Scientia Agricultura Sinica

Progress in Research of CRISPR/Cas9 System in Genome Targeted Editing in Plants

XIE Li-nan, SONG Feng-yan, ZHANG Yang

Scientia Agricultura Sinica. 2015, 48(9): 1669-1677. doi:10.3864/j.issn.0578-1752.2015.09.01

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When exogenous DNA was imported into plant cell by transgenic technology, DNA fragment will integrate into the genome by homologous recombination or nonhomologous recombination. In addition, the plants seedling will achieve corresponding target traits. Homologous recombination occurred when the exogenous DNA and the same or similar sequences in receptor cells recombined and integrated to the receptor cell’s chromosomes, so the sequence will be possible to precisely modified and transformed. However, in some cases, to avoid the fracture caused by DNA or chromosomal DNA degradation or the impact on the vitality, the two double-stranded DNA break ends will be joined without considering the sequence similarity by error-prone nonhomologous end joining. As a result, precise mutations control is more difficult to achieve because insertion and/or deletion and other variety of mutations are high-frequently occurred in non-homologous recombination than in homologous recombination. Unfortunately, the frequency of homologous recombination is very low in plants which results the undesirable genome editing. Site-specific nucleases make genome editing more efficient and more precise by the great improvement of the efficiency in the homologous recombination. Such nucleases, zinc finger nuclease (ZFN) and transcription activator-like effector nucleases (TALENs), have been demonstrated to efficiently produce a DNA double-strand break at target site and to induce genome modification in a variety of organisms including plants. However, some defects found in the specific application, such as off-target effects, specific combined site with genome affected by chromosomal location and adjacent sequence, furthermore, technical complexity and time-consuming for assemble limit its application. The clustered, regularly interspaced, short palindromic repeats(CRISPR) system is a prokaryotic adaptive immune system which widely exists in bacteria and archaea. As the result of the long-term evolution, the system can defense against the degradation of RNA to guide the invasion of the virus or phage DNA. Recent advances in the study demonstrated CRISPR/Cas typeⅡ system was a promising system of genome editing strategy with high efficiency, affordability and easy to engineer compared with ZFNs and TALENs. Many precise genome editing cases by CRISPR/Cas system were found successfully in human cells, zebra fish, mice and bacterial, including gene insertion, deletion, mutation in multiple sites or in specific locus. Although the application in plants is still in a relatively limited range, there is an outstanding prospect of CRISPR/Cas system in plant genetic engineering. A brief summary of the composition and principles was presented firstly. Then, the authors emphasis on citing numerous cases involved its application of exogenous and endogenous genes editing in cotyledon and dicotyledonous plants which demonstrate that the CRISPR/Cas9 system has become a powerful tool in plant genome engineering. Finally, the future of the genome technology application in agriculture and plant genetic engineering was discussed which will provide a reference for researchers in genetic modification.

A Combined Identification Method for the Verticillium Wilt Resistance in Cotton by Using Pathogen Toxin

WANG Ming, ZANG Li-li, FAN Kai, LI Feng, YUAN Shu-na, SHEN Hao, WANG Xue-de

Scientia Agricultura Sinica. 2015, 48(9): 1678-1688. doi:10.3864/j.issn.0578-1752.2015.09.02

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【Objective】 The Verticillium dahliae is the most harmful fungus for cotton plants and belongs to quarantine fungus. It is restricted to use Verticillium fungus to identify the Verticillium wilt resistance of cotton in the field. Some identification methods were compared in this research. In order to replace this traditional identification method, a new combined method by using Verticillium toxin instead of using its fungus was found in this paper. And this research provided an accurate, environment-friendly, convenient method to breed the cotton varieties with resistance to Verticillium wilt. 【Method】Method Ⅰ: The cotton was sowed in the greenhouse with conventional management and their roots were soaked in the toxin of Verticillium after 21 days. Then the disease indexes were counted after 72 h. Method Ⅱ: The antepenultimate leaves were collected at full-bloom stage of cotton, and they were cut to about 10-20 small leaf disc (the diameter is 1.5 cm) by hole puncher. Whereafter it was put into the Verticillium toxin, and counted the disease indexes and yellowing degree after 24 h. The final results were combined with two methods to evaluate the Verticillium resistance of candidate cotton lines. 【Result】This paper suggested that it would spend longer time in identifying the Verticillium wilt resistance by using dipping the root with toxin, sprinkling the spore suspension on root in non-bottom plastic film bottle and dipping the root with spore suspension. However, it would spend shorter time in identifying the Verticillium wilt resistance by using soaking the leaf disc with toxin and soaking the root with toxin. The best concentration of toxin in this research was 15 μg·mL^-1, when soaking the root with toxin was used to identify the Verticillium wilt resistance. Compared with the conventional method, the correlation coefficient was 0.94 (P＜0.01) in that concentration. When soaking the leaf disc with toxin was used to identify the Verticillium wilt resistance, the best concentration of toxin in this research was 18 μg·mL^-1. The antepenultimate leaves should be collected at full-bloom stage of cotton and the correlation coefficient was 0.92 (P＜0.01) compared with the conventional method. The combined method was a quick, accurate and environment-friendly method to identify the Verticillium wilt resistance of cotton. Compared with the conventional method, the correlation coefficient of combined approach was higher through the experimental results in 2013 and 2014. In 2013 and 2014, the correlation coefficient was 0.94 (P＜0.01) and 0.95 (P＜0.01), respectively.【Conclusion】 Using the combined method which includes soaking the leaf disc with toxin and soaking the root with toxin can identify the Verticillium wilt resistance accurately, environment-friendly and conveniently. It can replace the conventional method by using this combined method. The combined identification method was more reliable than the single identification method in identification.

