Scientia Agricultura Sinica

Comparative Analysis of Three Mutants of Divinyl Reductase Gene in Rice

WANG Ping-Rong, MA Xiao-Zhi, LI Chun-Mei, SUN Chang-Hui, DENG Xiao-Jian

Scientia Agricultura Sinica. 2013, 46(7): 1305-1313. doi:10.3864/j.issn.0578-1752.2013.07.001

Abstract ( 673 )

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【Objective】 The present study was aimed at investigating the differences of mutant phenotypes resulting from different mutations in base sequence of rice divinyl reductase gene OsDVR (Os03g22780), and evaluating application potential of this gene in rice breeding as a leaf color marker gene. 【Method】The yellow-green leaf mutants isolated from Indica rice variety G46B and japonica rice variety Nipponbare via ethyl methanesulfonate mutagenesis were detected using high-performance liquid chromatography (HPLC) to screen the mutants accumulating divinyl chlorophyll. Then genetic analysis, gene mapping and allelism test of the target mutants were performed. Subsequently, DNA sequencing of OsDVR gene in these mutants and alignment of the deduced amino acid sequences of homologous DVR proteins were conducted, and leaf photosynthetic pigments, plant phenotypes and main agronomic traits of the mutants were investigated. 【Result】590ys and 525ys, two novel mutants accumulating divinyl chlorophyll, were obtained by screening 53 yellow-green leaf mutants isolated from ethyl methanesulfonate mutagenesis. Their mutant phenotypes were all controlled by a pair of recessive nuclear gene and moreover, the mutant genes were all mapped on the chromosomal region harbouring the 824ys mutant gene reported. Furthermore, allelism test confirmed that the mutant genes of 590ys and 525ys were allelic to that of 824ys. So 590ys, 525ys and 824ys mutants were all OsDVR mutants. However, mutational sites of OsDVR mutant gene and their encoded protein product in the three mutants were different, which resulted in extremely significant differences of chlorophyll contents and compositions, plant phenotypes, main agronomic traits and yields per plant among the three mutants. Among them, 525ys mutant had slight yellow leaves, and its growth and development, main agronomic traits and yield per plant were affected relatively less, indicating that 525ys mutant gene can basically meet the requirements as a leaf color marker gene introduced into rice male sterile lines. 【Conclusion】Different mutations in base sequence of OsDVR gene could result in extremely significant differences of mutant phenotype and yield per plant. The OsDVR mutant gene could be applicable in hybrid rice breeding as a leaf color marker gene.

Cloning and Characterization of the BnbZIP1 Transcription Factor Gene from Ramie (Boehmeria nivea L.)

ZHOU Jing-Hua, JIE Yu-Cheng, XING Hu-Cheng, ZHONG Ying-Li, YU Wei-Lin

Scientia Agricultura Sinica. 2013, 46(7): 1314-1322. doi:10.3864/j.issn.0578-1752.2013.07.002

Abstract ( 536 )

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【Objective】The objective of this study was to clone the full-length cDNA of BnbZIP1 transcription factor gene from ramie, and the expression pattern and the bioinformatics of the sequence were analyzed, and the prokaryotic expression and the subcellular localization were analyzed.【Method】A full-length cDNA sequence was cloned by RT-PCR and RACE methods based on the unigene48047 in ramie transcriptome sequencing. Then the sequence was analyzed through bioinformatics methods and the expression patterns of BnbZIP1 were analyzed by using Real-time PCR in various tissues and under different stress conditions. A prokaryotic expression vector was constructed and the prokaryotic protein expression was induced with IPTG. Then a fusion expression vector containing EGFP was constructed to observe the subcellular localization. 【Result】The full-length cDNA sequence and the ORF of BnbZIP1 were 2 071 bp and 1 407 bp, which encoded 468 amino acids with predicted pI and molecular weight were 4.95 and 36.81 kD, respectively. Homology comparison analysis showed that the deduced BnbZIP1 amino acid sequence shares a 93% homology with bZIP gene (XP_002307972) in Populus trichocarpa. The relative molecular weight of recombinant protein induced by IPTG was 52 kD, which corresponded to the theoretical value. The results of subcellular localization analysis showed that the BnbZIP1 is located in nucleus. The results of real-time PCR suggested that the BnbZIP1 gene expressed in root, stem, shoot tip and blade, female flowers and male flowers, with the highest expression level in male flowers and the lowest in root. The BnbZIP1 gene was up-regulated by ABA, drought and high salt treatment.【Conclusion】The full-length cDNA sequence of BnbZIP1 from ramie was cloned and it had the typical bZIP transcription factor structural domain in plants, and BnbZIP1 gene responses to ABA, drought and high salt stress, which indicated that the BnbZIP1 genes might play an important role in stress response.

Effect of Nitrogen Application on Stem Lodging Resistance of Rice and Its Morphological and Mechanical Mechanisms

LI Guo-Hui, ZHONG Xu-Hua, TIAN Ka, HUANG Nong-Rong, PAN Jun-Feng, HE Ting-Hui

Scientia Agricultura Sinica. 2013, 46(7): 1323-1334. doi:10.3864/j.issn.0578-1752.2013.07.003

Abstract ( 724 )

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【Objective】 Lodging is one of the important constraints to high yield and stable production of rice. This study investigated the effects of nitrogen application on stem lodging resistance and its relationship with stem morphological and mechanical traits. The mechanisms of stem lodging resistance were explored. Results of the study will provide a theoretical basis for lodging resistance and high yield cultivation of rice. 【Method】 An indica rice cv. Yinjinruanzhan was grown in field experiments conducted with four nitrogen levels at Guangzhou. Lodging index and morphological and mechanical traits of basal culms were measured.【Result】As nitrogen input increased, plant height, gravity center height and basal internode length increased, basal internode plumpness declined, breaking resistance and elastic modulus decreased, lodging index increased and lodging resistance declined. Nitrogen treatment affected stem morphological and mechanical traits of rice. Lodging index was positively correlated with plant height, gravity center height and basal internode length, while negatively correlated with basal internode plumpness, breaking resistance and elastic modulus, and mostly significant. Positive correlation was found between lodging index and spikelet number per m2, while the correlation between lodging index and filled grain rate and 1000-grain weight were negative.【Conclusion】Nitrogen application affects stem lodging resistance through morphological traits such as plant height, gravity center height, basal internode length, basal internode plumpness, and mechanical traits such as stem bending moment and breaking resistance.

