Scientia Agricultura Sinica

Factors Optimization of Pollen Electroporation Transformation and Identification of Transgenic Wheat

ZHANG Xiao-Hong, ZHAO Xue-Jing, LI Bo, LI Fei-Fei, LIU Pei-Xuan, MIN Dong-Hong

Scientia Agricultura Sinica. 2013, 46(12): 2403-2411. doi:10.3864/j.issn.0578-1752.2013.12.001

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【Objective】The objective of this study was to establish a pollen electroporation transformation system for common wheat (Triticum aestivum L.) by optimizing the main parameters of transformation for obtaining transgenic wheat plants.【Method】The pollens of wheat were collected in the pollen germination buffer mixed with GUS gene and treated with electroporation, and then pollinated Jimai19 to obtain transgenic plants. The putative transformed plants were confirmed by PCR, Southern blot，histochemical staining of GUS positive reaction.【Result】The optimization parameters of pollen electroporation transformation were summarized: pulse field strength 6 kV•cm-1, operating on ice, pollen density 5×106 pollens/mL, and pollinating on the 5th day after castration. Southern blot proved that two T2 transgenic wheat lines showed positive signals of GUS gene. Meanwhile, histochemical staining showed that the blue color was detected in young roots, leaves of the transgenic plants.【Conclusion】Exogenous gene was integrated into the genome of common wheat by the pollen electroporation transformation method and stably expressed in progeny of transgenic plant.

Cloning and Functional Analysis of the Plasma Membrane Aquaporins Gene in Lolium perenne L.

ZHOU Chun-Lei, LI Ren, WU Xin-Xin, YANG Rong-Chao, ZHANG Hai-Jun, ZHANG Na, ZHAO Bing, GUO Yang-Dong

Scientia Agricultura Sinica. 2013, 46(12): 2412-2420. doi:10.3864/j.issn.0578-1752.2013.12.002

Abstract ( 588 )

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【Objective】To provide an insight into mechanism of drought resistance and cultivar development in Lolium perenne L., the sequence characteristics of the aquaporin gene LpAQP in L. perenne L. was analyzed and the expression profiling was studied.【Method】The full-length cDNA sequence of LpAQP was isolated by RACE. The obtained cDNA sequence and the deduced amino acid sequence was analyzed. In order to follow the intracellular localization of the protein, the GFP sequence was fused downstream to the LpAQP coding region and the fusion gene LpAQP::GFP was transferred into onion epidermal cells by biolistic method. The real time-PCR was adopted to study the expression profile of gene LpAQP. The southern blotting was used to analyze the copies number of the LpAQP. 【Result】The potential open reading frame (ORF) of LpAQP (GenBank Accession No. JX569791) was 867 bp, encoding a protein of 288 amino acids with a predicted molecular mass of 30.7 kD. Bioinformatics analysis demonstrated that LpAQP exhibited a typical structure with an internal symmetry showing two highly conserved Asn-Pro-Ala (NPA) motifs and six membrane-spanning domains, and possessing the MIP family signal consensus sequence. The LpAQP amino acids showed high identity with other plant species PIP subfamily by NCBI homology comparison analysis. Phylogenetic analysis indicated that LpAQP was clustered with the HvAQP from Hordeum vulgare and TaAQP from Triticum aestivum. The result of transient expression showed that LpAQP gene was probably located in the membrane. Southern blotting analysis indicated that LpAQP was a single-copy gene in L. perenne L. Real-time PCR analysis showed higher expression of LpAQP gene was observed in shoot than other tissues. LpAQP expression level was up-regulated firstly, then down-regulated under drought stress.【Conclusion】 An aquaporin gene LpAQP was cloned by RACE from L. perenne L. This gene is regulated by drought stress. This study will provide important information for the future research on the gene-expression regulation during drought stress.

Responses of Main Traits of Maize Hybrids and Their Parents to Density in Different Eras of China

LI Cong-Feng, ZHAO Ming, LIU Peng, ZHANG Ji-Wang, YANG Jin-Sheng, LIU Jing-Guo, WANG Kong-Jun, DONG Shu-Ting

Scientia Agricultura Sinica. 2013, 46(12): 2421-2429. doi:10.3864/j.issn.0578-1752.2013.12.003

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【Objective】The purpose of this study was to investigate the evolution characteristics of main agronomic traits and the responses to density of maize hybrids and their parents released in different eras, thus providing the critically important information for further developing the promising maize cultivars.【Method】Sixteen elite parents and their maize hybrids widely popularized and utilized in China released in 1960s, 1980s and 2000s were used to analyze the changes in main agronomic and physiology traits associated with yields in genetic improvement made during the past four decades in 2007-2008.【Result】The results showed that compared with 1980s and 1960s hybrids and their parents, the modern varieties had higher grain yield with the eras evolution, the grain yield increased by 25.7%, 35.8% and 16.3%, 29.7%, respectively. The 100-kenerl-weight increased significantly, but no significant difference was observed in heterosis index of maize hybrid under high density. However, plant height, ear position height and ear position height/plant height of hybrids released in different eras did not change significantly with eras evolution, while plant stem diameter increased, and leaf orientation value was higher significantly, the plant density-resistance and lodging-resistance enhanced significantly. Compared with 1980s and 1960s hybrids, the contemporary hybrids and their parents had obviously higher dry matter accumulation, post-anthesis dry matter accumulation/total dry matter accumulation and harvest index, especially the distribution ratio in post-anthesis was significantly increased of dry matter of contemporary hybrids. Also, the contemporary hybrids photosynthesis capacity (mean leaf index, chlorophyll contents, and mean net assimilation rate) had obviously advantages under high density, and 2000s hybrids had higher grain-leaf ratio, and leaf area per unit showed more contribution to their yields. The correlation analysis indicated that yield was positively correlated with 100-kernel weight, plant stem diameter and leaf orientation value, but not significantly correlated with plant height, ear height and plant height/ear height, positively significantly correlated with biomass, ratio of post-anthesis dry matter accumulation and mean net photosynthetic rate. The grain-leaf ratio and obtained grain yield of leaf area per unit of the contemporary hybrids positively significantly correlated with its grain yield, but not positively with their parents. So physical density-resistance of high-yielding maize population was significantly ameliorated.【Conclusion】These results demonstrated that maize grain yield enhancing mainly attributed by synergistically increased plant morphology and physiological density tolerance improvement in the progress of genetic improvement for maize in China.

