracea var. capitata L. E3. Then the encoding sequences of SRKj with subfragments (SRKjΔ1-SRKjΔ4) and the full length Exo70A1 with subfragments (Exo70A1Δ1-Exo70A1Δ3) were separately subcloned into the vector pGADT7 to generate the AD recombinant plasmids, after which the encoding sequence of ARC1 with subfragments (ARC1Δ1-ARC1Δ3) were respectively constructed into vector pGBKT7 to generate the BD recombinant bait plasmids. These recombinant plasmids were respectively cotransformed into the yeast competent cells of strain AH109, and then were planted on SD/-Leu-Trp-His-Ade/X-a-gal/25 mM 3-AT nutritional media to detect the growth and color change. A β-galactosidase assay was conducted. Finally, anin vitro binding assay was performed to confirm the interaction between SRK-ARC1 and ARC1-Exo70A1.【Result】A DNA sequence and restriction enzyme analysis suggested the recombinant plasmids were correct. The yeast AH109 cells were without an autonomous activation effect on the reporter gene MEL1. In the combinations of SRKj with ARC1Δ4, ARC1Δ8, and ARC1, the yeast AH109 cells could grow and turn blue on SD/-Leu-Trp-His-Ade/X-a-gal/25 mM 3-AT nutritional media with a transcription activation of the reporter genes HIS3, ADE2 and MEL1. With the extension of the amino acid sequence, a greater β-galactosidase activity was induced, in which the combinations of SRKj with ARC1 showed relatively high levels of β-galactosidase activity (15.98). In the combinations of ARC1-Exo70A1, Exo70A1Δ3 could interact with ARC1Δ1-ARC1Δ3. Their yeast cells could grow and turn blue on SD/-Leu-Trp-His–Ade/ X-a-gal/25 mM 3-AT nutritional media, activating the reporter genes HIS3, ADE2, and MEL1. With the extension of truncated ARC1 or Exo70A1, β-galactosidase activity showed a lower trend after the first increase. Meanwhile, ARC1Δ2 with Exo70A1Δ3 exhibited the highest levels of β-galactosidase activity in all combinations (25.07). In vitro binding assay further showed that SRKj could interact with ARC1Δ4, and ARC1Δ2 also could interact with Exo70A1Δ3. 【Conclusion】The kinase domain of SRK(SRKj) and the C-terminal ARM of ARC1 were the core interaction domains of SRK-ARC1. No interactions were detected for either truncating SRKj functional domains or ARM of ARC1. The Leucine zipper with coiled-coil of ARC1 and N terminal of Exo70A1 mediated the interaction between ARC1 and Exo70A1. The interaction strength of SRK-ARC1 was less than that of ARC1-Exo70A1.
    SNP Sites Developed by Specific Length Amplification Fragment Sequencing (SLAF-seq) in Sweetpotato
    SU Wen-jin, ZHAO Ning, LEI Jian, WANG Lian-jun, CHAI Sha-sha, YANG Xin-sun
    Scientia Agricultura Sinica. 2016, 49(1):  27-34.  doi:10.3864/j.issn.0578-1752.2016.01.003
    Abstract ( 617 )   HTML ( 9 )   PDF (1265KB) ( 999 )   Save
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    【Objective】 Single nucleotide polymorphisms (SNP) show high diversity in genomes and they are important markers for map construction and marker assistant selection (MAS). The development of high-throughput sequencing technology can provide the capacity to discover massive numbers of SNP sites in the genome. Polyploid crops have large size genomes along with large scale repetitive sequences, as a result, there are many challenges in developing SNP sites in the polyploidy crops.【Method】300 sweetpotato accessions were collected and sequenced by SLAF-seq technology. The scheme of the experiment was designed based on bio-informatics technology. Taking potato as the reference genome, specific size of DNA were chosen to construct the SLAF-seq library. After high-throughput sequencing, a great amount of sequences were obtained and used to obtain the polymorphism SLAF tags by software alignment, then found the distribution of specific SNP sites. 【Result】 The results coming from the alignment between the sequences of CK Arabidopsis and its reference genome indicated that the construction of SLAF library fitted well to the standard, with its paired-end mapped reads reaching 87.71%, normal digestion ratio reached 93.22%. With the help of sequencing alignment as well as other bio-informatics technologies, a total of 498.14 Mb reads were obtained,the total number of the reads of each sample varied from 441 595 to 2 731 920. Jishu4 had the largest number of reads with 2 731 920 reads. Arabidopsis had the smallest amount of data with 441 595 reads. The quality of Q30 changed from 88.37% to 90.67%, the Q30 of 3043 only reached 87.31%, while the Q30 of Ezi1 reached as high as 91.31%, the average value of Q30 of each sample was above 80%. The GC content of each sample varied from 37.23% to 38.09%; Sushu9 and Zheshu2 had extreme values with Sushu9 having a GC content of 39.80% , while Zheshu2 had 37.10%. The average value of each sample was 37.64% with the average level of GC content low enough to process the sequencing. In the end, 597 094 high quality SLAFs were produced, 260 000 were polymorphisms, accounting for 43.54%. The final results consisted of 795 794 SNP sites obtained based on the analysis of 260 000 polymorphic SLAFs.【Conclusion】A total of 498.14 Mb reads were obtained and produced 597 094 high quality SLAFs, of which 260 000 were polymorphisms. 795 794 SNP sites were developed based on SLAF-seq technology. This paper provided results that SLAF-seq technology can be well applied in developing SNP sites in sweetpotato, much more efficiently than markers like SSR, AFLP, or RAPD. The developed SNP sites can be further used for the analysis of population evolution and developing specific SNP markers.