Effects of Water-Nitrogen Management Patterns and Combined Application of Phosphorus and Potassium Fertilizers on Nutrient Absorption of Hybrid Rice Gangyou 725

SUN Yong-Jian, SUN Yuan-Yuan, XU Hui, YANG Zhi-Yuan, QIN Jian, PENG Yu, MA Jun

Scientia Agricultura Sinica. 2013, 46(7): 1335-1346. doi:10.3864/j.issn.0578-1752.2013.07.004

Abstract ( 620 )

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【Objective】In order to provide a scientific basis for high water-fertilizer use efficiency of rice, an experiment was conducted to study the effects of water-nitrogen management patterns and combined application of phosphorus potassium fertilizers on nutrient absorption and physiological characteristics of hybrid rice.【Method】Hybrid rice variety Gangyou 725 was used to investigate the effects of three water-nitrogen management patterns (submerged irrigation and optimized nitrogen (N) application, W1N1; controlled alternate irrigation and optimized N application, W2N1; and dry cultivation and optimized N application, W3N2) and different phosphorus (P)-potassium (K) combined applications on absorption of N, P, and K in rice, analyze their physiological characteristics, and study on correlations of N, P, and K absorption characteristics and its relationship with physiological index and grain yield. 【Result】The results showed that different water-nitrogen management patterns and P-K fertilizer combined application significantly affected N, P and K absorption, physiological characteristics and grain yield. W2N1 and suitable P90K90 combined application (P and K application rates both at 90 kg?hm-2) enhanced N, P, and K absorption at heading and maturity stages, increased the photosynthetic rate, superoxide (SOD) in flag leaves and bleeding intensity during grain filling, and activated malondialdehyde (MDA) in flag leaves, being the best model in this paper referred as the water-fertilizer regulating management model. Combined application of P90K180 resulted in superiority decrease of water-fertilizer regulating, yield increase not significant, and lowering the fertilizer use efficiency and the ratio of output to input. Compared with W1N1, W3N2 and suitable P90K180 had compensated for yield outputs. Correlation analysis indicated that there existed significantly positive correlations (r=0.644*-0.939**) between the amounts of N, P, and K, and between each of them and yield under different water-fertilizer regulation, and the amounts of different nutrients at heading and maturing stages and grain yield had a positive correlation with Pn, SOD in flag leaves, and root vigor (r=0.545-0.902**), and a high significant negative correlation with MDA in flag leaves (r=-0.612*--0.879**) at different growth stages at heading stage, with different correlation coefficients at different growth stages. According to the conditions above, it was important for delaying the leaf senescence, keeping root vigor, and increasing yield that the amounts of N, P, and K at heading stage and physiological metabolism of rice during 1-7 d after heading stage.【Conclusion】The results suggest rice plant growth could be improved and high grain yield and high fertilizer use efficiency could be achieved through integrating and optimizing water-fertilizer regulating techniques in rice production. W2N1 and suitable P90K90 combined application was considered to be optimum under the experimental conditions, optimum phosphorus (P)-potassium (K) combined application amount was P90K90 for W1N1, and P90K180 for W3N2.

Influence of Cultivation Methods and No-Tillage on Root Senescence at Filling Stage and Grain-Filling Properties of Eryou 498

YANG Zhi-Yuan, SUN Yong-Jian, XU Hui, QIN Jian, JIA Xian-Wen, MA Jun

Scientia Agricultura Sinica. 2013, 46(7): 1347-1358. doi:10.3864/j.issn.0578-1752.2013.07.005

Abstract ( 712 )

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【Objective】In this study, dry weight and bleeding sap of root at grain-filling stage and grain-filling properties of superior and inferior grains were studied to reveal some mark effects of different cultivation methods and no-tillage (NT) on root senescence and grain-filling of Eryou 498. 【Method】 Roots were obtained from paddy field with undistributed soil column, and grain-filling processes of wide and narrow row cultivation (WNR), triangle-planted system of rice intensification (TSRI), wide row and narrow space cultivation (WRNS) and seedling-throwing cultivation (ST) under NT and plow-tillage (PT) were fitted with Richard equation. 【Result】Compared with PT rice, dry weight of root of NT rice was smaller, but its root activity at early and middle grain-filling stages was larger than that of ZT rice, however, root activity of ZT rice at late grain-filling stage was much smaller because of the sharp drop which meant a bad ability in keeping root function, besides, grain yield of ZT rice was smaller than that of PT rice, too. TSRI with the outstanding root function of the four cultivation methods could not adjust to the NT conditions, however, WNR and ST adapted themselves to NT conditions greatly because of the interaction effects between cultivation methods and NT prompted root activity enormously. Cultivation measures not only impacted grain-filling processes greatly but also changed the grain-filling mode. WNR, WRNS and ST were of synchronous grain-filling mode under NT and asynchronous grain-filling mode under PT, however, TSRI was just the opposite of them under different tillage modes. Overall, grain-filling rate at middle grain-filling stage was larger than that at earty and late grain-filling stages, although middle grain-filling period was the shortest of the three, it still made a major contribution to grain-filling.【Conclusion】Cultivation methods and NT had a great influence on root senescence and grain-filling of Eryou 498. Based on root function, WNR and ST were more suitable for NT, however, TSRI showed better under PT. Because of the negative influence of natural environment of Sichuan basin, grain-filling of inferior grains of Eryou 498 was insufficient, so there was still a great yield potential if natural environment was proper, especially for ST under NT and TSRI under PT.

