Scientia Agricultura Sinica

Construction and Analysis of Normalized Full-Length cDNA Library of Upland Cotton Anthers

MA Jian-Hui-., LIU Ji-., FAN Shu-Li, SONG Mei-Zhen, PANG Chao-You, WEI Heng-Ling, YU Shu-Xun

Scientia Agricultura Sinica. 2013, 46(18): 3743-3749. doi:10.3864/j.issn.0578-1752.2013.18.001

Abstract ( 409 )

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【Objective】The objective of this study is to understand the anther development and make a firm foundation for sterile mechanism in upland cotton (Gossypium hirsutum L.). 【Method】A normalized full-length cDNA library from anthers including the full development stage was firstly constructed and 9 896 high-quality ESTs were sequenced in this study. The bioinformatics analysis and qRT-PCR were performed to deep analyze this cDNA library. 【Result】The storage capacity of this cDNA library is 1.2×107 and the average length of the inserted cDNA fragment was dispersed between 1 kb and 3 kb. These high-quality ESTs were further assembled into 6 643 unigenes with 779 bp in average length. Through bioinformatics analysis of these unigenes, many important biological processes were obtained during the anther development and 28 genes related with the biosynthesis of pollen wall were identified during anther development. The qRT-PCR was performed to check the expression pattern of these pollen wall biosynthesis related genes and all of them were high-expressed in early anther development especially in tetrad pollen period. 【Conclution】In this study, lots of anther expressed genes were firstly sequenced in upland cotton. Many important processes regulating anther development were obtained and many genes related with pollen wall biosynthesis were obtained.

Prodution of High-Amylopectin Potato Plants by Using ihpRNAi Technology

LIU Yu-Hui-12, WANG Li-3, YANG Hong-Yu-1, YU Bin-12, LI Yuan-Ming-4, ZHANG Jun-Lian-12, WANG Di-12

Scientia Agricultura Sinica. 2013, 46(18): 3750-3757. doi:10.3864/j.issn.0578-1752.2013.18.002

Abstract ( 453 )

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【Objective】 The objective of this study is to develop transgenic potato (Solanum tuberosum L.) plants with high-amylopectin starch in its tubers.【Method】RNA interference expression vector pBI121g-PgABI driven by Patatin was transformed into elite potato cultivar ‘Gannongshu 2’ by Agrobacterium-mediated transformation. The transgenic plants were identified by PCR, Southern-blotting, semi-quantitative RT-PCR and real-time quantitative PCR and the starch content of transgenic potatoes was determined.【Result】Ten transgenic potato lines were confirmed by PCR and Southern blot analysis that the target gene integrated into the plant genomes. Result of semi-quantitative RT-PCR indicated that the accumulation of mRNAs derived from GBSSI was inhibited significantly in all transgenic lines, which were not detectable in 6 tansgenic lines. Result of real-time quantitative PCR showed that the inhibition ratio was 66.27%-93.53%. There were significant changes of starch content in ten transgenic microtubers,of which the amylopectin content was up to 90.16%-98.84%, 10.31%-20.92% higher than the non-transgenic microtuber. A significant correlation was found between inhibition ratio of mRNA and amylopectin content of transgenic potato plants (r=0.937, P＜0.01). 【Conclusion】ihpRNAi technology can be used effectively in the production of high-amylopectin potato or pure-amylopectin potato by silencing endogenesis gene GBSSI.

Response of Maize Leaf Proteins Induced/Modulated by AM Mycorrhizal Inoculation and (or) Arsenic Stress

WANG Zhen-Hui-1, YUAN Kun-1, YANG Li-Fu-1, ZHANG Jun-Ling-2

Scientia Agricultura Sinica. 2013, 46(18): 3758-3767. doi:10.3864/j.issn.0578-1752.2013.18.003

Abstract ( 361 )

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【Objective】In this paper, proteomics were performed to analyze the changes in leaf proteins of maize in response to As additions and mycorrhizal inoculation.【Method】The leaf proteome of maize was analyzed using two-dimensional electrophoresis and mass spectrometry. A targeted proteomic approach was applied to determine the putative identity of some proteins induced/modulated by arsenic (As) and/or mycorrhizal inoculation.【Result】The proteomics aimed at comparing the leaf protein profiles of maize growing in substrate amended with arsenic or unamended. Seven differentially expressed protein spots were identified. Three of them were identified as unknown protein and the remaining four were 2-phosphoglycerate kinase, oxidoreductase, MAP3K delta-1 protein kinase and Os06g0262800. Another comparasion of leaf proteins between plants with arsenic stress and those with As stress plus mycorrhizal inoculation revealed that 22 spots were differentially displayed in response to mycorrhizal inoculation and 11 differentially expressed protein spots were identified. Four of them were identified as unknown protein and the other seven were oxygen-evolving enhancer protein 3-1, putative M protein, SNF1-related protein kinase 2.2, ATP synthase CF1 beta subunit, ATP synthase CF1 alpha subunit, pathogenesis-related protein 10, MAP kinase kinase and MEI1 protein, respectively. 【Conclusion】 The dry weights of maize plants with As addition were improved by mycorrhizal innoculations, root As concentrations were also decreased significantly. Changes in the amount and types of leaf protein expression in maize plants indicated that mycorrhizal inoculation might simulate the proteins associated with plant growth, nutrient uptake and resistance to produce responses under arsenic stress conditions. Therefore, it’s helpful to improve the resistance of maize plants to arsenic toxicity.

