Table of Content

16 January 2015, Volume 48 Issue 2

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CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS

The Allelic Variation of Lipoxygenase Genes in Bread Wheat Cultivars from the Yellow and Huai Wheat Areas of China

WU Pei-pei, SONG Shuang, ZHANG Fu-yan, CHEN Feng, CUI Dang-qun

Scientia Agricultura Sinica. 2015, 48(2):  207-214.  doi:10.3864/j.issn.0578-1752.2015.02.01

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【Objective】The objective of this study is to provide useful information for wheat breeding program, a total of 306 wheat cultivars or advanced lines from the Yellow and Huai valleys of China were used to identify LOX (Lipoxygenase) activity and allelic variations of LOX genes in this study.【Method】 Spectrophotometer and microplate were used to measure the LOX activity. The molecular marker Xwmc312 on chromosome 1AL and the functionalmarkers LOX16 and LOX18 on chromosome 4BSwere employed to identify the polymorphism of the LOX genes in Chinese wheat cultivars or advanced lines surveyed by combinations of PCR (Polymerase Chain Reaction) amplification and agarose and electrophoresis gels. 【Result】 The phenotyping results showed that the averaged LOX activity was 65.73 AU·min^-1·g^-1 and the standard deviation (SD) was 13.54 in the wheat cultivars surveyed from the Yellow and Huai valleys, and it ranged from 27.09 to 99.55 AU·min^-1·g^-1 and its coefficient of variation (CV) was 20.6%. In the wheat cultivars surveyed from the Yellow and Huai valleys, LOX activities of 7 wheat cultivars were less than 40 AU·min^-1·g^-1 and LOX activities of 8 wheat cultivars exceeded 90 AU·min^-1·g^-1. These cultivars could provide very important germplasms for improvement of LOX activities of currently popular wheat in wheat breeding program. Genotyping results indicated that three alleles Xwmc312₂₂₇, Xwmc312₂₃₅ and Xwmc312₂₄₇ were found at the QLpx.caas-1AL locus in these wheat varieties with 30.4%, 19.0% and 50.6%, respectively. At TaLOX-B1 locus, two alleles TaLOX-B1a and TaLOX-B1b were identified by the functionalmarkers LOX16 and LOX18 with 28.8% and 71.3%, respectively, in the bread wheat cultivars surveyed. Six allele combinations of LOX genes, i.e., TaLOX-B1a/Xwmc312₂₂₇, TaLOX-B1a/Xwmc312₂₃₅, TaLOX-B1a/Xwmc312₂₄₇, TaLOX-B1b/Xwmc312₂₂₇, TaLOX-B1b/Xwmc312₂₃₅ and TaLOX-B1b/Xwmc312₂₄₇, were found, with 7.2%, 9.5%, 12.1%, 23.2%, 9.5% and 38.6%, respectively, in the bread wheat cultivars surveyed.Analysis of association of LOX alleles and LOX activity indicated that the average of LOX activity of wheat cultivars with Xwmc312₂₃₅ is significantly higher than those of cultivars with Xwmc312₂₂₇ and Xwmc312₂₄₇, and the average of LOX activity of cultivars with TaLOX-B1a is significantly higher than those of cultivars with TaLOX-B1b (P＜0.05). Moreover, analysis of association of LOX allele combination and LOX activity showed that the LOX activity (76.803 AU·min^-1·g^-1) of the cultivars with TaLOX-B1a/Xwmc312₂₃₅ is significantly higher than those of other five LOX allele combinations, whereas the LOX activity (62.45 AU·min^-1·g^-1) of the cultivars with TaLOX-B1b/Xwmc312₂₂₇ genotype is significantly lower than those of other five combinations. 【Conclusion】It was concluded that LOX activities of most of the wheat cultivars from the Yellow and Huai valleys were middle type and the wheat cultivars with extremely low (less than 40 AU·min^-1·g^-1 ) or high (more than 90 AU·min^-1·g^-1 ) LOX activities were very scarce in the Yellow and Huai wheat regions. Amongst 6 different combinations of LOX genes, wheat cultivars with TaLOX-B1a/Xwmc312₂₃₅possessed relatively the highest LOX activity and wheat cultivars with TaLOX-B1b/Xwmc312₂₂₇ possessed relatively the lowest LOX activity in the Yellow and Huai wheat regions (P＜0.05). These results offered important gene germplasms for improvement of LOX activities of bread wheat in wheat breeding program. This study could also provide important information for wheat quality improvement in China.

Production of Transgenic Maize Germplasm with Multi-Traits of Insect-resistance, Glyphosate-Resistance and Drought-Tolerance

SUN Yue, LIU Xiu-xia, LI Li-li, GUAN Yun-yun, ZHANG Ju-ren

Scientia Agricultura Sinica. 2015, 48(2):  215-228.  doi:10.3864/j.issn.0578-1752.2015.02.02

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【Objective】 The objective of this experiment is to study the production of transgenic maize with triple resistance of insect, glyphosate and drought stress. 【Method】In this study, an expression vector harboring four target genes (cry1AcM, epsps, GAT and ZmPIS）was used to transform to maize elite inbred lines 9801 and Qi 319 (Q319) mediated by Agrobacterium, and produced transgenic maize plants with multiple modified traits. Based on herbicide resistance, PCR analysis, transcriptional analysis, western blot detection and insect resistance assay, six stable transgenic maize lines were selected from a large number of transgenic lines. Subsequently, the inbred lines 9801 and Q319 were used as the nontransgenic control to analyze the resistance of transgenic plants to Ostrinia furnacalis In view of different resistances to O. furnacalis of maize plants at different developmental stages, the resistance to O. furnacalis of transgenic plants at different developmental periods was tested indoor and in the fields by innoculating the worm of O. furnacalis, and kernels and the bracts of ears from transgenic plants at filling stage were used to feed the worm. In the herbicide resistance trials in fields, transgenic plants at the six-leaf stage were sprayed with 0.84 kg·hm^-2 glyphosate (commercial application dosage) to evaluate the resistance of transgenic plants. A spraying experiment with triple dose of glyphosate on the plants at three-leaf stage was also performed to evaluate the glyphosate-resistant levels of transgenic lines. To evaluate the resistance of drought stress, the morphological changes of transgenic plants were observed and their photosynthesis and chlorophyll fluorescence were measured on plants at the 10-leaf stage in a drought stress treatment. 【Result】Among six stable transgenic maize lines, L1-L3 come from inbred line 9801 and line Q1-Q3 come from inbred line Q319. The stable expression of the four transgenic genes in the transgenic lines was confirmed by using RT-PCR assay. The levels of cry1Ac protein in the different transgenic lines were determined by Western blot, which showed reliable expression in various organs of the transgenic lines. Six lines with higher resistance to the corn borer compared with wild type were selected through the corn borer resistance experiment in the plants at vegetative stage and filling stage. For herbicide resistance trials, the glyphosate resistance of transgenic plants showed more tolerant than that of wild type control. During drought stress, the transgenic plants had more strong photosynthetic capacity and photosystem II activity than that of the nontransgenic control plants under drought stress conditions. 【Conclusion】The introduction of the cry1Ac-M, epsps, GAT, and ZmPIS genes into maize elite inbred lines increased the insect-resistance, glyphosate-tolerance, and improved the drought-resistance of transgenic plants, and the transgenic plant are up to the level of extensive utilization in corn production. Six new corn transgenic lines with excellent complex traits were obtained in this study., glyphosate and drought under strictly controlled experimental conditions.

To Make Biofuel: Cutting the Lignin or Loosening Lignin’s Grip?

