Table of Content

01 August 2015, Volume 48 Issue 15

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CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS

Exploration of Elite Alleles of Agronomic and Fiber Quality Traits in Xinluzao Cotton Varieties by Association Analysis

NIE Xin-hui, YOU Chun-yuan, BAO Jian, LI Xiao-fang, HUI Hui, LIU Hong-liang, QIN Jiang-hong, LIN Zhong-xu

Scientia Agricultura Sinica. 2015, 48(15):  2891-2910.  doi:10.3864/j.issn.0578-1752.2015.15.001

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【Objective】Discovery molecular markers associated with agronomic and quality fiber traits in Xinluzao cotton varieties and identification of allelic variation related to these traits and typical materials carrying elite alleles will lay a foundation of mocular design breeding for Xinluzao cotton varieties. 【Method】Seventy-five pairs of SSRs with high polymorphism and uniform distribution on 26 chromosomes were used to scan polymorphism in fifty-one Xinluzao cotton varieties, programming language R was applied to make boxplot for phenotypic traits in different environments, molecular marker data and 15 phenotypic traits were ananlyzed by the method of MLM（mixed linear model）in TASSEL software package on the basis of population structure and linkage disequilibrium analysis, the loci of elite allelic variation and typical materials carrying elite alleles were identified based on phenotypic effect values. 【Result】Fifty-one Xinluzao cotton varieties were divided into four subgroups by analysis of population genetic structure. The BLUP（Best linear unbiased prediction）analysis on fifteen phenotypic traits showed that five traits (first fruit section height, first fruit section pitch number, lint percentage, fiber upper half mean length, short fiber) were very stable and ten traits (plant height, fruit section pitch number, leaf branch number, effective boll number, seed weight lint weight, micronaire value, fiber strength, fiber uniformity, fiber elongation) had stable changing trends in four environments. Through association analysis, a total of one hundred and seventeen allelic variation loci associated with agronomic traits (P＜0.05) were detected, the allelic variation loci with the maximum explanation rate for nine agronomic traits were BNL3650b (plant height, R²=11.78. First fruit section height, R²=20.80. First fruit section pitch number, R²=11.54), NAU3995c (fruit section pitch number, R²=14.86), BNL119b (leaf branch number, R²=9.7), NAU3995d (effective boll number, R²=14.98), BNL3255a (seed weight, R²=11.11), NAU1071a (lint weight, R²=10.15) and BNL663a (lint percentage, R²=12.42), repectively. A total of fifty-five allelic variation loci associated with fiber quality traits (P＜0.05) were detected, the allelic variation loci with the maximum explanation rate for six fiber quality traits were NAU1103b (fiber upper half mean length, R²=6.4), NAU1071a (Fiber strength, R²=7.57), BNL3140b (micronaire value, R²=12.06), BNL3650b (fiber uniformity, R²=13.47), BNL1421a (short fiber，R²=13.04) and BNL2960b (fiber elongation, R²=11.67). A total of thirty-nine loci (P＜0.01) significantly associated with agronomic (twenty-nine loci) and fiber quality (ten loci) traits were detected by association analysis, ranging from 6.45% to 20.8% for explanation rate of phenotypic variation; while, the average was 11.14%, and forty-seven loci related to more than two traits were detected. Seventeen typical materials with elite alleles were identified by phenotypic effect analysis. In this study, twenty-seven QTL reported in previous researches were detected, in which seven QTLs includingBNL3650 (FU), BNL3033 (MV), NAU3254 (FE), GH132 (LP), TMB1618 (FS), BNL1421 (FS,FU), and BNL119 (FE) associated with the same traits compared with the other reported results.【Conclusion】The original fifty-one Xinluzao cotton varieties have simple population genetic structure and low level of linkage disequilibrium, and the changing trend of phenotypic traits in two environmrnt was stable. The association analysis based on SSR revealed some elite alleles that related to agronomic and fiber quality traits and typical materials.

Negative Regulation of Anthocyanin Biosynthesis in Tomato by MicroRNA828 Under Phosphate Deficiency

JIA Xiao-yun, LIU Hui, SHEN Jie, LI Fang, DING Na, SUN Yan, GAO Chang-yong, LI Run-zhi

Scientia Agricultura Sinica. 2015, 48(15):  2911-2924.  doi:10.3864/j.issn.0578-1752.2015.15.002

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【Objective】 In this study, the responses of miR828 and its targets to phosphate deficiency and the role of miR828 in anthocyanin biosynthesis under phosphate deficiency were investigated by using wild-type and miR828 overexpressed transgenic tomatoes. 【Method】 The potential targets of miR828 were predicted and validated by psRNATarget and RLM-5′RACE, respectively. An alignment of deduced amino acid sequences of SlMYB7-like with orthologs from Arabidopsis thaliana (AtMYB7) and Antirrhinum majus (AmMYB330) was constructed using MegAlign of DNAStar. The phylogenetic tree of SlMYB7-like and the selected R2R3 MYBs was constructed using neighbor-joining method using MEGA5. The expressions of miR828 and SlMyb7-like in AC, MicroTom and LA1996 tomato seedlings were analyzed. The expression of miR828 in different tissues/organs of tomato (AC) was analyzed by qRT-PCR. Wild-type and miR828 overexpressed transgenic tomatoes were cultured under normal phosphate (KH₂PO₄ 3.4 g·L^-1) and phosphate deficiency (KCl 1.86 g·L^-1) for 15 d. The phenotypic changes and the expression of miR828, SlMyb7-like (SGN-U320618), several anthocyanin biosynthetic genes as well as the anthocyanin content were detected. 【Result】SlMyb7-like was validated to be a direct target of miR828. Protein sequence analysis showed that the SlMYB7-like protein shares the highest homology with the Arabidopsis subgroup 4 MYB7 (AtMYB7), and Snapdragon MYB330, having more than 80% sequence similarity. Phylogenetic analysis grouped SlMYB7-like in a clade with AtMYB7 and AmMYB330. SlMYB7-like contains the conserved amino acid motif ([D/E]L_X2[R/K]_X3L_X6L _X3R/DLIVRLHSLLGNRWSLIAGR), a signature feature common to subgroup 6 R2R3 MYBs (AtMYB75/90/113) that are involved in anthocyanin biosynthesis. The highest abundance of miR828 was detected in MicroTom seedlings, where the transcript of its target gene (SlMyb7-like) was accumulated the lowest. The expression of miR828 in different tissues of tomato tested was very low with relatively higher levels in buds, flowers and green fruits. Under the normal phosphate condition, the expression of anthocyanin biosynthetic genes in the miR828 over-expressed tomatoes decreased by 30%-60% and the content of anthocyanin decreased by 40%. Phosphate deficiency induced both the expression of miR828 and SlMyb7-like. The expression of SlMyb7-like, anthocyanin biosynthetic genes and the content of anthocyanin in the miR828 overexpressed tomatoes were lower than that in control plants under phosphate deficiency. The above data suggest that miR828 negatively regulates the anthocyanin biosynthesis in tomato under phosphate deficiency.【Conclusion】SlMyb7-like is a direct target gene of miR828. Both the expressions of miR828 and SlMyb7-like are induced under phosphate deficiency. MiR828 negatively controls anthocyanin pathway by repressing the expression of SlMyb7-like, and thereby negatively regulating anthocyanin biosynthetic genes in tomato under phosphate deficiency.