Canopy Structure and Photosynthetic Characteristics of High Yield and High Nitrogen Efficiency Summer Maize

JIN Li-Bin, ZHANG Ji-Wang, LI Bo, CUI Hai-Yan, DONG Shu-Ting, LIU Peng, ZHAO Bin

Scientia Agricultura Sinica. 2013, 46(12): 2430-2439. doi:10.3864/j.issn.0578-1752.2013.12.004

Abstract ( 657 )

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【Objective】Canopy structure and characteristics of high yield and high nitrogen efficiency summer maize were studied to discuss the physiological mechanism of achieving high yield and high nitrogen efficiency, which will provide a theoretical basis for high yield and high nitrogen efficiency cultivation of summer maize.【Method】Integrated management experiment (MT) and nitrogen treatments (NT) were designed to achieve high yield and high nitrogen efficiency and then reveal canopy structure and photosynthetic characteristics of summer maize by optimizing integrated agricultural management, such as sowing date, sowing methods, planting density, fertilizers applied date and levels, and so on.【Result】In NT, grain yield and light transmission of ear layer and ground floor achieved maximum at 184.5 kgN•hm-2 while the leaves area index (LAI) high value duration was long. Beyond this level, light transmission and net photosynthetic rate of ear layer decreased actually. In MT, the leaf area index (LAI) of further optimized combination of cropping systems and fertilizer treatment (Opt-2) always remained above 4.4 from twelve-leaf stage (V12) to six weeks after tasseling stage (6WAT) and its decrease speed was low in latter growing period; its light transmission of ear layer and ground floor, the internode length above ear, the uniformity of plant character was high, achieving grain yield of 10.91 t?hm-2 and nitrogen use efficiency of 54.97 kg?kg-1.【Conclusion】Grain yield did not increase as nitrogen fertilizer application increasing without limit and light transmission decreased when nitrogen fertilization was large. With an integration of agronomic measures and fertilizers management, the LAI high value duration of Opt-2 was longer. light transmittance of ear layer, the uniformity of plant character, the net photosynthetic rate, grain yield and nitrogen use efficiency were high. Consequently, Opt-2 achieved both higher yields and higher nitrogen use efficiency.

Effects of Waterlogging on Stem Lodging Resistance of Summer Maize Under Field Conditions

REN Bai-Chao, ZHANG Ji-Wang, LI Xia, FAN Xia, DONG Shu-Ting, LIU Peng, ZHAO Bin

Scientia Agricultura Sinica. 2013, 46(12): 2440-2448. doi:10.3864/j.issn.0578-1752.2013.12.005

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【Objective】Lodging is an important factor limiting grain yield of summer maize. This study was conducted to investigate the effects of waterlogging on the lodging resistance of summer maize under field conditions. 【Method】Two summer maize cultivars, Denghai605(DH605) and Zhengdan958(ZD958), were used as experimental materials. The field experiment was performed to explore the effects of waterlogging in different durations (3 and 6 days) on the morphology, rind penetration strength, bending property and microscopic structures of summer maize stalk at three-leaf stage (V3), six-leaf stage (V6), and ten days after the tasseling stage (10VT). 【Result】Results showed that the third internodes width and stalk rind penetration strength reduced significantly after waterlogging, and the decrease of the stalk rind penetration strength of two cultivars was the most in the treatment of waterlogging at V3 for 6 days, with 32.53% in DH605 and 42.73% in ZD958. Waterlogging at V3 and V6 decreased the stalk cortex thickness, the vascular bundle sheath thickness and vascular bundle number much more than waterlogging at 10VT, with the increase of waterlogging duration. Furthermore, waterlogging increased barrenness, and decreased grain yield, kernel number per ear and 1000-grain weight, and the response to different waterlogging treatments was waterlogging at V3>waterlogging at V6>waterlogging at 10VT and waterlogging for 6 days>waterlogging for 3 days.【Conclusion】Waterlogging had significant effects on grain yield and plant morphology. Waterlogging reduced the third internodes width, stalk rind penetration strength and bending property, the stalk cortex thickness, the vascular bundle sheath thickness and vascular bundle number, resulted in the decrease of the lodging resistance. It was most susceptible to be waterlogged at V3, followed by V6 and 10VT, and the effects decreased with the increase of waterlogging duration.

cDNA-AFLP Analysis of Differential Gene Expression in Pepper Inoculated with Endophytic Bacillus amyloliquefaciens Fy11

YANG Rui-Xian, FAN Xiao-Jing, QIU Si-Xin, CAI Xue-Qing, HU Fang-Ping

Scientia Agricultura Sinica. 2013, 46(12): 2449-2458. doi:10.3864/j.issn.0578-1752.2013.12.006

Abstract ( 521 )