Changes of Related Physiological Characteristics of Cotton Under Salinity Condition and the Construction of the Cotton Water Stress Index

ZHANG Lei, ZHANG Guo-Wei, MENG Ya-Li, CHEN Bing-Lin, WANG You-Hua, ZHOU Zhi-Guo

Scientia Agricultura Sinica. 2013, 46(18): 3768-3775. doi:10.3864/j.issn.0578-1752.2013.18.004

Abstract ( 425 )

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【Objective】To construct the cotton water stress index (CWSI) and determine the suitable water logistics approach, in order to provide theoretical basis for improving cotton yield and quality under salinity field.【Method】The two-year experiments were conducted with Sumian 12 (salinity-sensitivity) and CCRI-44 (salinity-tolerance) at Pailou experimental station of Nanjing Agricultural University. Seven kinds of salts (NaCl, Na2CO3, NaHCO3, CaCl2, MgCl2, MgSO4, and Na2SO4), were mixed into natural dried, sieved soils at an even molar ratio, forming soils with five levels of salinity, namely, CK (1.25 dS?m-1), 0.35% (5.80 dS?m-1), 0.60% ( 9.61 dS?m-1), 0.85% (13.23 dS?m-1), and 1.00% (14.65 dS?m-1), respectively. The effects of soil salinity rates on the water contents and temperature of cotton functional leaves were studied and quantitative relationships between leaf-air temperature difference and vapor pressure difference was determined.【Result】As the soil salinity rates increasing, the transpiration rate, water content and net photosynthetic rate of functional cotton leaves decreased and the leaf temperature increased. The lower equation of the cotton water stress index was set up in 1.25 dS?m-1 salinity rate (well watered), and the cotton water stress index based on the above lower equation under different salinity rates was constructed.【Conclusion】Comprehensive analysis the relationship between cotton water stress index and leaf water content and net photosynthetic rate under different soil salinity rates, and found that the cotton water stress index is a good indicator to detect cotton water stress in salinity field. So, it could be applied to monitor cotton water stress using cotton water stress index, in order to provide theoretical basis for determine suitable water logistics approach under salinity field.

Plasmid Elimination of Bacillus subtilis NJ-18 and Its Colonization on Wheat Roots

LU Jing-Le, YU Xin-Yan, HOU Yi-Ping, WANG Jian-Xin, ZHOU Ming-Guo, CHEN Chang-Jun

Scientia Agricultura Sinica. 2013, 46(18): 3776-3783. doi:10.3864/j.issn.0578-1752.2013.18.005

Abstract ( 389 )

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【Objective】Bacillus subtilis strain NJ-18, isolated from the soil of oilseed rape field, has a broad spectrum antibacterial activity and a great potential for biocontrol. In order to study the interaction between NJ-18 and plants, NJ-18 was marked with a green fluorescent protein (gfp) gene and its colonization ability on wheat roots was observed with a laser scanning confocal microscope. 【Method】 The plasmids of NJ-18 were cured by sodium dodecyl sulfate (SDS) and gfp was transformed into the strain with a chemical method. Colonization ability of NJ-18 on wheat roots was observed with a laser scanning confocal microscope. 【Result】 By a chemical method, gfp was sucessfully transformed into wild strain NJ-18 and C136, a plasmid cured strain from NJ-18. The transformation efficiency of C136 could be up to 3.42×105 cfu/μg of plasmid DNA and was about 10-fold of that of NJ-18. Compared with NJ-18, C136 had the same antibacterial activity, but its growth was inhibited in a certain extent. NJ-18 could attach on the pathogen microbial cells, resulting in mycelium enlargement and malformations. NJ-18 was able to colonize in the phloem and xylem of wheat roots on 7 days after inoculation. 【Conclusion】 The chemical transformation method is suitalbe for strain NJ-18. NJ-18 can colonize on wheat roots.

Genetic Diversity of Two Hypervariable Genes from Prophage Regions of ‘Candidatus Liberibacter asiaticus’ in China

TAN Jin-12, WANG Xue-Feng-2, SU Hua-Nan-12, LI Zhong-An-2, ZHOU Chang-Yong-2

Scientia Agricultura Sinica. 2013, 46(18): 3784-3792. doi:10.3864/j.issn.0578-1752.2013.18.006

Abstract ( 533 )

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【Objective】 Hypervariable genomic loci of prophage region were used to study population differentiation and genetic diversity of ‘Candidatus Liberibacter asiaticus’, the putative casual agent of citrus Huanglongbing (HLB). 【Method】 Two primer sets (Lap-TJ-F/Lap-TJ-R1 and Lap-TJ-F/Lap-TJ-R2), targeted hypervariable genomic regions of two prophage types (SC1 and SC2), respectively, were used for PCR detection and sequence analysis of 224 ‘Ca. L. asiaticus’ strains from different citrus producing areas in China. 【Result】 PCR amplicon polymorphisms and four amplion types (SC1-1, SC1-2, SC2-1 and SC2-2) were observed, SC1-1 and SC2-1 were the predominant types in Southwestern region and Guangdong-Guangxi region, respectively, and no dominant types were found in Fujian-Jiangxi-Zhejiang region. The sequence differences in SC1-1/SC1-2 were mainly caused by different combinations of two kinds of tandem repeats with 132 bp satellite sequence and 32 bp mini-satellite sequence, respectively. The diversity in SC2-1/SC2-2 likely derived from prophage gene rearrangement. 【Conclusion】Significant diversity in the prophage regions was investigated from ‘Ca. L. asiaticus’ strains collected from different geographical origins. Further studies on these genomic regions would be helpful for deciphering the genetic diversity of ‘Ca. L. asiaticus’ populations in China.

Molecular Cloning and Expression of Translationally Controlled Tumor Protein of Spodoptera litura (Fab.)