WANG Xiao-juan1,2, YANG Yang, ZHANG Xiao-qiang, JIANG Shao-jun, SONG Yu, ZHOU Hai-chen, JIN Liang

Scientia Agricultura Sinica. 2015, 48(2):  229-240.  doi:10.3864/j.issn.0578-1752.2015.02.03

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Biomass energy from renewable resources has been considered an alternative source of energy, and the growing contribution of biomass to the world energy also gives a new way to solve the problems such as global warming, the soaring fuel costs and environmental pollution. Lignocellulose is the main component of plant cell walls and is also one of the most abundant renewable resources on earth. Lignocellulosic biomass is principally composed of cellulose, hemicellulose and lignin, which can be converted into liquid biofuels such as bioethanol. The linkages between lignin and carbohydrates (hemicellulose and cellulose) are formed by ester, ether and glycosidic types of bonds, which formed a kind of covalently bonded aggregates called lignin-carbohydrate complex. The compositions of biomass and interaction of these components in the cell wall affect the hydrolysis of carbohydrates, and then determine the efficiency of lignocelluosic biomass utilization. Although lignin is one of the most abundant components in the lignocellulosic biomass besides polysaccharides, it has been considered as a physical barrier to prevent enzyme access to cellulose structure. Therefore, to improve the biomass production in the field and the effects of the factory conversions of biomass into biofuel are the two most concerned issues. That is, how to improve the utilization of lignin efficiency has been becoming a hot issue of biorefinery in biomass energy development. In this paper, the characteristics of development and sedimentary of lignin in plant cell walls, genetic improvement and genetic modification of lignin trait in bioenergy crops have been systematically reviewed, which is mainly from the point of view of cutting the lignin to enhance the overall lignocellulosic biorefinery. To select low-lignin-content energy crops, considerable genetic improvements can be expected through traditional breeding program or down regulation the levels of enzymes involved in the reactions specific for lignin monomer synthesis, which would reduce the amount of chemicals and energy used in pretreatment in lignocellulosic biorefinery. Furthermore, to explore the optimization of lignocellulose characters and the possibility of increased biofuel production rate, the technological means of biological refining pretreatment, transformation and biofuel production have been discussed for overcoming the cost barrier of lignocellulosic biomass utilization in the purpose of loosening lignin’s grip. Several different pretreatment and fractionation processes such as acid, alkaline and organic solvent hydrolysis can be used for treatment of lignocellulosic materials. And successful pretreatment can significantly improve the hydrolysis and increase the yield of bioethanol. In addition, the perspectives on hot issues and biofuel industry involved in studies of lignin were discussed.

TILLAGE & CULTIVATION·PHYSIOLOGY & BIOCHEMISTRY

Effects of Sulfur on Selenium Uptake in Wheat and Its Mechanism when Amended with Selenite

LIU Xin-wei, DUAN Bi-hui, ZHAO Xiao-hu, GUO Zai-hua, HU Cheng-xiao, ZHAO Zhu-qing

Scientia Agricultura Sinica. 2015, 48(2):  241-250.  doi:10.3864/j.issn.0578-1752.2015.02.04

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【Objective】This study was conducted to investigate the effects of sulfur (S) on the fractions and species of amended selenite in soil by applying S fertilizer, and explore the mechanism through which S controls the selenium (se) uptake of wheat. 【Method】Wheat (Triticum aestivum L. cv. Zhengmai 9023) was chosen as the test material, and underwent seedling stage soil cultivation and solution cultivation. A potted soil experiment was performed on wheat and the application amounts of S were 0, 150, and 300 mg?kg^-1, respectively, Sulfur was used as S source, Se source was selenite (Na₂SeO₃) and it was designated at 0 and 5 mg?kg^-1. The wheat seedlings were harvested on the 70^th day after sowing and soil samples were taken. The treatments started two weeks after the seedlings were planted in hydroponic experiment which the nutrient solution was made with 1/5 Hogland-Arnon formula. Different amounts of S were added in the experiment: 0, 0.1, and 2 mmol?L^-1; Se was designed at 10 µmol?L^-1. Se and S was supplied to plants in the form of sodium selenite (Na₂SeO₃), sodium selenate (Na₂SeO₄?10H₂O) and magnesium sulfate (MgSO₄?7H₂O), respectively. Wheat was harvested after 24 h. A fully interactional design was performed and each treatment was repeated four times in potted soil experiment and hydroponic experiment. The hydride generation atomic fluorescence spectrometer model HG-AFS-8220 was used to measure the Se content in shoot and root of wheat and soil. Based on the Se absorption of plant and the correlation of selenium content in soil, Se content in the soil was divided into different fractions and species. 【Result】Applications of both Se and S resulted in stimulation to wheat growth with appropriate amount. S was applied at 150 mg?kg^-1 significantly reduced the content and accumulation of Se in shoots and roots of wheat by 61.7% and 35.5% , 55.7% and 24.7%, respectively, but such reduction won’t change significantly with the increasing S application to soil. Application of S significantly decreased the pH value and obviously increased the organic matter content as much as 0.5 units and 0.78 g?kg^-1, respectively. Application of S significantly decreased soluble Se and increased Fe/Mn oxide-bound Se, also increased organic matter-bound Se and residual Se obviously, with no significant effect on exchangeable Se, showing that S application can prompted the passivation of Se in soil. Application of S also significantly decreased the content of various Se species in the soluble Se, and obviously increased the content of Se(IV) and significantly decreased the content of Se(VI) in the exchangeable Se, showing that application of S retarded the conversion of Se to the efficient species in soil. S had a profound effect on the intake of Se by wheat seedlings when Se(IV) and Se(VI) was applied. The Se content in shoot and root of wheat in the Se(VI)-treatment was 44.7 and 22.4 times, respectively, the content in Se(IV)-treatment, while with a S rate of 1 mmol?L^-1, their Se contents were less than 2.8 times and 51.8% of the Se(IV)-treatment, showing that S application significantly reduced the differences of absorption capacity in wheat between the treatments of Se(IV) and Se(VI). 【Conclusion】In addition to improving wheat growth, an appropriate amount of S fertilizer can reduce soil pH and increase organic content, causing soluble Se in soil to be Fe/Mn oxide-bound, organic matter-bound and residual Se, and retarding the conversion of Se(IV) to Se(VI) in the exchangeable Se, thus reducing Se uptake by wheat. Therefore, the content of Se in crops can be effectively controlled by increasing S in the high Se and deficient S area and reducing S in the deficient Se and high S area.

Study on Differences in Comparative Canopy Structure Characteristics and Photosynthetic Carbon Assimilation of Field-grown Pima Cotton(Gossypium barbadense) and Upland Cotton(G. hirsutum)

YAO He-sheng, ZHANG Ya-li, YI Xiao-ping, XUE Jun, LUO Yi, LUO Hong-hai, ZHANG Wang-feng

Scientia Agricultura Sinica. 2015, 48(2):  251-261.  doi:10.3864/j.issn.0578-1752.2015.02.05

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【Objective】The objective of this experiment is to study different characteristics of the canopy structure development, photosynthate accumulation and distribution between pima cotton and upland cotton, and to summarize the principle of yield formation which is of great significance to excavate yield potential and construct a high-yield cultivation technique system.【Method】Pima cotton cultivars Xinhai 22, and H 858 and upland cotton cultivars Xinluzao 13 and Xinluzao 33 were selected as experimental materials. Leaf area index, diffuse non-interceptance, canopy apparent photosynthetic rate, photosynthate accumulation and distribution were measured at different growing stages.【Result】Compared to pima cotton cultivar, the development of upland cotton canopy structure had a more dramatic change throughout the growth period, particularly at the early and late growing stages. Upland cotton cultivars had higher leaf area index, as well as the light absorption rate at the top of canopy. Upland cotton cultivars had lower canopy apparent photosynthetic rate and photosynthate accumulation. Pima cotton cultivar’s leaf area index was lower throughout the growth period, except for the late growth stage. The canopy apparent photosynthetic rate and photosynthate accumulation of pima cotton cultivar were higher than upland cotton cultivar at the full bolling stage and boll opening stage, respectively. However, the ratio of the weight of reproductive system to photosynthate accumulation was significant lower than upland cotton, which led to the significantly differences of economic coefficient and lint yield (upland cotton cultivar 3 000-3 500 kg·hm^-2, pima cotton cultivar 1500 kg·hm^-2).【Conclusion】The significant differences of photosynthate accumulation affected by canopy structure and photosynthetic capacity and photosynthate distribution were main factors causing the lint difference between upland cotton and pima cotton. Improving canopy structure and canopy apparent photosynthetic rate can further increase the production of upland cotton, while balancing the source-sink properties and promoting the conversion efficiency of photosynthate to boll-sink by increasing the plant population density and breeding early-maturing and big-boll varieties are of very important significance for improving the economic coefficient and excavating yield potential of pima cotton cultivar.