Cloning and Function Analysis of the MsNAC2 Gene with NAC Transcription Factor from Alfalfa

SHEN Yu-hua, XU Zhen-jun, TANG Li-hong, YANG Xiao-po, HUANG Wen-jie, WU Xiao-bin, ZHANG Wen-bo

Scientia Agricultura Sinica. 2015, 48(15):  2925-2938.  doi:10.3864/j.issn.0578-1752.2015.15.003

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【Objective】NAC transcription factors belong to a unique class of transcription factors in plants. The common characteristics of the NAC proteins are the presence of a conserved NAC domain, comprising about 150 amino acids in N-terminals and a highly variable transcriptional regulation region in C-terminals. Extensive studies have revealed that NAC transcription factors not only play important roles in plant growth and development, but also have functions in regulation of responses to biotic and abiotic stresses. The objective of this study is to analyze the sequence feature and expression patterns of MsNAC2 gene with NAC transcription factor from alfalfa (Medicago sativa L.) and to illustrate the stress-resistance regulation mechanism of alfalfa through over-expression in tobacco, and through transgenic technology to improve the resistance of alfalfa and lay a foundation for the research on improving the quality.【Method】 The full length cDNA of MsNAC2 gene was cloned by RT-PCR and RACE, whose nucleotides and amino acid sequence were analyzed by bioinformatics software. The relative quantitative expression of MsNAC2 was detected by real-time PCR technology. A fusion expression vector MsNAC2-GFP was constructed to identify the subcellular localization. And a plant over-expression vector pBI121-MsNAC2 was constructed and used to transform tobacco by agrobacterium-mediated method. The phenotypic and physiological performance of the transformants were characterized under various abiotic stresses to investigate the function of MsNAC2 on stress resistance in tobacco. 【Result】 The full-length cDNA of MsNAC2 was 1 358 bp and the open reading frame was 1 023 bp, which encoded MsNAC2 comprising 340 amino acids with a molecular weight of 39.4 kD. The N-terminal amino acid residue of MsNAC2 contained a typical NAC conserved domain, and the C-terminal amino acid residue was highly variable. Phylogenetic analysis demonstrated that MsNAC2 shared high homology with CsNAC (Citrus sinensis Osbeck), and both belong to ATAF subfamily of NAC family. Subcellular localization analysis revealed that MsNAC2 was expressed in the nucleus. Transcription and expression analysis by Real-time PCR showed that MsNAC2 was up-regulated by 250 mmol·L^-1 NaCl, 20% PEG6000, 0.1 mmol·L^-1 ABA and 4℃, and the expression of MsNAC2 in root was apparently higher than those in leaf. The seedling height, root length, fresh weight and dry weight and other growth indicators of the transgenic tobacco were higher than that of the wild type in the treatments of NaCl, PEG6000, and 4℃. The physiological analysis revealed that malonaldehyde (MDA) was lower in the transformants than that in the WT, 82.60%, 73.20% and 77.80% in the treatments of NaCl, PEG6000, and 4℃, while the concentrations of proline, SOD and POD were increased. 【Conclusion】A new NAC transcription factor MsNAC2 was cloned from alfalfa, which responded to cold, drought and ABA. And MsNAC2-over- expressed tobacco possessed better anti-salt, anti-drought and anti-cold activities than wild-type tobacco. Those showed that the MsNAC2 might play an important role in stress response which indicated that this gene might participate in regulation of plant abiotic stress response.

TILLAGE & CULTIVATION·PHYSIOLOGY & BIOCHEMISTRY

Effect of Nitrogen Application Rate on Agronomic, Photosynthetic Characteristics and Yield of Spring Foxtail Millet

ZHANG Ai-ying, GUO Er-hu, WANG Jun, FAN Hui-ping, LI Yu-hui, WANG Li-xia, WANG Xiu-qing, CHENG Li-ping

Scientia Agricultura Sinica. 2015, 48(15):  2939-2951.  doi:10.3864/j.issn.0578-1752.2015.15.004

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【Objective】The aim of the study is to determine the optimal amount of nitrogen application and to investigate the correlation between photosynthetic characteristics and yield of spring foxtail millet varieties through analyzing the performance of agronomic traits, photosynthetic characteristics and yield under different nitrogen levels.【Method】The experiment was carried out in a field-split plot design with three replicates with varieties as main plot and nitrogen fertilizer as secondary plot. Cultivars Changnong 35 and Jingu 21 were selected as the experimental materials, and the area of each plot was 15.0 m²(5 m×3 m) and the seedling density was 3×10⁵ plants/hm². The designed 6 levels of nitrogen were 0 kg·hm^-2 (N1), 45 kg·hm^-2 (N2), 90 kg·hm^-2 (N3), 135 kg·hm^-2 (N4), 180 kg·hm^-2 (N5) and 225 kg·hm^-2 (N6), respectively. Forty percent of the nitrogen was applied as base fertilizer, and 60% applied at jointing stage to booting stage. After plants heading, the SPAD (Soil and Plant Analyzer Development, SPAD) value of the top-three leaves was measured through SPAD-502 (Konica Minolta), and the Ci (intercellular CO₂ concentration, Ci), Pn (net photosynthesis rate, Pn) and E (transpiration rates, E) of the top-three leaves were measured using CIRAS-2 (PPSYSTEMS). 【Result】As the nitrogen levels increased, plant height, stem diameter and panicle length of spring foxtail millet exhibited a rising trend, and weight per panicle, grains weight per panicle and SPAD, E and Pn values of the flag leaf and the top-three leaves increased first and then decreased, however, 1000-grain weight had no significant difference in various nitrogen treatments. All of the above traits excepting 1000-grain weight reached to or tended to the highest value, meanwhile the yield tended to be stable at N level of 90 kg·hm^-2 for spring foxtail millet, which suggested that the N level of 90 kg·hm^-2 may be as the optimal amount of nitrogen application for spring foxtail millet. Furthermore, a regression equation between yield and various N levels was established and the maximum theoretical yield of spring millet was calculated. The results showed that there was no significant difference between the maximum theoretical yield and the observed one at N level of 90 kg·hm^-2 (P_{Changnong 35} = 0.5571, P_{Jingu 21}= 0.6632). Therefore, the N level of 90 kg·hm^-2 was confirmed as the optimal amount of nitrogen application for spring foxtail millet. The correlation analysis showed that the correlation coefficients between E, Pn of the flag leaf and yield and E, Pn of the top-three leaves and yield were 0.87, 0.86, 0.82, 0.83, respectively, and all reached a significant level.【Conclusion】 The optimal amount of nitrogen application was 90 kg·hm^-2 in South Central Shanxi ecological region and soil conditions, and E and Pn of the flag leaf and the top-three leaves were significantly positively correlated with the yield of spring foxtail millet.