LU Meng-Ling, YAN Chao, MAO Gen-Lin, XU Han-Hong

Scientia Agricultura Sinica. 2013, 46(18): 3793-3799. doi:10.3864/j.issn.0578-1752.2013.18.007

Abstract ( 441 )

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【Objective】 The objective of this study is to clone the full-length cDNA of TCTP from Spodoptera litura cells, as well as to analyze its sequence.【Method】The full-length cDNA of TCTP was obtained by RACE-PCR. A phylogenetic tree of TCTP protein in different species was constructed. TCTP was cloned in pET-32a (+) and was expressed as fusion protein in Escherichia coli BL21(DE3) cells.【Result】The full-length cDNA of SLTCTP (GenBank accession number: HQ896486.1) was 829 bp with an open reading frame of 519 and encodes 172 amino acids. According to Protparam on-line tools, the deduced protein was predicted to have a computed molecular mass of 19.67 kD. TCTP cloned in pET-32a (+) was expressed as fusion protein in E. coli BL21 (DE3) cells. In order to obtain much target protein of TCTP, optimal culture conditions of pET-32a (+)-TCTP were studied. Induced by 1mmol•L-1 IPTG for 4 h was the best optimized condition for recombinant protein expression. TCTP gene belonged to the TCTP protein family and had a high similarity with other species.【Conclusion】This study has provided a basis of method for further research on the function of SLTCTP and pest control.

Effects of Different Rotational Tillage Patterns on Soil Physical Properties in Rainfed Wheat Fields of the Loess Plateau

CHENG Ke-1, LI Jun-1, MAO Hong-Ling-2

Scientia Agricultura Sinica. 2013, 46(18): 3800-3808. doi:10.3864/j.issn.0578-1752.2013.18.008

Abstract ( 454 )

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【Objective】Effects of three different rotational tillage patterns, which were pairwise combined with no-tillage(N), subsoiling(S) and conventional tillage(C), on soil physical structure and stability were studied in rainfed wheat fields of the Loess Plateau.【Method】The experiments of N/S (no-tillage in the first year, subsoiling in the second year, again no-tillage followed by alternating the next year), S/C (subsoiling in the first year, conventional tillage in the second year, again subsoiling followed by alternating the next year) and C/N (conventional tillage in the first year, no-tillage in the second year, again conventional tillage followed by alternating the next year) rotational tillage were conducted in rainfed wheat fields of Weibei highland in summer fallow period from 2007 to 2012, soil bulk density, aggregates amount, size distribution and fractal dimension were observed and examined by dry and wet sieving methods.【Result】The results showed that, compared with pretreatment, soil bulk density of N/S, S/C and C/N rotational tillage treatments in harvest period had no significant differences at 0-60 cm. Soil aggregates properties changed significantly in three different rotational tillage treatments, N/S was higher in the macro-aggregate content (R0.25) and stability rate of water-stable aggregates than S/C and C/N treatments at 0-10 cm; N/S was also higher in mean weight diameter(MWD) of water-stable aggregates than S/C and C/N treatments at 0-30 cm. The fractal dimension of aggregates among these treatments were significantly different at 10-30 cm by dry sieving and at 0-10 cm by wet sieving, its value under N/S was lower than S/C and C/N treatments.【Conclusion】 The three different rotational tillage patterns had no significant difference in soil bulk density. N/S conservation rotational tillage pattern increased soil macro-aggregate content and stability, improved dryland soil structure. S/C and C/N rotational tillage patterns disturbed soil layer because of every other year plowing, so its soil structure had no significant improvement.

Characteristic of Soil Available Trace Elements on Abandoned Cropland in the Loess Hilly Region

ZHANG Chao-12, LIU Guo-Bin-12, XUE Sha-12, XIAO Lie-1

Scientia Agricultura Sinica. 2013, 46(18): 3809-3817. doi:10.3864/j.issn.0578-1752.2013.18.009

Abstract ( 461 )

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【Objective】 The research was made to provide a scientific guidance for the sustainable development of ecological restoration and the evaluation of soil quality by studying the effect of abandoned cropland on soil trace elements in the hilly-gully region of the Loess Plateau. 【Method】 The present study investigated the changes of four available trace elements at different slope aspects during succession on abandoned cropland in the loess hilly region. 【Result】 The results showed that during the vegetation succession on abandoned cropland, the content of organic C, total N, available Mn, available Fe, available Zn in this shady slope presented a significant regular variation due to the high soil moisture favorable vegetation conditions, showing no significant difference at the early stage of succession while increased with the increasing years at the later stage. Available Cu exhibited a decrease trend with years. Organic C, total N, and four available trace elements in the sunny slope fluctuated with the increasing years. The density of four trace elements differed significantly at different succession stages. Compared with the shady slope, available Fe and Zn density in the sunny slope was higher in the first 10 years while was lower 10 years later, available Cu density was slightly lower in the first 5 years and was higher thereafter. 【Conclusion】Although the content of four soil trace elements in the cropland increased after abandoned for 20 years, it is still in the extremely lower level, thus natural recovery to improve the status of trace elements in this region probably need more time.

Effects of Ammonium and Nitrate Nitrogen on Growth and Properties of 15N Distribution of Apple Trees

LI Jing, JIANG Yuan-Mao, MEN Yong-Ge, LI Hong-Na, ZHOU Le, WEI Shao-Chong

Scientia Agricultura Sinica. 2013, 46(18): 3818-3825. doi:10.3864/j.issn.0578-1752.2013.18.010

Abstract ( 441 )

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【Objective】 The study was carried out to investigate the growth and properties of 15N allocation and distribution in field grown apple trees with dwarfing interstock. 【Method】 The effectiveness of different forms of ammonium and nitrate nitrogen on growth and properties of 15N distribution of apple trees were studied by using 15N tracer on 1-year-old apple tree (Fuji/SH6/Malus hupehensis Pamp Rehd.).【Result】The results showed that 46 days after fertilization the utilization rate of 15NO3-15N was 3.43%, which significantly higher than 15NH4-15N, and also the growth vigor and root-shoot ratio. The 15NO3-15N partition ratio of base stock was 27.91%, which was also significantly higher than 15NH4-15N partition ratio of base stock of 25.13%. The nitrogen efficiency of NO3--N reduced till 118 days after fertilization, the growth vigor was significantly lower than NH4+-N，and there was no significant difference between the utilization rate of 15NO3-15N and 15NH4-15N. 15NO3-15N partition ratio of base stock (31.19%) was still significantly higher than 15NH4-15N partition ratio of base stock (25.10%). 【Conclusion】 Fertilizer efficiency of nitrate nitrogen was much faster than ammonium nitrogen, but there was no advance in long-term cultivation conditions.