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【Objective】The objective of this study is to analyze extensive transcription profiling of pepper seedlings inoculated with an endophytic bacterial strain, Bacillus amyloliquefaciens Fy11, and elucidate the molecular mechanism of endophytic Bacillus and plant interaction.【Method】cDNA-AFLP technique was used to conduct transcription profiling of pepper seedlings across five sampling time points after inoculation with Fy11. The validation of cDNA-AFLP expression patterns was analyzed by qRT-PCR.【Result】 A total of 18 620 transcript derived fragments (TDFs) were obtained using cDNA-AFLP with 256 primer pairs, 353 (1.89%) displayed altered expression patterns after inoculation. Two-hundred and fifty seven differentially expressed TDFs produced reliable sequences after cloning and sequencing. Two-hundred and twenty nine expressed sequence tags (ESTs) of unigenes were obtained after assembling, of which 144 showed up-regulated and 85 down-regulated. Blastx analysis and functional annotations were then performed and the results revealed that the 156 ESTs had predicted gene products mainly implicated in energy (10.92%), metabolism and disease/defense (each accounted for 8.73%), signal transduction (7.42%), transporter (6.99%), cell structure (6.55%), transcription (5.68%), cell growth (3.93%), protein destination and storage (3.49%), protein synthesis (2.18%), secondary metabolism and intracellur traffic (each accounted for 1.75%). The 65 ESTs (28.38% of the sequenced total 229 ESTs) had no match to known genes, and the 8 ESTs (3.49% of the sequenced total 229 ESTs) were highly homologous to unknown functional proteins. Ten differential genes related to disease/defense, transcription, and signal transduction were chosen for further qRT-PCR expression patterns, which confirmed the cDNA-AFLP profiles. 【Conclusion】 Endophytic bacteria and plant interaction involved in multifaceted biochemical and physiological reactions, including concerted regulation of the genes involved in different pathways, like disease/defense, transcription, protein metabolism, signal transduction, as well as abiotic stresses. These results provide information for further elucidation of molecular mechanism of endophytic bacteria and plant interaction.

Photosynthetic Physiology of Odontothrips Damaged Medicago sativa

KOU Jiang-Tao, SHI Shang-Li, HU Gui-Xin, ZHOU Wan-Hai, YAO Tuo

Scientia Agricultura Sinica. 2013, 46(12): 2459-2470. doi:10.3864/j.issn.0578-1752.2013.12.007

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【Objective】Photosynthetic physiological response mechanism of alfalfa to thrips and compensational mechanism of thrip damage were studied.【Method】Thrip resistant clone R-1 and susceptible clone I-1 were used to investigate the changes of gas exchange parameter and kinetic parameter of chlorophyll fluorescence of differently damaged leaves of R-1 and I-1 at initial flower stage under continuous thrips damage.【Result】As the increase of leaf damage grade, the chlorophyll content and water use efficiency (WUE) increased, net photosynthetic rate (Pn) increased firstly, then decreased, while intracellular CO2 concentration (Ci), transpiration rate (Tr) and stomatal conductance (Gs) decreased. Light compensation point (Lcp), dark respiration rate (Rd), apparent quantum yield (AQY), light saturation point (Lsp), maximum net photosynthetic rate (Pnmax) increased firstly and then decreased in R-1, and for I-1, Lcp and Rd increased, AQY and Lsp decreased, Pnmax increased firstly and then decreased. Under the same damage level, Pn, AQY, Lsp and Pnmax of R-1 were higher than I-1, while Rd and Lcp were lower than I-1. As the increase of leaf damage grade, F0 of R-1 decreased firstly and then increased, photosynthetic efficiency of PSII (ФPSII), non-photochemical quenching coefficient (NPQ), photochemical quenching coefficient (qP), potential activity of PSII (Fv/F0) and original light energy transformation efficiency of PSII (Fv/Fm) increased firstly, then decreased, F0 of I-1 increased, while NPQ and qP increased firstly, then decreased, ФPSII, actual Fv/F0, and Fv/Fm decreased. Under the same damage grade, R-1 was found with lower F0 and higher ФPSII, qP, Fv/F0 and Fv/Fm, compared with I-1. Comparatively, gas exchange parameter, light response parameter and kinetic parameter of chlorophyll fluorescence fluctuations of R-1 were all less than that of I-1. 【Conclusion】Alfalfa PSII reaction center was injured due to thrip damage, which resulted in a decreased photosynthetic efficiency, indicating that R-1 was with a higher photosynthetic efficiency and a more effective photosynthetic compensation efficiency under minor thrip damage compared with I-1, as well as a relatively stronger resistance against thrip damage.

Analysis of Temporal and Spatial Variation of Soil Nutrients in the Winter Wheat-Summer Maize Rotation Field

CUI Bei, WANG Ji-Hua, YANG Wu-De, CHEN Li-Ping, HUANG Wen-Jiang, GUO Jian-Hua, SONG Xiao-Yu, FENG Mei-Chen

Scientia Agricultura Sinica. 2013, 46(12): 2471-2482. doi:10.3864/j.issn.0578-1752.2013.12.008

Abstract ( 624 )

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【Objective】The objective of this study was to explore the temporal and spatial variation rules of soil nutrients in winter wheat-summer maize rotation field and the influence of long-term rotation on soil nutrients.【Method】The temporal and spatial variability characteristics of soil properties on the wheat-corn rotation at the National Precision Agriculture Experimental Station were evaluated using GIS and geostatistical methods with soil nutrient classification standard in Beijing.【Result】Results showed that, from 2000 to 2007, the study area was seriously lack of potassium and phosphorus nutrients with downward trend, the soil total nitrogen content was high in some areas, but it also had the downward trend. Organic matter content slightly increased within a state of equilibrium. All soil nutrients were ranged from 11.00% to 51.71%, which belonged to medium degree of variation. With the passage of time, the effective phosphorus and organic matter, total nitrogen and alkali solution nitrogen spatial distribution law of soil available phosphorus (AP), alkali-hydrolyzable nitrogen (AN), total nitrogen (TN) and organic matter (OM) were stable. Although the spatial distribution law of available potassium (AK) in 2001 was different from the ones in 2000 and 2007, the spatial distribution of AK in 2000 and 2007 was similar. These showed that the spatial distribution of soil nutrients has a certain stability.【Conclusion】The study area had high nutrient content in the southern and northeast region, a little content in the central region. When the soil physical characteristics had big change, space structure characteristics of soil nutrients will be affected by certain influence; but when field cultivation management was relatively consistent over a long period of time, structural factors (especially soil characteristics and climate) will play the leading role.