The Transitional Cultivation Patterns of Winter Oilseed Rape in China and the Corresponding Nutrient Management Strategies

WANG Yin, LU Jian-wei

Scientia Agricultura Sinica. 2015, 48(15):  2952-2966.  doi:10.3864/j.issn.0578-1752.2015.15.005

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Both direct-sowing and transplanting are the dominated methods to establish winter oilseed rape in the current Yangtze River Basin, China, which play important roles in the development of national oilseed rape industry and edible oil supply security. With the altered cultivars, production targets, local labor conditions, and productivity levels, cultivation patterns and corresponding technical practices had transformed and developed at different historical stages. The present paper reviewed the development processes of cultivation pattern and corresponding technique in winter oilseed rape production since the foundation of China. Direct-sowing, the main cultivation pattern at the beginning and transplantation, was started from 1960s, and then transplantation was fully adopted in large scale during 1980s to 1990s. Nowadays, direct-sowing and transplantation cultivations have co-existed in winter oilseed rape production of China. Correspondingly, nutrient management practices have progressed from farmyard manure application to N and P chemical fertilizers application, and then converted to advocate the balanced application of N, P, K, and B fertilizers, and now a high-yielding and high efficiency technique system is found for transplanted oilseed rape. There are significant differences in the cultivation characters and growth progresses between direct-sowing oilseed rape (DOR) and transplanting oilseed rape (TOR), their growth stages, plant density, population structure and individual morphology were emphatically compared in this paper. The DOR showed generally shorter growth stages, weaker plants and lower individual yield, compared with TOR. However, DOR played group effects under the higher plant density and had stronger abilities to absorb nutrient and water due to the greater root system, and therefore showed a potential of high yield and high efficiency. At present, the abundance-deficiency nutrient indices and recommended fertilization system of TOR have been established in Yangtze River Basin, and nutrient management strategy has also been completed. The main points included determining fertilizer application rates based on soil fertility and target yield level, ensuring balanced nutrient supply and combined application of organic and inorganic fertilizers, adopting split application of N and K fertilizers (basal fertilizer﹕overwintering fertilizer﹕bolting fertilizer = 60%﹕20%﹕20%) to match the growth and nutrient uptake, aiming to strengthen individual plant and obtain high yield. In view of the rapid development but lagging nutrient management practice of DOR, the differences in nutrient responses, absorption and distribution, requirement and utilization between DOR and TOR were emphatically compared and discussed. The results indicated that DOR were more sensitive to nutrient deficiency and insufficient nutrient supply leaded to weak individual growth and population decline, its transport efficiencies for biomass and nutrient were lower, and it showed higher requirement for P and K than TOR. The nutrient management strategy of “promotion in early and stabilization in late” for DOR was presented according to the existing researches, and it included: ensuring P and K supply based on the rational and balanced nutrients application, applying organic fertilizer and returning straw to the field, and paying more attention to the role of N fertilizer in the development of individual and population. The N fertilizer as basal should be reduced and seedling topdressing need to be increased to improve plant growth at early stage, and appropriate topdressing before flowering are also required to ensure population density and yield formation. The recommended N fertilizer application practice of DOR is basal fertilizer﹕seedling fertilizer﹕overwintering fertilizer﹕bolting fertilizer = 40%﹕30%﹕15%﹕15%. Moreover, nutrient management should be adopted with other cultivation practices, e.g. selecting early-maturing and density-resisting varieties, adopting mechanization precision sowing technique, adopting specific long-acting formula fertilizer or controlled release fertilizer, increasing appropriately plant density to reduce fertilizer rate, compensate late-sowing, and improve sink and source, and enhancing plant protection to control diseases, pests and weeds. Furthermore, the main problems in current researches and the key points in future studies for DOR were also proposed in this paper, in hope of providing references for further improvement of cultivation practices and scientific fertilization technique.

PLANT PROTECTION

Multi-Disease Resistance Evaluation of Chinese Advanced Winter Wheat Lines for the National Regional Test

LIU Tai-guo, QIU Jun, ZHOU Yi-lin, XU Shi-chang, CHEN Huai-gu, LIU Yan, GAO Li, LIU Bo, ZHENG Chuan-lin, CHEN Wan-quan

Scientia Agricultura Sinica. 2015, 48(15):  2967-2975.  doi:10.3864/j.issn.0578-1752.2015.15.006

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【Objective】The objective of this study is to evaluate the multi-disease resistance of candidate cultivars and advanced lines which were attended the National Wheat Regional Test (NWRT) to stripe rust, leaf rust, stem rust, powdery mildew, eyespot of wheat and Barley yellow dwarf virus (BYDV) at adult stage in the past fifteen years, and the results of the study could provide a foundation for the breeding, release and application, reasonable arrangement of resistant cultivars, and food security of wheat in China.【Method】From 1999 to 2014, there were 1 815 advanced lines of NWRT inoculated with mixture races of CYR32, CYR33 of Puccinia striiformis f. sp. tritici, with PHT and THT of Puccinia triticina, 21C3 and 34C2 of Puccinia graminis f. sp. tritici, more than 15 mixture virulences of Blumeria gramins f. sp. tritici, strong virulence isolates of Fusarium graminearum and Rizoctonia cerealis, GAV strain of BYDVat the adult plant stage.【Result】There were 250 cultivars or advanced lines showed resistance to three or more diseases out of the 7 evaluated diseases. Out of the 1 815 advanced lines, 310 lines were approved to be released by the National Crop Variety Approval Committee (NCVAC). The frequency of the moderately resistant (MR) and highly resistant (HR) lines was (39.8±21.7) %, while it was (39.7±27.9)% for the frequency of moderately susceptible and highly susceptible lines. Similarly, the frequencies of resistant lines (MR and HR) to wheat Fusarium head blight, sheath blight, stem rust and powdery mildew were (31.5±27.5)%, (31.1±35.6)%, (48.2±25.6)% and (25.0±14.6)%, respectively, while the lines showing susceptible to these diseases were (68.5±27.5)%, (62.2%±38.6)%, (31.3±20.7)% and (70.7±14.6)%, respectively. Nevertheless, it was only (9.9±3.8)% of wheat lines showed leaf rust for resistance and (70.4±15.1)% was susceptible. In addition, except Henong 831 showed moderately resistance, there were no any lines showed resistance to BYDV. 【Conclusion】It is very difficult to find out resistant cultivars due to lack of resistance sources, while it is much more easier for stripe rust and powdery mildew. Gansu and Sichuan provinces are the principal origins of resistant cultivars to wheat stripe rust and powdery mildew in the released cultivars. There were less lines showed resistance to wheat leaf rust.

Molecular Cloning and Function of the Yellow Gene from Nilaparvata lugens

WANG Bo, YAO Yun, XU Ze-wei, LIN Xin-da

Scientia Agricultura Sinica. 2015, 48(15):  2976-2984.  doi:10.3864/j.issn.0578-1752.2015.15.007

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【Objective】The objectives of this study are to clone NlYellow from the brown planthopper (Nilaparvata lugens), explore the expression profiles during the development in adults and in different tissues, then study its function through RNA interference.【Method】Primers were designed by online version of primer 3 software and NlYellow was cloned, then the nucleotide sequence was translated into amino acid sequence. The alignment of amino acids residues was conducted using N. lugens Yellow and others searched from GenBank. Also a Neighbor-Joining method was used to construct a phylogenetic tree. Quantitative real-time PCR (qRT-PCR) was applied to measure the relative expression levels of N. lugens at different developmental stages (embryo, 1st-5th instar nymphs and adult) and in different tissues (head, thorax, abdomen, leg, wing, midgut, ovary, and testis). NlYellow dsRNA at 0.4 µg was injected into 5th instar nymphs and the adult phenotypes were observed. 【Result】The NlYellow was cloned, and through sequence alignment it was found that the NlYellow was more similar to the ApYellow (98%), but it was less conserved when compared with that of Drosophila melanogaster and Bombyx mori.Phylogenetic analysis showed that it clustered with Acyrthosiphon pisum,too. NlYellow’s expression was variable in the embryo, the expression level in 1st, 3rd, 4th and 5th day were lower than other days. Furthermore, its expression levels were higher in 3rd and 5th instar nymph than others (P＜0.05). Tissue-specific expression analysis elucidated that NlYellow expressed in all tissues including head, thorax, abdomen, leg, wing, midgut and testis in male, but the first four tissues had higher expression level (P＜0.05). On the contrary, NlYellow was only expressed in head, thorax, leg, and wing in female. Furthermore, its expression level in short wing adult was higher than that of long wing adult (P＜0.05). Also in short wing adults, male’s expression level was higher than female (P＜0.05). Using RNA interference to knock down the NlYellow expression, it was found that the color of the whole body turned into yellow, especially in thorax, abdomen and legs.【Conclusion】NlYellow might involved in insect pigmentation and change of the body colour.