Effects of Zinc Deficiency on Photosynthetic Rate and Chlorophyll Fluorescence Characteristics of Apple Leaves

FU Chun-Xia, ZHANG Yuan-Zhen, WANG Yan-An, FAN Xiao-Dan, YAN Yu-Jing, ZHANG You-Peng

Scientia Agricultura Sinica. 2013, 46(18): 3826-3833. doi:10.3864/j.issn.0578-1752.2013.18.011

Abstract ( 568 )

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【Objective】The purpose of this study is to explore the effects of zinc deficiency on the chlorophyll content, photosynthetic rate and chlorophyll fluorescence characteristics of apple tree leaves, and to reveal the damage mechanism of Zn deficiency to the photosynthetic system of apple leaves.【Method】Normal trees without Zn deficiency symptoms and rosette trees with typical Zn deficiency symptoms (called “little leaf”) in field of ‘Red Fuji’ apple (rootstock Malus hupehensis Rehd) were used as materials, and the zinc content, chlorophyll content, net photosynthesis rate and fluorescence parameters of apple leaves under different Zn stress levels were measured.【Result】Under Zn deficiency, the chlorophyll content, single leaf area and specific leaf weight of apple leaves decreased significantly; the stomatal conductance decreased, intercellular CO2 concentration increased, net photosynthetic rate and water use efficiency also decreased. As the aggravation of Zn deficiency, the original fluorescence Fo rose while Fv/Fo, ΦPSⅡ, qP and Fv’/Fm’ decreased under Zn deficiency. The relative variable flurescence at J-step(VJ) and the ratio of variable fluorescence Fv to the amplitude Fj-Fo at the K-step(Wk) increased, the quantum yield for electron transport further than QA¬ (ETo/ABS), the density of active PSⅡ reaction centers per cross section(RC/CSo) and the performance index on absorption (PI) basis decreased.【Conclusion】Non-stomatal limitation was one of the reasons for the reduction of photosynthesis rate under zinc deficiency. Zn deficiency first caused the damage of oxygen evolving complex (OEC) and then the reactive center of PSⅡ, followed by the inhibited electron transfer of donor side and receptor side of the reaction center at the same time, which finally affected the absorption, transmission and utilization of light energy in apple leaves.

Lenticel Blotch Pit and Ca Nutrition, Distribution and Localization in ‘Fuji’ Apple Fruit

DONG Yu-12, FENG Yun-Xiao-1, GUAN Jun-Feng-1

Scientia Agricultura Sinica. 2013, 46(18): 3834-3841. doi:10.3864/j.issn.0578-1752.2013.18.012

Abstract ( 550 )

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【Objective】 In order to investigate the content and micro-zone distribution of Ca, K and Mg in different parts of apple fruits with lenticel blotch pit and healthy fruits, and to analyze the relationship between Ca localization and lenticel blotch pit occurrence, thus, to clarify the cause of lenticel blotch pit in apple. 【Method】Apple (Malus domestica cv. Spur Fuji) with lenticel blotch pit and healthy fruits were used to determine the content and micro-zone distribution of Ca, K and Mg in fruit using atomic absorption spectrometry and X-ray energy spectra, respectively, and the cellular Ca localization was observed using ultracytochemical method.【Result】Ca content in peel tissue of apple fruit with lenticel blotch pit was significantly lower than that in healthy fruit. However, there were no significant difference in K and Mg contents between lenticel blotch pit fruit and healthy one. The results of X-ray microanalysis showed that the energy spectra intensity peak of Ca and K in peel tissue of healthy fruit were significantly higher than that of the one with lenticel blotch pit, but in the pulp tissue, the peaks of Ca and K were higher and lower in healthy fruit than that with lenticel blotch pit, respectively. And, the highest of K peak appeared in the zone of lenticel blotch pit. Numerous of Ca grains with uniform distribution were deposited in the vacuole of peel and pulp tissue in the healthy fruit. However, less Ca grains were observed in fruits with lenticel blotch pit, and especially, lots of Ca grains were showed at outside of tonoplast in the zone of lenticel blotch pit in peel tissue, and more, the cells shapes were irregular with lower inclusions, and formed some different sizes of vesicles.【Conclusion】Ca content in peel tissue of fruits with lenticel blotch pit was significantly lower than that in the healthy fruits. A lot of K accumulated in zone of lenticel blotch pit in fruit flesh. Intracellular Ca was reduced and got out of the cell in fruits with lenticel blotch pit, which revealed that Ca involved in and influenced the occurrence of the lenticel blotch pit.

Effects of Beer Sludge Compost on Growth and Physiological Characteristics of Apple Seedlings with Different Application Methods

TONG Shao-Wei-1, SHI Lian-Hui-12, LIU Deng-Min-1, JIANG Yuan-Mao-23, SHU Huai-Rui-23, HU Yu-Tong-1, WEI Mei-Yan-1

Scientia Agricultura Sinica. 2013, 46(18): 3842-3849. doi:10.3864/j.issn.0578-1752.2013.18.013

Abstract ( 393 )

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【Objective】 The aim of this experiment was to research the best application pattern of beer sludge compost on apple trees and yield-increasing mechanism. 【Method】 The effects of beer sludge compost treated with three application methods including blending(H), mulching (F), blending and mulching (HF) on the growth of apple trees, leaf physiological characteristics and the root morphology. 【Result】The test results were as follows: The beer sludge compost with different application methods could significantly promote apple seedlings growth. H, HF treatments could significantly enhance dry stem diameter, branch diameter, leaf area per leaf, and leaf area per plant. Chlorophyll a, Chlorophyll b, carotenoid content and Pn were significantly enhanced, chlorophyll a/b and transpiration rate were significantly reduced compared with CK. Beer sludge compost could enhance leaf antioxidant enzyme activities and reduce harm, H and HF treatments were better than F. Root length, root surface area, root volume, root fresh weight, and root number were significantly higher than CK with H, F, HF treatments. Apple seedlings roots growth could be induced and promoted with beer sludge compost, and H and HF treatments were better. The root vigor of H, F, and HF were, respectively, increased by 53.68%, 40.50%, and 77.72% compared with CK. 【Conclusion】The beer sludge compost was applied with three different methods could promote the growth of apple trees, and improve the physiological indexes of leaf and root. In the whole, HF treatment integrated the advantages of H and F treatments, and it was the most effective application method.