Response of Alfalfa Root Traits to Fall Dormancy and Its Effect on Winter Hardiness

LIU Zhi-ying, LI Xi-liang, LI Feng, TAO Ya, LIU Lei, WANG Zong-li, SUN Qi-zhong

Scientia Agricultura Sinica. 2015, 48(9): 1689-1701. doi:10.3864/j.issn.0578-1752.2015.09.03

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【Objective】Fall dormancy of alfalfa (Medicago sativa L.) is an adaptive strategy in response to change of temperature and lighting in the growth environment in late autumn. Roots, as the main functional organ of alfalfa, play a vital role in surviving the winter. Although previous studies have shown that there are significant correlations between fall dormancy and cold resistance, the overwintering characteristics of alfalfa roots on cold resistance are not clearly understood. The aim of this study was to determine the role of fall dormancy on winter hardiness in consideration of aspects of root traits.【Method】 Eleven alfalfa standard check cultivars were chosen and grown in semi-arid temperate regions, which is an appropriate area for alfalfa cultivation in China. Using the standardized major axis (SMA) method, the allometric scaling of different root traits in response to fall dormancy in the process of overwintering was studied. Furthermore, using partial least square regression (PLSR) and other methods, the master regulators of the effects of alfalfa with different fall dormancy types on cold resistance in root phenotypic traits were analyzed.【Result】The results showed that with increasing fall dormancy rating, from one to 11, the collar diameter and lateral root number of wintering alfalfa significantly decreased (P＜0.05). More dormant alfalfa was accompanied with enhanced storage phenotypic characteristics. However, taproot diameter and lateral root diameter significantly increased (P＜0.05) as alfalfa became more non-dormant. Tradeoffs were found between lateral root number and other lateral root traits (P＜0.05). Affected by fall dormancy ratings, root traits performed notable allometric scaling of different parts, mainly in collar depth, lateral root position, and lateral root number. Significant differences (P＜0.05) existed between allometric scopes and one (P＜0.05). Therefore, different root traits had differentiation strategies during the overwintering process. There were significantly negative correlations between the alfalfa winter survival rate and fall dormancy rating. The negative correlation could be fitted by logistic curve but not by a linear equation. Alfalfa cultivars with a fall dormancy (FD) rating of 1-5 tended to have a higher winter survival rate (＞95%), 6-8 FD tended to have a sharply decreased winter survival rate (50%-70%), and 9-11 rarely survived in winter (＜5%). Collar diameter and lateral root number were positive contribution factors in the alfalfa winter survival rate (P＜0.05), but the taproot diameter and lateral root diameter, negative contribution factors, were not main factors that decided the winter hardiness in alfalfa (P＜0.05). 【Conclusion】Root traits of alfalfa with different fall dormancy ratings performed differentiation strategies in response to low-temperature stress in winter, followed by allometric relationships among different traits. Alfalfa enhanced its winter survival rate mainly through increasing collar diameter and lateral root number, and did not depend on the absolute size of other root phenotypic traits. Overall, during wintering, adaptive changes of root traits of alfalfa with different fall dormancy ratings are a primary pathway to improve winter survival.

Progress in Research of Yield Formation of Ratooning Rice and Its High-Yielding Key Regulation Technologies

XU Fu-xian, XIONG Hong, ZHANG Lin, ZHU Yong-chuan, JIANG Peng, GUO Xiao-yi, LIU Mao

Scientia Agricultura Sinica. 2015, 48(9): 1702-1717. doi:10.3864/j.issn.0578-1752.2015.09.04