Solanum tuberosum L.; Correlation Among Endogenous Hormone Content, Threshold and Dormancy Periods in Processing Potato Varieties Minitubers During Storage

LIU Yue-shan, LI Cheng, WANG Dong-xia, XU Gang, WANG Yu-ping, CHENG Li-xiang, ZHANG Jun-lian, WANG Di, ZHANG Feng

Scientia Agricultura Sinica. 2015, 48(2):  262-269.  doi:10.3864/j.issn.0578-1752.2015.02.06

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【Objective】The dormancy breaking and sprouting of processing potato varieties seed minituber are very important for potato production because of economic purpose. The relationship amongendogenous phytohormone levels, threshold values and dormancy periods were investigated under different storage temperatures and storage periods. The present researches will provide not only a technical parameter for storage, but also a suitable storage conditions for processing potato varieties seed minituber. 【Method】The contents of endogenous phytohormones GA₃, IAA and ABA in seed minitubers were determined using HPLC after storage at three temperatures (4℃, 10℃ and 20℃) during 0-150 d. The effects of endogenous phytohormone levels on the processes of minituber dormancy breaking under different storage days and temperatures were analyzed. The linear relationship between the level of dynamic balance of endogenous phytohormone and the dormancy periods were deduced.【Result】The periods of dormancy breakage were significantly shorter in Shepody and Atlantic cultivars with the increase storage temperature from 4℃ to 10℃ and 20℃ throughout the storage period. The dormancy period of Atlantic was about 15 d shorter than Shepody under 10℃ and 20℃ storage. The contents of GA₃ were significantly increased throughout storage period. After 150 d of storage, the contents of GA₃ were increased by 1.7 and 1.9 times under 10℃, by 1.9 and 2.0 times under 20℃ compared to 4℃ in Atlantic and Shepody, respectively. The contents of IAA were significantly increased during 90-150 d. After 150 d of storage, the contents of IAA were significantly increased by 1.5 and 1.2 times under 10℃, by 1.9 and 1.5 times under 20℃ compared to 4℃ in Atlantic and Shepody, respectively. The contents of ABA were significantly decreased throughout the storage period. The levels of ABA were decreased by 7.2 and 6.4 times under 10℃, respectively. The ratios of (GA₃+IAA)/ABA were significantly increased throughout the storage period. The correlation analysis indicated that there existed a strong positive linear relationship between the ratios of (GA₃+IAA)/ABA and the percentage of sprouting minitubers during storage. The linear relationship showed that the threshold values of ratio of (GA₃+IAA)/ABA were 52.02 and 48.49 in Shepody and Atlantic cultivars at time point of dormancy breakage, respectively. 【Conclusion】The result suggested that the changes in the level of dynamic balance of endogenous phytohormones GA₃, IAA and ABA in minitubers were affected by the storage period and temperature. The threshold value of ratios of (GA₃+IAA)/ABA and conclusions drawn can be an important technical parameter and can be used to control the minitubers dormancy breaking and sprouting in processing potato varieties.

PLANT PROTECTION

Complete Genome Analysis of a PVY^NTN-NW Recombinant Isolate from Yulin of China

GAO Fang-luan, CHANG Fei, SHEN Jian-guo, XIE Lian-hui, ZHAN Jia-sui

Scientia Agricultura Sinica. 2015, 48(2):  270-279.  doi:10.3864/j.issn.0578-1752.2015.02.07

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【Objective】 Potato virus Y (PVY) is a typical member of the genus Potyvirus in the family Potyviridae. It is one of the most destructive pathogens affecting potato and tobacco and causes significant economic losses worldwide. The objective of this study is to determine the genomic structure of an isolate of PVY from Yulin of China and its phylogenetic relationship with reported PVY strains.【Method】The complete genome of the ShX14 was amplified and sequenced from overlapping fragments using 11 pairs of primers designed from the conserved regions of the known PVY isolates. Genomic structure and recombination events of the isolate were evaluated by various bioinformatics approaches. Phylogenetic tree was reconstructed by maximum likelihood (ML) method using nucleotide sequences of coding regions. In addition, phylogeny-trait association analysis was used to evaluate the relationship between PVY isolate and the reported stains. 【Result】The complete sequence of ShX14 had 9 724 nucleotides, excluding the 3′- terminal poly (A) tail. It contains a single open reading frame of 9 186 nucleotides and encodes a polyprotein of   3 061 amino acids. An additional protein, termed ‘PIPO’, is also translated by +2 nucleotide frame shifting within the P3 cistron. The isolate shares 98%-99% nucleotide identity and 98%-100% amino acid identity with HN2 and SYR-NB-16 (PVY^NTN-NW strain, SYR-I genotype), respectively. Similar to the PVY^NTN-NW (SYR-I) genomic structure, three recombination breakpoints were identified at nucleotide positions 2 318, 5 674 and 8 385 in the P1, HC-Pro/P3 and the 5′-terminus of CP gene respectively with high confidence. Phylogenetic analysis indicated that ShX14 was clustered together with HN2 and SYR-NB-16, suggesting that it shared high sequence homology with PVY^NTN-NW (SYR-I) strain. Association index (AI), parsimony score (PS) and maximum monophyletic clade (MC) all indicated that ShX14 was strongly associated with the PVY^NTN-NW (SYR-I) group. Three fragments of about 1 000, 600 and 400 bp in size were also amplified from the isolate by a multiplex RT-PCR, consisting with the expected band of the PVY^NTN-NW (SYR-I) strain. Overall, these analyses strongly indicate that ShX14 is likely to be a PVY^NTN-NW strain(SYR-I). 【Conclusion】 ShX14 is a N×O recombinant isolate, classified to PVY^NTN-NW strain (SYR-I). It will provide useful information in the further study of the biology of this pathogen.

Application of Orthogonal Design and Uniform Design in Physical Stability Promotion of Clothianidin Suspension Concentrate

LI Bei-xing, WANG Wei-chang, ZHANG Da-xia, WANG Kai, GUAN Lei, LIU Feng

Scientia Agricultura Sinica. 2015, 48(2):  280-292.  doi:10.3864/j.issn.0578-1752.2015.02.08