Effects of Organic Fertilizer and Biochar on Root System and Microbial Functional Diversity of Malus hupehensis Rehd.

CHEN Wei-1, ZHOU Bo-3, SHU Huai-Rui-2

Scientia Agricultura Sinica. 2013, 46(18): 3850-3856. doi:10.3864/j.issn.0578-1752.2013.18.014

Abstract ( 529 )

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【Objective】 Effects of organic fertilizer and biochar on root system and microbial functional diversity in soil planted with the biennial seedling Malus hupehensis Rehd were studied. Meanwhile, the effect of increasing soil carbon on soil quality and plants were evaluated to provide a theoretical basis for sustainable development of orchard. 【Method】The biennial seedlings of M. hupehensis Rehd was planted in a pot experiment. The root system of plants and microbial functional diversity were analyzed by adding different amounts of organic fertilizer and biochar【Result】The quantity and area of fine absorbing root, the population of bacteria in soil and rhizosphere, the FDA enzyme activity and the microbial functional diversity were improved by application of organic fertilizer or biochar, and combined application of both were proved to be even more effective. Biochar was predominant in increasing area of fine absorbing root, while disadvantaged in improving microbial functional diversity, compared with organic fertilizer. The area of fine absorbing root in 10% organic fertilizer and 6% biochar, 10% organic fertilizer and 3% biochar, 10% organic fertilizer, 6% biochar and 3% biochar was 6.6,10, 2.5, 3.3 and 3.1 times than that of CK.The population of bacteria, actinomycetes and fungi in soil of treatments were 3.32-10.23, 1.2-1.97 and 3.24-5.26 times as much as the control group. The largest amount of rhizosphere actinomycetes was obtained after application of organic fertilizer, while application of 3% rhizosphere biochar corresponded to the largest amount of rhizosphere fungi. 【Conclusion】The root system of plants and microbial functional diversity in soil can be improved by increasing soil carbon, which is beneficial to soil fertilizer and sustainable development of agriculture.

A Solution for Improving Vegetable Circulation Traceability Precision Based on Barcode-RFID Correspondence

QIAN Jian-Ping-1, WU Xiao-Ming-1, FAN Bei-Lei-1, YANG Xin-Ting-1, SUN Lian-Xin-2, CHEN Ming-2

Scientia Agricultura Sinica. 2013, 46(18): 3857-3863. doi:10.3864/j.issn.0578-1752.2013.18.015

Abstract ( 403 )

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【Objective】 The aim of the study is to improve the low traceability precision in vegetable circulation process affected by aggregation and splitting in different package units. The improvement will pave a way to establish quality safety traceability system from farm to table. 【Method】 In the paper, barcode and RFID were adopted for identifying different package units. A portable circulation information acquisition system was developed and the vegetable enterprise management information system was updated.【Result】The relationship of RFID Tag ID and traceability codes on small packages was implemented and the correspondence of circulation process information was collected. Consequently, traceability precision in circulation process was improved on the relationship and correspondence. The solution was applied in the traceability management of a vegetable enterprise in Shandong province. The application test results showed that the traceability barcode reading rate in the range of less than 20 cm as an important stage in the solution was above 95%, and the correct correspondence rate as the other stage was 100%. 【Conclusion】 Therefore, the solution can be applied in order to implement single product or single batch traceability in circulation process.

Discrimination of Kiwi Fruit According to Geographic Origin Based on Organic Compound Fingerprint Analysis

MA Yi-Yan, GUO Bo-Li, WEI Yi-Min, ZHAO Hai-Yan, WEI Shuai

Scientia Agricultura Sinica. 2013, 46(18): 3864-3870. doi:10.3864/j.issn.0578-1752.2013.18.016

Abstract ( 461 )

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【Objective】The organic compounds characteristics and content differences in kiwi fruit from different regions were analyzed, and the validity of indicators for kiwi fruit origin discrimination was assessed, the aim is to explore the feasibility of organic compound fingerprinting in distinguishing the geographical origin of kiwi fruit.【Method】The contents of VC, VE, soluble sugar, total acid, and total sugar were determined in ninety-three samples from Shaanxi (Zhouzhi, Meixian), Sichuan (Muchuan) and Hu’nan (Yongshun) provinces of China, and analysis of variance (ANOVA), multiple comparison analysis, and discriminant analysis (DA) were applied in data analysis. 【Result】The contents of the organic compounds were significantly different in kiwi fruit from different regions. The content of VC was the highest, and those of VE, soluble sugar, and total sugar were the lowest in the samples from Zhouzhi and Meixian, Shaanxi provinces. While the contents of VE, soluble sugar, and total sugar were the highest, and that of total acid was the lowest in the samples from Muchuan, Sichuan provinces. Besides, the content of total acid was the highest and VC was the lowest in the samples from Yongshun, Hu’nan provinces. A linear discriminant analysis procedure produced a successful assignation (92.2% original classification, and 90.0% cross validation)of kiwi fruit according to the region. 【Conclusion】Kiwi fruit samples from different regions have their unique quality characteristics. It is a potential technology that determine the geographical origin of kiwi fruit using organic compound fingerprint analysis.