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The development of ratooning rice is a vital way to achieve a full utilization of the solar-thermal resources in autumn, and promote the profit of rice field. On the basis of the reported data and the research results of the author’s study for more than 20 years, the research reviews and the research progress of yield formation mechanism and key control techniques of ratooning rice were summarized. The main results include that: (1) Compared with the lower regeneration buds, the mid-upper regeneration buds of main crop had an earlier heading date, a fewer number of leaves, a faster speed of leaf emergence, shorter growth period, higher bearing panicle rate and grain filing percentage. The mechanism of a large number of regeneration buds’death after full heading of main crop was mainly due to main photosynthate allocation to grains and fewer to regeneration buds during the main crop grain filling period. Regeneration bud growth depends upon not only the improved light condition at the base of main crop plant population, but also the biomass supply. The difference in ratooning ability among cultivars varied with the leaf-grain ratio of main crop. The higher the leaf-grain ratio at heading stage, the more the photosynthetic matter remained in the basic stems for ratooning rice growing at harvesting date of main crop, as a consequence, the better ratooning ability was gained as well. There was a high and significant negative correlation between the ratooning ability and the spikelets per panicle among varieties. (2) The ratooning ability could be divided into 4 stages based on the sink-source characteristics of the main crop and grain yield of ratooning rice. The high-yielding cultivars for main crop and ratooning rice would have the following sink-source traits:160-190 spikelets per panicle, the ratio of leaf area to grain weight 0.0737-0.0827 cm² per mg, panicles 232.12×10⁴-249.40×10⁴per ha, grain filling percentage 81.54%-85.74%, 1000-grain weight 28.58-30.07 g, and grain weight 4.13-4.43 g per panicle. (3) The reason why N applying for bud development (NABD) was beneficial to increasing grain yield of ratooning rice is that N application delayed the functional leaves senescence of main crop at the late grain filling stage, improved nitrogen content of leaf and net photosynthetic rate of main crop, increased ratio of leaf photosynthetic products flowing into panicle, reduced the amount of photosynthetic products from initial storage in main stem input panicle, relatively improved stem-sheath dry weight and enhanced ratooning ability of main crop. The difference of the effects of NABD on ratooning rice varied with rice cultivars. By applying more NABD at an earlier stage, for the main crop with large panicle (higher spikelets per panicle), the grain yield of ratooning rice could be increased. The optimum date of NABD is about at full heading of the main crop. The method of estimation of efficient rate of NABD using chlorophyll meter reading (SPAD value) of flag leaf at the full heading stage of main crop in mid-season hybrid rice was found. The protection basal leaves of main crop by controlling sheath blight and enhancing root activity through reduction of paddy soil moisture posed an important assurance of high yield for ratooning rice. There was a significant and positive correlation between ripe degrees of main crop and ratooning ability. The optimum harvesting date is at complete ripe date for grains of main crop. The appropriate height for cutting seedlings is at 5-7 cm above 2nd node from the top of mother stem. (4) Aiming at the main factors restraining ratooning rice production, for example, lower temperature at heading stage of ratooning rice, a big yield gap existed among ratooning rice production areas, and the bad effect that machine harvesting main crop had on ratooning rice production, solutions to above the problems were put forward accordingly. (5) The research focus on theory and technology of ratooning rice in the future was pointed out. First, multi-factor interaction mechanism of ecological conditions and agronomic practices on canopy characteristics after full heading stage of main crop and regeneration bud growth. Second, the main cause of regeneration bud stagnation after main crop harvesting and its regulation approach. Third, the technology approach for improving nitrogen use efficiency of ratooning rice. Fourth, the study on the techniques of hybrid rice-ratooning rice’s adaption to machinery transplanting and harvesting.

Determination of Phenolic Acids in Rice by Ultra-High Performance Liquid Chromatography

ZHANG Na, WANG Guo-xiang, Abacar Jose Daniel, LIU Zheng-hui, DING Cheng-qiang,TANG She, LI Gang-hua, WANG Shao-hua, DING Yan-feng

Scientia Agricultura Sinica. 2015, 48(9): 1718-1726. doi:10.3864/j.issn.0578-1752.2015.09.05

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【Objective】 Phenolic acids in rice grain are the main source for natural antioxidants. This study aims to develop an ultra-high performance liquid chromatography method for the determination of phenolic acids in brown rice and milled rice, and analyze the tissue distribution of phenolic acids in rice grain. 【Method】Standard compounds of the target phenolic acids, including gallic acid, protocatechuic acid, gentisic acid, 2,5-dihydroxybenzoic acid, 4-hydroxybenzoic acid, chlorogenic acid, vanillic acid, caffeic acid, syringic acid, ρ-coumaric acid, ferulic acid, sinapic acid, isoferulic acid, 2-hydroxycinnamic acid, and trans-cinnamic acid, were purchased from Sigma-Aldrich. The liquid-chromatographic separation was performed on Agilent ZORBAX Eclipse Plus C18 (2.1 mm×50 mm, 1.8 μm-Micron) column kept at 30℃, using acetonitrile/acetic acid/water mixture as the mobile phase with a flow rate of 0.5 mL·min^-1 through gradient elution: 0-1 min, 8%-10%B; 1-2.5 min, 10%-13%B; 2.5-5.5 min, 13%B; 5.5-6 min, 13%-21%B; 6-6.5 min, 21%-27%B; 6.5-7.5 min, 27%-50%B; 7.5-9 min, 50%-100%B; 9-12 min, 100%B; 12-12.5 min, 100%-8%B. And the detection wavelength was set at 280 nm and 325 nm. 【Result】 Results showed that a good separation of 14 phenolic acids was achieved within 8 min. Eleven phenolic acids were detected in brown and milled rice by mass spectrometry including gallic acid, gentisic acid, 4-hydroxybenzoic acid, vanillic acid, syringic acid, ρ-coumaric acid, ferulic acid, sinapic acid, isoferulic acid, trans-cinnamic acid. With eight of them can be used for quantitative analysis, including 4-hydroxybenzoic acid, vanillic acid, syringic acid, ρ-coumaric acid, ferulic acid, sinapic acid, isoferulic acid, trans-cinnamic acid. The proposed method exhibited a linear range of 5-220 μg·mL^-1(R²=0.9994-0.9999), with limits of quantification ranging from 0.002 to 0.03 μg·mL^-1, recovery rates from 84.11% to 114.43%. A significant difference existed in the distribution of 4-hydroxybenzoic acid, vanillic acid, syringic acid, ρ-coumaric acid, ferulic acid, sinapic acid, isoferulic acid, trans-cinnamic acid between brown (B) and milled rice (M). With 4-hydroxybenzoic acid, vanillic acid, syringic acid, ρ-coumaric acid, ferulic acid, sinapic acid, trans-cinnamic acid mainly concentrated in the outer endosperm, and the ratio of M to B was 4.52%-16.72%. By contrast, a considerable part of isoferulic acid accumulated in the endosperm, having a M/B value of 45.86%. 【Conclusion】 It was proved that the proposed method is simple, rapid, accurate, reliability, and should be used for the determination of phenolic acids in rice grain as well as other cereal grains.