Molecular Cloning and Expression Pattern Analysis of a Protein Disul?de Isomerases (SpLPDI) in Spodoptera litura

LI Yan, WANG Zhong-Kang, CHEN Huan, FENG 二Yan, YIN You-Ping

Scientia Agricultura Sinica. 2013, 46(7): 1359-1369. doi:10.3864/j.issn.0578-1752.2013.07.006

Abstract ( 478 )

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【Objective】The objective of this study is to analyze the mRNA expression characteristics and immune related function of protein disulfide isomerase gene (SpLPDI) from Spodoptera litura, and to provide a foundation for further study of S. litura immune molecular mechanism in response to Nomuraea rileyi infection.【Method】The full-length cDNA of SpLPDI was amplified by specific primers based on the EST from the fatbody suppression subtractive hybridization (SSH) library and SMART RACE cloning technology and further analysed by bioinformatics software. The expression patterns of SpLPDI in different tissues were analyzed by semi-quantitative RT-PCR and quantitative PCR. The mRNA expression of SpLPDI upon N. rileyi challenge was studied by qPCR and the protein characteristics by prokaryotic expression was analyzed.【Result】The full length cDNA of SpLPDI was cloned which was 2 367 bp, including 1 485 bp ORF encoding 494 amino acid proteins. The SpLPDI protein had two thioredoxin domains and an ER retention signal (KDEL) with a signal peptide of 17 amino acid residues containing an a-b-b′-a′ domain organization. The result of multiple alignment indicated that a, a′ domains and ER retention signal were highly conserved. Phylogeny analysis showed that SpLPDI had a closest genetic relationship with Bombyx mori while a distant genetic relationship with Cryptosporidium parvum and Aspergillus niger. Expression profile analysis showed that SpLPDI was expressed in all detected tissues, but highest expressed in hemocytes, followed in fatbodies, and lowest in heads. Induction expression analysis showed that mRNA levels of SpLPDI were significantly increased in fat bodies and midguts upon N. rileyi challenge but the rate of increase in fat bodies higher than that in midgut. SpLPDI was successfully expressed in E. coli, producing a soluble fusion protein. 【Conclusion】The full length cDNA of SpLPDI was cloned from S. litura and changes of PDI mRNA expression were observed in different tissues of S. litura after N. rileyi injection. It suggested that SpLPDI might be related to immune response of S. litura to N. rileyi.

Screening of Surfactants for Promoting the Efficiency of Pesticide Used in Paddy Field

XU Guang-Chun, GU Zhong-Yan, XU De-Jin, XU Xiao-Long

Scientia Agricultura Sinica. 2013, 46(7): 1370-1379. doi:10.3864/j.issn.0578-1752.2013.07.007

Abstract ( 583 )

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【Objective】The objective of this study is to filter out suitable surfactants based on improving the pesticide application efficiency in paddy field among 10 surfactants of 3 surfactant-types, organosilicone, alkylphenol polyoxyethylene and azone.【Method】Surface tension of 10 surfactant solutions was measured followed by national standard (GB 5549-1990) and the critical micelle concentration (CMC) of surfactant was also measured according to the change of surface tension of surfactants solutions. Then the proper surfactant solutions spraying in paddy field were selected on the basis of surface tension reducing efficiency, initial contact angle on rice leaf surface and maximum retention (Rm) by micro-weighing method.【Result】The surface tension values of PTS and NP-15 solutions at CMC were both more than the estimated critical surface tension (CST) values of rice leaves and the initial contact angles (＞100°) on rice leaves were indicative of poor wetting and adhesion. GSS, KNS and GJZ could be wetting on rice leaf surface, but the initial contact angles (＞90°) exhibited that droplets of solutions were hard to adhere to rice leaf surface and easy to roll or run off the rice leaf surface. The rest 5 surfactants at suitable concentrations could be used in paddy field and one of them was Silwet 408 showed good wetting and adhesion on rice leaf surface. At 30°, 45° and 60° inclination, its maximun run-off points ((14.33±0.27), (12.44±0.58) and (10.27±0.40) mg•cm-2) and Rm ((7.98±0.37), (6.84±0.40) and (5.23±0.23) mg•cm-2) of rice leaf surface were significantly greater than TX-10. After adding Silwet 408 at concentration of 125.0 mg•L-1 in water and three pesticides (chlopyrifos, validamycin and imidacloprid) solutions at recommended concentrations, values of surface tension of solutions decreased to 20.77-23.12 mN•m-1 and the initial contact angles reduced to 28.4°-67.1°. Excepting the chlopyrifos solution at recommended concentration, Rm of other solutions increased significantly. 【Conclusion】 The 5 surfactants tested at suitable concentrations can be used in paddy field. At the concentration of 125.0 mg•L-1, Silwet 408 solution is most suitable for spraying in paddy field.

Effects of Nitrogen Fertilizer Regulation and Soil Surface Mulching on Nitrogen Use by Winter Wheat and Its Residue and Leaching in Dryland Soil

LI Qiang, WANG Chao-Hui, DAI Jian, LI Fu-Cui, LI Meng-Hua, ZHAO Hu-Bing, CAO Qun-Hu

Scientia Agricultura Sinica. 2013, 46(7): 1380-1389. doi:10.3864/j.issn.0578-1752.2013.07.008

Abstract ( 601 )

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【Objective】Since water deficiency and unreasonable nitrogen (N) fertilization are the key factors impacting crop production in dryland area of Northwest China, it is of great importance to work on fertilization and cultivation management for increasing crop yield, water and fertilizer efficiency in this area.【Method】Long term field experiments were carried out to study the effects of topdressing at early spring, N fertilizer regulation, ridge-mulching and furrow seeding, seeding density and straw mulching during summer fallow on winter wheat yield, N uptake, residual nitrate-N after crop harvest and its leaching, and soil N mineralization during summer fallow.【Result】Results showed that compared with conventional fertilization, topdressing at early spring, N fertilizer regulation and topdressing at early spring, N fertilizer regulation and ridge mulching with furrow seeding, N fertilizer regulation and ridge mulching with furrow seeding at high seeding density increased winter wheat yield by 15.7%, 22.2%, 38.6% and 22.4%, N uptake by 25.0%, 32.0%, 36.4% and 6.4%, and N harvest index by 3.8%, 3.3%, 5.7% and 6.8%，but decreased residual nitrate-N in current season by 30.9%, 20.0%, 59.5% and 68.2%, and also decreased leaching of nitrate-N to deep soil layers below 60 cm by 27.5%, 24.6%, 42.3% and 24.3% during summer fallow. 【Conclusion】Thus, topdressing at early spring, N fertilizer regulation and topdressing at early spring, N fertilizer regulation and ridge mulching with furrow seeding, N fertilizer regulation and ridge mulching with furrow seeding at high seeding density increased winter wheat yield while promoting plant N uptake and decreasing residual nitrate-N in Weibei arid areas. The practice of N fertilizer regulation and ridge mulching with furrow seeding combined with covering soil surface by straw mulching during summer fallow increased the water storage in summer fallow while preventing nitrate-N leaching to deep soil, thus is recommended in dryland area of Northwest China.