Changes of the Construction of Myofibrillar Proteins in Chinese Traditional Bacon During Processing

CAO Jin-Xuan-1, ZHANG Yu-Lin-1, HAN Min-Yi-2, JIANG Ya-Ting-1, PAN Dao-Dong-1, 欧Chang-Rong-1

Scientia Agricultura Sinica. 2013, 46(18): 3871-3877. doi:10.3864/j.issn.0578-1752.2013.18.017

Abstract ( 466 )

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【Objective】The objective of the study was to analyze the configuration and microenvironment changes of the myofibrillar proteins in Chinese traditional bacon at different processing points. 【Method】 Raman spectroscopy was used to analyze the changes of amide I, III belt and S-S, C-C and C-N stretching vibration bands and compare the intensity changes of I760, I850/I830, respectively. 【Result】 The content of α-helix decreased significantly accompanied by an final increase in β-sheet during the processing period, while the β-turns and random coil had no obvious change. The intensity of main chains of C-C（α-helix）and C-N stretching vibration was weaken. The intensity of native conformation of disulfide bond decreased during the processing. The normalized intensity of I760 decreased significantly after 2 days when samples were dry-cured, and then increased significantly during the drying process, while the I850/I830 ratio was a significantly decreased.【Conclusion】The main chain and secondary structure of myofibrillar proteins changed during the whole process period, but the hydrophobicity of amino acid residues was enhanced just in the drying process.

Effects and Molecular Mechanism of Nicotinamide on Attenuating Apoptosis of Rosiglitazone-induced Porcine Preadipocytes

PANG Wei-Jun, XU Ru-Xiang, XIONG Yan, SUN Shi-Duo, YANG Gong-She

Scientia Agricultura Sinica. 2013, 46(18): 3878-3886. doi:10.3864/j.issn.0578-1752.2013.18.018

Abstract ( 458 )

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【Objective】This study was performed to explore the effect and molecular mechanism of nicotinamide (NIC) and rosiglitazone (RSG) on apoptosis of porcine preadipocytes for finding a novel method to control adipogenesis through regulation of preadipocyte apoptosis.【Method】Cells were cultured in high glucose DMEM medium containing 0, 0.2, 0.4, 0.6, 0.8 and 1.0 mmol•L-1 RSG. After being treated for 48 h, cells were stained with Hoechst33258 and Annexin-V-FITC/PI and observed under microscope. Simultaneously, cells were harvested for analysis of flow cytometry and collection of total protein to detect protein levels of several genes related with apoptosis. Based on the above results, cells were further co-cultured in DMEM medium containing 0, 0.1, 0.2 and 0.3 mmol•L-1 NIN with 1 mmol•L-1 RSG. After being treated for 48 h, cells were stained and observed. Cells were harvested for analysis of flow cytometry and collection of total, cytoplasmic and nuclear proteins for Western blotting analysis.【Result】RSG induced preadipocyte apoptosis by a dose-dependent way and reduced cell number. The levels of Bcl-2 and cleaved Caspase-3 increased, whereas the level of Bax decreased in higher concentration of RSG. Moreover, 0.1（P＜0.05）、0.2（P＜0.01）and 0.3 mmol•L-1 （P＜0.01）NIC significantly attenuated RSG-induced apoptosis of preadipocytes. The levels of Bcl-2 and cleaved Caspase-3 were down-regulated, whereas the level of Bax was up-regulated in treatment with 0.3 mmol•L-1 NIN + 1 mmol•L-1 RSG. Compared with the control (1 mmol•L-1 RSG + 0 mmol•L-1 NIC), in treatment with 0.3 mmol•L-1 NIC + 1 mmol•L-1 RSG, Sirt1 was transfered from cytoplasm into nucleus and the level of cleaved Caspase-3 was reduced.【Conclusion】NIC attenuated RSG-induced apoptosis of porcine preadipocytes via cytoplasm/nucleus transposition of Sirt1 and down-regulating the level of nuclear cleaved Caspase-3.

The Expression of Paternal Imprinting Genes in Bovine Early Stage Embryo

ZHANG Chun-Qiang, ZHAO De-Chao, HAN Zhi-Ling, ZHANG Wen-Guang, ZHANG Jia-Xin

Scientia Agricultura Sinica. 2013, 46(18): 3887-3893. doi:10.3864/j.issn.0578-1752.2013.18.019

Abstract ( 457 )

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【Objective】The objective of the experiment is to investigate the expression pattern of paternal imprinting genes in bovine early stage embryo.【Method】The expression profile of paternal imprinting genes Gnas, Grb10, and Xist were detected in bovine embryo derived from in vitro fertilization (IVF) and parthenogenetic activation (PA) by real-time PCR. 【Result】 There were no difference in imprinted expression profile of Gnas between IVF and PA embryo. Grb10 expression was significantly higher during 2-cell stage in PA embryo than that of IVF embryo, however, no difference was observed in expression of Grb10 between IVF and PA embryos from 4-cell satge to blastcyst stage. Xist expression was similar between PA and IVF embryo from 2-cell to 8-cell stage. But in the later stage, Xist expression was higher significantly in PA embryo than that in IVF embryo. 【Conclusion】Gnas, Grb10 and Xist were expressed differently in bovine IVF embryo and PA embryo from 2-cell stage to the blastocyst stage, and they might play an important role in bovine embryo development.