Effect of Nitrogen Fertilizer Application on Yield, Nitrogen Absorption and Soil Nitric N in Super-High-Yield Summer Maize

WANG Yi-Lun, LIU Tian-Xue, ZHAO Peng, ZHANG Xu, TAN Jin-Fang, LI Chao-Hai

Scientia Agricultura Sinica. 2013, 46(12): 2483-2491. doi:10.3864/j.issn.0578-1752.2013.12.009

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【Objective】The objective of this study is to provide a scientific and rational nitrogen fertilizer amount for super-high-yield summer maize (≥12 000 kg•hm-2). 【Method】field experiments were conducted to study the effect of nitrogen fertilizer application on grain yield, nitrogen metabolism, nitrogen absorption, use efficiency of nitrogen fertilizer and soil nitric N on the farm of super-high-yield summer maize in 2007 and 2008. 【Result】The results showed that, by applying nitrogen fertilizer, the yields increased by 6.76%-9.62% and 5.21%-9.80%, respectively in 2007 and 2008. With the increased nitrogen levels, the production of summer maize increased at first and then decreased. Nitrogen applied at 300 kg•hm-2 had the highest yield and incomes, increased the activity of nitrate reductase and sucrose phosphate synthase, and promoted the assimilation and accumulation of nitrogen to maintain soil nitrate nitrogen balance, and the recovery efficiency and agronomic efficiency of the applied N were 16.12% and 3.69 kg•kg-1, respectively. The economic optimum nitrogen application rate was 255.4 kg•hm-2. 【Conclusion】Considering the yield, economic benefits, nitrogen metabolism, nitrogen accumulation, nitrogen use efficiency and soil nitrogen balance, the proper nitrogen application rate for super-high-yield summer maize is about 255-300 kg•hm-2 in clay loam fluvo-aquic soil in north Henan province.

Effects of Tillage Practices on Soil Erosion and Phosphorus Loss in Sloping Cropland of Purple Soil

HE Xiao-Ling, ZHENG Zi-Cheng, LI Ting-Xuan

Scientia Agricultura Sinica. 2013, 46(12): 2492-2500. doi:10.3864/j.issn.0578-1752.2013.12.010

Abstract ( 583 )

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【Objective】The characteristics of soil erosion and phosphorus losses caused by rainfall on the scattered sloping cropland in purple soil during the maize maturity were studied in order to provide a theoretical evidence for the prediction, assessment and control of soil nutrient loss and coordinate land management to improve the ecological environment in purple soil area.【Method】The simulated rainfall experiments and runoff plot experiments were employed to investigate the characteristics of surface erosion, subsurface runoff and phosphorus loss among different tillage practices(flat planting, longitudinal ridge and cross ridge) during the maize maturity. Rainfall intensity is 1.7 mm•min-1, rainfall duration for 40 mins.【Result】Tillage practices had no significant influence on the phosphorus concentration in surface runoff，longitudinal ridge produced maximal amount of surface erosion and surface phosphorus loss. Meanwhile，it produced minimal subsurface runoff and subsurface phosphorus loss. Conversely, cross ridge produced minimal surface erosion and surface phosphorus loss，but the subsurface runoff was extremely serious. Although, the total of subsurface runoff were less compared with surface runoff under every tillage practice, but the phosphorus content was very high, the total phosphorus concentration all reached 0.2 mg•L-1, which were about 1.3 times higher than the surface runoff.【Conclusion】On the scattered sloping cropland, longitudinal ridges should not be adopted in normal agricultural production. Cross ridge was proven to be highly effective in control of both soil erosion and P losses, but attention needs to be paid to ridge repairing and protecting. In the whole process of erosion, the surface runoff was a large portion of the total runoff and phosphorus losses, and phosphorus loss in runoff was mainly in a form of dissolved phosphorus.

Identification, Evolution and Expression Analysis of the LBD Gene Family in Tomato

WANG Xiao-Fei, LIU Xin, SU Ling, SUN Yong-Jiang, ZHANG Shi-Zhong, HAO Yu-Jin, YOU Chun-Xiang

Scientia Agricultura Sinica. 2013, 46(12): 2501-2513. doi:10.3864/j.issn.0578-1752.2013.12.011

Abstract ( 855 )

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【Objective】Identification of LBD genes from tomato genome, and analysis of phylogeny，gene structure, chromosome location, phylogenetic and tissue expression pattern analysis of LBD family genes in tomato will be useful to the functions identification of plant LBD genes.【Method】Based on tomato genome database and bioinformatic method, tomato LBD family genes were identified and the genes were sequenced. A phylogenetic tree was created using the MEGA5 program. Gene structure and chromosomes location were done by Perl-based program, MapDraw and GSDS. Expression pattern of LBD genes at different development stages was analyzed based on the existing microarray database.【Result】A total of 46 LBD genes were systematically identified from tomato and classified into 2 classes (class I and class II), then was classified into 5 subfamilies (Ia, Ib, Ic, Id and II) according to the gene structure and conserved domain phylogeny relationship. They were distributed on 10 of tomato chromosomes, suggesting that they have an extensive distribution on the tomato chromosomes. Most of the LBD genes had differential expression pattern and response to external stimulation. 【Conclusion】Forty-six LBD family genes in tomato were identified by genome-wide screening. They were classified into 2 classes, 5 subfamilies and distributed on 10 chromosomes with different expression patterns in different tissues and developmental stages. They had special response to external stimulation. These results are helpful for the functional analysis of LBD genes in plants.