Improving Nitrogen Use Efficiency by Combined Use of Slow Release Nitrogen Fertilizer with Urea for High Yielding Winter Wheat-Summer Maize Rotation System

SI Xian-Zong, HAN Yan-Lai, WANG Yi-Lun, LIU Meng-Meng, TAN Jin-Fang

Scientia Agricultura Sinica. 2013, 46(7): 1390-1398. doi:10.3864/j.issn.0578-1752.2013.07.009

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【Objective】 The efficient and simplified fertilization technology for crop rotation of winter wheat and summer maize high yield system was studied. 【Method】 Based on the soil conditions and cultivation management measures in field, in which grain yields of winter wheat and summer maize were up to 22 500 kg•hm-2 in one year, effect of slow release nitrogen combined with conventional urea application in one base and one topdressing mode on yield, above-ground portion nitrogen accumulation rate, nitrogen use efficiency and soil inorganic nitrogen dynamic indexes in winter wheat and summer maize rotation cycle in two years location test were studied, and compared with no nitrogen fertilizer (CK) and conventional urea application in one base and three topdressing mode (CK1).【Result】The results of two years experiments showed that there were no significant influence on yield, above-ground portion nitrogen accumulation rate, nitrogen use efficiency and nitrogen agronomic efficiency in treatment of 100% slow release urea (T1), however, it provided higher nitrogen level in winter wheat at the jointing and after filling stage, compared with CK1. Meanwhile, yield, above-ground portion nitrogen accumulation rate, nitrogen use efficiency and nitrogen agronomic efficiency in treatments of 80%-20% slow release-conventional urea combination (T2) and 60%-40% slow release-conventional urea combination (T3) were higher than CK1. In summer maize, yield, above-ground portion nitrogen accumulation rate, nitrogen use efficiency and nitrogen agronomic efficiency in treatments T1, T2 and T3 were higher than CK1, with the highest in the treatment T2. Inorganic nitrogen concentrations of winter wheat in treatment T1 was higher than CK1 at the after filling stage. In summer maize, inorganic nitrogen concentrations in treatment T2 was also higher than other treatments at the silking stage.【Conclusion】In this experiment, yield, above-ground portion nitrogen accumulation rate, nitrogen use efficiency and nitrogen agronomic efficiency of 80%-20% slow release-conventional urea combination and 100% slow release urea were higher than CK1, and yields of winter wheat and summer maize in the two treatments were up to 22 500 kg•hm-2, it realized the target of high yield and high efficiency and simplified fertilization in one base and one topdressing mode under winter wheat and summer maize rotation system.

Influence of C/N Ratio on Degradation of Oxytetracycline and Composting Parameters During Chicken Manure Composting

WANG Gui-Zhen, LI Zhao-Jun, ZHANG Shu-Qing, MA Xiao-Tong, LIANG Yong-Chao

Scientia Agricultura Sinica. 2013, 46(7): 1399-1407. doi:10.3864/j.issn.0578-1752.2013.07.010

Abstract ( 609 )

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【Objective】 The effects of C/N on the OTC degradation and the physicochemical characters and maturity of chicken composts during chicken feces composting were studied.【Method】 The indoor aerobic composting method was used in the present paper. Three groups of composting materials with C/N ratios of 21.6, 25.5 and 32.8 were prepared through combinations of chicken manure and wheat straw. To illustrate the effects of C/N on OTC degradation and composting process, OTC contents of chicken feces and the selected parameters such as pile temperature, pH, EC, contents of NO3--N, and NH4+-N, and germination index(GI) in composting process were investigated. 【Result】 It was found that C/N has significant effects on the degradation of OTC, and the order of OTC degradation was T2＞T3＞T1. The degradation of OTC in chicken feces during composting could be well described by the first-order kinetic model, and the correlation coefficients ranged from 0.9431 to 0.9967. The effects were found of C/N on pile temperature during composting. The high C/N treatments had higher speed of temperature rising and the shorter time for temperature rising than the low C/N treatment. The highest pile temperatures were significantly higher than that of the low C/N treatment. Effects were also found of C/N on the soluble nitrogen contents of piles during composting. For example, the content of NO3--N of three treatments were increased by 78.50%, 62.37%, and 59.34% at the end of composting, compared with their values at the beginning of composting. High C/N treatment could significantly reduce the biotoxicity of compost. For example, the treatments with C/N ratios of 25.5 and 32.8 had high GI values (The GI values are much higher than 80%).【Conclusion】The highly initial C/N ranging from 25.5-32.8 could significantly decrease the OTC in chicken feces and be benefit for the chicken feces maturity.

Expression and Functional Analysis of VpMYBR1 Gene Resistant to Uncinula necator from Vitis pseudoreticulata

HOU Hong-Min, WANG Hao, YIN Xiang-Jing, YAN Qin, WANG Yue-Jin, WANG Xi-Ping

Scientia Agricultura Sinica. 2013, 46(7): 1408-1418. doi:10.3864/j.issn.0578-1752.2013.07.011

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【Objective】 The VpMYBR1 gene was cloned from Vitis pseudoreticulata resistance to powdery mildew clone ‘Baihe-35-1’ leaves infected by Uncinula necator, and its expression model and function were analyzed in this study. This study will provide a theoretical basis for revealing the grape powdery mildew resistance mechanism.【Method】VpMYBR1 was isolated by RT-PCR and rapid amplification of cDNA ends (RACE) approaches, and its expression patterns under different treatments and in different tissues were analyzed by using semi-quantitative RT-PCR and real-time quantitative PCR. The plant expression vector of this gene was constructed, and then transformed into Arabidopsis thaliana by Floral Dip method. The disease resistance identification of transgenic plants was analyzed by infecting powdery mildew, trypan blue staining and analyzing the expression of Arabidopsis defense marker genes.【Result】The assembled VpMYBR1 (GenBank ACC no. HQ284197) cDNA sequence was 539 bp in full length including a 228 bp ORF which encodes a polypeptide of 75 amino acids containing a single Myb-like DNA-binding domain. The VpMYBR1 was expressed in leaves, stems, inflorescence, tendril, and fruit of ‘Baihe-35-1’. The expression of VpMYBR1 was strong in leaves and weak in inflorescence and fruit. The VpMYBR1 was induced by U. necator during 6 to 12 hpi in three grapevine genotypes (V. pseudoreticulata clone ‘Baihe-35-1’, ‘Hunan-1’ and V. quinquangularis clone ‘Shang-24’), the maximum induction of VpMYBR1 was the greatest in powdery mildew resistant genotypes ‘Baihe-35-1’, reaching more than 28-fold. In addition, the VpMYBR1 expression of ‘Baihe-35-1’ induced by defence signal molecular (SA and MeJA) was significantly higher than that of ‘Hunan-1’ and ‘Shang-24’. Then the VpMYBR1 gene was transformed into A. thaliana by Floral Dip method to make further study for verifying its function. The results showed that the expression of VpMYBR1 improved the resistance against the powdery mildew by hypersensitive reaction.【Conclusion】 The results of the study indicate that VpMYBR1 is a founding member of a small family of proteins containing a novel Sant/myb domain which is likely to participate in the regulation of the resistance to U. necator. This research has provided a new candidate gene for improving the disease resistance of V. vinifera by transgenic technology.