Regulation of Nitrogen on Protein Expression of Summer Maize (Zea mays L.) Leaves at Filling Stage

WANG Xiang-yu, WEI Shan-shan, DONG Shu-ting, LIU Peng, ZHANG Ji-wang, ZHAO Bin

Scientia Agricultura Sinica. 2015, 48(9): 1727-1736. doi:10.3864/j.issn.0578-1752.2015.09.06

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【Objective】The objective of this experiment is to explore the regulating effect of nitrogen on protein expression of leaf of summer maize under the field production condition.【Method】Denghai 618 was selected as an experimental material, different nitrogen treatments were designed to investigate the photosynthetic rate, the activity of nitrate reductase (NR), superoxide dismutase (SOD) and peroxidase (POD), malondialdehyde (MDA) content and soluble protein content in maize ear leaves after tasseling. The total proteins from harvested sample at the stage of 20 days after tasseling were extracted by the trichloroacetate (TCA)-acetone precipitation method and the protein profiles of maize ear leaves were set up by two-dimensional electrophoresis (2-DE). The proteins in maize leaves responding to nitrogen were identified by ImageMaster-2D Elite 7.0 software. The functions of these differentially expressed proteins were identified by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF MS) analysis and NCBInr database searching.【Result】After tasseling, the photosynthetic rate, activities of nitrate reductase (NR), superoxide dismutase (SOD) and peroxidase (POD), and soluble protein content were significantly decreased, and malondialdehyde (MDA) content was significantly increased. The photosynthetic rate, NR, SOD and POD, and soluble protein content were significantly increased, and MDA content was significantly decreased in nitrogen supply treatment when compared to nitrogen limited treatment. There were 1 086 and 1 170 protein spots were found after 2-DE analysis in nitrogen limited and nitrogen supply treatment, respectively. After a comparative proteomics analysis and MALDI-TOF/TOF mass spectrometry (MS), 29 differentially expressed proteins were found, including 25 which were successfully identified. They were all up-regulated in nitrogen supply treatment except spots 55 and 1089. They were mainly involved in energy, defense-related pathways, protein synthesis, metabolism, protein transportation and storage, cell growth, secondary metabolism, transcription and unknown.【Conclusion】Nitrogen supply can significantly improve the photosynthesis, carbohydrate catabolism, defense-related pathways, protein synthesis, protein transportation and storage and secondary metabolism in maize leaves at filling stage.

Effect of Sophora alopecuroides Alkaloids on Tomato Growth and Fruit Quality

XIONG Xin, GUO Shu-qi, LI Lin, MA Zhi-qing, ZHANG Xing

Scientia Agricultura Sinica. 2015, 48(9): 1737-1746. doi:10.3864/j.issn.0578-1752.2015.09.07

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【Objective】The objective of this study is to investigate the effect of Sophora alopecuroides alkaloids on tomato growth and development for evaluating its biological function and providing a basis for its application.【Method】 Avermectins was chosen as the contrast agent. Tomato plants were sprayed with different concentrations (333.0, 166.5 and 111.0 mg?L^-1) of S. alopecuroides alkaloids and the changes of morphology, fruit quality and yield were measured. Tomato plant height, stem diameter, the number of leaves, the length-width rate of the maximal leaves and the chlorophyll content were measured in a pot experiment. The effect of S. alopecuroides alkaloids on tomato yield, soluble sugar content, titratable acid content, vitamin C content and nitrate content were investigated in a field plot experiment.【Result】S. alopecuroides alkaloids could regulate the plant growth and fruit quality. The pot experiment showed that the effect on stimulating growth of tomato seedlings was most obviously at concentration of 166.5 mg?L^-1. Compared with the blank control (water treatment), 7th day after each spray, the relative growth rate of tomato plant height increased by 55.07%, 103.03% and 60.60%, respectively, net increase of stem diameter of tomato plant increased by 31.54%, 225.80% and 185.45%, respectively, but the alkaloids did not significantly influence leaf morphological factors. At concentration of 333.0 and 166.5 mg?L^-1, after spraying S. alopecuroides alkaloids, the chlorophyll content in treated leaves increased by 6.16%-13.79% and 6.89%-17.12%, respectively, but in 111.0 mg?L^-1 treatment the chlorophyll content decreased by 0.60%-9.40% compared with blank control. Field plot experiment showed that tomatoes treated by 333.0, 166.5 and 111.0 mg?L^-1 of S. alopecuroides alkaloids, the single fruit weight increased by 13.07%, 20.92% and 9.15%, respectively, on the first cluster compared with the blank control, did not have a significant difference on the second cluster, and increased by 12.23%, 20.86% and 17.27%, respectively, on the third cluster. At concentration of 166.5 mg?L^-1, the tomato yield was 16.48% more than blank control, and there was no significant increase in other treatments. Fruit quality results showed that at concentrations of 333.0 and 111.0 mg?L^-1, the soluble sugar content declined obviously compared with the blank control, but it did not have a significant difference in tomatoes treated by 111.0 mg?L^-1 of alkaloids. At concentrations of 333.0, 165.6 and 111.0 mg?L^-1, the mean of titratable acid content in tomatoes increased by 37.01%, 23.88% and 27.16%, respectively, compared with the blank control. At concentrations of 333.0 and 166.5 mg?L^-1, the vitamin C content decreased by 28.06% and 21.91%, respectively, on the first cluster, decreased by 27.37% and 26.34%, respectively, on the second cluster, but increased by 7.28% and 7.69%, respectively, on the third cluster, and showed a trend of increase at first and then decrease. At concentration of 111.0 mg?L^-1, the vitamin C content declined on both the first and second clusters, but did not have a difference on the third cluster compared with the blank control. The nitrate content all increased compared with the blank control, and the nitrate content accumulated more as the concentration increased, but it was still far below the tomato nitrate allowance in China. 【Conclusion】At the field commonly used concentration (166.5 mg?L^-1), S. alopecuroides alkaloids could stimulate seedling growth and increase the yield of tomato remarkably, and it had no adverse influence on fruit quality.