Optimization of Fermentation Process of Beauveria bassiana of SDDZ-9 Against Frankliniella occidentalis

ZHANG Lu-lu, WU Sheng-yong, WANG Shuai-yu, LI Juan, LEI Zhong-ren

Scientia Agricultura Sinica. 2015, 48(15):  2985-2994.  doi:10.3864/j.issn.0578-1752.2015.15.008

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【Objective】Entomopathogenic fungus Beauveria bassiana has been widely applied as an important biocontrol agent for controlling many insect pests, while the poorly large-scale production technology of B. bassiana is one of the key issues which may consequently restrict its wide application. Study on the liquid fermentation process research of B. bassiana can improve the liquid fermentation of biomass, thereby providing high-quality fermentation broth for solid liquid fermentation stage and improving spore production of solid fermentation. The study may serve as a basis for the large-scale production technology of B. bassiana. 【Method】The liquid fermentation of B. bassiana is affected by several factors, such as temperature, volume of liquid, rotate speed, initial conidia concentration, pH and so on. Firstly, Plackett-Burman (PB) design was used to screen the main factors which affected the liquid fermentation. Secondly, the method of the steepest ascent path was applied to identify the central point of response surface and approach the optimal region. The climbing direction and the change range were determined by gradient direction of experimental value changes and the effect value of each factor, respectively. Lastly, the central composite design (CCD) was adopted to identify the optimal liquid fermentation concentration by setting up a multiple quadratic regression equation, analyzing and evaluating the correlations among these factors which affect the mycelium mass of B. bassiana SDDZ-9, one of the most virulent strains against western flower thrips (Frankliniella occidentalis. 【Result】Temperature, volume of liquid, rotate speed and initial conidia concentration were the main factors on mycelium mass. The change of pH within certain limits had less influence on this strain of liquid fermentation. Without considering pH, when the fermentation conditions of temperature, volume of liquid, rotate speed and initial concentration were 27.08℃, 49.72 mL/250 mL of triangular flask, 205.45 r/min, and 1.122×10⁷ conidia/mL, the maximum mycelium mass was produced after 48 h of fermentation. Using the culture conditions to produce conidia, the mycelium mass obtained was about 14.769 g·L^-1, which was consistent with the predicted biomass. After the solid state fermentation, the yield achieved about 20.16 g·kg^-1 and 1.84×10¹¹ conidia/g.【Conclusion】The spore production of B. bassiana could be significantly improved under optimized fermentation conditions. The results may provide reference for large-scale liquid fermentation of B. bassiana SDDZ-9.

SOIL & FERTILIZER·WATER-SAVING IRRIGATION·AGROECOLOGY & ENVIRONMENT

Effect Degree of Fertilization Practices on Soil Organic Carbon and Fraction of Croplands in China—Based on Meta-Analysis

CAI An-dong, ZHANG Wen-ju, YANG Pin-pin, HAN Tian-fu, XU Ming-gang

Scientia Agricultura Sinica. 2015, 48(15):  2995-3004.  doi:10.3864/j.issn.0578-1752.2015.15.009

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【Objective】 Fertilization practice is one of the key regulating factors on soil organic carbon in cropland. The objectives of this study were to explore the effect size of fertilization practices on total and mineral-associated organic carbon in topsoil under different cropping system and cropland uses, which has important implications for the equilibrium and sustainable management of soil organic carbon in agricultural ecosystem.【Method】By using the data collected from published literatures, a data set (286) with the same soil organic carbon fractionation method from independent research was built up. Meta-analysis method was implied to quantify the effect degree of fertilization practices (chemical fertilizers and organic manure) on the content of soil total organic carbon and mineral-associated organic carbon fraction under different cropping systems, cropland uses, and inherent soil properties (e.g., texture).【Result】Compared with no fertilizer treatment, fertilization practices significantly increased soil total and mineral associated organic carbon content by 39.4% and 27.7%, respectively. The increasing rate of applying organic manure (58.4% and 41.9%) was 3.4 and 5.2 times higher than that of chemical fertilizers application (13.4% and 8.0%), respectively. Generally, the effect degree of fertilization practices on the total soil and mineral associated organic carbon was significantly different among cropping systems, cropland uses, and soil textures. The effect size of organic manure application on total soil organic carbon (58.5%) and chemical fertilizers application on mineral associated organic carbon (10.7%) with mono-cropping were significantly higher than that with double cropping (55.6% and 7.3%), whereas there was no significant difference under chemical fertilizers application on total soil organic carbon (13.3%-13.8%) and organic manure application on mineral associated organic carbon (42.6%-43.5%) between these two cropping systems. For different cropland uses, the application of organic manure and chemical fertilizers in upland resulted in significantly higher increased rate (15.8% and 59.7%) than that in paddy fields (10.0% and 43.3%) on total and mineral associated organic carbon. However, application of chemical fertilizers did not significantly increase total or mineral associated organic carbon content in paddy field. As for soil textures, the increased rate of applying organic manure on total soil organic carbon (64.4%) and that of chemical fertilizers on mineral associated organic carbon (15.6%) in sandy soil with low content of soil organic carbon were significantly higher than that for loam and clay soil, whereas there was no significant difference for that between loam and clay soil with a mean value of 8.0%.【Conclusion】 Overall, applying organic fertilizer including chemical fertilizers combined with organic manure has a great significance to the accumulation and sustainable management of soil organic carbon and fertility, especially for mono-copping system and sandy soil.