Isolation of Flowering Locus T Ortholog and the Effects on Blooming of Cymbidium faberi

SUN Chong-Bo, XIANG Lin, LI Xiao-Bai, QIN De-Hui, LI Bo-Jun, GUO Fang-Qi, WU Chao

Scientia Agricultura Sinica. 2013, 46(7): 1419-1425. doi:10.3864/j.issn.0578-1752.2013.07.012

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【Objective】The objective of this study was to analyze the function of Flowering locus T(FT) on blooming in Cymbidium faberi. 【Method】 The full-length cDNA sequence of CfFT was obtained by RT-PCR and RACE cloning technology. The expression patterns of CfFT in different tissues and flower buds of different developmental stages were analyzed by real-time quantitative RT-PCR. To investigate the function of CfFT, an expression vector was constructed for transformation into tobacco by using agrobacterium-mediated method.【Result】In flowers, CfFT was expressed more in young flower buds than in mature flowers and was predominantly expressed in young ovary. Ectopic expression of CfFT in transgenic tobacco plants showed novel phenotypes by flowering earlier than wild-type plants. Real-time quantitative RT-PCR analysis suggested that expression levels of CfFT closely related with the flowering time of different 35S::CfFT transgenic tobacco lines. Further analysis of the flower time related genes indicated that the expression of genes LEAFY (LFY), APETALLA1 (AP1), FRUITFULL (FUL) and SEPALLATA1 (SEP1) were closely related with the expression of FT in 35S::CfFT transgenic tobacco plants.【Conclusion】Ectopic expression of CfFT gene promote flowering in transgenic tobacco.

Analysis of Frozen Beef Protein Oxidation Effect During Thawing

LI Yin, SUN Hong-Mei, ZHANG Chun-Hui, BAI Yue-Yu, WANG Zhen-Yu

Scientia Agricultura Sinica. 2013, 46(7): 1426-1433. doi:10.3864/j.issn.0578-1752.2013.07.013

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【Objective】The protein oxidation effects of frozen beef during thawing were investigated. The purpose of this study was to provide a scientific basis for meat fresh-keeping thawing and quality control.【Method】The frozen hindquarter samples of grassland yellow beef were thawed using low-variable temperature and high relative humidity thawing method (test group, temperature 2℃→6℃→2℃，RH＞90%) and air thawing method (control group, 4℃), respectively, then the quality of the thawing meat was compared.【Result】Compared to the control group, the degree of meat oxidation of the test group was lower, the appearance of beef was fresher; the degree of myofibrillar protein oxidation was lower (The content of carbonyl was lower 0.75 nmol•mg-1 protein and sulfydryl was lower 13.11 nmol•mg-1 protein than the control group), cooking loss, drip loss, protein content of drip were significantly lower than the control group. The results of the SDS-PAGE and DSC showed that thawing could result in protein aggregation, degradation and denaturation, and the control group was more serious than the test group. The scanning electron microscopy (SEM) results showed that thawing could destroy the microstructure of muscle, but the muscle fiber bundles of the control group were damaged more seriously.【Conclusion】 During thawing protein oxidation could result in browning, protein aggregation, degradation and denaturation, damaging the structure of muscle fiber, lowering the WHC, all of these changes would result in drip loss and deterioration of meat quality. The low-variable temperature and high relative humidity thawing could significantly reduce the oxidation of protein, prevent the deterioration of beef quality and achieve fresh-keeping thawing.

Construction of the Recombinant Adenovirus of FABP4 Gene of Qinchuan Cattle

WEI Sheng-Juan, ZAN Lin-Sen, WANG Hong-Bao, CHENG Gong, JI Shu-Han, WANG Hong, FU Chang-Zhen, JIANG Bi-Jie

Scientia Agricultura Sinica. 2013, 46(7): 1434-1440. doi:10.3864/j.issn.0578-1752.2013.07.014

Abstract ( 740 )

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【Objective】The aim of this study was to construct the recombinant adenovirus vector with FABP4 gene of Chinese Qinchuan cattle, so as to provide a basis for studying FABP4 gene functions and mechanisms at cell level.【Method】The primers were designed according to the FABP4 mRNA sequence in GenBank, and the gene was cloned by RT-PCR and then sequenced. The bovine gene fragments containing both FABP4 gene and restriction enzyme were inserted into a shuttle vector pAdTrack-CMV to construct the recombinant shuttle vector pAdTrack-CMV-FABP4. After identifying by digestion and sequencing, pAdTrack-CMV-FABP4 plasmid was linearized by PmeⅠ, and then it was transformed into E. coli BJ5183 competent cells containing backbone vector pAdeasy-1 to obtain recombinant vector by homologous recombination. Then the positive plasmid was linearized by PacⅠ, and transfected into HEK 293A cells for virus packing, amplification and titer testing by TCID50.【Result】The results of enzyme digestion, sequencing and regular PCR detection showed that the recombinant overexpression vector containing FABP4 CDS region was successfully constructed. The infectious titer of the virus AD-FABP4 was 1.58×109 PFU•mL-1.【Conclusion】In this experiment, the recombinant adenovirus vector carrying FABP4 gene was constructed successfully and high titer adenovirus was acquired.