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【Objective】The objective of this study is to discuss the advantages and disadvantages in application of two multi-factor experimental designs in SC (suspension concentrate) formula. Effectively providing reference for the application of orthogonal design and uniform design in pesticide formulation was also expected. 【Method】 Clothianidin 30% SC was prepared based on orthogonal design and uniform design after the wetting-suspending agents with favorable wettability were selected by applying flow point method. The effect of wetting-suspending agent and viscosity modifier on the water separation rate after hot storage (WSRHS), precipitation rate after centrifugation (PRC), viscosity, fluidity, dispersibility, the size distribution (including D₁₀, D₅₀ and D₉₀) and suspensibility of the samples before and after hot storage were investigated. The main effect plots were used to analyze the results of orthogonal design while stepwise regression and partial least squares (PLS) were used to analyze that of uniform design. Finally, validation tests were conducted to assess the properties of optimized formulas.【Result】The viscosity of all samples prepared based on orthogonal design and uniform design ranged from 144.50 to 317.84 mPa·s and all the samples exhibited favorable fluidity and dispersibility. Particle size became larger after hot storage due to ostwald ripening. The D₁₀, D₅₀ and D₉₀ were 0.56-1.00, 0.88-1.53 and 1.77-2.68 μm for samples before storage and 0.76-1.02, 1.12-1.56 and 2.07-3.25 μm for samples after hot storage, respectively. Little change was observed for the suspensibility even after hot storage, of which the suspensibility ranged from 91.88% to 96.39% before storage and 91.91% to 96.13% after hot storage. Given the favorable performance the authors had measured which could satisfy the general requirements of SC quality control, only WSRHS and PRC were mainly optimized in this study. The ANOVA (analysis of variance) results of orthogonal design indicated that the dosage of T2700, xanthan gum and veegum had significant negative effects on the WSRHS and PRC which meant that both the WSRHS and PRC decreased with the increasing dosage of the three adjuvants. However, the dosage of NR1601 showed no significant impact on the two dependent variables. The viscosity of optimized sample based on orthogonal design was 229.6 mPa·s, while its fluidity and dispersibility were favorable. The suspensibility of the optimized sample before and after hot storage were (94.76±0.70)% and (93.50±0.20)%, respectively. The WSRHS of the optimized formula was (4.23±0.19)%, while the PRC was less than 10%. As for uniform design, PLS square model have a good predictability in predicting WSRHS, for which the sample WSRHS of the optimized formula was (2.55±0.03)% and the PRC being (4.36±0.21)%. The viscosity of optimized sample was 324.16 mPa·s, while its fluidity and dispersibility were favorable. The suspensibility before and after hot storage were (93.19±0.09)% and (92.77±0.22)%, respectively. The particle size was small and the size distribution was narrow. PLS linear model exhibited favorable predictability in predicting PRC. The PRC and WSRHS of the optimized formula were (7.75±0.14)% and (5.24±0.19)%, respectively. The predictability of stepwise regression models was poor for both the two properties. What’s more, the optimized formula was not optimum formula, of which the WSRHS was (9.51±0.20)% and the PRC was (16.63±0.19)%. Both the double screening stepwise regression and PLS were capable of optimizing the WSRHS and PRC at the same time while validation test confirmed that the former model matched the final results better, but unfortunately, even optimized formula of the former model was not optimum. 【Conclusion】 Although orthogonal design required a large test number, the data analysis method was simple and easy to grasp. Combining the main effect plots and ANOVA results, the preparation of clothianidin 30% SC was effectively optimized. Although the robustness of uniform design was poorer than orthogonal design, it required fewer experiments, which could significantly decrease financial cost. Ideal formula could also be obtained provided that the complicated statistics and analysis methods were mastered.

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

Effects of Returning Stalks into Field on Soil Humus Composition  of Continuous Cropping Cotton Field

LIU Jun, JING Feng, LI Tong-hua, HUANG Jin-hua, TAN Jian-xin, CAO Jing-jing, LIU Jian-guo

Scientia Agricultura Sinica. 2015, 48(2):  293-302.  doi:10.3864/j.issn.0578-1752.2015.02.09

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【Objective】The objective of the experiment is to study the effect of straw incorporation on soil humus component content in the continuous cropping cotton field.【Method】This experiment was conducted in the long-term continuous cropping plots from the experiment station of Shihezi University. There were ten treatments in this continuous cropping plots, six treatments’ cotton straw were returned into the soil, and the other treatments’ were removed. There were three replicates per treatment, initial soil background values for each plot were similar. The six treatments which had the cotton straw included six continuous cropping plots, such as 5, 10, 15, 20, 25 and 30 years. The other four treatments were 1, 5, 10 and 15 years continuous cropping plots (they were marked CK1, CK5, CK10 and CK15, respectively). Cotton variety ‘Xinluzao 46’ in was grown cotton field, and the way of planting adopted was the ‘30+60+30’ configuration, and drip irrigation under mulch was used. The planting density was 198 000 plants per hectare. During the growth period, 11 times of drip irrigation were conducted, the total drip irrigation norm was 5 400 m³·hm^-2, total nitrogen application rate was 495 kg·hm^-2, 30% was used as basic fertilizer, others were fertilized with water. The other managements were the same as the general field management. 【Result】 Results showed that stalk returning could increase the soil humic acid content significantly in three soil layers. Besides, with the time of continuous cropping increasing, the humic acid content was remarkably raised, especially the humic acid content was most abundances in 20-40 cm soil layer. Compared with the 5, 10, 15, 20, 25 years continuous cropping, the soil humic acid content in 30 years increased by 139.90%, 86.68%, 93.33%, 58.60% and 22.86%, respectively. However, after continuous cropping and straw incorporation, the time of continuous cropping had no effect on fulvic acid content in soil, and the fulvic acid content was always (2.79 ± 0.19) g·kg^-1 in the 0-20 cm soil layer, (2.56 ± 0.10) g·kg^-1 in the 20-40 cm soil layer and (1.77 ± 0.15) g·kg^-1 in the 40-60 cm soil layer. Moreover, the fulvic acid content in the 0-20 cm soil layer and 20-40 cm soil layer was significantly higher than in the 40-60 cm soil layer. In addition, Straw returning could significantly increase the humin content in three different soil layers, humin content in the 0-20 cm soil layer was greater than in the 20-40 cm and in the 40-60 cm soil layers. In the different years of straw returning, C_HA /C_FA values＞1, and PQ＞0.5, C_HA/C_FA values and PQ values were increased as the time of continuous cropping increasing, besides, the 5-year continuous cropping was the minimum and the 30-year continuous cropping was the maximum. Compared with 0-20, 20-40 and 40-60 cm soil layers in the 5-year continuous cropping, C_HA/C_FA values of 30 years increased by 120.04%, 116.39% and 112.91%, respectively, and the PQ values of 30 years were increased by 37.15%, 36.44% and 36.81%, respectively. 【Conclusion】Straw returning increased the soil humic acid, humin and fulvic acid contents in the long-term continuous cropping cotton field, besides, it kept them in a stable dynamic level, but also significantly improved the C_HA/C_FA and PQ values, so that the quality of the soil humus transformation went far towards a good direction.

A Study of the Optimized Model of N, P, K Fertilization on Muskmelon in Desert Oasis Area

XUE Liang, MA Zhong-ming, DU Shao-ping

Scientia Agricultura Sinica. 2015, 48(2):  303-313.  doi:10.3864/j.issn.0578-1752.2015.02.10

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【Objective】 The purpose of the study was to provide a reasonable fertilization basis for developing yield potential, reducing production costs and preventing environmental pollution from over use of chemical fertilizer.【Method】Two consecutive two-year field experiments of nitrogen(N), phosphorus (P) and potassium (K) fertilizers was conducted, by adopting the 312-D optimized saturation design method in the years of 2012 and 2013, and the up relationships between balanced N, P and K fertilization and melon yield, soluble solids content (SSC) and economic benefit parameters of muskmelon were studied.【Result】Compared with no fertilizer treatment, melon yield increased by 16.4%-66.0%, and SSC content increased by 6.7 per cents in 2012 and 6.6 per cents in 2013. At average level in any of the two in the NPK three factors , with N amount increased from 0 to 360 kg·hm^-2, the yield increased by 31.2% in 2012 and 31.5% in 2013, and SSC improved by 2.9 per cents in 2012 and 2.5 per cents in 2013. When P application from 0 to 180 kg·hm^-2, the yield improved by 5.8% in 2012 and 7.5% in 2013, and SSC improved by 0.6 per cents in 2012 and 1.1 per cents in 2013. When K amount increased from 0 to 90 kg·hm^-2, the yield improved by 5.5% in 2012 and 5.1% in 2013, and SSC improved by 1.4 per cents in 2012 and 1.7 per cents in 2013. The yield, SSC and economic benefit increased then decreased as N, P, K rates increased, but the order of its effect on yield and SSC and benefit was followed as N＞K＞P. The interaction effects of N×P and N×K on the yield and economic benefit were obvious but the effect of P×K was not. The N×P played a positive role on the SSC but no effect on SSC. The N×K was negative on yield and benefit but positive on SSC. Applying N prohibited yield and SSC when low P or K amount was used, otherwise the N had an obvious increase role when high N rate was applied. The minimum of fertilizer threshold for SSC was higher for yield, therefore high yield was obtained when an optimized cultivation program was desighed. 【Conclusion】According to the optimal fertilization effect function model and recommendation, the optimal N, P₂O₅ and K₂O rates ranged from 260.2 to 263.6 kg·hm^-2, 133.0 to 134.1 kg·hm^-2, and 87.5 to 88.5 kg·hm^-2, in which muskmelon yield and SSC and benefits were over 43 500 kg·hm^-2, 13% and 68 445 yuan·hm^-2, respectively.