Profiling of Differential Expressed MicroRNA in Intramuscular Fat and Subcutaneous Fat of Simmental and Bioinformatic Analyses of miR-27b Target Gene

WANG Hai-Yang, ZHENG Yue, LI Hui-Xia, HAN Zhao-Yu, WANG Gen-Lin

Scientia Agricultura Sinica. 2013, 46(18): 3894-3900. doi:10.3864/j.issn.0578-1752.2013.18.020

Abstract ( 435 )

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【Objective】 In order to explore the role of microRNA in the growth and development process and its regulatory mechanism in intramuscular fat, the profiling of differential expressed microRNA in intramuscular fat and subcutaneous fat of Simmental were analyzed by microarray and real-time PCR (qRT-PCR). The target genes and signal pathway of miRNA-27b were predicted by bioinformatics software and database.【Method】Microarray of bovine microRNA was employed to detect the differentially expressed microRNA between intramuscular adipose tissue and subcutaneous adipose tissue. Then the expression of four selected microRNAs (miR-140, miR-145, miR-143 and miR-27b) in intramuscular adipose and subcutaneous adipose was also validated by qRT-PCR method. Target scan algorithm was used to predict target gene of miR-27b, and the result of gene set was analyzed by bioinformatics of GO annotations, GO term enrichment and KEGG enrichment methods. 【Result】 It was revealed that there were 88 microRNAs with significant differential expression between bovine intramuscular fat and subcutaneous fat (P<0.01). The expression of miR-140, miR-145, miR-143 and miR-27b which were also detected by qRT-PCR was in high concordance with microarray results. The results of bioinformatics indicated that target genes of miR-27b were mostly enrichment in MAPK, Wnt, Hedgehog, TGF-beta, GnRH signaling pathway, especially in MAPK pathway. 【Conclusion】There are specific microRNA expression profiles in bovine intramuscular adipose tissue and subcutaneous adipose tissue. Some differentially high expressed microRNA in intramuscular adipose tissue such as miR-27b may play important roles by distinct pathway in bovine intramuscular adipogenesis.

Phylogenetic Analysis of Avain-like H1N1 Swine Influenza Virus in Shaanxi Province

REN Juan-Juan, WANG Jing-Yu, LIU Hong-Yan, LIU Wan-Hua, WU Ning, QIU Yuan-Hao, TANG Pan, JIANG Yue-De

Scientia Agricultura Sinica. 2013, 46(18): 3901-3913. doi:10.3864/j.issn.0578-1752.2013.18.021

Abstract ( 426 )

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【Objective】The prevalence and transmission of swine influenza in Shaanxi Province were studied.【Method】Nasal/tracheal swap samples were randomly collected from apparently healthy pigs in farms and slaughterhouses in Shaanxi province between October 2011 and May 2012. The samples were firstly separated by SPF chick embryos, and the HA-positive samples were identified as swine influenza virus using RT-PCR. Then, the full genomes of these isolates were sequenced and used to re-construct phylogenetic trees with software MEGA 4 and analyze genetic variations. 【Result】 Six H1N1 influenza viruses were isolated and identified among 500 samples, with isolation rate of 1.2%. The full length genomes were obtained from six isolates, with nucleotide similarity of 98.3–99.1% . Phylogenetic analysis showed that six H1N1 virus isolates were located into the lineage of avain-like H1N1 swine influenza viruses. The nucleotide similarities were 97–99% between six isolates and SIVs from Jiangsu, Liaoning, Shandong, and Hong Kong. The mutated nucleotides of eight gene fragments for six isolates were detected at receptor binding sites and potential glycosylation sites. These genetic variations will affect antigenicity, host adaptability and virulence of the viruses.【Conclusion】 The H1N1 swine influenza virus were firstly isolated from apparently healthy pigs in Northwestern China, and six full length genomes of these isolates were obtained. Phylogenetic analysis suggested six isolates were avain-like H1N1 swine influenza virus. Mutations were also detected in some amino acid sites of these isolates.

Dipotassium Glycyrrhizinate Against MDV on CEFs by Promoting MDV-Infected Cells Apoptosis in vitro

CONG Xiang-Ming-1, NIU Li-1, BAI Xi-Yun-1, WU Cai-Hong-2, SUN Yao-Gui-1, HE Jun-Ping-1, BAI Yuan-Sheng-3, LI Hong-Quan-1

Scientia Agricultura Sinica. 2013, 46(18): 3914-3921. doi:10.3864/j.issn.0578-1752.2013.18.022

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【Objective】The aim of the study is to test the antiviral activity and mechanism of dipotassium glycyrrhizinate (DG) against Marek’s disease virus (MDV) in vitro.【Method】 Using MTT test and standard plaque reduction assay on chick embryo fibroblast cells (CEFs), the antiviral effect of DG against MDV was observed by antiviral assay, time-of-addition assay and virucidal assay, and the antiviral mechanism was tested by cell apoptosis assay.【Result】Antiviral assay showed that DG could reduce the damage of MDV to CEFs , EC50 was (893.5±36.99) µg•mL-1，SI＞3.36 and the inhibition ratio was 94.4%, at the maximum with no-cytotoxic concentration. Time-of-addition assay and virucidal assay demonstrated that DG could inhibit the viral replication in the full replication cycle and inactivated MDV particles in non time-dependent manner. Moreover, DG could promote MDV-infected cells apoptosis in vitro.【Conclusion】DG could inhibit MDV on CEFs by promoting MDV-infected cells apoptosis in vitro.

Expression of RVNP in the Silk Gland of Transgenic Silkworm (Bombyx mori)

YOU Zheng-Ying-1, WEI Hao-13, RUAN Xi-Zhen-2, YE Lu-Peng-1, WANG Shao-Hua-1, ZHOU Ji-Yong-2, ZHONG Bo-Xiong-1

Scientia Agricultura Sinica. 2013, 46(18): 3922-3929. doi:10.3864/j.issn.0578-1752.2013.18.023

Abstract ( 468 )

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【Objective】In order to obtain a potent technology which can produce high value RVNP protein and then lay a foundation for the production of vaccines, the silk gland bioreactor was employed as an expression system in this study.【Method】The nucleoprotein gene of rabies virus ERA strain (RVNP) was cloned by RT-PCR, and the RVNP was firstly connected to the downstream of middle silk gland-specific Ser1 promoter, and then inserted into pBA3EGFP, an expression vector which contained an EGFP reporter gene. By using the transgenic method, the silk gland bioreactor of piggyback-mediated transgenic silkworm was obtained. 【Result】Twenty-six broods of transgenic silkworms (G1) were obtained and the GFP-positive percentage of broods was 59.09%. Further PCR identification indicated that RVNP had been integrated into silkworm genome, and Western blot analysis showed that the protein might be attached to the sericin which was secreted into the cocoon with the spinning of transgenic silkworms. 【Conclusion】Transgenic silkworm strain able to express exogenous RVNP protein was obtained. The present system is expected to become a high efficiency, low-cost and stable technical system to produce genetic engineering rabies vaccine.