    TILLAGE & CULTIVATION·PHYSIOLOGY & BIOCHEMISTRY
    Relationship of NSC with the Formation of Branches and Spikelets and the Yield Traits of Indica Hybrid Rice in Different Planting Methods
    TIAN Qing-lan, LIU Bo, ZHONG Xiao-yuan, ZHAO Min, SUN Hong, REN Wan-jun
    Scientia Agricultura Sinica. 2016, 49(1):  35-53.  doi:10.3864/j.issn.0578-1752.2016.01.004
    Abstract ( 593 )   HTML ( 7 )   PDF (891KB) ( 750 )   Save
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    【Objective】The objective of this experiment was to explore the effects of planting methods on the accumulation and distribution of non-structural carbohydrates (NSC) and the differentiation and retrogress of branches and spikelets, and to make clear the relationship of NSC in the panicle differentiation stage with differentiation and retrogress of branches and spikelets and the relationship of the accumulation of NSC after heading with yield and its form factors. 【Method】On the basis of the early two years experiments, using a split plot field experiment research was done on the accumulation and distribution of NSC before and after heading, and the regulation and differences of the differentiation and retrogress of branches and spikelets on different parts of panicle which under three planting methods included mechanized direct-seeding (MD), mechanized transplanting (MT), and artificial transplanting (HT) of two combinations of indica hybrid rice. 【Result】(1) The stem-sheath had an obvious advantage to the young panicle about the competition of NSC in the panicle differentiation stage. MT garnered more NSC in heading, and transported more NSC to grain with a higher efficiency in the grain filling stage, making it gain more distribution of NSC in maturity. (2) The main differences among these planting methods were in the survived and retrograded percentage of secondary branches and the differentiated third branches. MT had more numbers of the survived and differentiated secondary branches and the survived and differentiated secondary spikelets so that it had more hole branches and spikelets. The retrograde of secondary branches and primary spikelets were respectively concentrated on the lower part and the upper part of panicle. Numbers of secondary branches and secondary spikelets were lower part>middle part>upper part. Numbers of survived secondary spikelets in different parts of panicle of MT were higher than HT and MD. (3) The higher accumulation of NSC in 12 d, 4 d and 0 d before heading were not beneficial to the differentiation and retrogression of branches and spikelets, but the accumulation of young panicle had a significant or extremely significant positive correlation of most characteristics of spikelets in 16 d to 8 d before heading, so this stage was the key stage of forming big panicle. The distribution of NSC after heading was mainly through the effect of the distribution of NSC in leaves and panicle on yield. There was a close contact between the yield and characteristics of the branches and spikelets. Thousand grain weight and effective panicles per unit area had a significant or extremely significant negative correlation of the characteristics of branches and spikelets, numbers of grains per panicle, and the setting percentage and yield had a significant or extremely significant positive correlation with the characteristics of the branches and spikelets. (4) Fyou498 had a higher exportation rate of the NSC of stem-sheath and a higher contribution rate of the NSC of stem-sheath to panicle than Yixiangyou2115, and most characters of branches and spikelets of Fyou498 were significantly or extremely significantly higher than Yixiangyou2115. The number of grains per panicle and the setting percentage of Fyou498 were extremely significantly higher than Yixiangyou2115, so its yield was higher. 【Conclusion】There were large differences of the accumulation and distribution of NSC and the characters of branches and spikelets among different planting methods, and also the varieties. MT cooperates big panicle varieties has a higher yield potential.
    Proteomics Analysis of Grain Position Effects During Early Developmental Stages of Maize
    YU Tao, LI Geng, LIU Peng, DONG Shu-ting, ZHANG Ji-wang, ZHAO Bin, BAI Han
    Scientia Agricultura Sinica. 2016, 49(1):  54-68.  doi:10.3864/j.issn.0578-1752.2016.01.005
    Abstract ( 516 )   HTML ( 3 )   PDF (3859KB) ( 957 )   Save
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    【Objective】In order to understand the molecular mechanism of maize grain position effects, the function of differential expression proteins between upper and middle grains during the early developmental stages of maize were studied by using an approach of plant proteomics.【Method】Denghai661(DH661) with significant grain position effects was used as experimental material and planted at 90 000 plants/hm2 in a field. Upper and middle grains were harvested after flowering artificial saturation pollination at 0, 3, 6, and 12 d. The total proteins were extracted by the trichloroacetate (TCA)-acetone precipitation method, and the protein profiles were set up by two-dimensional gel electrophoresis (SDS-PAGE). The upper grain gel was compared to middle grain gel as a reference at 0, 3, 6, 12 d, respectively. The differential expression proteins between the upper and middle grains were analyzed with Image master 2D 7.0. The functions of these differentially expressed proteins were identified by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF) analysis and NCBI database searching.【Result】After high density planting, grain ears were detected at more than 1000 clear spots at early developmental stages. After a comparative proteomics analysis and MALDI-TOF/TOF mass spectrometry (MS), 66 protein spots were found significantly differentially expressed between upper and middle grains during the early developmental stages, of which 52 protein spots were successfully matched in the NCBI database and the identification rate was about 78.8%. These proteins were involved in grain respiration and energy metabolism (10 spots, 19%), stress and defense (9 spots, 17%), protein metabolism (9 spots, 17%), nitrogen metabolism (6 sports, 11%), cell differentiation and proliferation (5 spots, 10%), transcription and translation (5 spots, 10%), secondary metabolism (3 spots, 6%), and other function categories. Analyzing related proteins expression differences in abundance, compared with the middle grains, most of proteins expressed abundance in the upper grains involved in cell differentiation and proliferation, respiration and energy metabolism were significantly lower at one or more of the time periods, indicating that the upper grains’ ability of endosperm cell proliferation and respiratory energy metabolism significantly reduced. Meanwhile, the upper grains’ proteins involved in stress and defense of a variety of antioxidant enzyme system, glyoxalase I, and four molecular chaperone proteins involved in protein metabolism were at a low level expression at the early development, indicating that the upper grains have weaker defense capability and less stable protein structure to stress conditions. In addition, compared with the middle grains, the upper grains alanine aminotransferase, and S-adenosine methionine synthetase 1 involved in nitrogen metabolism were down regulated within 6 to 12 days after pollination, indicating that the upper grains had a weak nitrogen assimilation ability and influence the subsequent amino acid synthesis and protein metabolism process.【Conclusion】Compared with the middle grains, the upper grains’ proteins involved in cell differentiation and proliferation are at low levels of expression and have weaker respiratory and energy metabolism vitality, resulting in the upper grains having lower sink sizes and sink activities. Additionally, when responding to oxidative stress and other stress conditions, because of the low level of expression of antioxidant enzymes and the molecular chaperone proteins, the regulatory capacity of upper grains is less than the middle grains. Differentially expression of alanine aminotransferase and SAMS also may be an important cause of grain position effects.