HORTICULTURE

Effect of Water and Fertilizer Coupling on Quality, Yield and Water Use Efficiency of Tomato Cultivated by Organic Substrate in Bag

WANG Peng-bo, LI Jian-ming, DING Juan-juan, LIU Guo-ying, PAN Tong-hua, DU Qing-jie, CHANG Yi-bo

Scientia Agricultura Sinica. 2015, 48(2):  314-323.  doi:10.3864/j.issn.0578-1752.2015.02.11

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【Objective】 This study aims at discovering the relations of water and fertilizer with the quality, yield and water use efficiency of tomato cultivated by organic substrate in bag, so as to provide a theoretical basis for the high-efficient production of tomato in greenhouse. 【Method】 Tomato ‘Jinpeng 1’ was used to investigate the effects of water and fertilizer coupling on quality, yield and water use efficiency of tomato cultivated by organic substrate in bag, meanwhile, synthetically evaluated the quality of tomato by principal component analysis and analyzed the cost and profit of different treatments . 【Result】 The results indicated that the content of NO^-₃^-N and soluble protein in tomato fruits increased with the increasing of fertilize under the same irrigation levels, but the content of Vc, lycopene and soluble sugar showed a trend of first increase and then decline. Under the same fertility condition, the content of NO₃^--N, Vc, soluble protein and soluble sugar gradually reduced with the improvement of substrate moisture, and that lycopene content of AW level was the highest. The yield of tomato cultivated by organic substrate increased as the amounts of water and fertilizer increased, the effects of fertilizer, water and their interactions on yield of tomato were very significant, and the orders of the factors were water, fertilizer, their interactions in turn. The irrigation water use efficiency of tomato cultivated by organic substrate in bag showed a trend of first increase and then decline with the increasing of irrigation and fertilizer, the effects of fertilizer and water on irrigation water use efficiency were highly significant, while their interactions had no significant influence on irrigation water use efficiency of tomato. The net income of HFHW was the highest and had no significant differences with AFAW, HFAW and AFHW, while the net income of LFLW was substantially below others. 【Conclusion】 Considering the quality, yield, water use efficiency, conserving resources and sustainable production, the AFAW is the optimal choice without degrading much of quality and net income of tomato.

Hormone Levels and Gene Expression Analysis of Chrysanthemum Cultivar ‘Puma Sunny’ Under Low Light Intensity

HAN Shuang, CHEN Su-mei, JIANG Jia-fu, FANG Wei-min, GUAN Zhi-yong, CHEN Fa-di

Scientia Agricultura Sinica. 2015, 48(2):  324-333.  doi:10.3864/j.issn.0578-1752.2015.02.12

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【Objective】 In the study, ethylene accumulation rate, GA levels, IAA content and correlative gene expression levels in leaves of chrysanthemum under low light condition during shade avoidance symptoms was assayed, to explore the shade tolerant mechanism.【Method】Chrysanthemum ‘Puma Sunny’ was assayed under 55% irradiance (control group) and 15% irradiance (low light treatment). GA and IAA contents on 0, 2, 4, 6 and 8 d were measured by high performance liquid chromatograph Agilent 6890N GC system, the relative expression level was measured on 0, 2, 4, 6 and 8 d using GAPDH as reference gene. The concentrations of GA3 were set at 0.5, 1.0, 2.0 and 4.0 mmol·L^-1, with a proper the amount of ethanol dissolution, the final concentration ethanol was 1%. The sample was treated on 4 d and 8 d using the above ground spraying. The concentrations of PAC were set at 10, 20, 50 and100 μmol·L^-1, with a proper amount of ethanol dissolution, the final concentration of ethanol was 1%. The results showed that 2.0 mmol·L^-1 GA₃, and 50 μmol·L^-1 PAC showed impacts on plant growth significantly. After treatment on 0 d and 7 d, the sample was collected on 14 d for analysis of gene expression.【Result】The ethylene level increased markedly on 2 and 4 d, especially on 4 d, and it was 2.04 times more than that of CK. After 6 d, the ethylene level between CK and treatment was not significant. The plant ethylene generation rate under 15% low light conditions was higher than that of 55% irradiance. The gibberellin content in leaves increased significantly on day 2, 4 and 6 compared with the control, the peak appeared on the 6 d, reached 4700.56 ng·g^-1, 2.57 times higher than the control, then decreased quickly, and down to control level on the 8 d. With the prolonging of treatment time, the content of auxin in control group showed an increasing trend, weak light treatment group decreased. On the 8 day, the auxin content in treatment group was 223.95 ng·g^-1 FW, and that in the control group was 489.77 ng·g^-1 FW. The IAA content of the control groupment was 1.12 times higher than that of the treatment group. In the second day of weak light treatment, the expression level of PHYA increased rapidly, 2-6 d with a smaller velocity, 6-8 d kept unchanged, but the control group began to decline from the second day. The change of the expression of PLYA in the treatment group from 4 to 8 day was higher than that of control group. The expression level of PIF4 of the control and the treatment kept the same change trend, from 0 to 6 day the difference was that on 4-8 day the treatment group was higher than the control, especially on the sixth day, it was 1.85 times higher than the control. COP1 gene in control group showed a slow decreasing trend while treatment group increased first and then decreased, the peak appeared on the second day, and on 8 d down to the control group. In the whole process, GAI in the treatment group tended to increase continuously, the control group that in increased slightly on 0-4 day and then declined rapidly. The expression of IAA1 in the treatment group and control group on the 0-2 d increased slightly, and that in the control group on the 2-4 d decreased rapidly. After 2 days, the expression level of IAA1 changed little. Irradiance at 15% induced the PHYA, COP1, IAA1 and PIF4 gene expression. Irradiance at 15% increased GAI transcript level. Exogenous GA inhibited the expression of COP1 and PIF4 expression and down regulated GAI and IAA1 level. PAC inhibited the expression of COP1, PIF4, GAI and IAA1. 【Conclusion】All these data indicated that the expression of PHYA, COP1, IAA1 and PIF4 wasinduced by low light, the GAI transcript level increased under low light. GA and ethylene involved in shade avoidance process of chrysanthemum.

STORAGE·FRESH-KEEPING·PROCESSING

Characteristics of Sucrose: Sucrose 1-Fructosyltransferase in Garlic

WEN Ming, BU Li-wei, LUO Zi-yun, WANG Jia-wei, DONG Fen, HUANG Xue-song

Scientia Agricultura Sinica. 2015, 48(2):  334-342.  doi:10.3864/j.issn.0578-1752.2015.02.13