Effects of Short-Term Exposure to High Temperature on the Survival and Fecundity of the Brown Planthopper (Nilaparvata lugens)

LI Gan-jin, XU Xian-hao, ZHANG Hai-liang, ZHU Min, CUI Xu-hong

Scientia Agricultura Sinica. 2015, 48(9): 1747-1755. doi:10.3864/j.issn.0578-1752.2015.09.08

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【Objective】The objective of this study is to clarify the effects of short-term exposure to high temperature on the survival and fecundity of the brown planthopper (Nilaparvata lugens Stål). The results will provide a theoretical basis for the prediction of occurrence and treatment of the population of N. lugens.【Method】Newly-eclosed N. lugens adults (within 24 h of eclosion) were used in the experiments. Five adult pairs were randomly collected and placed in a glass tube in a water bath at 25℃, and the experiments were carried out, at an increasing temperature with a change of 0.1℃?min^-1from 25℃ to 33, 35, 37, 39, 39.5 and up until 40℃, and kept at the target temperature for 2 h. The error range was 0.02℃. After treatment, tubes were moved into the phytotron to recover for 1 h, and the survival rate of N. lugens was examined. Adults rawed in the phytotron were used as the control group. 【Result】Within the range of 33-37℃, the survival rate and fecundity of N. lugens were not significantly different from the control. Within the range of 39-40℃, the survival rate of N. lugens were decreased. After exposure to 39.5℃ for 2 h, the survival rate of male and female adults was less than 50%. After exposure to 40℃ for 2 h, the female adult survival rate dropped to 4.5%, and that of the male adults was 6.7%. All of the adults died within 24 h. After exposure to 33-39.5℃, the gradient of the male adult survival curve decreased abruptly than female. The preoviposition period was extended from 3.5-6.3 days with the increasing temperature from 33-39.5℃. The preoviposition period was 3.5 d at 25℃, 4.4 d at 35℃, 4.4 d at 39℃ (P＜0.05), and 6.3 d at 39.5℃ (P＜0.01), respectively. Furthermore, high temperature had a negative effect on the fecundity. The average fecundity decreased dramatically after exposure to high temperature for 2 h. F₁ survival to adulthood was significantly reduced when adults were exposed to 33-39.5℃. The survival rate dropped from 80.4% to 61.8%-75.5% as the temperature increased. Female adults ceased oviposition activities after the exposure to 40℃. The oviposition rhythms and the number of eggs of F₁ hatched per day also changed in these heat shock treatments, with prolonged oviposition period and delayed F₁ peak hatching time after exposure to 39.5℃.【Conclusion】The survival and reproduction of N. lugens significantly decreased after exposure to temperatures of 39-40℃. When the exposure temperature exceeds 39℃, it has the potential threats to the survival and fecundity of N. lugens.

Chiral Stability of Dinotefuran in Organic Solvents and Water

CHEN Zeng-long, CHEN Xiu, DONG Feng-shou, LIU Xin-gang, XU Jun, ZHENG Yong-quan

Scientia Agricultura Sinica. 2015, 48(9): 1756-1763. doi:10.3864/j.issn.0578-1752.2015.09.09