Component Characteristics of Soil Labile and Recalcitrant Nitrogen Under Different Long-Term Fertilization Systems in East China

YU Wei-shui, LU Chang-ai, LI Gui-hua, WU Hong-liang, ZHAO Ya-wen, WANG Bi-sheng, MENG Fan-hua

Scientia Agricultura Sinica. 2015, 48(15):  3005-3014.  doi:10.3864/j.issn.0578-1752.2015.15.010

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【Objective】Labile nitrogen (Lab-N) and recalcitrant nitrogen (Rec-N) are two important components of soil nitrogen, and their ratio has an effect on nitrogen turnover and sequestration properties. Therefore, it is important to study the characteristics of Lab-N and Rec-N under different long-term fertilization systems for better nitrogen management and soil fertility improvement. 【Method】Lab-N, Rec-N and their ratio were studied by particle size-density separation method under four long-term fertilizer treatments, namely no fertilizer (CK), chemical fertilizer (NPK), chemical fertilizer combined with straw (NPKS), and chemical fertilizer combined with manure (NPKM), of three upland soils (Gongzhuling black soil, Zhengzhou fluvo-aquic soil and Qiyang red soil) and Wangcheng paddy soil that collected from east China. 【Result】 The average Lab-N in upland soils was 0.15 g·kg^-1, much lower than that of paddy soil (0.22 g·kg^-1). While Lab-N/TN in upland soils was much higher than that in paddy soil. After 23 years, the Total N (TN) of the upland soils under CK treatment decreased significantly (7.5%-9.7%) compared with the start of the experiment, while in the paddy soil the TN increased by 11.5%. The TN contents in all soils under NPK treatment were significantly higher than that of CK treatment. The Lab-N of red soil under NPK treatment was significantly lower than that of CK treatment, while for other soils there were no significant differences under two treatments. The TN contents of all soils under NPKS treatment increased significantly compared with CK treatment, while there was no significant difference in the Lab-N and Lab-N/TN between black soil and paddy soil. The Lab-N of red soil under NPKS treatment decreased significantly compared with CK treatment, while the Lab-N of fluvo-aquic soil increased significantly. The TN, Lab-N and the Lab-N/TN in the upland soils under NPKM treatment increased significantly. And the black soil increased the most by 85.0%, 106.0% and 4.2%; there was no significant difference in the Lab-N and Lab-N/TN in the paddy soil between NPKM and CK treatments. Both the Rec-N and the TN in four soils under four different treatments were in the order of NPKM＞NPKS＞NPK＞CK. The Rce-N/TN under NPKM decreased significantly compared with CK treatment. 【Conclusion】 Both the Lab-N and the Lab-N/TN in upland soils were more susceptible to fertilizer application compared with paddy soil. The TN, Lab-N and Lab-N/TN all increased significantly under NPKM treatment, and the improvement effect was in the order of NPKM＞NPKS＞NPK.

HORTICULTURE

Selenium Content of Main Deciduous Fruits from China and Its Dietary Exposure Assessment

NIE Ji-yun, KUANG Li-xue, LI Zhi-xia, PANG Rong-li, YANG Lian, CHEN Qiu-sheng, LI An, ZHAO Xu-bo, XU Wei-hua

Scientia Agricultura Sinica. 2015, 48(15):  3015-3026.  doi:10.3864/j.issn.0578-1752.2015.15.011

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【Objective】Detection and dietary exposure assessment of selenium content of 6 main deciduous fruits from China (including apple, pear, peach, grape, jujube, and kiwifruit) will provide reference for production and consumption of these fruits in China. 【Method】 A total of 760 samples of above 6 fruits were collected from the main fruits producing areas, including Anhui, Hebei, Henan, Jiangsu, Liaoning, Shaanxi, and Xinjiang. Selenium in samples was detected by hydride generation atomic fluorescence spectrometry. Risk of selenium daily intake of Chinese adults and lactating women from these fruits (including mean intake, maximum intake, median intake, and minimum intake) were assessed by upper tolerable limit (UTL), upper intake limit (UIL), and adequate intake (AI), respectively. 【Result】Selenium content of 760 samples were 0 (not detected)-38.0 μg·kg^-1with an average of 4.3 μg·kg^-1. 78.2% samples contained selenium below 5 μg·kg^-1, and 14.2% samples were selenium-rich with selenium over 10 μg·kg^-1. There were differences in selenium content among samples, and the variance coefficient of selenium content was up to 80.0% (jujube), 110.0% (grape), 116.8% (pear), 125.7% (kiwifruit), 126.0% (apple), 148.2% (peach), and 136.5% (total). The mean selenium contents in different fruits were in the order of jujube (7.3 μg·kg^-1)＞grape (6.4 μg·kg^-1)＞peach (5.5 μg·kg^-1)＞kiwifruit (5.3 μg·kg^-1)＞pear (4.7 μg·kg^-1)＞apple (1.3 μg·kg^-1). Mean selenium content of pear and that of peach from Shaanxi, as well as that of grape from Shaanxi and Xinjiang, were significantly higher (at 5%) than that of the same fruits from the other provinces. Mean selenium daily intake of Chinese from the above 6 fruits were 0.032 μg·d^-1 (kiwifruit), 0.120 μg·d^-1 (apple), 0.183 μg·d^-1 (grape), 0.197 μg·d^-1 (jujube), 0.213 μg·d^-1 (peach), 0.222 μg·d^-1 (pear), and 0.968 μg·d^-1 (total), respectively. Risk assessment results showed that selenium daily intake of Chinese from the above 6 fruits were safe, and the risk indexes were all under 100% with the range of 0.001%-99.702% (adults) and 0.001%-31.847% (lactating women). 【Conclusion】Generally, both the levels of selenium and the ratio of selenium-rich production of the above 6 fruits were low. Mean selenium content of jujube and that of apple was the highest and the lowest, respectively, while there was no significant difference between the other 4 fruits. There was a significant difference in fruit selenium content among some provinces. For Chinese, the selenium intake risk from the above 6 fruits to lactating women was lower than those to adults. In short, selenium daily intakes of Chinese from the above 6 fruits were safe with no negative effects on their health. And selenium-rich fruit production is one of the effective ways to increase Chinese dietary intake of selenium.

Pectin Polysaccharide Degradation in Relation to the Texture Softening in Pear Fruit

QI Xiu-dong, WEI Jian-mei, GAO Hai-sheng, JIA Yan-ru, ZHANG Hai-e

Scientia Agricultura Sinica. 2015, 48(15):  3027-3037.  doi:10.3864/j.issn.0578-1752.2015.15.012

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【Objective】The aim of this study is to investigate the relationship between the characteristics of cell wall pectin polysaccharide degradation and textural softening and storability of pear fruit, these results will further demonstrate the mechanism of fruit softening and provide a basis for improving fruit quality and developing better storage technology.【Method】Take the fruits of ‘Yali’ and ‘Jingbaili’ pears as the materials to compare the difference of the changes of pectin polysaccharide content, the molecular weight distribution and the related enzyme activity between two pear cultivars with different storabilities and textural properties during growth and softening.【Result】During development, in ‘Yali’ fruit, the content of both covalent soluble pectin (CSP) and ironic soluble pectin (ISP) increased rapidly, which were significantly higher than that of ‘Jingbaili’ fruit, and the water soluble pectin (WSP) content of ‘Yali’ fruit increased slowly which was lower than that of ‘Jingbaili’ fruit. Moreover, both WSP and CSP components were transferred from the low molecular weight to the high molecular weight in ‘Yali’ fruit. During storage, in ‘Yali’ fruit, the CSP showed a high and constant content with a slow increase in the content of WSP, which all maintained the higher molecular weight. But in ‘Jingbaili’ fruit, the CSP content decreased rapidly and a rapid increase showed in WSP and ISP content, and the pectin components were shifted from higher molecular mass to the lower ones. Moreover, changes of CSP, WSP and ISP contents were obviously related to fruit firmness only during development stage in ‘Yali’ fruit, but in ‘Jingbaili’ fruit, the changes of the contents of the three pectin compositions showed significant correlations with firmness loss mainly at stage of storage. The results indicated that it showed mainly the significant differences of pectin degrading enzymes activity between the two pear cultivars during storage, which the activities of polygalacturonase (PG), pectinmethylesterase (PME), β-galactosidase (β-Gal) and α-arabinofuranosidase (α-Af) and the increased rate in ‘Jingbaili’ fruit was significantly higher than that of ‘Yali’ fruit. Among them, β-Gal and α-Af activities increased rapidly just after harvest, while the increase date of PG and PME activities were relatively lagged and the correlations of the changes of β-Gal and α-Af activities with the changes of firmness and pectin components were stronger than that of PME and PG in ‘Jingbaili’ fruit, while in ‘Yali’ fruit only the change of α-Af activity had a significant relationship with the ISP content during storage. In summary, the pectin polysaccharide degradation showed significant differences between ‘Yali’ and ‘Jingbaili’ pear fruits and the related-factors played different roles at different softening stages, thus resulting in significantly different softening and storage characteristics.【Conclusion】The pectin polysaccharide fractions showed obvious accumulation of large molecular component and slowly degradated with fruit ripening in ‘Yali’ fruit, while the larger molecular components accumulated during fruit development degradated rapidly into smaller ones with fruit softening in ‘Jingbaili’ fruit. The insoluble pectin content and its molecular mass distribution may be an important index for measuring storability of pear fruit. Both β-Gal and α-Af played stronger roles in promoting ‘Jingbali’ fruit softening.