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【Objective】 Enzymologic characteristics of sucrose: sucrose 1-fructosyltransferase (1-SST) in garlic (Allium sativum L.) was investigated to provide a basis for the preservation, processing and further quality improvements of fresh garlic.【Method】1-SST was fractional precipitated by ammonium sulfate with different saturations of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 100%, successively, in order to determine the fraction of the ammonium sulfate which 1-SST is in. 1-SST catalyses in fructan biosynthesis. After that, the 1-SST was further researched on the catalytic activity under different temperatures, pH, ionic strength and substrate concentrations. Here sucrose was used as substrate. The 1-SST enzymatic activity was revealed through production of 1-kestose which was analyzed by high performance liquid chromatography-evaporation light-scattering detection (HPLC-ELSD). Chromatographic separation of 1-kestose was achieved by the Prevail Carbohydrate ES column (250 mm×4.6 mm, 5 μm) at a flow rate of 1 mL·min^-1 under 20 MP . The mobile phase consisted of a combination of B water and A acetonitrile. The gradient elution was applied as followings: 75% A : 25% B at 0 min, 65% A : 35% B at 15 min, 50% A : 50% B at 30 min, 75% A : 25% B at 35 min, 75% A : 25% B at 40 min. The column temperature was held at 40℃. ELSD impactor was off and drift tube temperature was kept at 90℃. The air was used as carrier gas for ELSD detection with a flow rate of 2.5 L·min^-1.【Result】Fructose, glucose, sucrose, 1-kestose, nystose, 1F-fructofuranosylnystose were separated in 20 min. Thus the HPLC-ELSD method was proved a reliable and highly sensitive method for determining the 1-kestose. As early described, 1-kestose was produced when sucrose incubated with 1-SST and used to represent the enzymatic activity of 1-SST. Only 1-kestose was produced with 50% (w/w) sucrose solution as substrate. This phenomenon indicated that the 1-SST preserved very high substrate specificity under the above experiment conditions. Garlic 1-SST mainly existed in the fraction precipitated with 30%-40% ammonium sulfate solution while exhibited very weak enzymatic activity in the fractions precipitated with 0-30%, 40%-70% ammonium sulfate solution and no 1-SST was detected in the fraction precipitated with 70%-100% ammonium sulfate. Garlic 1-SST from the fraction precipitated with 30%-40% ammonium sulfate solution was further investigated. The optimal temperature was 35℃. The enzymatic activity was decreased dramatically when the temperature reached 40℃ which indicated this enzyme is heat labile. Therefore, high temperature should be avoided for studying the garlic 1-SST. Its optimum pH value was 5, which was similar to the 1-SST from Hordeum vulgare, Cichorium intybus L., Helianthus tuberosus Colombia and Lolium rigidum, Agave americana L. This result shows that the conformation of 1-SST activity site from different plants could be similar. The enzymatic activity of 1-SST was dependent on the sucrose concentration in the range of 100-600 mg·mL^-1 which was increased with the substrate concentration below 500 mg·mL^-1. However, the enzyme was saturated at the high concentration of substrate (over 500 mg·mL^-1). This result fits into the Michaelis-Menten Equation. For Na⁺, Cl^- ion, 1-SST enzymatic activity was reversed with the ion strength. Michaelis constant of 1-SST was 10⁴ mmol·L^-1 at 35℃.【Conclusion】 In this study, The effect of different temperatures, pH, ionic strength and substrate concentration on the 1-SST enzymatic activity was investigated. This mechanism will help to understand the change of fructan, species and content of saccharides during processing and storage.

ANIMAL SCIENCE·VETERINARY SCIENCERE·SOURCE INSECT

Cloning, Tissue Expression and Imprinting Analysis of PHLDA2Gene in Dazu Black Goat

SU Lu-fang, PENG Xue-qiang, JIANG Cao-de

Scientia Agricultura Sinica. 2015, 48(2):  343-351.  doi:10.3864/j.issn.0578-1752.2015.02.14

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【Objective】The objectives of this study are to clone goat PHLDA2 (pleckstrin homology-like domain, family A, member 2), to analyze its tissue expression and imprinting status, and to reveal its relationship with placental traits, thereby providing data for further investigation on the gene functions in goat placenta.【Method】Partial cDNA sequence of PHLDA2 was cloned for Dazu black goats according to the conserved regions among human, cattle and pig sequences, and the full-length cDNA of this gene was further cloned with 5′- and 3′-RACE. Sequence characteristics of goat PHLDA2 were analyzed using online softwares, such as ORF Finder and ProtParam. Real-time PCR was applied to examine tissue expression of goat PHLDA2 in 10 tissues, including heart, liver, spleen, kidney, skeletal muscle, lung, tongue, fat, brain and placenta. Imprinting status of the gene in placenta was investigated by expressed SNP method. And, linear regression estimation was used to reveal the correlation between PHLDA2 expression and placental traits.【Result】A full-length cDNA of 935 bp was cloned for goat PHLDA2. The cDNA contains 62 bp 5′-UTR, 450 bp 3′-UTR and 420 bp coding sequence, and encodes a protein of 140 amino acids in total. The molecular weight of goat PHLDA2 protein is 15638.7 Da, and its theoretical isoelectric point is 9.18. The secondary structure of the protein contains α-helix (37.14%), β-turn (9.29%), extended strand (23.57%) and random coil (30%). The result of BLAST showed that goat PHLDA2 gene was located on goat chromosome 29, and that it had high sequence similarity with the homologues of cattle (91%)，pig (90%), macaque (90%), horse (90%) and human(89%). Furthermore, goat PHLDA2 has the closest phylogenetic relationship to the cattle gene in both nucleotide and amino acids sequences. Different expressions of goat PHLDA2 were detected among tissues (P < 0.01), with the highest mRNA level in the placenta, undetectable level in the spleen, and much less and no significant difference in the other tissues (P > 0.05). Imprinting analysis demonstrated that this gene was expressed from the maternal allele in goat placenta, heart and liver tissues, but biallelically expressed in the lung, brain and skeletal muscle. In addition, the expression levels of goat PHLDA2 were significantly correlated with placental weight (R²=0.855. P <0.01). 【Conclusion】 The sequence characteristics and imprinting status of PHLDA2 are conserved in goats, although it has a tissue-specific imprinting. The gene is an important candidate gene for goat placental growth and functions.

Fusion Expression of Non-Structural Proteins 3a and 3b of Porcine Transmissible Gastroenteritis Virus and Influence on Cell Cycle

LIANG Ya-bing, ZHANG Qi, CHANG Rong, TONG De-wen, XU Xin-gang

Scientia Agricultura Sinica. 2015, 48(2):  352-361.  doi:10.3864/j.issn.0578-1752.2015.02.15

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【Objective】 Non structural protein (nsp) 3a and 3b genes of porcine transmissible gastroenteritis virus (TGEV) were cloned and expressed in eukaryotic cell. Expression of nsp 3a and 3b proteins and their influence on cell cycle were studied.【Method】 Two pairs of primers used to amplify TGEV SX strain’s nsp 3a and 3b genes were designed according to the archived TGEV strain nucleotide by Primer 5.0. TGEV SX strain’s nsp 3a and 3b genes were cloned by RT-PCR, and ligated into eukaryotic expression vector pEGFP-N1, respectively. The recombinant plasmids were named as p3a-EGFP-N1 and p3b-EGFP-N1. The recombinant plasmids p3a-EGFP-N1 and p3b-EGFP-N1 were transfected into swine intestinal epithelial cells (IEC) using lipofectamine 2000. Expression of nsps 3a and 3b were confirmed by confocal microscopy. Transcription of TGEV 3a and 3b genes were analyzed by RT-PCR and the expression of nsp 3a and 3b was analyzed by Western blotting assay. The effects of proteins on cell cycle were investigated by flow cytometry assay. Transcription level of cyclin A and GRP78 were analyzed by qRT-PCR assay, and the expression level of GRP78, Cyclin A and Cyclin B1 were analyzed by Western blotting assay. 【Result】 The DNA fragments of 3a gene (213bp) and 3b gene (732bp) of TGEV SX strain were cloned successfully. The 3a gene of SX strain shared 97.4%—100% homology of nucleotide sequences and 98.6%—100% homology of amino acid. 3b gene shared 98.3%-99.9% homology of nucleotide sequences and 100% homology of amino acid with those of TGEV strains. Western blotting assay showed that 3a-GFP and 3b-GFP fusion proteins were in agreement with the predicted MW of 35kD and 54kD, respectively. The confocal microscopy analysis showed that 3a-GFP and 3b-GFP fusion proteins were distributed in the whole cell. Flow cytometry assay showed that the percentage of cells in G2/M phase was more than the control cells (IEC and IEC-GFP) in TGEV nsp 3b expression cells. The transcription level of Cyclin A was higher in 3b-GFP-expressing cells compared with the control cells by qRT-PCR assay. At the same time, the protein level of Cyclin A was significantly increased and Cyclin B1 level was down-regulated in TGEV 3b-GFP expressing cells by western blot assay. TGEV nsp 3a had no influence on cell cycle. The mRNA level of GRP78 was higher in 3a-GFP expressing cells than the control cells by qRT-PCR assay, and the expression level of GRP78 was up-regulated by TGEV nsp 3a compared with the control cells by western blot assay. Moreover, TGEV nsp 3b had no influence on GRP78 expression in IEC. 【Conclusion】The expressed nsp 3a and 3b of TGEV distributed in the whole transfected cells. Nsp 3a induced ER stress by up-regulated GRP78. Nsp 3b caused cell cycle arrested at G2/M phase by up-regulated Cyclin A and down-regulated Cyclin B1.