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【Objective】The objective of this study is to investigate the chiral stability of dinotefuran racemate (Rac-dinotefuran) and enantiomers (S-dinotefuran and R-dinotefuran) in methanol, ethanol, isopropanol, ethyl acetate, acetonitrile, dichloromethane, ultrapure water and β-cyclodextrin solution under xenon lamp, natural light, and dark, determine chiral neonicotinoid dinotefuran used extensively in China, and to accurately evaluate its efficacy and environmental safety.【Method】The samples were collected periodically during the whole experimental process. Dinotefuran enantiomers were analyzed by high performance liquid chromatography/diode array detector (HPLC/DAD) with amylose tris-3, 5-dimethylphenylcarbamate as chiral stationary phase and n-hexane/methanol/ethanol (85/10/5,v/v/v) as mobile phase. The ultraviolet detection wavelength of the DAD was identified at 270 nm. The column temperature was set at 30℃ and the injection volume was 20 µL. The retention times of S-dinotefuran and R-dinotefuran were 8.3 and 9.7 min. The quantification of dinotefuran enantiomers was made by using external standard method.【Result】No transformation was observed between S-dinotefuran and R-dinotefuran in methanol, ethanol, isopropanol, ethyl acetate, acetonitrile, dichloromethane, ultrapure water and β-cyclodextrin solution under xenon lamp, natural light and dark conditions. Dinotefuran did not undergo enantioselective degradation by the student’s paired t-test for the Rac-dinotefuran in all the solvents. The enantiomer fractions (EF) were 0.4746-0.5116. However, Rac-dinotefuran, S＞acetonitrile＞ethyl acetate≈isopropanol ≈ethanol＞methanol＞ultrapure water＞β-cyclodextrin solution. The degradation of dinotefuran enantiomers in the organic solvents was faster than in the aqueous solutions and the estimated half-lives were 3.3-3.6 h and 1.2-2.3 h, respectively. The photodegradation followed first-order kinetics mode and the correlation coefficients were ranged from 0.9550 to 0.9959. Rac-dinotefuran, S-dinotefuran and R-dinotefuran showed no obvious degradation in dichloromethane, acetonitrile, ethyl acetate, isopropanol, ethanol, methanol, ultrapure water, and β-cyclodextrin solution under natural light and dark during the whole experimental process. The measured concentration and the corresponding relative standard deviation were 9.5-10.4 mg?L^-1 and 1.0%-3.2% under natural light, whereas they were 9.5-10.5 mg?L^-1 and 1.4%-2.8% under dark. 【Conclusion】Dinotefuran enantiomers were chiral configuration stability in methanol, ethanol, isopropanol, ethyl acetate, acetonitrile, dichloromethane, ultrapure water, and β-cyclodextrin solution under xenon lamp, natural light and dark condition conditions at (25±2)℃. The qualitative and quantitative analysis, efficacy and environmental safety assessment were accurate under these conditions.-dinotefuran and R-dinotefuran were degraded rapidly in the solvents under xenon lamp. The order of the degradation rate was dichloromethane

Effect of Conservation Tillage on Microbial Respiration of Black Soil

JIA Shu-xia, SUN Bing-jie, LIANG Ai-zhen, CHEN Xue-wen, ZHANG Shi-xiu, WEI Shou-cai, LIU Si-yi, CHEN Sheng-long, ZHANG Xiao-ping

Scientia Agricultura Sinica. 2015, 48(9): 1764-1773. doi:10.3864/j.issn.0578-1752.2015.09.10

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【Objective】 In this study, soil microbial activity and biomass carbon under 13-year conservation tillage (no-till) were assessed in a black soil agro-ecosystem in northeast China in order to determine the effect of tillage treatment on soil organic carbon and soil microbial biomass, which would provide a theoretical basis for valuing the ‘sink’ or ‘source’ function of soil carbon pool. 【Method】 The present study was conducted as part of a long term tillage experiment on the continuous maize (Zea mays L.), tillage treatments consisted of no-tillage (NT), and mouldboard plough (MP). The NT treatment had no soil disturbance except planting, crop residues were left on soil surface after harvest. The MP treatment included one fall mouldboard plough (about 15 cm in depth) after maize harvest, one disking (7.5 to 10 cm in depth) in spring and field cultivation (ridging in June). Soil respiration without roots represented soil microbial respiration, which was measured biweekly from 14 June 2012 to 25 September 2013 using LI-8100 automated soil CO₂ flux system (LI-COR Inc., Lincoln, NE, USA), soil microbial biomass and the number of colony forming units of bacteria (cfu_b), fungi (cfu_f), and actinomyces (cfu_a) were measured during the soil microbial respiration was the highest. 【Result】 It was found that the range of soil microbial respiration was 0.42-3.35 μmolCO₂·m^-2·s^-1under NT and 0.48-3.24 μmolCO₂·m^-2·s^-1under MP during growing season, the average soil microbial respiration was similar between NT and MP (8.8%), while the total soil CO₂ emission during the growing season under NT was 10.0% (2012) and 4.3% (2013) higher than MP (P＜0.05). The cfu_b, cfu_f and cfu_a at 0-5 cm under NT were significantly higher by 125.7%, 112.4%, and 53.3% than MP, respectively, and cfu_f at 5-10 cm, 10-20 cm and 20-30 cm under NT was 105.3%, 159.4% and 114.7% higher than MP, and. Soil temperature at 0-5 cm and 5-10 cm under NT were 2.8% and 5.8% higher than MP in June, respectively. Soil microbial respiration showed a significant seasonal pattern similar to soil temperature, the highest rate occurred in summer (July or August), followed by spring and fall. Although tillage treatment did not influence the seasonal dynamic of soil microbial respiration, the highest rate of soil microbial respiration under MP was a half-month behind the NT. There was a significant exponential relationship between soil microbial respiration and soil temperature at 5 cm and 10 cm soil depth, exponential model at 10 cm produced better fitness than 5 cm, the temperature sensitivity (Q₁₀) at 5 cm under NT was 10.8% higher than MP. The combined exponential model indicated that soil temperature and water content (SWC) could jointly explain 65% (MP) and 81% (NT) of variation in soil microbial respiration.【Conclusion】The results suggested that the soil microbial activity at surface soil (0-5 cm) under NT was higher than MP, which is contributed to the higher SOC content under NT, the increase of the weighted average of SOC at 0-30 cm suggested that NT appears to be a better tillage practice to SOC sequestration in Northeast China.