miR-133a Targets BIRC5 to Regulate Its Gene Expression in Chicken

WANG Xing-Guo, SHAO Fang, GONG Dao-Qing, LU Xiang-Yun, GU Zhi-Liang

Scientia Agricultura Sinica. 2013, 46(7): 1441-1447. doi:10.3864/j.issn.0578-1752.2013.07.015

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【Objective】This study was conducted to investigate the expression pattern of miR-133a in various chicken tissues, and find whether baculoviral IAP repeat containing 5 (BIRC5) is the target gene of miR-133a.【Method】The bioinformatics methods were used to predict the target genes of miR-133a. Real-time PCR was used to detect the expression patterns of miR-133a of various tissues in chicken. Dual-luciferase reporter assays and site mutation assays were used to verify whether BIRC5 is a target gene of miR-133a. 【Result】Two hundred and eighty-seven genes of 11891 genes from 3′UTR database were predicted as target genes of miR-133a. The expression patterns of miR-133a in various chicken tissues showed that miR-133a was highly expressed in muscle especially in skeletal muscle. Reporter assays showed that BIRC5 was the target gene of miR-133a and the site mutation assays validated the target site of miR-133a in BIRC5. 【Conclusion】 miR-133a is a miRNA related with skeletal muscle development of chicken, and BIRC5 is a bona fide target gene in chicken.

Proteome Comparison of Honeybee (Apis mellifera ligustica) Worker Venom Between Collected from Venom Glands and Electrical Stimulated

LI Rong-Li, ZHANG Lan, HAN Bin, FANG Yu, FENG Mao, ZHOU Tian-E, LI Jian-Ke

Scientia Agricultura Sinica. 2013, 46(7): 1448-1462. doi:10.3864/j.issn.0578-1752.2013.07.016

Abstract ( 556 )

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【Objective】The objective of this study is to investigate bee venom composition and difference by comparison of bee venom collected from venom glands and electrical stimulated of Italian bees (Apis mellifera ligustica), and to provide a theoretical basis for pharmaceutical application.【Method】The proteome between bee venom collected directly from the venom glands and artificially electrical stimulation were compared using gel-based (one-dimensional gel electrophoresis, 1-DE, two-dimensional gel electrophoresis, 2-DE) and gel-free proteomics approaches, mass spectrometry, and bioinformatics.【Result】In the gland venom, 19, 11 and 14 proteins were identified by 1-DE, 2-DE and shotgun analysis, respectively, which correspond to 30 nonredundant proteins. They were mainly classified as bee venom toxins (50%), antioxidant systems, protein folding and molecular transporters (50%). In construct, in electrical stimulated venom, 12, 3 and 7 proteins were identified, respectively, by 1-DE, 2-DE and shotgun analysis. They were classified into 14 nonredundant proteins, which the major components were venom toxins (93%). Notably, phospholipase A2-like protein was identified for the first time in two forms of bee venom and peptidyl-prolyl cis-trans isomerase was identified only in the gland venom for the first time. The abundance of apamin preproprotein and secapin in the gland venom were significantly higher than those in the electrical stimulated venom. However, phospholipase A-2, venom dipeptidyl peptidase IV precursor, venom allergen acid phosphatase and mast cell degranulating peptide had higher abundance in the electrical stimulated venom than those in the gland venom.【Conclusion】 The venom collected from venom glands contains more protein species, but the abundance of the toxin proteins in electrical stimulated venom are no less than the venom collected from the glands. As the pharmacological components are mainly contained in the toxin proteins, the electrical stimulated venom is a convenient and effective way for utilization. The identified new proteins significantly extend the knowledge of bee venom composition. The result may provide a theoretical and practical basis for future rational use of the honeybee venom.

Molecular Characteristics of Extended-Spectrum β-Lactamases in Clinical Isolates of Proteus mirabilis from Poultry

PAN Yu-Shan, YUAN Li, WU Hua, LIU Jian-Hua, LIU Zhen-Zhen, ZHAO Yi-Shuang, HU Gong-Zheng

Scientia Agricultura Sinica. 2013, 46(7): 1463-1469. doi:10.3864/j.issn.0578-1752.2013.07.017

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【Objective】This experiment was conducted to explore the genotpye of extended-spectrum β-lactamases (ESBLs) gene and the genetic environment of blaCTX-M carried by Proteus mirabilis. 【Method】The isolates were characterized by isolation and identification, antimicrobial susceptibility testing, confirmation of ESBLs, resistant genes screening, and conjugation experiments. PCR mapping was performed to investigate the genetic environment of blaCTX-M. 【Result】 Among the 21 P. mirabilis clinical isolates from poultry, 10 (47.6%) isolates exhibited extended-spectrum β-lactamase (ESBL) phenotypes. The most common types of ESBL gene screened by PCR and sequencing experiments were blaCTX-M-14 (n=6) , blaOXA-1 (n=6), followed by blaCTX-M-65 (n=4). The blaOXA-10 (n=1) was also detected, while blaSHV was not detected. Nucleotide sequence analysis revealed that insertion sequence ISEcp1B elements present in the upstream region of blaCTX-M genes in all ESBL-producing P. mirabilis, and IS903D was also detected in downstream of CTX-M-9 group genes.【Conclusion】This is the first report the blaCTX-M-65 gene was found in P. mirabilis in China, and the results showed that CTX-M-tpye ESBL in P. mirabilis is no longer an unusual phenomenon in poultry origins.

Effects of Vitamin D3 on Growth Performance, Slaughter Performance, Carcass Quality and Tibia Quality of Geese

WANG Di, WANG Bao-Wei, GE Wen-Hua, ZHANG Ming-Ai, YUE Bin, CHEN Miao-Lu, WANG Jiao, MENG Ling-Feng

Scientia Agricultura Sinica. 2013, 46(7): 1470-1480. doi:10.3864/j.issn.0578-1752.2013.07.018