    Functional Traits of Maize Stems as Supporting Organs and Their Plasticity
    YANG Jin-zhong, LIANG Shu-min, LI Na-na, LIU Yong-hua, HAO Jian-ping
    Scientia Agricultura Sinica. 2016, 49(1):  69-79.  doi:10.3864/j.issn.0578-1752.2016.01.006
    Abstract ( 598 )   HTML ( 4 )   PDF (427KB) ( 585 )   Save
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    【Objective】Plant stems function as supporting organs among many other functions, however, information on their load-bearing capacity has seldom been reported. The objectivs of this study were to: (1) define some stem functional traits; (2) examine effects of abiotic and biotic factors on these traits; (3) feature plasticities of these traits; and (4) explore their potential implications. 【Method】Three stem functional traits, namely, linear mass density (MD), ratio of load to self-weight (RLSW), and ratio of load to linear density (RLMD), were proposed and examined in 6 field experiments with maize (Zea Mays) as a model plant. MD = internode weight / its length, RLSW = internode mass load / its weight, RLMD = internode mass load / MD, where the mass load of an internode is the sum of the weights of all organs and tissues above the internode. The plasticity of a trait was represented as plasticity coefficients, which were computed after the manner of variation coefficients, and where variance components were estimated from the component models of expected mean squares for the experiment’s data. The 6 field experiments were conducted in Taigu, Shanxi. The treatments of these experiments are as follows: (1) the combinations of 5 sites by 2 cultivars; (2) the combinations of 11 sampling timings by 2 cultivars; (3) the combinations of 4 plant densities by 3 sampling timings; (4) 4 plant densities from 2.4 to 6.0 plants per square meter; (5) the combinations of 3 nitrogen fertilizer rates by 2 fertilization timings; and (6) high plant density without fertilization versus low plant density with fertilization. All 3 traits were subject to ANOVA, and means were separated by means of Least Significant Difference. The profile of MD along node ranks was fitted with a negative logarithm equation. 【Result】MDs of internodes varied from 0.052 to 0.72 g DW·cm-1 and followed a straight line equation of a negative logarithm of node ranks. RLSWs of internodes ranged from 7 to 51, and RLMDs of internodes from 122 to 260 cm. If including load of ear weights, these ratios for the first node below the ear jumped to 246 and 3225 cm at their maximum values, respectively. These 3 stem functional traits showed statistically significant differences among genotypes and geographical sites. In the late duration of the kernel filling stage, MDs generally went down. MDs decreased with plant density, but RLSWs remained stable within a large range of plant densities. Higher nitrogen fertilizer rates increased MDs, but did not affect RLMDs, compared with lower ones. The plasticity of stem functional traits ranked as: MD>RLSW>RLMD, and the biomass investments for supporting were modulated by the optimization strategy. 【Conclusion】These findings showed that the stem functional traits proposed were able to feature the load-bearing of stems, and could delineate the biomass investments in the stem structure of plants and the investment efficiency, and may improve the understanding of biomass partitioning within whole plants.
    Optimizing of Agrobacterium-Mediated Transformation of Switchgrass Cultivars
    LIU Yan-rong, CEN Hui-fang, YAN Jian-ping, ZHANG Wan-jun
    Scientia Agricultura Sinica. 2016, 49(1):  80-89.  doi:10.3864/j.issn.0578-1752.2016.01.007
    Abstract ( 430 )   HTML ( 2 )   PDF (2639KB) ( 577 )   Save
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     【Objective】 To establish a high-efficiency plant regeneration and transformation procedure of switchgrass.【Method】mature seeds of three different switchgrass (Panicumvirgatum L.) varieties were used as explants for callus induction. After six weeks of callus induction, the phenotype of the callus was observed under a dissecting microscope, we discarded the out-layer sponge-like, watery callus and selected the core-like, highly compact callus that lies in the middle of callus clump for further subculture, after the sub-culture was in the dark for three weeks, calluses grew fast, compact and grainy like were selected for further culture, calluses generated from one seed as a cell line and were kept together. This type of callus was highly regenerable, and was characterized as Type Ⅱ callus of monocot plant. After two rounds of selection, enough type Ⅱ callus could be obtained for plant regeneration and transformation. To optimize the procedure of Agrobacterium-mediated transformation, transformation efficiencies under three different Agrobacterium-infection procedures were evaluated. They are vacuum-drying treatment (VD): That was in a 50 mL centrifuge tube, the calluses that merged in Agrobacterium suspension were given a vacuum treatment (-0.8 Mpa) for 10 min, then slight agitated in a incubator-shaker at 80 r/mim, 28 for 20 min. Then drainage the bacteria and dry on a sterilized filter paper tower, the calluses were transfer onto two layers of filter paper soaked with 500 mL sterile water in a petri dish for co-cultivation. Cold combined with vacuum-drying treatment (CVD): calluses were given a cold treatment on ice in a solution of 3% maltose and 300 μmol·L-1 of glutamine (Gln) for 20 min before conducted as VD process. A slightly different procedure was used instead of sterile water MP liquid medium was used for cocultication. Osmotic treatment combined with the cold and vacuum-drying treatment (PVD): 6% maltose was used in CVD process. The transgenic transformation efficiencies were evaluated by GUS staining assay and PCR tests.【Result】A regeneration efficiency of over 95% was reached for the type Ⅱ callus selected from the lowland switchgrass cultivar “Alamo” and “Performer”; 50% was reached for upland cultivar “Blackwell”. The transformation rate of switchgrass cultivar “Alamo” reached to 72% under CVD procedure, significantly higher than that was obtained under the procedure VD (53%) and PVD (44%). Under CVD procedure, transformation efficiency of lowland cultivars “Performer” reached to 96.7%; for the upland cultivar “Blackwell”, the transformation efficiency reached to 5.6%. This was the first time of obtaining the transgenic plant of the upland cultivar “Blackwell”.【Conclusion】Highly regenerative type Ⅱ calluses can be obtained from different ecotypes of switchgrass with a stereo-microscope. Agrobacterium- mediated transformation efficiency could be significantly improved under a CVD procedure for infection. This was the first report on how to select the type Ⅱ callus derived from mature seeds of switchgrass. A high-efficiency plant regeneration and transformation procedure that was suitable to lowland and upland switchgrass cultivars was developed, which made a foundation for switchgrass genetic improvement and functional gene research. The method presented here is also helpful in establishing a plant regeneration and transformation system for other recalcitrant monocot.
    PLANT PROTECTION
    Development of Detection Method for Sweet potato feathery mottle virus (SPFMV) and Sweet potato chlorotic stunt virus (SPCSV) Through Fluorescence Quantitative RT-PCR
    LU Hui-xiang, Lü Chang-wen, WU Zheng-dan, LUO Kai, YIN Wang, YANG Hang, WANG Ji-chun, ZHANG Kai
    Scientia Agricultura Sinica. 2016, 49(1):  90-102.  doi:10.3864/j.issn.0578-1752.2016.01.008
    Abstract ( 554 )   HTML ( 3 )   PDF (2400KB) ( 539 )   Save
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    【Objective】 Sweet potato virus disease (SPVD), which could cause more than 90% of yield loss, is one of the most harmful diseases of the sweet potato. The objective of this study is to establish a rapid detection method of SPVD, provide a useful tool for the early warning of SPVD and epidemiological investigation, based on assay of co-infection of Sweet potato feathery mottle virus (SPFMV) and Sweet potato chlorotic stunt virus (SPCSV).【Method】 The leaves of sweet potato cultivars/lines with typical SPVD symptoms were collected, and the co-infection of SPFMV and SPCSV were tested by typical symptoms analysis and Nitrocellulose Membrane Enzyme-Linked Immunosorbent Assay (NCM-ELISA). The partial genes encoding coat protein (CP) of SPFMV and heat shock protein70 (HSP70) of SPCSV were cloned from the diseased leaves. Fluorescence quantitative PCR (qRT-PCR) was used to detect SPFMV CP and SPCSV HSP70 by using primers designed according to the conservative regions, and by using sweet potato ubiquitin (UBI) and histone (H2B) as the reference genes. The rapid and accurate protocol for SPFMV and SPCSV detection were established bycomparison with the symptoms and virus detection results by NCM-ELISA, and the detection results were also compared among leaves collected from different sweet potato cultivars/lines, among different plants collected from the same cultivar/line, and among top young leaves and mature leaves in the middle collected from the same plant. 【Result】 Symptoms analysis suggested that infected leaves from different cultivars/lines might exhibit different typical symptoms, and the symptoms of infected leaves from different plants of the same sweet potato cultivar/line were similar but exhibited differentiation. Cluster analysis showed that there were at least two types of SPFMV strains (EA and RC) and one type of SPCSV strain (WA) existing in the tested sweet potato leaves. Among the 15 tested cultivars/lines, 10 cultivars/lines were detected to be co-infected by SPFMV and SPCSV by using NCM-ELISA detection and qRT-PCR method. The results obtained from qRT-PCR detection corresponded to that from NCM-ELISA testing, indicating that the qRT-PCR detection method developed in this study could accurately reflect the co-infections degree of SPFMVand SPCSV in the sweet potato plants. Differential transcriptional levels of SPFMV CP andSPCSV HSP70 were detected in diseased leaves from different cultivars/lines or different plants of the same cultivar/line, and even in top young leaves and mature leaves in the middle from the same plant by using qRT-PCR. The detected transcriptional levels of SPFMV CP in the tested diseased leaves were higher than and were 3-556 times of that ofSPCSV HSP70. In addition, qRT-PCR showed a transcriptional level of SPCSV HSP70 in two of four cultivars/lines which were detected to be only infected by SPFMV but not infected by SPCSV by NCM-ELISA detection, indicating the present qRT-PCR detection method was more sensitive and accurate when compared to the NCM-ELISA method. 【Conclusion】 The appropriate PCR primers for qRT-PCR detection of SPVD were selected and a rapid method for SPVD detection was developed, which could provide a useful tool for the monitoring and early warning of SPVD. The characteristic of co-infection of two viruses and differentiation in typical symptoms among different cultivars/lines should be comprehensively analyzed and targeting approach should be concerned when a new monitoring or warning system of SPVD was established.