SPECIAL FOCUS: THE IMPACT OF INFLUENZA VIRUSES ON HUMAN AND ANIMAL HUSBANDRY

Guidance: The Impact of Influenza Viruses on Human and Animal Husbandry

WANG Xiu-rong, CHEN Hua-lan

Scientia Agricultura Sinica. 2015, 48(15):  3038-3039.  doi:10.3864/j.issn.0578-1752.2015.15.013

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Genome Sequencing and Genetic Analysis of H4N8 Subtype Avian Influenza Virus Isolated from Duck

ZHAO Qing-qing, LI Qun-hui, ZHU Jie, ZHONG Lei, LIU Jing-jing, GU Min, WANG Xiao-quan, LIU Wen-bo, LIU Xiu-fan

Scientia Agricultura Sinica. 2015, 48(15):  3040-3049.  doi:10.3864/j.issn.0578-1752.2015.15.014

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【Objective】 Based on the difference of hemagglutinin (HA) and neuraminidase (NA), avian influenza viruses (AIVs) are classified into 16 hemagglutinin (HA) and 9 neuraminidase (NA) subtypes. According to the differences in pathogenicity, AIVs can be divided into highly pathogenic avian influenza virus (HPAIV) and low pathogenic avian influenza virus (LPAIV). H4 AIVs are low pathogenic influenza viruses which are generally produced asymptomatic infections in poultry. But H4 AIVs also has potential threats to both poultry and mammals. Strengthening investigation on H4 subtype avian influenza viruses is important for the study of evolution of AIVs. The objective of this experiment is to investigate the molecule characteristics and genetic evolution of H4 subtype avian influenza virus. 【Method】One H4N8 subtype avian influenza virus, designated as A/duck/Nanjing/1102/2010 (H4N8) (DK/NJ/1102), was isolated from a live poultry market in eastern China during epidemiological surveillance in 2010. The complete genome sequences of the strain was sequenced and analyzed. The virus was identified by HA/HI test and RT-PCR test. The gene was cloned into pGEM-Teasy vector for sequencing, respectively. BLAST the nucleotide identity in GeneBank. The genome sequences of H4 subtype influenza viruses available in GeneBank and some other reference sequences were downloaded for genetic analysis .【Result】The results showed that the HA gene of DK/NJ/1102 had the highest nucleotide sequence identity of 98.9% with A/duck/Mongolia/274/2007(H4N3), and the amino acid sequence at the cleavage region of the HA gene was “P-E-K-A-S-R-G”, which is typical for low pathogenicity AIVs. NA gene had the highest nucleotide sequence identity of 98.8% with a duck-origin virus A/Duck/Eastern China/n91/2009 (H3N8) isolated from eastern China, whereas PB1, PA and NP genes were all mostly related with H1 subtype avian influenza viruses. The M gene shared the greatest nucleotide sequence identities (over 99.9%) with A/wild duck/Korea/CSM4-12/2009(H5N1) while the NS gene was most closely related to H7N7 subtype AIV isolated from Korea in 2009 and there was no amino acids missing in NS1 protein at 80-84 sites.【Conclusion】These data indicated that the genome composition of the strain A/duck/ Nanjing/1102/2010(H4N8) was complicated, it may be a reassortant whose genes originated from different subtypes of AIV.

Development of RT-PCR Technique for Detection of H7N9 Subtype Avian Influenza Virus

WANG Yun-he, BAO Hong-mei, SUN Jia-shan, LI Yan-bing, XU Xiao-long, WANG Zi-long, SHI Jian-zhong, ZENG Xian-ying, WANG Xiu-rong, CHEN Hua-lan

Scientia Agricultura Sinica. 2015, 48(15):  3050-3055.  doi:10.3864/j.issn.0578-1752.2015.15.015

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【Objective】On March 2013, the National Health and Family Planning Commission of China announced that human infections with H7N9 subtype AIV had occurred in Shanghai and Anhui province, China. Because this novel reassortant (H7N9) virus had not previously been seen in either animals or people, the situation raises many urgent questions and global public health concerns. In this study, two pairs of RT-PCR primers were designed to target the haemagglutinin (HA) and neuraminidase (NA) genes of H7N9 virus, and a reverse-transcription PCR assay to rapidly detect the novel H7N9 subtype avian influenza virus was developed and evaluated. 【Method】 H7 and N9 primers were chosen based on the conserved regions of sequences that were designed and analyzed using DNASTAR and oligo 6.0. one-step RT-PCR assay for the detection of H7N9 virus was established with RNA extracted by using one-step access RT-PCR kit. The specificity of the RT-PCR assay was evaluated with H7N9 influenza virus as positive control and Newcastle disease virus, infectious bronchitis virus, infectious bursal disease virus and other avian respiratory viral pathogens as negative control. After specificity test by using clear background influenza virus and other pathogens, the detection limits of the assay were assessed with serial 10-fold dilutions of H7N9 influenza virus (10^6.5 EID₅₀·mL^-1). The specificity assays were evaluated using influenza A viruses of various genetic backgrounds and other avian pathogen. The sensitivity assays were determined using viral RNA extracted from serially diluted AIV-infected allantoic fluid. In addition, a blinded experiment was carried out to validate the accuracy of this assay in comparison with the results of real-time fluorescence RT-PCR. 【Result】The H7 HA can be detected by this assay, while other H1-H6 and H8-H15 subtype HA, as well as other avian pathogens were detected negative in specificity assay. Similarly, only the N9 NA related to the novel H7N9 virus was detected, the other N1-N9 NA were detected negative. Results of 10-fold dilution series of allantoic fluid by one step RT-PCR assays showed that detection limit of the assay was approximately 1.4×10^2.5 EID₅₀ per reaction. Furthermore, the assays showed clinical specificity for identification cloacal swabs of H7N9 virus.【Conclusion】The RT-PCR assay established in this study can be used as a referee method for early diagnosis of the avian-origin influenza A (H7N9) virus infection.