Expression and Genotype Analysis of VvmybA1 in Chinese Wild Grapes and Interspecific Hybrids

JIAO Jian, LIU Chong-Huai, FAN Xiu-Cai, ZHANG Ying, SUN Hai-Sheng, JIANG Jian-Fu, LI Min

Scientia Agricultura Sinica. 2013, 46(12): 2514-2525. doi:10.3864/j.issn.0578-1752.2013.12.012

Abstract ( 505 )

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【Objective】The aim of this study is to detect the genotypes of Chinese wild grapes and the hybrids of V. amurensis and V. vinifera and compare the VvmybA1 gene sequences in different wild grapes, reveal the molecular mechanism of no anthocyanin collected in white-fruited Xiongyuebaiputao by analysising the expression and genotype of VvmybA1 in the interspecific hybrids (V. amurensis ×V. vinifera).【Method】The specific primers were designed to detect the genotype of different wild grapes by the means of molecular cloning and absolute quantitative real-time PCR. Using Real-time PCR methods, analysis the expression of VvmybA1 and UFGT in the the hybrids of V. amurensis and V. vinifera during veraison. 【Result】①There was no VvmybA1a allele detected in Chinese wild grapes. ②Comparing sequences of VvmybA1 gene in the different species of Chinese wild grapes with thoses of V. vinifera, extensive base deletion, insertion and substitution exist in promotor region, intron region and the third coding exon, and exhibits rich genetic diversity between species of Chinese wild grapes. Furthermore，there are several unique bases or mutations in the VvmybA1 gene of wild grapes which could be selected to be the molecule marker of distinguishing different species and cultivars. ③VvmybA1a allele has been discovered in Suxiaohong and the hybrids of V. amurensis × V. vinifera, including Gongniang 1, Beihong and Xiongyuebaiputao. In addition, Xiongyuebaiputao is the homozygous genotype of VvmybA1a. ④ RT-PCR analysis showed high expression of VvmybA1 and UFGT in Gongniang 1 during veraison, but they did not express in Xiongyuebaiputao during the entire mature period.【Conclusion】The result proved that VvmybA1a allele didn’t exist in Chinese wild grapes but existed in V. vinifera and its interspecific hybrids uniquely. High consanguinity was speculated between Suxiaohong and V. vinifera. Different VvmybA1 gene sequences of species in Vitis were obtained and compared with each other. The reason why Xiongyuebaiputao has no anthocyanin collected is that the homozygous genotype of VvmybA1a lead to the no expression of VvmybA1.

The Annealing Modification and Characterization of Wheat Starch

WANG Yi-Jian, DU Xian-Feng, XU Yang, GUO Li

Scientia Agricultura Sinica. 2013, 46(12): 2526-2533. doi:10.3864/j.issn.0578-1752.2013.12.013

Abstract ( 629 )

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【Objective】Annealing can change the physical properties of wheat starch, and the annealing conditions were optimized according to gelatinisation temperatures (To, Tp, Tc) of annealed wheat starches. The correlation of morphological characteristics and annealing was also studied. 【Method】Differential scanning calorimetry (DSC) and DPS data analysis software were used to study the gelatinisation temperatures, and the effects of moisture content, annealing time and annealing temperature on the gelatinisation temperature of native and annealed wheat starch were also investigated. Besides, the influence on the morphology and size of the annealed wheat starch granules was discussed by using Scanning Electron Microscope (SEM) and Laser Particle Size Analyzer (LPA). 【Result】The results showed that the optimum condition of the wheat starch was annealed at 80% moisture content for 3 d at 45℃. Annealing changed the surface of wheat starch granules without destroying its shape, and more obvious influence on A-type granules than B-type granules. At the same time, annealing increased starch granule diameter.【Conclusion】Annealing leads to elevation of starch gelatinisation temperatures and sharpening of the gelatinisation range, and there is a significant positive correlation between the changes of wheat starch granules and annealing temperature.

Advance in Research on Risk Assessment of Aflatoxin in Agricultural Products

LI Pei-Wu, DING Xiao-Xia, BAI Yi-Zhen, ZHOU Hai-Yan, YIN 南Ri

Scientia Agricultura Sinica. 2013, 46(12): 2534-2542. doi:10.3864/j.issn.0578-1752.2013.12.014

Abstract ( 632 )

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Aflatoxins are genotoxic and carcinogenic. For their serious impact on quality and safety and international trade of China’s agricultural products, especially on peanut and severe threat to both animal and human health, aflatoxins has been subjected to the extensive concern of the society. Domestic and foreign risk assessment researches of aflatoxins were comprehensively reviewed, and development of risk assessment of aflatoxin, technical difficulties and future trends were proposed for promoting the further development of risk assessment work of aflatoxins in China in the future.