    Development of a RT-LAMP Assay for Detection of Grapevine virus A
    ZHANG Yong-jiang, XIN Yan-yan, LI Gui-fen, QIAN Yi-ke
    Scientia Agricultura Sinica. 2016, 49(1):  103-109.  doi:10.3864/j.issn.0578-1752.2016.01.009
    Abstract ( 438 )   HTML ( 8 )   PDF (721KB) ( 612 )   Save
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    【Objective】Grapevine virus A (GVA) is one of the most important pathogens causing grapevine rugose wood complex, which is mainly transmitted by grafting using seedlings with GVA in the field. The production using free-GVA grape seedlings is the radical measure for the control of GVA disease, while the simple and sensitive detection method is the effective guarantee for the free-GVA seedlings screening. The objective of this study is to carry out the study of reverse transcription loop- mediated isothermal amplification technology (RT-LAMP), and to establish the RT-LAMP specific method for the detection of GVA.【Method】 Six specific primers for GVA detection including GVA-FIP (5′-CTTACAGCCACGCTCAGAGTCC-CGTGGGAAG TTGGTTGTGT-3′), GVA-BIP (5′-GCCCGTCAAAGGGGCTACAC-TCATAGGCGTTCTGTGCGA-3′), GVA-F3 (5′-AGAAGATG GGGATAGACCCG-3′), GVA-B3 (5′-CCGCCATTAACACGAGGAA-3′), GVA-LF (5′-ATCCTTCCCACCAGCTCGG-3′) and GVA-LB (5′-TCAGGCAGATGTGTGAACCT-3′) were designed using GVA coat protein (CP) gene sequences after a comparison analysis and design using primer design software Primer Explore 4.0. Different RT-LAMP reaction temperatures including 59, 61, 63 and 65℃ were experimented in order to determine the optimal reaction temperature according to the appearing order of the amplification curve within 1 h. The total RNAs of three other grape-infecting viruses including Grapevine rupestris stem pitting-associated virus (GRSPaV), Grapevine leafroll-associated virus (GLRaV), Grapevine fleck virus (GFKV) and the negative control (healthy grape leaf named NC) were used in this study to determine the specificity of the RT-LAMP method. The total RNA of GVA was ten-fold serially diluted including 101, 102, 103, 104, 105 and 106 dilutions and used as the templates for the sensitivity comparison between RT-LAMP and conventional RT-PCR. The RT-LAMP result could be judged by a real-time amplification curve, the curve of the positive sample appeared at the turbidity meter while no curve was observed in the negative samples. The RT-LAMP result could be also judged by a dye color reaction, the color of the positive sample was green and the color of the negative sample was orange after adding SYBR Green I in the reaction liquid.【Result】The faster and specific RT-LAMP method for the detection of GVA was developed, and the optimal reaction temperature was 65℃. The method could obtain an amplification curve only in 27 min, while the detection results could be obtained in 1.5 h using conventional RT-PCR. Specificity experiments indicated that the amplification curve could only be obtained from the total RNA of GVA and the color of the reaction liquid changed to green, which showed positive; while the other three viruses and the healthy control did not appear on the amplification curve and the color of the reaction liquids were orange, which showed negative. RT-LAMP detection results could be directly observed by the naked eye, which was easier than the results judgment of the conventional RT-PCR. Sensitivity experiments indicated that RT-LAMP could detect 101, 102, 103, 104 and 105 diluent total RNA templates of GVA, while the conventional RT-PCR could only detect 101, 102, 103 and 104 diluent total RNA templates of GVA, which showed the sensitivity of the former was 10 times higher than the sensitivity of the latter. 【Conclusion】The RT-LAMP method developed in this study could be used to detect GVA rapidly and specifically, which provided technical support for the screening of GVA-free grape seedlings and was suitable for the detection and identification of GVA in the entry quarantine, seedling breeding and field monitoring works by quarantine, research and production organizations.