Effects of Catch Crops on Reducing Soil Nitrate Accumulation and Cucumber Growth in Greenhouse Vegetable Production System

PENG Ya-jing, HAO Xiao-ran, JI Yan-zhi, WANG Lin, REN Cui-lian, JU Xiao-tang, ZHANG Li-juan

Scientia Agricultura Sinica. 2015, 48(9): 1774-1784. doi:10.3864/j.issn.0578-1752.2015.09.11

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【Objective】In intensive vegetable growing areas in China, the traditional high water and fertilizer inputs led to high soil nitrogen accumulation and nitrogen leaching into the deep layers of soil or groundwater, thus resulting in contamination of groundwater. Growing catch crops could control and reduce migration deep nitrate-nitrogen accumulation in soil to groundwater, therefore, this study was conducted to investigate the effects of different catch crops species on reducing nitrate accumulation in soil profile and crop growth in the next season, select the appropriate catch crop species. 【Method】The field experiment was conducted in the traditional cropping systems of greenhouse in North China Plain. In the fallow period of vegetables, deep roots-catch crops including sweet corn, amaranth, and sweet sorghum were planted in order to employ their root systems, fast growing and large amount of nitrogen uptake to promote soil nitrate nitrogen consumption, reduce nitrate accumulation in the root layer of soil profile and nitrate leaching of soil profile. Fallow field plots were also designed. Soil, plant and root samples were collected and measured, and the reduction effects of different catch crop species were analyzed.【Result】The results indicated that among the three species, sweet corn was the best in biomass production, N uptake, and root length density. From the effect of soil profile NO₃^--N reduction, sweet corn was the best among them, the reduction of NO₃^--N was 153.76, 605.70, and 56.3 kg·hm^-2 in 2008, 2009, and 2010. In the first quarter, the previous crop of cucumber was the highest in yield, N uptake and biomass production, and there was a significant difference with others in yield and N uptake. In the second and third quarters, there were no too clear difference in yield, N uptake and biomass production. Catch crops had little effect on vegetable production, but also reduced the accumulation of soil NO₃^--N. The N deficient ullage of sweet corn-cucumber was larger in 0-200 cm soil layer of the N apparent balance, which showed that sweet corn could obviously reduce the accumulation of soil NO₃^--N in the N apparent balance. Planting catch crops could obtain economic and ecological profits, and farmers obtained higher economic profits from sweet corn, amaranth and sweet sorghum, the values were 39 467, 497 and 16 522 yuan/hm², respectively.【Conclusion】Catch crops planted in greenhouse in summer could not only reduce nitrate accumulation in the root layer of soil profile, but also have little effect on cucumber production and obviously reduced the accumulation of soil NO₃^--N. In greenhouse vegetable crop rotation system, the introduction of catch crops is feasible, sweet corn is a better catch crop species.

Phenolic Composition and Antioxidant Activities of Grapefruit Varieties Cultivated in China

ZHANG Gui-wei, ZHANG Qiu-yun, JIANG Dong, XI Wan-peng, ZHOU Zhi-qin

Scientia Agricultura Sinica. 2015, 48(9): 1785-1794. doi:10.3864/j.issn.0578-1752.2015.09.12

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【Objective】 The polyphenols in grapefruit (Citrus paradis Macfad.) of different varieties were identified and characterized. The antioxidant activities of polyphenol extracts from different samples of each variety were compared. The aim of the study is to obtain the scientific data for improvement of Citrus breeding, grapefruit resources development and utilization. 【Method】The composition and content of phenolic compounds in pulps of nine major grapefruit varieties cultivated in China were determined by High Performance Liquid Chromatography (HPLC), and the antioxidant activities of these grapefruits were evaluated by using 2,2-diphenyl-1-picrylhydrazyl radicals (DPPH), ferric reducing antioxidant power (FRAP), and 2,2′-azino-bis (3-ethylbenzthiozoline- 6)-sulphonic acid (ABTS) methods. The correlation between antioxidant capacity and phenolic content was measured with Pearson correlation coefficient. 【Result】 Results showed that naringin and neohesperedin were the predominant flavanones in grapefruit, the naringin contents varied from 1 180.58 μg·g^-1 to 3 065.33 μg·g^-1, Huoyan had the highest content of naringin, while neohesperedin contents varied from 94.05 μg·g^-1 DW to 637.24 μg·g^-1 DW, Jiwei had the highest content of neohesperedin. Tangeretin was the major polymethoxylated ?avone, varying from 0.56 μg·g^-1 DW to 4.66 μg·g^-1 DW, which reached the highest content in Aoluobolangka, and gallic acid was the major phenolics acid, varying from 60.42 μg·g^-1 DW to 385.53 μg·g^-1 DW, which reached the highest content in Sibeitimaxu. Jiwei was rich in eriocitrin (111.23 μg·g^-1 DW), while Maxu rich in hesperidin (130.90 μg·g^-1 DW). Huoyan, Ruihong, Sibeitimaxu, and Hongmaxu had abundant gallic acid (＞300 μg·g^-1 DW), whereas Jiwei contained rich chlorogenic acid, caffeic acid and ferulic acid. The overall antioxidant potency composite index (APCI) revealed wide variations in the grapefruits investigated (36.70-97.50). Except for Aoluobolangka and Jiwei, the other seven grapefruit cultivars presented high APCI (＞80). Naringin, neohesperedin, hesperetin, nobiletin, tangeretin and chlorogenic acid showed a significant positive correlation with antioxidant abilities. 【Conclusion】 The pulps of nine major grapefruit varieties cultivated in China were rich in polyphenols. Different varieties showed obvious variations and characterized by their composition and content of phenolic compounds. Naringin, tangeretin and gallic acid were the predominant phenolic compounds of nine grapefruit varieties studied. Significant variations in polyphenols contents and antioxidant abilities were found between the tested varieties. Huoyan, Ruihong, Sibeitimaxu and Hongmaxu are good natural source of gallic acid, while Jiwei is the natural source of chlorogenic acid, caffeic acid and ferulic acid. Among the nine grapefruit varieties investigated in the study, Fenhongtangmuxun, Maxu, Huoyan, Ruihong, Sibeitimaxu and Hongmaxu not only contained rich phenolic compounds, but also presented high antioxidant activities. It was concluded that these cultivars may be used as a good natural antioxidant as well as an important basic material of functional foods and biomedicinals.