Effects of HMGCS1 Gene on Skeletal Muscle Growth and Development and Regulated by miR-18a/b of Pig

SUN Wei, TANG Zhong-Lin, TAN Lin, LEI Chu-Chao, LI Kui

Scientia Agricultura Sinica. 2013, 46(12): 2543-2549. doi:10.3864/j.issn.0578-1752.2013.12.015

Abstract ( 542 )

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【Objective】The purpose of the present research was to explore whether HMGCS1 impacts the skeletal muscle growth and development of pig and validate its target gene.【Method】In this study，the expression of HMGCS1 in different tissues and the expression change during Longissimus dorsi muscle development (28 stages) was investigated by real-time qPCR.【Result】The result showed that the HMGCS1 gene was strongly expressed in lung of adult pig, and the expression level of HMGCS1 gene in embryonic period 33 d and 65 d were higher than those in other periods in Tongcheng pigs, and lower expression at postnatal 20 d, 30 d, 40 d and 60 d. The luciferase activity was lower in miR-18a/b minics than in the control.【Conclusion】The results of study indicated that HMGCS1 regulates skeletal muscle growth and development by miR-18a/b, and is a potential candidate target gene of meat production traits.

Study on Network Database Platform of Hill-Black Swine Breeding Data

YANG Liang, XIONG Ben-Hai, 吕Jian-Qiang , CHANG Le, SUN Xiu-Kun

Scientia Agricultura Sinica. 2013, 46(12): 2550-2557. doi:10.3864/j.issn.0578-1752.2013.12.016

Abstract ( 445 )

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【Objective】 The aim of the study is to preserve resource data and to increase efficiency of breeding and genetic improvement of Hill-Black breeding pigs. 【Method】 Based on the North Hill Black pigs production process from progenitor to parents and to its commodity pigs, the pigs in breeding farm were divided into breeding boar, mothball boar, breeding sow, mothball sow, grew boar, grew sow, finishing boar, finishing sow and so on, and the standards and specifications of essential information of breeding pigs were designed. 【Result】 The study constructed pig breeding data management and analysis platform by using. Net and SQL Server 2008 network database technology, and realized remote network database management of essential breeding process information or data, and it could analyze some derived indexes including calving interval, low production sow and high production sow so on on-line, and it also realized intelligent alert of various production events such as mating, parturition, weaning, transfer group and culling.【Conclusion】 Regardless of how complicated in types, quantity and generation reproduction of the Hill-Black breeding pigs, it is possible to do a integrated data management, analysis and intelligent decision-making by developing a network computing system in terms of a series of databases, business logics and corresponding models.

Polymorphism of Chicken Prolactin Receptor Gene and It’s Association with Broodiness and Egg Reproduction Traits

DU Xiao-Hui, LI Cong-Yan, LIU Jun-Ying, NIU Wei-He, ZHANG Xi-Quan

Scientia Agricultura Sinica. 2013, 46(12): 2558-2565. doi:10.3864/j.issn.0578-1752.2013.12.017

Abstract ( 531 )

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【Objective】 In order to reveal the relationship between bloodiness and egg reproduction of Chinese indigenous chicken and prolactin receptor (PRLR) gene,which closely related to reproductive traits among amimals.【Method】Using the PCR-SSCP method and 576 Ningdu Yellow chicken, the experiment studied the polymorphism of PRLR gene and its association with nesting and egg production traits.【Result】The results confirmed previous studies that SNP1 (T10862C, exon 3) and SNP2 (T25670C, exon 6) had no significant association with broody traits (P＞0.05). The effect of SNP2 on egg production of 300 d (EN-300 d) was significantly (P＜0.05). The new SNP3 (G30716A, intron 8) significantly influenced the age at first egg (AFG) (P＜0.05). The novel SNP4 (A31900G, exon 10) had an extremely remarkable (P＜0.01) impact on the nesting rate, significant (P＜0.05) impact on the total broody days and EN-300 d. The differences among different haplotypes constructed by the four SNPs, in AFG reached an extremely significant level (P＜0.01), in other traits were no significant differences (P＞0.05). 【Conclusion】 PRLR gene polymorphism was associated with broodiness and egg production traits, especially the effect of SNP4.

Studies on Native Grass Silage of Different Plant Communities in Meadow Steppe

WANG Hong-Mei, SUN Qi-Zhong, HUA Mei

Scientia Agricultura Sinica. 2013, 46(12): 2566-2575. doi:10.3864/j.issn.0578-1752.2013.12.018

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【Objective】 The silage quality and feasibility of sixteen plant communities in meadow steppe were studied in this paper. 【Method】 Fresh grass from 16 sites in meadow steppe were loaded after cutting into polyethylene bag to be vacuumed and sealed. After 60 days, the silage fermentation quality(pH value, content of ammonia nitrogen（NH3-N）, lactic acid（LA）, acetic acid（AA）, propionic acid（PA）, butyric acid（BA）and total acid（TA）) and chemical composition(Buffering capacity（BC）, content of dry matter（DM）, crude protein（CP）, water-soluble carbohydrate（WSC）, neutral detergent fiber（NDF）and acid detergent fiber（ADF）) were analyzed. 【Result】 Many of the raw materials were suited to silage, because of their lower buffering capacity and higher content of dry matter and water-soluble carbohydrate. The contents of dry matter, WSC, NDF and ADF in each silage were lower than them in each raw materials, except in sites 5 and 14. But the change of CP content in silage had no trend compared with raw materials. There were significant differences in pH value, NH3-N/TA, contents of four organic acids and total acid and contents of nutrient compositions (DM, CP, WSC, NDF and ADF) among 16 silage materials(P＜0.05). The silage pH value was from 4.56 to 5.69, with lower value of NH3-N/TA.【Conclusion】 The rating of silage fermentation quality could be divided into optimal(1, 8, 9, 13, 14, 15), good(2, 10, 12), medium (5, 16) and bad(3, 4, 6, 7, 11).