The Adaptation of H9N2 Subtype AIV in Mouse and Analysis of Amino Acid Mutation

DING Jie, GAO Yu-wei, SANG Xiao-yu, CHENG Kai-hui, YU Zhi-jun, ZHANG Kun, CHAI Hong-liang, WANG Tie-cheng, XIA Xian-zhu, HUA Yu-ping

Scientia Agricultura Sinica. 2015, 48(15):  3056-3063.  doi:10.3864/j.issn.0578-1752.2015.15.016

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【Objective】H9N2 subtype avian influenza virus can spread between the guinea pig after passages in guinea pigs continuously for 9 generations, showing that the capacity of H9N2 subtype avian influenza virus infection in mammals as well as the spreading capacity between mammals are still very strong. Thus, this experiment using this strain virus A/Chicken/Jinan/Li-2/2010 (H9N2), JN, to study the molecular basis of H9N2 subtype influenza virus variation, and screen the pathogenicity related amino acid sites.【Method】An H9N2 subtype AIV was serial passaged in the lung of mice to acquire the variant strains JN-P9-2-M1, the mice were dissected, the lungs were removed, after grinding and centrifugation, the mice of next generation were inoculated intranasally, after nine generations of spreading, MDCK cells were used for virus multiplication. Then the full-length sequences of JN-P5-2-M1 and JN-P9-2-M1eight segments were amplified, cloned and analyzed. Amino acids encoded by each gene were deduced, compared to JN primary virus (P0) , and the nucleotide and amino acid changes in virus passages were obtained. The mice were dissected, the lung, liver, spleen, kidneys, brain and intestines were obtained, and the virus titer of tissues was titrated. Mice were anesthetized, and each virus dilution was used to inoculate intranasally three mice for detecting the survival rate and morbidity of mice. Lungs of mice were collected, then the pathological and immunohistochemical staining was made to compare the JN and JN-P9-2-M1 virus. 【Result】 JN-P9-2-M1 had high pathogenicity to mice, its MLD₅₀ was 10^3.5EID₅₀, when had a 10⁶EID₅₀, 10⁵EID₅₀, 10⁴EID₅₀ dose in mice, its survival rate was 0, at least 1000 times higher than JN, the JN in mice was not lethal. When the dose of JN-P9-2-M1 was 10⁶-10³EID₅₀ for mice, the body weight was significantly reduced, and the clinical symptoms were obvious. After inoculation for 3-8 d the mice showed listless, hair messy, shortness of breath, arched, etc., but when the mice were inoculated with 10⁶EID₅₀ JN virus, the mice body weight change rates were similar to the negative group. JN-P5-2-M1, JN-P9-2-M1, and JN could binding to SAa-2,6Gal receptor, like B influenza virus. JN virus was detected only in the lungs of mice, but JN-P9-2-M1 not only in the lungs of mice but also could be detected in liver, spleen, kidney and brain. 【Conclusion】JN-P9-2-M1’s pathogenicity in mice significantly improved, compared with the original drug increased by at least 1 000 times. The three amino acid sites PB2 E627K, HA N313D, and HA N496S might be the reasons of preliminary improvement of the virus virulence in mice, and the two acid sites PA L342I and NA N218T might be possible to further improve the virus virulence in mice. 

Electrochemical Luminescence Immunoassay for the Detection of H9 Subtype Avian Influenza Virus

QI Wen-bao, LI Fang, LI Hua-nan, HUANG Li-hong, HE Jun, MU Guang-hui, LUO Kai-jian, LIAO Ming

Scientia Agricultura Sinica. 2015, 48(15):  3064-3070.  doi:10.3864/j.issn.0578-1752.2015.15.017

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【Objective】 H9 subtype avian influenza is an important zoonosis. Their six internal genes (PB2, PB1, PA, NP, M, NS) of H7N9 and H10N8 subtypes influenza viruses were derived from endemic H9N2 influenza viruses circulating in poultry. The objective of the study is to establish a special detecting method for H9 subtype avian influenza virus by Electrochemical Luminescence Immunoassay (ECLIA). This ECLIA is significant for influenza surveillance.【Method】The monoclonal antibody and rabbit polyclonal antibody anti-H9 subtype AIV were firstly labeled with [Ru (bpy)₃]²⁺ and biotin, respectively. And the labeled efficient was evaluated by MPI-E system and HABA. The second step is the reaction between samples and monoclonal antibody symbolized with [Ru(bpy)₃]²⁺. Then combine the antigen-antibody complex with rabbit polyantibodies labled by biotin-streptavidin linkage system. The chemiluminescence detection can be conducted within the electrochemical analysis system after addition of tripropylamine as substrate. The best working concentrations of the labeled monoclonal antibody and rabbit polyclonal antibody anti-H9 subtype AIV were optimized. The sensitivity, specificity and repeatability were tested. Three days and 5 days after challenging, 88 clinical samples were detected by ECLIA and chicken embryo isolation method, and the result was compared and analyzed.【Result】The efficiency of the monoclonal antibody symbolized with [Ru(bpy)₃]²⁺ was 1﹕21, and the efficiency of the rabbit polyantibodies labled by biotin-streptavidin linkage system was 1﹕6. Both labeled antibodies were active in IFA and Western blotting. The detection cutoff value was 28.3, with a suspicious interval of 23.4-33.2. Negative and positive coefficients of variant were both less than 10%. The LOD (limit of detection) was 5×10⁴ EID₅₀. ECLIA can specially detect H9 subtype AIV, no reaction with other influenza viruses (H1, H3, H4, H5 and H6) and avian pathogens (NDV, IBV and IBDV). The accordance rate of ECLIA with chicken embryo isolation method was 86.4%.【Conclusion】The ECLIA could be an available tool for diagnosis and control of H9 subtype avian influenza.

Phylogenetic Analysis and Molecular Characteristics of an H1N1 Subtype Swine Influenza Virus

XU Hui-yang, XU Bang-feng, CHEN Yan, SUI Jin-yu, YANG Huan-liang, YIN Hang, YANG Da-wei, QIAO Chuan-ling, CHEN Hua-lan

Scientia Agricultura Sinica. 2015, 48(15):  3071-3078.  doi:10.3864/j.issn.0578-1752.2015.15.018

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【Objective】 The objective of this study is to understand the molecular epidemiology of swine influenza virus, to provide scientific references for prevention of animal influenza in China. 【Method】Nasal swab samples collected from pigs were inoculated into SPF chicken embryos for virus isolation. The HA positive samples were further purified and proliferated in SPF chicken embryos. Eight gene fragments of the isolated virus were amplified by RT-PCR and then sequenced by Applied Biosystems 3500xL Genetic Analyzer. Nucleotide homology was analyzed by using DNASTAR. The phylogenetic trees of the genes were constructed using MEGA 6.0. 【Result】 The isolated virus was identified as an H1N1 subtype influenza virus, designated as A/swine/Zhejiang/245/2013(H1N1). Nucleotide homology analysis showed that eight gene fragments of this strain were highly homologous with the respective gene of the viruses recently isolated from China. Phylogenetic analysis revealed that the strain belonged to the avian-like swine H1N1 lineage, no gene reassortment occurred. Amino acid sequence at the HA cleavage site was IPSIQSR↓G, which had characteristics of low-pathogenic influenza viruses. And its receptor-binding sites contained 190D and 225E, which prefer sialic acid (SA)-2,6-Gal-terminated saccharides that are abundant in mammal upper respiratory epithelium. Among the six glycosylation sites, four sites were located in HA1 section and the rest in HA2. The isolates contained 271T, 627E and 701N in the polymerase subunit PB2, which had been shown to be determinants of virulence and host adaptation. The M2 proteins of the isolates have the mutation (S31N), a characteristic marker which may confer resistance to amantadine and rimantadine antivirals.【Conclusion】The sustained existence and continual mutation of avian-like swine H1N1 influenza virus suggested that intensive swine influenza surveillance should be carried out in the future.