    SOIL & FERTILIZER·WATER-SAVING IRRIGATION·AGROECOLOGY & ENVIRONMENT
    Characteristics of Field Water and Nitrogen Leaching in a Haplic Luvisol Soil Based on Large Lysimeter
    ZHANG Yi-tao, WANG Hong-yuan, LIU Hong-bin, REN Tian-zhi
    Scientia Agricultura Sinica. 2016, 49(1):  110-119.  doi:10.3864/j.issn.0578-1752.2016.01.010
    Abstract ( 472 )   HTML ( 1 )   PDF (730KB) ( 461 )   Save
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    【Objective】The aim of this article was to determine clear characteristics of input and leaching of water and nitrogen under a typical farmland planting system in the North China Plain, and clarify the effect of N fertilizer on leaching water quality, which was eventually to provide a scientific basis for reducing nitrogen leaching from the source and prevention farmland of non-point source pollution. 【Method】 The experiment was set in the “Key Experimental Station on Ecological Environment of Drab Fluvo-aquic Soil in Changping, Ministry of Agriculture” for maize monoculture from 2007 to 2012, including three nitrogen gradients (no nitrogen CK, optimize nitrogen T1 and traditional nitrogen T2). A large lysimeter was used to monitor the process of leaching of soil water and nitrogen during maize growth, at the same time the field microclimate stations were used to monitor the process of precipitation, while detailed irrigation events were also recorded. 【Result】The result showed that the precipitation and irrigation mainly occurred in April to October of every year. And leaching mainly occurred in May to November, which was later than the precipitation and irrigation. Annual precipitation was different among the five years (134-525 mm). Precipitation in 2009 was less with no irrigation and resulted in no leaching events, while total water input was higher than 500 mm/year in other years leading to leaching events. Leaching occurrence frequency and water amount in the treatment of nitrogen application decreased compared to no nitrogen application, CK had 37 leaching events with a water volume of 422 mm while T2 had 31 leaching events with a water volume of 310 mm. Total nitrogen in irrigation and precipitation was 157 kg·hm-2 and the dissolved total nitrogen was 106 kg·hm-2, while annual total nitrogen in irrigation and precipitation was different among the five years (7.53-34.1 kg·hm-2). For one year, the more nitrogen in irrigation and precipitation, the more nitrogen leaching for the same treatment. Nitrate-N in dissolved total nitrogen was the main type of nitrogen, the ratio of nitrate-N in the leaching water increased with the increase of the nitrogen application. No matter in single or annual leaching events both dissolved total nitrogen and nitrate-N in leaching water of T2 was significantly higher than that of CK and T1. Average nitrate-N concentration of CK and T1 in all leaching events were lower than 2.0, 5.0 mg·L-1 respectively, while the average nitrate-N concentration of 31 leaching events of T2 within five years was 29.5 mg·L-1. There were 15 leaching events in which the nitrate-N concentration was higher than Class III of groundwater (20 mg·L-1), of which the maximum concentration was 79.0 mg·L-1. 【Conclusion Irrigation and precipitation was the main cause of leaching, and the more water input, the more leaching. Nitrate-N was the main type of nitrogen in leaching water. In different years, nitrogen leaching had a positive relationship with dissolved total nitrogen in precipitation and irrigation. The more dissolved total nitrogen involved in precipitation and irrigation, the more nitrogen leaching. In one year, the amount and concentration of nitrate-N leaching increased with the increase of the nitrogen application rate. So nitrogen application rate must be reduced to decrease nitrogen leaching and ensure the water nitrate content does not exceed the criteria. However, the effect of the total nitrogen involved in precipitation and irrigation on nitrogen leaching when identifying the optimal nitrogen rate should be considered.
    Characteristics of Soil Carbon Emission and Water Utilization in Wheat/Maize Intercropping with Minimal/Zero Tillage and Straw Retention
    HU Fa-long, CHAI Qiang, GAN Yan-tai, YIN Wen, ZHAO Cai, FENG Fu-xue
    Scientia Agricultura Sinica. 2016, 49(1):  120-131.  doi:10.3864/j.issn.0578-1752.2016.01.011
    Abstract ( 570 )   HTML ( 4 )   PDF (451KB) ( 868 )   Save
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    【Objective】 This study exploited the potential of intercropping and conservation tillage on optimizing carbon emission and water utilization, and integrated them into one cropping system to promote their respective and coordination effect. Thereby, to establish an effective intercropping system with double-wins on carbon emission reduction and water conservation. 【Method】 The study employed wheat (Triticum aestivum)/maize (Zea mayz) intercropping, which was cultivated as a long-term and large-scale cropping mode in Hexi irrigation areas, as the research object. The field experiment was conducted by integrating minimal/zero tillage into wheat/maize intercropping and established four tillage and straw retention patterns, which were: (a) no-till with 25 cm wheat stubble standing in the field (NTSSI, stubble standing), (b) no-till with 25 cm height of wheat straw chopped and spread evenly on the soil surface (NTSI, straw mulching), (c) tillage with 25 cm height of straw was incorporated in the soil (TISI, straw incorporated), and conventional tillage (CTI, tillage without straw retention). Meanwhile, the study also applied conventional-tilled sole wheat (SW) and sole maize (SM) as the controls, and investigated the conditions of soil respiration and crop water consumption in various treatments. An EGM-4 system (Environmental gas monitor-4, UK, PP system) was used to measure soil respiration and there from calculating the carbon emission during the growing season. Two methods of water use efficiency were determined, i.e., based on grain yield (WUEGY) and carbon emission (WUECE, that is carbon emission per unit of water) in order to make a proper assessment of water use condition in the intercropping system. 【Result】 Among the three cropping systems, the sole maize had the highest soil respiration rate (1.57 μmol CO2·m-2·s-1), which was almost twice as higher as the intercropping (0.83 μmol CO2·m-2·s-1), while among four straw retention patterns, NTSI had the lowest soil respiration, which was decreased by 20.4% in 2011 and 11.9% in 2012 as compared to CTI. Accordingly, carbon emission of NTSI was reduced by 12.4% compared to CTI. Integration of no-till and straw mulching into wheat/maize intercropping effectively lowered the crop water consumption while improving grain yield and WUEGY. Compared to CTI, the water consumption of NTSI was reduced by 4.1%, while grain yield and WUEGY was respectively increased by 29.7% and 15.6%. Furthermore, the carbon emission per unit of water was decreased by 5.9%, although carbon emission efficiency was promoted by 28.2%. 【Conclusion】 The results suggested that the integration of no-till and straw mulching into wheat/maize intercropping optimized the relationship between grain yield, carbon emission, and crop water consumption in this cropping system. The cropping mode could promote more water to produce more grain yield, meanwhile reducing the carbon emission during the crop production process. Consequently, it achieved a win-win effect on coordinating carbon emission reduction and efficient use of soil water in a wheat/maize intercropping system.