Immunoregulatory Effects of Compound Ammonium Glycyrrhizin Soluble Powder on Liver Injury Induced by Enrofloxacin and LPS in Chickens

GUO Fan-Xi, LIU Teng-Fei, GENG Zhi-Xia, JIANG Fan, YU Zu-Gong

Scientia Agricultura Sinica. 2013, 46(12): 2576-2583. doi:10.3864/j.issn.0578-1752.2013.12.019

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【Objective】 The present study was aimed at the effects of compound ammonium glycyrrhizin soluble powder (CAG) on proinflammatory cytokines production and T-cell subsets deviation in peripheral blood in chicken liver injury induced by enrofloxacin and lipopolysaccharide crude extract(LPS).【Method】A total of 104 animals were allocated randomly into 5 groups named I- V: I normal group, II liver injury group(model group), III prevention group, IV high dosage treatment group and V low dosage treatment group. Group II- IV were administered with enrofloxacin(100 mg•kg-1) once a day for three days and on the third day administered with LPS (4 mL•kg-1) at the same time. Group III received prior administration of CAG at the dosage of 40 mg•L-1 ( by ammonium glycyrrhizin, the same as follows) in drinking water for three days before establishment of liver injury model. Groups IV and V received CAG at a dosage of 22.5 and 7.5 mg•kg-1 twice a day for three days after LPS treatment. Every eight chickens were sacrificed at 6, 24 and 48 h following post-treatment with LPS and their blood samples were collected for detection.【Result】Compared with group I, the group II levels of serum ALT, AST, TNF-α, IL-1 and IL-6 were significantly higher at 6, 24, 48 h post-treatment with LPS and CD3(+), CD4(+) and CD8(+) T-lymphocytes were significantly lower at 6 and 24 h higher at 48 h post-treatment with LPS. When compared with group II , there existed a significant fall in the levels of serum ALT ,TNF-α, IL-6 at 6 h, AST at 6 h and 24 h and the number of CD3(+), CD4(+) and CD8(+) T-lymphocytes at 48 h in group III. In group IV, the levels of ALT, AST, TNF-α, IL-6 at 6 h and 24 h , IL-1 and CD3(+) , CD4(+) and CD8(+) T-lymphocytes at 48 h decreased significantly and CD3(+) T-lymphocytes at 6 h and 24 h , CD4(+) T-lymphocytes at 24 h increased obviously. In group V, there was a significant fall in CD8(+) T-lymphocytes at 48 h and no obvious other differences were detected. The liver histopathological changes got a better improvement in group IV than others.【Conclusion】The novel liver injury induced by enrofloxacin and LPS effects might be related to TNF-α, IL-1 and IL-6 production and CD3(+), CD4(+) and CD8(+) T-cell deviation in peripheral blood. CAG prevents the liver injury through inhibiting TNF-α, IL-1 and IL-6 production and regulating the number of T-cell subsets in peripheral blood by bi-directional regulation and the efficacy might be related to dosage and treatment time.

Cloning and Functional Study of ZmSCL7 in Zea mays

GUO Peng, XING Xin, JIN Hua, DONG Yan

Scientia Agricultura Sinica. 2013, 46(12): 2584-2591. doi:10.3864/j.issn.0578-1752.2013.12.020

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【Objective】The aim of this study is to clone SCARECROW-LIKE 7 (SCL7）gene, to analyze its molecular mechanisms and promote their applications in breeding. 【Method】 The total RNA from the leaves of tobacco was used as the template to design the degenerate primers based on homology cloning strategy, and then the full-length cDNA sequence of zmSCL7 was obtained through a combined reverse transcription-PCR (RT-PCR). Bioinformatics method was used to analyze the sequence characteristics of this gene. Northern blot was used to investigate the expression pattern. Western blot was used to investigate the transgenic analysis. Chlorophyll content, Fv/Fm, MDA and proline content were investigated for functional verification of salt resistance.【Result】The results indicated that cDNA of ZmSCL7 was 1 653 bp and contained a single open reading frame of 550 amino acid residues. Northern blot indicated that the mRNA accumulation of ZmSCL7 was induced by low temperatures, salt stress, abscisic acid (ABA) and H2O2. Additionally, compared with the control, transgenic ZmSCL7 tobacco, germination was inhibited less. Chlorophyll content, Fv/Fm, proline content increased and MDA content decreased. 【Conclusion】ZmSCL7, a transcription factor, was induced by a variety of stress and could increase salt tolerance by overexpression in tobacco.

Analysis of Quantitative Expression of the Flowering-Regulating Transcription Factor CONSTANS Gene in Brassica napus L.

ZHENG Ben-Chuan, ZHANG Jin-Fang, LI Hao-Jie, CHAI Liang, CUI Cheng, JIANG Jun, PU Xiao-Bin, NIU Ying-Ze, JIANG Liang-Cai

Scientia Agricultura Sinica. 2013, 46(12): 2592-2598. doi:10.3864/j.issn.0578-1752.2013.12.021

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【Objective】The expression of CONSTANS(CO) homologous genes in B. napus was characterized. 【Method】Materials D626-6 and D125-5 with different growth periods were used to characterize the expression level of CO gene. A pair of specific primers was designed based on Bn1CON19 cDNA sequence, and a pair of Real-time fluorescence quantitative specific primers was designed according to the obtained cDNA sequence. The Real-time fluorescence quantitative analysis was conducted by using SYBR Green I dye method.【Result】 The results showed that the highest expression levels were appeared in the leaves of B. napus L., followed by the buds and stems. The expression levels in the morning and the evening were higher than those at noon. At different growth stages, the highest expression levels were appeared at bolting stage. The early-maturing material had a significantly higher express level than the late-maturing material in leaves and buds at bolting stage. 【Conclusion】 Result of the study indicated that the CO gene might play an important role in growth period and flower formation process in B. napus L..