¹H NMR-based Serum Metabolomics Analysis of Dairy Cows with Milk Fever

SUN Yu-hang, XU Chu-chu, LI Chang-sheng, XIA Cheng, XU Chuang, WU Ling, ZHANG Hong-you

Scientia Agricultura Sinica. 2015, 48(2):  362-369.  doi:10.3864/j.issn.0578-1752.2015.02.16

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【Objective】 This trial was designed to screen small molecule metabolites with differences in quantity in the serum of cows with milk fever (MF), and to clarify the changes of metabolites in cows suffering from MF at the molecular level and from the angle of material, energy metabolism. 【Method】 Thirty-two multiple holstein cows with similar age, parity, body condition and milk yield at 0 day after parturition were selected, and then assigned into two groups according to their serum calcium concentration and the clinical symptoms. Among them, 24 cows were assigned to the healthy control group (Group1, serum calcium concentration> 2.5 mmol·L^-1, without any other symptoms) and 8 MF group (Group2, serum calcium concentrations <1.4mmol·L^-1, with obvious clinical symptoms of MF). Blood  at 10mL was collected from the jugular vein from each of the 32 cows before feeding and miking in the morning, placed in a centrifugal tube, centrifuged at 4℃, 1 500×g for 20min, and then the serum obtained by centrifugation was separately loaded in a 1.5 mL EP tube at -80℃ until the test. After thawing, 400 μL serums was taken from every EP tube, added 200 μL buffered salt solution, mixed well, and then 550 μL supernatant was extracted into 5 mm NMR tube after centrifugation to collect signals using the 500 MHz NMR spectrometer. Then MestReNova and Topspin softwares were applied on the Fourier transform of the signal, the correction of zero, the baseline and phase, removed water and urea peaks, and the integration of one-dimensional map for segmentation, and mapping information into a TXT file format to facilitate subsequent data analysis, and then all compounds were identified using the Chenomx software. Finally, the SIMCA-P software was used for the multivariate statistical analysis, including principal component analysis (PCA) and orthogonal signal correction-partial least squares-discriminant analysis (OSC-PLS-DA), combined with the P values obtained from one-way ANOVA of NMR data performed in SPSS software and Loading plot, eventually small molecule metabolites with differences in performance were filtered out. 【Result】 In this study, serum metabolic profiles and loading plot from MF group and control group were successfully obtained. PCA result showed that all samples were within 95% confidence interval, without excluding, and the contribution rate of the primary component was low, in which PC1 = 26.2%, PC2 = 16.7%, the different variables between the two groups can not be selected. OSC-PLS-DA result showed that the five orthogonal signal corrections were conducted to remove those components which displayed no significant grouping and maximize the differences between groups. Compared with the healthy control group, 9 differentially expressed serum metabolites were screened, which included 4 upregulated compounds, such as β- hydroxybutyrate, acetone, pyruvate and lysine, 5 downregulated compounds, such as glucose, alanine, glycerol, creatine phosphate and gamma-Aminobutyrate. They were mostly sugars and amino acids, formed an energy conversion network diagram between each, involved in various energy metabolism pathways in the body. Of which, the more specific metabolites, such as creatine phosphate, directly provided energy for the body, decrease of which may be associated with the performance of muscle weakness and paralysis on the cow. In addition, the gamma-Aminobutyrate(GABA) of cows with MF was decreased, and had clinical manifestations of depression, even coma, which was coincided with human performance of depression, but the relationship between decrease of GABA spirit of the cow and MF needs to be further confirmed by investigators. 【Conclusion】 ^1ders.H NMR technique can be applied to screen small molecule metabolites in cow serum with differences in quantity and the difference was significant. In this study, metabolites in cows with MF showed some pathological features of negative energy balance and fat mobilization, suggesting that MF is associated with energy metabolism disor

Molecular Cloning and Expression Analysis of a Soluble  Trehalase Gene Tre-1 in Bombus hypocrita

QIN Jia-min, LUO Shu-dong, LIAO Xiu-li, HUANG Jia-xing, HE Shao-yu, WU Jie

Scientia Agricultura Sinica. 2015, 48(2):  370-380.  doi:10.3864/j.issn.0578-1752.2015.02.17

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【Objective】The objective of this study is to clone full-length cDNA sequence of the soluble trehalase gene (Tre-1) in Bombus hypocrita, predict the physicochemical properties, clarify its expression in different tissues and developmental stages, and to understand the function of this gene in the development of B. hypocrita.【Method】 Degenerate primers were designed by using Primer Premier 5.0 software according to the conservative sequences of Tre-1 in B. terrestris and B. impatiens to get the corresponding conservative fragment in B. hypocrita. Gene-specific primers were designed according to the conserved fragment. The rapid-amplification of cDNA ends (RACE) method was used to clone B. hypocrita 5′ and 3′ sequences, then the open reading frame (ORF) was amplified by the specific primers based on the initiation codon and termination codon near 5′ and 3′, respectively. Different fragments were spliced by BioEdit to obtain the full-length cDNA. The full-length cDNA was analyzed by a variety of bioinformatics softwares such as ExPASy, SignalP 4.1, NetOGlyc 1.0 sever, ClustalW and MEGA 5.0. Finally, the relative expression of Tre-1 in different tissues and developmental stages in B. hypocrita were analyzed by real-time PCR and 2^-ΔΔCt method.【Result】The full-length cDNA of B. hypocrita Tre-1 is 3 129 bp, and designated as BhTre-1 (GenBank number: KJ025078). The bioinformatics analysis suggested that BhTre-1 has an ORF of 1 743 bp, a 5′ (untranslated region) UTR of 441 bp and a 3′ UTR of 945 bp. The ORF of BhTre-1 encodes a polypeptide of 580 amino acids with a predicted molecular weight of 67.16 kD, and an isoeletric point value of 5.95. Thededuced amino acid sequence has six predicted sequences of Asn-Xaa-Ser/Thr, a signature peptide, a highly conserved glycine-rich region (GGGGEY), and two conserved signature motifs (PGGRFKEFYYWDSY and QWDFPNAWPP), but no transmembrane domain. Homology comparison found that BhTre-1 has the greatest similarity to B【Conclusion】The cDNA sequence of BhTre-1 was successfully cloned from B. hypocrita, and the properties were similar with Tre-1 of other insects. BhTre-1 hasthe highest expression in midgut. In addition, the expression of BhTre-1 of adult worker was higher than at larva and pupa stages,and the expression of BhTre-1 increased first and then decreased. This study indicated that the expression patterns in different tissues and developmental stages of B. hypocrita, which will lay a foundation for biological functional research of BhTre-1.. terrestris BtTre-1 (99%)and B. impatiens BiTre-1 (98%), it also has greater similarity to Tre-1 of Apis mellifera and A. florea which have 78% sequence homology. The phylogenetic tree analysis showed that BhTre-1 was first clustered with BtTre-1, BiTre-1, AmTre-1 and AfTre-1. The results agreed with the sequence homology analysis. Tissue-specific expression results indicated that BhTre-1was expressed in all the major tissues, it had the highest expression in midgut, then Malpighian tubules, and lower expression in other tissues. The expression of BhTre-1 in adult worker was higher than at larva and pupa stages, and it increased from the 1st day after emergence, and had the maximum expression in the 15-day-old adults, then, it decreased with the age.