    HORTICULTURE
    Establishment of Non-Destructive System for Fruit Quality Grading of ‘Bingtang’ Sweet Orange and Its Application on Packing Line
    LI Na, YANG Xing-xing, DAI Su-ming, LI Rong-hua, JIANG Hang, LUO Yue-xiong, Alessandra Gentile, DENG Zi-niu
    Scientia Agricultura Sinica. 2016, 49(1):  132-141.  doi:10.3864/j.issn.0578-1752.2016.01.012
    Abstract ( 501 )   HTML ( 2 )   PDF (438KB) ( 1055 )   Save
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    【Objective】 The inconsistency between external appearance and the internal quality of ‘Bingtang’ sweet orange (Citrus sinensis Osbeck) fruit has significantly reduced its marketing value. The present research intended to establish the classification standard for fruit quality and the non-destructive technologies for quality analysis and quality rating of ‘Bingtang’ orange, and then to quickly sort out the online products with different total soluble solids (TSS) and acidity. 【Method】 Fruit TSS and acid content were analyzed by sampling from the main production areas with the conventional destructive methods for a long period. The correlation among the external and internal key fruit quality parameters was calculated, and then the relationship between fruit diameter vs. TSS and acid content was determined. A standard for fruit quality classification of ‘Bingtang’ sweet orange was established. Based on the standard, the NIR transmission spectrum at 710-960 nm was utilized for fruit quality analyses, and repeated correction and verification were performed in comparison with the conventional quality analysis data to evaluate the accuracy in non-destructive fruit quality analysis and sorting. This non-destructive system was run on packing line, and the rating products were investigated in the market including the quantity, price and sold volume of different classes of ‘Bingtang’ sweet oranges. 【Result】 (1) Classification standard of ‘Bingtang’ sweet orange was established. Fruit size classification was set up based on fruit diameters: large 68-74 mm, medium 62-67 mm, and small 56-61 mm. And fruits were further classified into 4 classes based on the TSS and acid content, i.e. Super class: Birx≥14 and acidity≤0.4%, Class I: 12≤Birx<14 and acidity≤0.4%, Class II: Birx ≥14 and 0.6%≥acidity>0.4%, and Marketable class: Birx<12 and acidity≤0.4%. (2) Following repeated analyses of single fruits, non-destructive analytical standards for TSS and acidity were formulated. In the 1st TSS testing experimentation, the testing range was set up between 10.1-14.9 Brix and resulted in only 26% of conformity between the non-destructive and the conventional quality analyses; when the TSS testing range extended to 10.1-16.2 Brix in the 2nd experimentation, 90% of conformity was obtained; when the TSS lowered to 9.8 Brix (testing range of 9.8-16.2 Brix) in the 3rd TSS test, the conformity remained as high as 90%. For the acidity analysis, the testing range was set up between 0.1%-1.26% at first, only 64% of the conformity was obtained; when the testing range expanded to 0.1%-1.37%, up to 94% of conformity was realized; if the testing range of acidity increased further to 1.53%, the result was 92% of conformity indicating no improvement was gained. (3) The application of the technique on packing line resulted in a sorting speeds up to 10 oranges per second, and the sorted fruits had a highest price of 48 yuan per kg. On the packing ling, annual sorting output reached 4 500 tons, valuing 91.22 million yuan.【Conclusion】The results indicated that the established non-destructive system was accurate in TSS and acidity analysis and the analytical standards covered the full range of TSS and acidity contents of all the commercial fruits of ‘Bingtang’ sweet orange. The classification with the fast, high-quality and non-destructive online determination of the internal quality of ‘Bingtang’ sweet orange on a large scale combined with the grading standard of fruit size would ensure an unified internal and external quality of the products.
    Cloning, Subcellular Localization and Expression Analysis of AsAG from Sugar Apple (Annona squamosa L.)
    LIU Kai-dong, LI Hai-li, ZHONG Shu-ting, YUAN Chang-chun, CHEN Yan, LIU Jin-xiang, ZHONG Jun-di
    Scientia Agricultura Sinica. 2016, 49(1):  142-154.  doi:10.3864/j.issn.0578-1752.2016.01.013
    Abstract ( 402 )   HTML ( 4 )   PDF (2714KB) ( 711 )   Save
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    【Objective】This paper aims to isolate the cDNA of AGAMOUS from sugar apple (Annona squamosa L.) and to study their subcellular localization and expression level, in order to lay a foundation for further exploration of the flower development mechanism and solve the problem of flower abnormality. 【Method】 Total RNA was extracted from mature flowers of sugar apple. Polymerase chain reaction (PCR) combined with 5′ RACE, and 3′ RACE technology were used to clone the full length cDNA. The sequencing data were assembled by DNAMAN software. BLASTn and BLASTp in NCBI were used to conduct the similarity analysis. DNAMAN was used to analyze the amino acid sequences and MEGA 5.1 was used to create the phylogenetic tree; the protein secondary structure and the structure of 3D modeling was predicted by SOPMA and Phyre2 programs. Recombinant plasmid was introduced into onion epidermal cells and tobacco epithelial cells. Green fluorescence was monitored under a laser scanning confocal microscope. Quantitative real-time PCR (qRT-PCR) was performed to determine the expression pattern of AsAG in different developmental stages of flower and different flower organs. The expression levels of AsAG responsing to different hormone treatments were also determined by qRT-PCR. 【Result】 A full-length cDNA sequence of the homologous AGAMOUS gene was cloned from sugar apple. Sequence analysis showed that the AsAG gene contains a 669 bp open reading frame (ORF) encoding 222 amino acids. The sequence was submitted to GenBank, and the registration number is KT159768. The predictive secondary structure showed that AsAG protein was made up of 59.46% alpha-helix, 8.11% beta-turn, 14.41% extended strand and 18.02% random coil. The amino acid sequences shared 79%-84% in homology compared with Phoenix dactylifera (XP 008781978.1), Asparagus virgatus (BAD83772.1), Arabidopsis thaliana (AT4G18960), Elaeis guineensis (XP 010912706.1), Magnolia delavayi (AFH74390.1), Dendrobium nobile (ABQ08574.1) and Magnolia wufengensis (AEO52692.1). The deduced amino acids sequence contained a highly conserved MADS domain and a secondary conserved K domain. The molecular weight and an isoelectric point of this protein were 25.7 kDa and 9.15, respectively. The protein was stable protein and had no signal peptide. Subcellular localization assays showed that the AsAG protein was located in the nucleus. The qRT-PCR results suggested that AsAG gene showed a high transcription level in flower. The AsAG gene could be detected during the whole period of flower development. Interestingly, AsAG gene showed a high transcription level in the stage of flower bud Ⅳ. The expression level in the stamen was higher than that in the pistil, sepal and petal. The expression level of perfect flower was higher than deformity flower. After 2 h and 4 h treatments with GA and ABA signal molecules in flower buds, the expression patterns of AsAG showed negative regulation by GA, positive regulation by ABA. 【Conclusion】AsAG gene may participate in the pistil and stamen development and hormone signal response in sugar apple.