Agronomic Traits Development and Evolution of Winter Wheat in Huanghuai Basin

WU Xiu-Ting-12, ZHANG Zheng-Bin-1, XU Ping-1, QI Ya-Juan-12, WANG Yu-Ying-3, HE Xiu-Ping-3, GAO Hui-Ming-3

Scientia Agricultura Sinica. 2013, 46(18): 3930-3938. doi:10.3864/j.issn.0578-1752.2013.18.024

Abstract ( 457 )

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【Objective】The objective of this research was to analyze the evolutionary regularity of wheat agronomic traits and study the adaptation of winter wheat to climate change and the artificial high yield breeding.【Method】The agronomic traits of diploid, tetraploid, hexaploid wheat and modern wheat varieties were recorded in different growth periods and the yield traits were measured after harvesting. 【Result】 The results showed that the tillering number and leaf number tended to reduce in wheat evolution. In the evolution of diploid → tetraploid → hexaploid, the plant height tended to increase; from the wild hexaploid wheat to modern wheat varieties, the plant height and biological yield per plant tended to decrease. In the returning green-heading period, the evolution of total leaf area per plant was diploid＜tetraploid＜hexaploid＜landraces＜modern wheat varieties, on the contrary, in the flowering-grain filling stage, the evolution of total leaf area per plant was hexaploid＞tetraploid＞landraces and modern wheat varieties＞diploid. The biological yield per plant tended to decrease while the grain yield and harvest index tended to increase.【Conclusion】These results indicated that the wheat wild species kept the genetic character and growth pattern of adapting to low temperature and grew slowly. Under the natural climate warming selection and high yield plant type breeding selection, modern wheat varieties tended to mature earlier with fewer tillering number to avoid cold damage and more dry matter transported to the grains from leaves.

Detection of Sweet potato chlorotic stunt virus Strain WA by Reverse Transcription Loop-Mediated Isothermal Ampli?cation Reaction

QIAO Qi, ZHANG Zhen-Chen, QIN Yan-Hong, ZHANG De-Sheng, TIAN Yu-Ting, WANG Shuang, WANG Yong-Jiang

Scientia Agricultura Sinica. 2013, 46(18): 3939-3945. doi:10.3864/j.issn.0578-1752.2013.18.025

Abstract ( 426 )

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【Objective】The objective of this study is to develop a one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for rapid and sensitive detection of Sweet potato chlorotic stunt virus strain WA (SPCSV-WA). 【Method】Four RT-LAMP primers, designed on the basis of the coat protein (CP) gene sequences of SPCSV-WA, and template RNA from infected leaves were used for one-step RT-LAMP which were carried out under isothermal conditions at 65℃ for 1 h. RT-LAMP products were analyzed by electrophoresis in agarose gels followed by staining with SYBR green I. A fragment from the RT-LAMP product was then cloned and sequenced to con?rm the spect?city of the assay. The RT-LAMP, and meanwhile compared with the conventional RT-PCR assay, was performed to detect SPCSV-WA from 14 sweet potato samples. 【Result】The RT-LAMP showed a high specificity and the detection limit was 101 copies/µL RNA template. A fragment cloned from the RT-LAMP product indicated that the nucleotide sequence cloned was SPCSV-WA CP. Fourteen sweet potato samples testing results of RT-LAMP were consistent with that of RT-PCR. 【Conclusion】 The RT-LAMP described in this study represents a very sensitive, speci?c and rapid assay for the detection of SPCSV-WA.

The Effect of LEP and STAT5a Genes on Milk and Growth Traits of Laoshan Dairy Goat

LIU Yuan-Feng-1, WANG Gui-Zhi-1, LI Qiu-Mei-2, CAO Hai-Yang-1, PI Xiu-Shuang-1, WANG Jian-Min-1

Scientia Agricultura Sinica. 2013, 46(18): 3946-3954. doi:10.3864/j.issn.0578-1752.2013.18.026

Abstract ( 416 )

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【Objective】 The objective of this experiment is to explore the genetic variation and interaction of LEP and STAT5a genes in Laoshan dairy goat and its association with production traits. 【Method】 There are 174 lactating ewes of Laoshan dairy goats, of which 89 goats having complete records of milk yield are from dairy goat breeding farm and 84 goats are from farm house-holds.DNA pooling technology and candidate genes by direct sequencing technology were used to detect the polymorphisms in exon 2, exon 3 of LEP gene and exon 10, exon 16 of STAT5a gene. The method of least squares was used to analyze the correlation effects of polymorphic loci associated with milk composition, milk yield at 300 d and growth traits. 【Result】 There was no polymorphism in exon 2,3 of LEP gene and in exon 10,16 of STAT5a gene. A mutation from A to G was found in intron 2 of LEP gene, which had a significant effects (P＜0.01 or P＜0.05) on milk yield at 300 d, rump width of goats from farm and body height of goats from farm house-holds. Model and groups had a significant effect (P＜0.05) on milk traits and growth traits. There were two linked mutations on intron 9 of STAT5a gene, which had a significant effects on body length, heart girth (P＜0.01) and had no significant effect on milk traits (P＞0.05) in all groups. It had a significant effects on heart girth (P＜0.01) of goats from farm house-holds.【Conclusion】 Results of this study showed that the candidate genes LEP and STAT5a have polymorphisms in Laoshan dairy goat. There was no significant interaction between the two genes. The locus of L393 has a significant effects on milk yield at 300 d and rump width of goats from breeding farm, and on body height of goats from farm house-holds. The loci of S163/S206 have a significant effect on body weight, body length and heart girth of all groups, and on heart girth of goats from farm house-holds. So they could provide valuable genetic markers for milk yield and some growth traits of Laoshan dairy goat.

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