RESEARCH NOTES

Comparation of SSR Molecular Markers Analysis of Annual Ryegrass Varieties in DUS Testing

HUANG Ting, MA Xiao, ZHANG Xin-quan, ZHANG Xin-yue, ZHANG Rui-zhen, FU Kai-xin

Scientia Agricultura Sinica. 2015, 48(2):  381-389.  doi:10.3864/j.issn.0578-1752.2015.02.18

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【Objective】Annual ryegrass is a world-cultivated forage, but frequent exchange of breeding material causes intervarietal genetic differences become smaller due to the cross-pollination characteristics, therefore, cultivar identification using traditional agronomic traits is becoming increasingly difficult. Efficient, rapid identification of cultivar plays an important role in breeding, and also provides a scientific basis for DUS testing and intellectual property protection.【Method】In this study, the cultivar identification among 6 backbone varieties of annual ryegrass was conducted by using 20 pairs of SSR primers from 165 for 3 bulk of DNA samples each attracted from 30 random individual plants equally based on strong stability, high polymorphism and co-dominant characteristics of SSR markers, and the specific bands of bulked samples appeared at least twice could be recorded in amplification profile. And the combination of 30 individuals and bulked samples using SSR markers, and the comparation of amplification profiles were used to identify 6 varieties of annual ryegrass.【Result】Comparative analysis of 6 varieties of annual ryegrass using SSR markers between individuals and bulked samples by using 3 primer pairs (13-07A, 12-10F and 15-08C) showed that the amplification bands of bulked samples were more obvious and more bands than individuals, but also some diffuse bands. And the frequency bands more than 40% in individuals appeared the same bands in bulked samples. The results of identification of 6 varieties of annual ryegrass using 3 bulked samples showed that, 20 primer pairs amplified 143 legible bands, comprising 127 polymorphic bands, and the polymorphism ratio reached 88.81%, and the amplified brands to each pair of primers were 5 to 11, and the average amplified brand, polymorphic bands, and polymorphism information content (PIC) were 7.15, 6.35 and 0.571, respectively. Although some primers polymorphic information content is low, high stability, i.e. three groups had almost same amplification results. The genetic similarity coefficient of 6 varieties varied from 0.4755 to 0.7552, the largest genetic similarity coefficient was between Abundant and Aderenalin, and the smallest genetic similarity coefficient was between Changjiang No.2 and Tetragold. The 6 varieties of annual ryegrass were divided into three groups on the average genetic similarity coefficient of 0.615, one included Angus1, Abundant, Aderenalin and Double Barrel, one was ChangjiangNo.2, and one was Tetragold. Nine of 20 SSR primer pairs could directly identify 6 varieties of ryegrass, however, the other primer pairs could only identify 3 or 4 varieties, the identification ability could be improved by combining with different primer pairs.【Conclusion】For cross-pollinated plants, polymorphism is not the only criterion to measure the effectiveness of primers, the stability of primers is also an important indicator. By comparative analysis of the 2 methods, bulked samples using SSR markers is better than the method of individuals. The screened 20 pairs of SSR primers could clearly distinguish 6 varieties of ryegrass, indicating that the use of SSR markers is feasible and efficient for cultivar identification.

Detection of Embellisia allii Using Real-Time Quantitative PCR Based on Glyceraldehyde-3-Phosphate Dehydrogenase Gene

WANG Ying-chao, GAN Qin-hua, LI Yan, JI Ying, WU Xing-hai, SHAO Xiu-ling

Scientia Agricultura Sinica. 2015, 48(2):  390-397.  doi:10.3864/j.issn.0578-1752.2015.02.19

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【Objective】Morphological identification method is mainly used for detection and identification of Embellisia allii (Campanile) Simmons. The method is time-consuming in identification, moreover, the spore of garlic bulb canker is very similar to other species of the genus Embellisia and other genera, but is also very difficult to distinguish. Rapid and effective molecular diagnostic methods are needed. The objective of this study is to develop a real-time quantitative PCR (qPCR) technique for detection of the E. allii sensitively and accurately.【Method】From the NCBI GenBank database, 15 kinds of glyceraldehyde-3-phosphate dehydrogenase (gpd) gene nucleic acid sequences of the garlic bulb canker and other similar species diseases were selected, according to primer design software Primer express, a probe and three pairs of primers were elected theoretically. Based on the experimental results of the above-mentioned primers, specific primers and TaqMan probes to E. allii were screened. While the reaction system renaturation-extension temperature of the reaction parameters were optimized, three temperature gradients of 56, 58, 60℃ were designed. The garlic bulb canker pathogen genomic DNA in the nucleic acid analyzer UV quantification was performed 10 times serial dilutions in order to test the probe sensitivity. The TaqMan qPCR detection system was established with optimized reaction conditions.【Result】The species-specific primer pair Emb21/Emb32 and probe Emb were designed based on gpd gene for E. allii, and E. allii could be detected specifically from the 6 tested strains with qPCR based on gpd gene, only the target fungi could be specifically detected positive, the blank control and other strains were detected negatively. About the optimization of refolding- extended temperature reaction parameters, △Rn fluorescence signal at 58℃ was the maximum while the value of the fluorescence signal was lower at 56, 60℃. After repeated tests, the optimal annealing-extended temperature was determined as 58℃. In the test of the sensitivity, the garlic bulb canker pathogen DNA concentration was diluted to 140 pg, resulting better graphics; when the system template concentration is diluted to 14 pg, the resulted graph was poor. The detection sensitivity of qPCR for DNA concentration was about 140 pg.【Conclusion】The qPCR detection system was established for E. allii. This detection technology can specifically detect E. allii from the 6 tested strains and the detection sensitivityis140 pg.The assay of qPCR based on gpd gene was specific and sensitive, and the detection method is suitable for E. allii rapid detection.

Rapid Karyotype Analysis of Cucumber Varieties Based on Genomic in situ Hybridization

ZHANG Yun-xia, LOU Qun-feng, LI Zi-ang, WANG Yun-zhu, ZHANG Zhen-tao, LI Ji, CHEN Jin-feng

Scientia Agricultura Sinica. 2015, 48(2):  398-406.  doi:10.3864/j.issn.0578-1752.2015.02.20

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【Objective】Using genomic in situ hybridization (GISH), metaphase chromosome analysis was performed for two variants of cucumber (C. sativus L.，2n = 2x = 14) [cultivated cucumber (C. sativus var. sativus) and wild cucumber (C. sativus var. hardwickii)] to quickly establish the chromosome karyotype of cucumber varieties, which will provide useful information for molecular cytogenetic research of cucumber. 【Method】Genomic DNA from cultivated cucumber ‘9930’ isolated using CTAB method and 45S rDNA were labeled with Dig-dUTP or Bio-dUTP through nick-translation method, and used as probes and hybridized onto the metaphase chromosomes of cultivated cucumber itself and wild cucumber using genomic in situ hybridization technique. Individual chromosome of two cucumber variants was analyzed based on the GISH banding, and 45 rDNA signals as well, and the karyotypes of these two variants were constructed. 【Result】 The GISH results showed that cucumber genomic DNA probes exhibited distinguished signal patterns on the chromosomes of two variants. The unique distribution and intensity of signals were observed on each chromosome, rather than the even distribution of signals across all chromosomes. For C. sativus var. sativus ‘9930’, GISH produced obvious signals at the both ends of chromosomes and pericentromeric heterochromatin regions on all other chromosomes except for chromosome 6 which only gave signals at the end of short arm and pericentromeric heterochromatin region. The signal patterns from each chromosome exhibited an obvious difference. 45S rDNA loci were mapped adjacent to centromeric regions on chromosomes 1, 2, 3, 4 and 7, among which three pairs of strong signals and two pair of weak signals were observed. However, the signal patterns from wild cucumber showed an obvious difference compared with that from ‘9930’. The GISH signals were observed only at one end of chromosomes 1, 2, 4 and 5, and pericentromeric heterochromatin regions of all chromosomes. 45S rDNA signals were observed only on the chromosomes 1, 2 and 3, in which chromosome 1 produced extremely strong signals and chromosomes 2 and 3 gave very weak signals. These results indicated that unique signal patterns for cucumber variants could be produced by GISH using cucumber genomic DNA. Each chromosome was characterized unequivocally according to the signal patterns, and a karyotype for each cucumber variant was constructed. Compared with the distribution of cucumber repetitive sequences from previous reports, the GISH patterns were found to locate mainly in tandem repetitive blocks of cucumber chromosomes. 【Conclusion】Results of this study showed that the cucumber genomic in situ hybridization could quickly reveal the distribution of genomic tandem repetitive sequences at one time, and could be efficiently applied to rapid karyotype analysis of different variants. It was also found that significant differentiation of repetitive sequences happened among cucumber variants.