    STORAGE·FRESH-KEEPING·PROCESSING
    Detection of Endpoint Temperature of Chicken and Fish by Near-Infrared Spectroscopy
    LIU Gong-ming, SUN Jing-xin, LI Peng, XU Xing-lian, ZHANG Wan-gang, HUANG Ming, ZHOU Guang-hong
    Scientia Agricultura Sinica. 2016, 49(1):  155-162.  doi:10.3864/j.issn.0578-1752.2016.01.014
    Abstract ( 332 )   HTML ( 4 )   PDF (2375KB) ( 559 )   Save
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    【Objective】Endpoint temperature (EPT) of heat treated meat products is a determinant factor to control food-borne diseases. EPT of cooked meat and meat products has been determined using different methods including determination of enzyme activity, coagulation test and Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) etc. However, these methods are time consuming, destructive and labor intensive. The objective of this study was to investigate if near-infrared spectroscopy (NIR) in combination with partial least squares (PLS) data analysis can be used to determine the EPT in cooked meat.【Method】The meat samples (chicken and fish) were heat treated until they reached a final internal temperature of 50 to 90 (5 intervals) at the rate of 1·min-1. The cooked samples were removed from the waterbath and cooled immediately in ice water to 4. The 144 samples in total (the number of chicken and fish was 77 and 67 respectively) were homogenized for 1 minute and deposited at 4 before measurement. Then NIR reflectance spectra were collected using a scanning monochromator equipped with a rotating sample cup with a quartz window between 11 000 and 4 000 cm-1 at 8 cm-1 intervals. Each sample was scanned for three times. Each spectrum, an average of 64 scans, was recorded over the selected wavenumber range. Randomly selected 108 spectra from chicken and fish samples were assigned to a calibration set while remaining 36 spectra were used for a validation set. The calibration sets were used for establishing the calibration model and the validation sets were used to test model prediction ability.The spectra was processed using the method of Standard Normal Variate, first-order differential and Norris Derivative. The principal component number was determined by the cross-validation mean square error (RMSECV). The prediction mean square error (RMSEP), correlation index (r) and the standard deviation (σ) were used to evaluate prediction ability of the calibration model.【Result】By RMSECV, the principal components of the chicken and fish were 9 and 11 ascertained through RMSECV value (1.59% and 0.96%) and r value (0.9844 and 0.9936). The lower the RMSECV and lower the r values, the more accurate is the calibration model. As can be observed in the study, prediction of EPT using the NIR spectra showed very high correlation (r=0.9966 for chicken and r=0.9832 for fish) with a prediction error (RMSECV) of 3.02% and 2.94%, respectively.【Conclusion】The results showed that NIR spectroscopy has the potential for use in the food processing industry and food inspection to ensure that all meat products have reached the recommended EPT, keeping it safe. And the model established in this paper has a good prediction ability of EPT for meat products, which is a non-destructive, simple and reliable technique to monitor the ?nal quality of a product.
    Storage and Transportation Characteristic of Different Moisture Paddy Rice Dealt with Dynamic Temperature and Humidity
    CHEN Yin-ji, JIANG Wei-xin, CAO Jun, DAI Bing-ye, DONG Wen
    Scientia Agricultura Sinica. 2016, 49(1):  163-175.  doi:10.3864/j.issn.0578-1752.2016.01.015
    Abstract ( 387 )   HTML ( 1 )   PDF (1163KB) ( 525 )   Save
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    【Objective】 Safe storage and transportation of rice is very important because China is the major rice producer and consumer in the world. Through laboratory simulation of dynamic temperature and humidity conditions, the rice quality and moisture migration law of the dynamic storage and transportation conditions are studied. The objective of this paper is to provide reference for the construction of dynamic storage and transportation of rice. 【Method】 Through dynamic temperature and humidity simulation experiment, five moisture content paddy rice (14%, 16%, 18%, 20%, and 22%) were stored and transported for three months respectively at low temperature (fluctuates around 10℃), middle temperature (fluctuates around 20℃) and high temperature (fluctuates around 30℃). During the storage and transportation, the changes of microbial growth, texture quality, fatty acid value and low field nuclear magnetic resonance (NMR) data were measured to clarify the influence of temperature and moisture content on paddy rice during storage and transportation. 【Result】 Aerobic plate count increased with prolonged time, the increasing trends of aerobic plate count in different temperatures and moisture content for paddy rice were different. Aerobic plate count increased with rising temperature. The mould number raised with prolonged storage and transportation time and the mould number increased significantly under middle and high temperature. The higher temperature was, the more obviously mould number increased; mould happened at lower levels when 14% and 16% moisture content paddy rice storage and transported at low, middle and high temperature. The mildew occurrence of 18% and higher moisture content paddy rice was significantly higher than 16% and 14% moisture content paddy rice during 15 days storage and transportation at middle and high temperatures. The hardness and springiness of cooking rice increased gradually with prolonged time. The higher initial moisture content was, the faster the hardness rose (P<0.01). The hardness had a significant negative correlation with the initial moisture content of paddy rice; the influence of initial moisture content on springiness was significant (P<0.01). The adhesiveness of paddy rice reduced with prolonged time, the influence of temperature and moisture content on adhesiveness was significant (P<0.05). The fatty acid value and the initial moisture content were positively correlated (P<0.01), the higher initial moisture content was, the more obviously fatty acid value of paddy rice increases. The influence of temperature on fatty acid value was particularly apparent, the higher temperature was, the faster fatty acid value rises. The low field nuclear magnetic total semaphore t (T2w) decreased with the increase of storage and transportation time, T2w total semaphore and moisture content exhibited significant linear positive correlation (P<0.01). As magnetic resonance imaging (MRI) exhibits, moisture mainly concentrates on epidermis and aleurone layer when paddy rice was stored and transported 15 days. After 30 days storage and transportation, surface moisture losses and the moisture distribution became more uniform. 【Conclusion】 The short storage time (15 or 30 days) and transportation of relative high moisture (14% or 16%) paddy rice can improve rice main the texture’s quality. The initial moisture content of paddy rice should be controlled under 16% during a long time transportation with dynamic variation of the temperature and humidity (around low and middle temperature).
    ANIMAL SCIENCE·VETERINARY SCIENCERE·SOURCE INSECT
    Expression and Regulation of Sall4 and Screening Core Regulation Region of Sall4 Promoter
    WANG Ya-xian, YANG Fan, WANG Hua-yan
    Scientia Agricultura Sinica. 2016, 49(1):  176-185.  doi:10.3864/j.issn.0578-1752.2016.01.016
    Abstract ( 405 )   HTML ( 5 )   PDF (4301KB) ( 454 )   Save
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    【Objective】Sall4 (spalt-like transcription factor 4), as a zinc finger transcription factor, plays an important role in establishing and maintaining the pluripotency of embryonic stem cells. To investigate the expression pattern and regulatory mechanism of Sall4, we cloned porcine Sall4 promoter and functionally analyzed the core regulatory domains of this promoter. 【Method】A 2.1 kb fragment of porcine Sall4 promoter was cloned from pig genomic DNA by PCR and was used to construct reporter vectors. The pE2.1 vector was transfected into different kinds of cells to determine the expression of fluorescent protein for cellular specificity. The Sall4 expression in different tissues was detected by real-time RT-PCR. The core regulatory elements of Sall4 promoter, which share homology to known transcription factor binding sites, were analyzed by GPminer, Methprimer and JASPER program. Sall4 promoter and pluripotent factors, including Oct4, Sox2, Klf4, c-Myc, Esrra/b and Smad were co-transfected into 293T cells to determine the activation of Sall4 promoter. A series of deletion fragments with different sizes of Sall4 promoter were produced by PCR and inserted into pGL3-Basic vector. After transient transfection into 293T, dual luciferase assay was used to measure promoter activities. 【Result】 Sall4 has specific expression in pluripotent cell type P19, reproductive cell type CHO and cancerization cell types 293T and Hela. The real-time fluorescent quantitative RT-PCR results showed that the expression of porcine Sall4 was significantly higher in ovary and testis tissues and porcine iPS cells, but there was low expression in brain, heart, liver and muscle tissues, suggesting that Sall4 is a key factor maintaining pluripotency of stem cells. Bioinformatics analysis of Sall4 promoter by JASPER and GPminer programs indicated that cis-acting elements like TATA box, GC box and CAAT box, and the potential binding sites for Oct4, Sox2, Klf4, Myc, Esrra/b, Stat3 and Smad were found within the promoter sequence. We found that Oct4, Sox2 and TGF-beta signal pathway could significantly activate Sall4 promoter, in which Sall4 promoter activity increased three times compared with the control. However, Klf4 could repress Sall4 promoter activity, showing a negative regulatory role. Deletion constructs of pL2.1, pL1.0 and pL0.5 were constructed based on PCR method. The result of dual luciferase assay indicated there was no significant differences in promoter activity between pL2.1 and pL1.0 though pL2.1 retains more than a 1 kb fragment. However, the promoter activity of pL1.0 which is 486 bp larger than pL0.5 increased two times compared to pL0.5. Additionally, the promoter activity in pL0.5 was 8 times over that of the control. These results demonstrate that two core regulatory elements of Sall4 promoter are located within -367 bp to -852 bp region and -1 bp to -366 bp region. 【Conclusion】Sall4 promoter has been cloned and showed tissue-specific expression. Numerous potential transcription factor binding sites and core promoter regulatory region on Sall4 promoter regulated the expression of Sall4 gene. The interaction between these transcription factor and Sall4 gene would be essential for transcriptional regulation of porcine Sall4 gene in pluripotency stem cells.