RESEARCH NOTES

Expression and Function Analysis of the Transcription Factor GmMYB111 in Soybean

XU Ling, WEI Pei-pei, ZHANG Da-yong, XU Zhao-long, HE Xiao-lan, HUANG Yi-hong, MA Hong-xiang, SHAO Hong-bo

Scientia Agricultura Sinica. 2015, 48(15):  3079-3089.  doi:10.3864/j.issn.0578-1752.2015.15.019

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【Objective】 A gene encoding MYB transcription factor, designated GmMYB111, was cloned, its basic biological functions and expression pattern were identified in soybean and yeast cells. 【Method】 A MYB transcription factor GmMYB111 was obtained from salt stress-related digital expression profiling (DGEP) data analysis. cDNA sequence of GmMYB111 was isolated and cloned using cDNA from soybean salt-treated roots by RT-PCR method. A homology search was performed using GmMYB111 protein sequence as a query, and protein sequences of high similarity with GmMYB111 from other species were obtained. Using MEGA5.05, multiple sequence alignments between GmMYB111 protein and its homologous ones from other species were done and a phylogenetic tree of homologous species was constructed. The induced expression and tissue-specific expression profiles of target genes in soybean with abiotic stress were detected by real-time fluorescent quantitative PCR. The subcellular localization of GmMYB111 was analyzed using Arabidopsisprotoplast transformation system, and its transcriptional activity and in vivo binding activity were determined by yeast hybrid system. 【Result】GmMYB111 gene, a significantly upregulated gene (27 folds) in response to salt stress, was obtained based on the preliminary digital expression profiling (DGEP) data related to salt stress in authors laboratory. Using RT-PCR method, fragment of this gene was cloned from cultivated soybean root. Sequence alignment revealed that its sequence was consistent with that from the published Williams82 genome database. Bioinformatics analysis indicated that the deduced amino acids had common characteristics of MYB transcription factors with two MYB domains of R2 and R3 at the N-terminal and an acidic amino acid-rich transcriptional activation domain at the C-terminal. Phylogenetic tree analysis suggested that the encoded protein had the closest genetic relationship with GmMYB76, GmMYB12a, and MtMYB61. The expression of GmMYB111 in soybean was induced by high salt, drought, chilling, and ABA treatments, respectively. Real-time fluorescent quantitative PCR detection results showed that the induced GmMYB111 was upregulated by high salt and cold stress, and was first upregulated and followed by a down-regulation by drought stress. There was a wave-like up- and down-regulated expression of GmMYB111 inducted by ABA treatment. Analysis of temporal and spatial expression showed that GmMYB111 was nearly expressed in all detected tissues, and its expression level was relatively high at seedling and low at maturing stage. From the perspective of different tissues, GmMYB111 showed the highest expression in stem, leaf, and flower, relatively low in root, and no expression in pod. Subcellular localization results showed that GmMYB111 was located in the nucleus which belongs to a typical transcription factor, yeast hybrid assay indicated that GmMYB111 had transcriptional activation functions and could bind to the cis-acting element TAACTG motif. 【Conclusion】GmMYB111 is a typical R2R3-MYB transcription factor, with transcriptional activation function and DNA binding activity. Its expression in soybean may be related to the abiotic stress and ABA signal transduction pathway. GmMYB111 is speculated to regulate the soybean response to abiotic stresses by regulating the expression of downstream genes.

Suitability Evaluation of Different Varieties of Chicken for Cooking Process

WANG Chun-qing, LI Xia, ZHANG Chun-hui, LI Xue-ke, DU Gui-hong, LI Hai, XIE Xiao-lei

Scientia Agricultura Sinica. 2015, 48(15):  3090-3100.  doi:10.3864/j.issn.0578-1752.2015.15.020

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【Objective】Different varieties of chicken were selected as the research objects, to analyze the raw meat and cooked meat quality of broilers so as to provide references for the selection of specific varieties of chicken for processing and cooking. 【Method】 The quality indices of raw and cooked meat were measured by 16 and 9 evaluation indices, respectively. The correlation between raw meat and cooked meat was analyzed by correlation analysis. Factors were analyzed to screen the evaluation indices of raw meat for cooking and processing suitability. Then analytic hierarchy process (AHP) was used to decide the indices’ weight. The comprehensive evaluation scores of ten chickens were calculated by the method of “Reasonable-Satisfaction” (RS), and combined with cluster analysis to divide into different grades. Regression analysis was used to verify the accuracy of the model, and the regression equation was established by sensory evaluation scores (the dependent variable) and comprehensive evaluation scores (the independent variable). 【Result】Ten chicken showed a significant difference in basic quality indices (P＜0.05). Shearing force, hardness, spring and chewiness increased after cooking, but sarcomere length shortened, and sarcomere shrinkage of local variety hens (30.41%) was significantly greater than that of other 9 breeds of chickens (P＜0.05), and that of Bantam was only 4.99%. The quality indices of raw and cooked meat showed greater coefficient of variation (CV) among different chicken varieties. In the raw meat, the CV of fat content and fibre density were 60.51% and 48.62%, respectively; but in the cooked meat, that of sarcomere shrinkage, chewiness and shearing force were 48.33%, 34.64% and 20.51%, respectively, so the 10 breeds of chicken can be representative varieties. The result of correlation analysis showed that the indices of fat content, L*, shearing force, cooking loss, hardness, chewiness, sarcomere length and shrinkage were significantly correlated with that of cooked meat (P＜0.01). The indexes of shearing force, hardness and chewness of raw meat were in highly significantly correlation coefficients which were 0.59, 0.62 and 0.48, respectively, and it indicated that the quality of raw meat had an important effect on that of the production. Cumulative proportion of five principal factors was 90.09%, and it contained 90.09% information of the total amount in the factor analysis. The fat content, shearing force, hardness, chewiness, sarcomere length and shrinkage were chosen as evaluation indices of cooking process suitability evaluation by factor analysis from 16 indices of raw meat because of their proportions to five principal factors were great and their weight was 0.0603, 0.1045, 0.0920, 0.2223, 0.0416, 0.1898 and 0.2896, respectively by AHP. And the comprehensive evaluation model was Y=0.0603×M_f+0.1045×M_s+0.0920×M_h+0.2223×M_c+0.0416×M_sl+0.1898×M_sk+0.2896×M_vs, which could count the comprehensive evaluation scores of the ten chickens. The result of cluster analysis showed that the ten chickens could be divided into three classes, which were based on the comprehensive evaluation scores, among the 10 varieties of chicken, Tsingyuan chicken, Spring chicken and AA broiler chicken are most suitable for cooking, the local variety hens are unsuitable for cooking, and the other chickens are more suitable for cooking. Results obtained from regression analysis showed that the regression equation was y=1.899x-0.510, which indicated that the comprehensive evaluation model was acceptable (R²=0.887), and could be used to evaluate the suitability of unknown varieties.【Conclusion】Among the 10 varieties of chicken, Tsingyuan chicken, Spring chicken and AA broiler chicken are most suitable for cooking, the local variety hens was unsuitable for cooking, the other chickens are more suitable for cooking. In this study, based on the raw meat, the suitability evaluation model for cooking processing was set up and could also provide a two-way theoretical support.