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【Objective】This experiment was conducted to study the effects of dietary vitamin D3 level on growth performance, carcass quality and tibia quality of geese aged 0 to 15 weeks.【Method】Three hundred and sixty 1-day-old Qingnonghui liver goose were selected and randomly assigned into 6 treatment groups with 6 replicates for each treatment and 10 in each replicate．The geese were fed with basal diet supplemented with 0, 200, 400, 800, 1 600, and 3 200 IU•kg-1 vitamin D3, respectively. Geese were raised for fifteen weeks and their feed intake and weight were recorded to get the growth performance. One or two geese were selected and slaughtered from each replicate to measure slaughter performance, carcass quality and tibia development at the end of 4 and 15 weeks.【Result】Osteomalacia rate of the controlled group was 36.67% between weeks 0 to 4 and 26.67% between weeks 5 to 15, which was remarkably higher than that of treatment groups. There was no clinical poisoning symptoms when addition of vitamin D3 reached 3 200 IU•kg-1. Compared with the controlled group, the daily gain and daily feed intake of geese aged 0 to 4 weeks were significantly increased by supplementation of 400 IU•kg-1 vitamin D3 (P＜0.05)．Adding vitamin D3 could enhance the daily gain of geese aged 5 to 15 weeks significantly (P＜0.05), but there was no significant difference among the treatment groups. Supplementation of 800 IU•kg-1 vitamin D3 could significantly improve the slaughter rate (P＜0.01) and significantly increase the half net carcass rate of geese aged 15 weeks (P＜0.05). Adding vitamin D3 significantly affected the color of pectoral muscle (P＜0.05). BMD, bone weight, tibia calcium and tibia phosphorous of geese aged 4 weeks and BMD, bone weight and bone ash of geese aged 15 weeks were significantly improved by supplementation of vitamin D3(P＜0.05). 【Conclusion】 Vitamin D3 plays a regulatory role in the growth performance, slaughter performance, carcass quality and tibia quality of geese. Osteomalacia rate of geese increased notably without the addition of vitamin D3. The optimal vitamin D3 level in geese diet should be 471.70 IU•kg-1 between 0 and 4 weeks, and 548.63 IU•kg-1 between 5 and 15 weeks.

Effects of Drought Stress on Water and Photosynthetic Physiology Activities of Near-Isogenic Indica Rice Lines at Flowering Stage

YANG Yong-Jie, ZHANG Cai-Xia, SONG Jian, XIONG Jie, WANG Xi, ZHANG Xiu-Fu, FU Guan-Fu, TAO Long-Xing

Scientia Agricultura Sinica. 2013, 46(7): 1481-1491. doi:10.3864/j.issn.0578-1752.2013.07.019

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【Objective】An indica near-isogenic rice lines were treated with drought stress at flowering stage to study water and photosynthetic physiology changes of flag leaf. By analyzing the relationships among agronomic phenotype characters, water and photosynthetic physiology changes and fertility of rice, the authors try to accumulate data for drought tolerance evaluation of rice.【Method】The rice materials were suffered with drought stress begin from heading of main shoot for 15 days, water and photosynthetic physiology parameters were measured after treatment.【Result】The results showed that the indica near-isogenic rice represented various drought tolerance, and there was no correlation between drought tolerance and agronomic phenotype characters, neither with physiological activities of rice under well watered condition. However, under drought stress at flowering stage, the correlation coefficient between drought resistance indexes and changes of water content of flag leaf, water potential of flag leaf, stomatal conductance of flag leaf were 0.614**, 0.514** and 0.541**, respectively. This indicates that rice drought tolerance has a correlation with changes of water content, water potential and stomatal conductance. In addition, except the correlation coefficient between drought resistance indexes and changes of Fv/Fm of flag leaf (0.470*), there was no correlation between rice drought tolerance and photosynthetic physiology.【Conclusion】In summery, the changes of water physiology parameters could be used as indicators for screening rice with drought tolerance.

Accumulation Characteristics of Cadmium-Accumulated Rice Cultivars with High Cadmium Accumulation

ZHENG Tao, LI Ting-Xuan, ZHANG Xi-Zhou, YU Hai-Ying, WANG Yong

Scientia Agricultura Sinica. 2013, 46(7): 1492-1500. doi:10.3864/j.issn.0578-1752.2013.07.020

Abstract ( 611 )

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【Objective】It is important to remediate Cd-polluted soils and select the cultivars with high Cd accumulation ability. The characteristics of cadmium-accumulated rice cultivars with high cadmium accumulation, and the purification ability to Cd-polluted soil were studied to provide a theoretical basis for restoration of Cd-polluted soil.【Method】Two Cd high-accumulation cultivars (Lu17-T2171 and Wujin4B) were screened earlier, with common variety Luhui17 as contrast. A pot culture experiment was carried out in a net room on the farm of Sichuan Agricultural University, Sichuan province, China in 2010, including soil culture experiment with Cd treatments of 0 mg•kg-1, 2 mg•kg-1, 10 mg•kg-1, and 50 mg•kg-1. The differences between cultivars in purification ability of rice to the soil Cd were discussed.【Result】As the concentration of Cd increased, the biomass of Cd high-accumulation cultivars was also increased. Biomass of Lu17-T2171 increased by 33.96% and 19.51%, respectively, at tillering stage and flowering stage with Cd50, compared with Cd0; while that Wujin4B increased by 54.71% and 15.22%, respectively. High-accumulation cultivars with stronger absorptive capacity to Cd at tillering stage. Under the condition of Cd50, Cd contents in shoot and root of Lu17-T2171 reached 87.24 mg•kg-1 and 400.59 mg•kg-1, respectively, which were 2.38 and 2.86 times higher than Luhui17, while those of Wujin4B reached 102.26 mg•kg-1 and 384.77 mg•kg-1, respectively, 2.79 and 2.86 times higher than Luhui17. Enrichment factor of both rice cultivars decreased as Cd concentration increase, moreover, the high-accumulation cultivars were apparently higher than common cultivars. High-accumulation cultivars with stronger ability to accumulate cadmium at flowering stage. Under the condition of Cd50, Cd accumulations in shoot and root of Lu17-T2171 reached 1 847.20 μg/pot and 892.06 μg/pot, respectively, 2.91 and 2.74 times higher than Luhui17, while those of Wujin4B reached 1 895.37 μg/pot and 783.42 μg/pot, respectively, 2.98 and 2.41 times higher than Luhui17. Moreover, the migration rate of high-accumulation cultivars at flowering stage was obviously higher than that of common cultivars. The purification rate in plant and shoot of high-accumulation cultivars was apparently higher than that in common cultivars. Under the condition of Cd50, the purification rate in plant and shoot of Lu17-T2171 reached 5.07% and 3.42% at flowering stage, 2.86 and 2.90 times higher than Luhui17, while those of Wujin4B reached 4.96% and 3.51%, 2.79 and 2.98 times higher than Luhui17. 【Conclusion】 Cd high-accumulation rice cultivars showed a high absorptive capacity and accumulation ability to Cd at tillering stage and flowering stage. Thus, it can be taken as potential restoration material for Cd-polluted farmlands.