    Effects of Constant Moderate Temperatures on the Diversity of the Intestinal Microbial Flora of Broilers
    PENG Qian-qian, WANG Xue-min, ZHANG Min-hong, FENG Jing-hai, ZHEN Long, ZHANG Shao-shuai
    Scientia Agricultura Sinica. 2016, 49(1):  186-194.  doi:10.3864/j.issn.0578-1752.2016.01.017
    Abstract ( 375 )   HTML ( 13 )   PDF (469KB) ( 663 )   Save
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    【Objective】The objective of this study is to provide useful information for healthy breeding on broilers. Ambient temperature is one of the important factors affecting the poultry intestinal flora; as the ambient temperature changes, poultry normal intestinal flora will be affected. This study was carried out to investigate the effects of constant moderate temperatures on structural diversity of cecal bacteria in broilers. 【Method】One hundred and forty four 22-day-old Arbor Acres (AA) broilers were assigned to three environment chambers, each chamber contained six cages with eight birds per cage (four males and four females), with each cage used as a replicate. The pre-test period lasted for 7 days and broilers were kept at 21 and 60% relative humidity. When broilers were 29-day-old, the temperatures of each environmental chamber were gradually regulated to 21, 26 and 31, respectively, while maintaining the relative humidity at 60% and both kept constant until the end of the experiment. The trial period lasted for 14 days. On day 21 and 42, one birds from each replicate were randomly selected and killed, cecal contents were aseptically collected, placed in a centrifugal tube, rapidly frozen in liquid nitrogen, and stored at -80. The bacterial community and diversity in the ceacal digesta of broilers at the 7 and 14 days of constant moderate temperatures was studied by using 16S rDNA-based denaturing gradient gel electrophoresis (DGGE). 【Result】 (1) The number of PCR-denaturing gradient gel electrophoresis bands of 26 and 31 group was lower than 21 group, and 31 group was lower than 26, at 31, broiler cecal DGGE bands at the 14 days are significantly lower than at the 7 days. (2) The dendrograms and Shannon-Weaver indices revealed that diversity of the intestinal microbial flora at 26 and 31 group was less than at 21; at 31,14 days are significantly lower than at the 7 days; (3) DGGE profiles stripe sequence analysis showed that Alistipes timonensis and Barnesiella viscericola were predominant in the 21, 26 and 31 groups, The special bacteria in 21, 26 and 31 groups were Ruminococus faecis, Lutispora thermophila and Faecalibacterium prausnitzii respectively, 31 group were Gemmiger formicilis.【Conclusion】Compared with 21, continuous treatment with constant moderate temperature (26 and 31) can significantly impact the community structure and diversity of intestinal flora in broilers, and the extent of effect varies with the different temperatures.
    Identification of Bmhairy as the Target of bmo-miR-7 and Its Transcriptional Expression Profiles in the Silkworm (Bombyx mori)
    LIU Shi-ping, WU Xiao-yan, ZHANG Dan-yu, HUANG Ya-xi, WANG Wei, ZHAO Ping, XIA Qing-you
    Scientia Agricultura Sinica. 2016, 49(1):  195-204.  doi:10.3864/j.issn.0578-1752.2016.01.018
    Abstract ( 398 )   HTML ( 1 )   PDF (3004KB) ( 723 )   Save
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    【Objective】microRNAs are a class of non-coding RNAs with about 22 nucleotides and post-transcriptionally regulate the target genes responsible for the metabolism, growth, development and some other important life activities of organisms. The objective of this study is to clone Bmhairy, characterize the expression profiles of Bmhairy and bmo-miR-7 in the silkworm (Bombyx mori) and investigate whether Bmhairy is the target gene of bmo-miR-7. 【Method】The total RNA isolated from the embryo of B. mori was used to synthesize the first strand of cDNA of Bmhairy, which has served as the PCR template for producing CDS (coding DNA sequence) and 3′UTR. The expression patterns of bmo-miR-7 and Bmhairy were compared based on the results from fluorescence quantitative PCR and the previous microarray data. The binding sites of bmo-miR-7 within the 3′UTR of Bmhairy were predicted with the online tools, RNAhybrid and MiRTif, followed by experimental confirmation through the luciferase reporter gene assay in the BmE cells in vitro.【Result】The homologue of Drosophila hairy in B. mori was cloned, and named as Bmhairy, which consists of two introns and three exons; its CDS is 654 bp, encoding 217 amino acids, and its 3′UTR is 366 nt. The predicted protein comprises three domains, namely bHLH, ORANGE and WRPW, which are highly conserved in vertebrates and invertebrates. Bmhairy is most similar to the homologue in the Lepidoptera insect, Danaus plexippus. Bmo-miR-7 was highly expressed in the embryo and testis of the day 3 of 5th instar larva of B. mori. However, Bmhairy was highly expressed in the adult moth and in the head of the day 3 of 5th instar larva. Combined use of RNAhybrid and MiRTif revealed a target site of bmo-miR-7 within the 3′UTR of Bmhairy, which was supported by luciferase report gene assay in the BmE cell lines cotransfected with bmo-miR-7 mimics and Bmhairy 3′UTR luciferase reporter vector. 【Conclusion】The expression profile of Bmhairy showed opposite changes to that of bmo-miR-7 in the embryo and adult stages of B. mori as well as in the head and testis of the day 3 of 5th instar larva. Bmhairy is one target of bmo-miR-7 based on the results from the prediction and luciferase report gene assay and hopefully might lay a base for further study of the roles of bmo-miR-7 and Bmhairy as well as their relationship.