Localization of STK2 of Setosphaeria turcica in the Genome, Characterization of Its Protein Structure and Expression in Eukaryotic Cells

GONG Xiao-Dong, FAN Yu, LI Po, YANG Yang, ZHANG Chang-Zhi, TIAN Lan, ZHANG Xiao-Yu, FAN Yong-Shan, HAN Jian-Min, GU Shou-Qin, DONG Jin-Gao

Scientia Agricultura Sinica. 2013, 46(12): 2599-2606. doi:10.3864/j.issn.0578-1752.2013.12.022

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【Objective】The objectives of this study is to determine the location in genome, and to investigate the protein structure of STK2 of Setosphaeria turcica. Meanwhile, a eukaryotic expression vector of STK2 was constructed and expressed in Pichia pastoris to obtain secreted protein in extracellular. 【Method】 The location of STK2 and protein structures were analyzed using bioinformatics method. Primers were designed according to multiple cloning sites in vector pPIC9K and the ORF sequence of STK2. A recombinant eukaryotic expression vector was constructed and transformed into host yeast strain GS115 by electroporation. The aiming protein expressed in yeast cell through induction and was identified by SDS-PAGE. 【Result】 The identity of STK2 was 91433, locating between 1561986 and 1563262 in the positive-strand of scaffold_3 in the genome of S. turcica. Stk2 shared characteristic conserved domains of MAPK-like protein kinase. α-helices and random coils were its main elements of the secondary structure of Stk2, β strands were lesser and mainly located in N ends in this structure. The 3D structure was composed of a smaller N-terminal lobe and a bigger C-terminal lobe. The eukaryotic expression vector of pPIC9K-STK2 was successfully constructed, and the transformants were screened according to their resistance. A protein, with its molecular weight of about 41 kD, was obtained after induced by methanol, while no corresponding proteins were found in control, suggesting STK2 had been secreted to extracellular of the yeast cells.【Conclusion】STK2 is located between 1561986 and 1563262 in the positive-strand of scaffold_3 in the genome of S. turcica. The characteristics of Stk2 were analyzed, and showed that Stk2 shared all of conserved domains of MAPK kinase. The results also revealed that STK2 was successfully expressed in yeast cell and soluble protein was got in the experiment. These results will lay a foundation for the preparation of Stk2 polyclonal antibody and for the study of the functions of STK2.

The Virus-Free Technology of Bulblets of Chongming Daffodil (Narcissus tazetta var. chinensis) in vitro

YANG Liu-Yan, ZHANG Yong-Chun, LI Yu-Xiu, DAI Fu-Ming

Scientia Agricultura Sinica. 2013, 46(12): 2607-2614. doi:10.3864/j.issn.0578-1752.2013.12.023

Abstract ( 519 )

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【Objective】The objective of this experiment is to explore a simple-operate method to eradicate viruses of Chongming Daffodil (Narcissus tazetta var. chinensis), and detect the effect of virus-free technology.【Method】The treatments of DHT+thermotherapy and DHT+virazole were used to eliminate viruses in bulblets in vitro of Chongming Daffodil, and the effect of treatments were tested by indicator plant and RT-PCR.【Result】Thermotherapy treatment had effect on the growth and survival rate of bulblets in vitro. Thermotherapy with DHT treatment decreased the content of viruses，and when the concentration of DHT was less than 15 mg•L-1, there was less inhibitory effect on growth. Virazole at 30 mg•L-1+DHT 30 mg•L-1 had good inhibitory effect on NMaV, and there was a very low level of virus detected by RT-PCR. NDV and NCLV had higher sensitivity to two antiviral compounds, and could be mostly eradicated.【Conclusion】There was a negative effect on thermotherapy treatment. DHT with thermotherapy treatment could decrease the content of viruses detected by indicator plant. The content of NMaV in bulblets in vitro treated with 30 mg L-1DHT +30 mg L-1virazole was reduced to a very low level, and the treatment had no impact on bulblet’s survival. NDV-free and NCLV-free plants were obtained by the treatment of combination with 10 mg L-1DHT and 10 mg L-1virazole, this result was proved by RT-PCR detection.

Effect of Different Ripening Stages on Antioxidant Activity of Soluble Extracts from Mozzarella Cheese with Various Strains

MA Ling, PENG Deng-Feng, LI Hui, WANG Xiao-Wen, LIU Hui-Ping

Scientia Agricultura Sinica. 2013, 46(12): 2615-2624. doi:10.3864/j.issn.0578-1752.2013.12.024

Abstract ( 504 )

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【Objective】 This study was conducted to examine the antioxidant activity of extracts with sterile water, pH 4.6 acetic acid buffer solution and 12% TCA for Mozzarella cheese in different mature periods in order to investigate the proper ripening periods for eating from the facet of antioxidant activity.【Method】Extracts of different ripening periods of Mozzarella cheese with combined starter were prepared and their scavenging DPPH capacity, reduction power, scavenging hydroxyl radical and superoxide anion were determined by using sterile water, pH 4.6 acetic acid buffer solution and 12% TCA as extraction solvent, respectively. 【Result】The results showed that soluble extracts had a certain antioxidant capacity. In the ripening of 50 days, most of the antioxidant indexes of various extracts from Mozzarella cheese with starters AB, AB+LPC, AB+BI were higher than in other ripening days, but cheese with AB+LA showed higher antioxidant activity both in the ripening of 40 d and 50 d. The proper eating stage was various with distinctive starters, for Mozzarella cheese with AB, AB+LPC and AB+BI starter, at the age of 50 d, while AB+LA between 40 and 50 days of ripening. 【Conclusion】 Antioxidant activity was various with different extracts, peptide contents in the extracts of 12% TCA were the highest. The proper mature stage for eating of Mozzarella cheese was different with different starters in consideration of the antioxidant activity.

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