Pheromonal Activity of Rectum Extracts from Chinese Citrus Fly (Bactrocera minax)

XIAO Wei, WU Ke-Ming, GONG Qing-Tao, ZHAO Zhi-Mo, HE Lin

Scientia Agricultura Sinica. 2013, 46(7): 1501-1508. doi:10.3864/j.issn.0578-1752.2013.07.021

Abstract ( 622 )

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【Objective】The objective of this study is to make clear days that emerged adults of Chinese citrus fly (Bactrocera minax) need to reach sexual maturity, to obtain rectum extracts from adults fly and assay their pheromonal activity.【Method】Under a microscope, morphology and relative location of rectum in digestion system of adult flies were observed. Rectums from both males and females of 5-, 10- and 15-day-old adult flies were extracted with n-hexane. Male and female bodies with rectum excised were also extracted. EAG responses of male and female adults to rectum extracts and body extracts were recorded. Field attractiveness of rectum extracts from 15-day-old male and female adults were also investigated. 【Result】The rectum of B. minax was located at the distal part of digestion system, between hind gut and anus. The rectum was morphologically similar as that of other species in Bactrocera. EAG responses of male and female adults to heterosexual rectum extracts were the highest among all extracts. Meanwhile, male and female adults also showed relatively high EAG responses to homosexual rectum extracts. In field tests, 15-day-old female rectum extracts attracted significantly more adults than any one attractant of hydrolyzed protein lure, sugar solution with wine and vinegar mixture, and 15-day-old male rectum extracts. Meanwhile, even attractiveness of 15-day-old male rectum extracts was much lower than that of 15-day-old female rectum extracts, male rectum extracts still attracted a small number of adults. Sex ratio of adults attracted by 15-day-old female rectum extracts was 0.82﹕1, while any of other attractants attracted more females than males. 【Conclusion】About 15 days after emergency, Chinese citrus flies reaches sexual maturity. With similar morphology and location in digestion system as other species in Bactrocera, the rectum of B. minax also probably secrets and stores pheromonal substances in its rectal gland. Moreover, pheromones in female rectum are attractive to male and female adults in the field.

Preparation and Identification of the Monoclonal Antibody Against Chlorothalonil

WANG Ling-Ling, ZHI Ai-Min, YANG Yan-Yan, SONG Chun-Mei, WANG Kun, CHAI Shu-Jun, HOU Yu-Ze, DENG Rui-Guang, ZHANG Gai-Ping

Scientia Agricultura Sinica. 2013, 46(7): 1509-1515. doi:10.3864/j.issn.0578-1752.2013.07.022

Abstract ( 556 )

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【Objective】 The objective of this study is to generate high sensitivity and high specificity monoclonal antibody against chlorothalonil and identification of its immunological characteristics. 【Method】 The active group hydroxyl was introduced to chlorothalonil (CTN) and formed CTN derivatives (CTN-OH) by chemical modification. The artificial immunogen CTN-BSA and coating antigen CTN-OVA were obtained by using the method of mixed N, N-carbonyl diimidazole (CDI) to conjugate CTN-OH with bovine serum albumin (BSA) and ovalbumin (OVA), respectively. Ultraviolet (UV) and sodium dodecyl sulfate polycrylamide gel electropheresis (SDS-PAGE) were used to identify CTN-BSA. Then BALB/c mice were immunized. The titer and sensitivity of polyclonal antibody was detected by indirect ELISA and blocking ELISA after four times immunization. The high titer and high sensitivity mice were selected for cell fusing. High sensitivity and high specificity monoclonal antibody was prepared after many times subcloning, and immunological characterization was characterized. The preparation of ascites was carried out using in vivo induction method.【Result】UV and SDS-PAGE results showed that CTN-BSA artificial antigen was synthesized successfully. Indirect ELISA showed a high titer above 1﹕104 of the antiserum of all the three BALB/c mice. The sensibility of antiserum of No.2 mouse was the best, which the IC50 was 93.593 ng•mL-1. Four hybridoma cell lines 1E8, 1F4, 2A10, and 2E2 were screened after cell fusing, the indirect ELISA titers of the mAb were 1﹕1.6×103, 1﹕6×102, 1﹕6×102, and 1﹕6×102 in supernatant and 1﹕5.12×105 in ascites. The special mAb showed good sensitivity with IC50 of 21.6685 ng•mL-1 to chlorothalonil.【Conclusion】The CTN artificial antigens were synthesized successfully and high sensitivity and specificity monoclonal antibodies were obtained. It laid a foundation for the CTN immunology fast detection methods.

The Extraction and Isolation Method of Nitrosyl Hemochromogen

SUN Wei-Qing, ZHOU Guang-Hong, XU Xing-Lian

Scientia Agricultura Sinica. 2013, 46(7): 1516-1522. doi:10.3864/j.issn.0578-1752.2013.07.023

Abstract ( 588 )

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【Objective】The purpose of the experiment is to obtain high purity nitrosyl hemochromogen (NH). 【Method】Seven kinds of different polarity organic solvents were used to extract and isolate the NH. The UV and EPR spectral characteristics of extracted solution and the hue of meat residue after being extracted NH were analyzed. 【Result】 NH is insoluble in petroleum ether completely. Acetone can dissolve NH efficiently and the extracted solution is relatively stable. Extraction rate of ethyl acetate is not high, but which can keep the pink of NH. The EPR spectrum of NH extracted by acetone was showed the signal information of free radical not of the NH because of the presence of the lipid. For these results, a three-step gradational extraction and isolation procedure with petroleum ether/acetone/ethyl acetate step by step was established based on the characteristics of UV and EPR spectroscopy of NH solution. The diced sample was extracted with 3 times the petroleum ether ( boiling range is 30-60℃) in the dark, anaerobic conditions for 36 h, discard the petroleum ether, and then add 1.5 times the acetone extraction for 10 min, filter to obtain bright red solution. The acetone was removed and the NH was isolated with ethyl acetate again.【Conclusion】It indicated that the three-step gradational extraction procedure with petroleum ether/acetone/ethyl acetate step by step is an effective method for isolation of NH.

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