Genetic Diversity Analysis and Molecular Identification Card Construction of Chinese Cymbidium Germplasms Based on SRAP Markers

TANG Yuan-jiang, CAO Wen-jing, WU Kun-lin

Scientia Agricultura Sinica. 2015, 48(9): 1795-1806. doi:10.3864/j.issn.0578-1752.2015.09.13

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【Objective】 The genetic diversity of Chinese cymbidium germplasms was studied, meanwhile, the molecular fingerprinting and ID card were established in order to provide technique support for their identification at molecular level, and to lay the root for their exploitation, conservation, utilization and innovation in the future. 【Method】 139 samples of Chinese cymbidium gemplasm containing cultivated and wild species were collected from south China and its neighborhood. Their genomic DNA was extracted by the modified method of CTAB. 208 pair SRAP primers was from random combinations with 13 pieces F-primer and 16 pieces R-primer, which each pair of SRAP primers was initially screened for amplification of a specific product from genomic DNA of two cultivars, one from ‘Songmei’ and the other from ‘Daxun’. The PCR amplification products were separated on 6% nondenaturing polyacrylamidegel electrophoresis. The electrophoresis pattern was artificially analyzed, and total bands, polymorphic bands and percentage of polymorphic bands(PPB) were counted. The polymorphism information content(PIC) were counted according to Botstein’s formula. The observed number of alleles (Na), effective number of alleles (Ne), Nei’s genetic diversity diversity index (H) , and Shannon’s information index (I) all were estimated by software POPGENE32. The UPGMA clustering analysis was performed by software NTSYS-pc2.10e, and attained the genetic similarity coefficient value. The method of primer combinations was used to construct a digital fingerprinting of 139 samples of Chinese cymbidium germplasm. 【Result】 17 pair polymorphic primers were screened from 208 pair SRAP primers, and a total of 489 bands were amplified by these primers, of which 484 bands were polymorphic and PPB was 98.89%, observed number of alleles (Na), the effective number of alleles (Ne), Nei’s genetic diversity diversity index (H) , and Shannon’s information index (I) were respectively 2.00, 1.49, 0.30, 0.45. The genetic similar coefficient of 139 Chinese cymbidium ranged from 0.51 to 0.91, and the cluster analysis based on UPGMA showed that these germplasm can be divided into six major groups at similarity coefficient of 0.70. All of the samples could be effectively distinguished by different primer combinations. The specific molecular identity for 139 samples of Chinese cymbidium germplasm were established with 99.99% probability of confidence. However, Chinese cymbidium germplasm can be divided into four sections according to their genetic relationship, section one of which is C. goeringii germline, only consists of one species, section two mainly includes C. ensifolium and C. sinense germline, section three consists of C. faberi, C. kanran andhybrid species, section four includes C. longibractium and C. lianpan. In terms of genetic relationship among them, section four is the nearest with section three, section two comes second, is farthest with section one.【Conclusion】 A higher level of genetic diversity exists in Chinese cymbidium germplasm examined, it was unique and efficient that the molecular identity system of 139 samples was first established based on 17 pair primer combinations of SRAP. The SRAP technique can provide a powerful tool for Chinese cymbidium germplasm identification.

Lipid and Microstructure Changes of Small Packaged Rice During Storage

WANG Li-feng, CHEN Chao, YANG Jing-hua, CHEN Jing-yi, WANG Hai-feng, JU Xing-rong

Scientia Agricultura Sinica. 2015, 48(9): 1807-1817. doi:10.3864/j.issn.0578-1752.2015.09.14

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【Objective】Due to the loss of protection of endosperm, rice prones to aging during storage, mildew and other issues, which resulted in edible quality deterioration or even lost edible value. The aim of this study was to explore the reasons for the deterioration of rice quality by measuring the content of total acids and free fatty acids, changes in fatty acid composition and content, microscopic structure of cross-sectional endosperm cells or starch granules of rice, and to provide reference for storage technology of rice. 【Method】The No. 99 japonica rice packaged in woven, natural airtight anoxia and vacuum was simulated storaged in artificial climate chamber at a temperature of 15, 25, and 30℃ under 60% RH for 180 d, then the quality indicators and morphology of rice were measured every month. The aging mechanism of rice and variation of starch granules morphology caused by lipids and cross-sectional structure of endosperm after deterioration were explored by determining the variation of fatty acid and total acid value, the changes of fatty acid composition and content were analyzed by gas chromatography, and the cross section of rice endosperm cell morphology was observed by scanning electron microscopy.【Result】It was found from the study that the free fatty acids and total acid contents of rice packaged in woven and natural airtight anoxia increased sharply in initial process while decreased with a serious deterioration of the quality. The free fatty acids and total acid contents of rice packaged in vacuum maintained at a relatively stable level while the storage temperature had no significant effect on its content. The most abundant fatty acids were linoleic acid, oleic acid and palmitic acid,which accounted for more than 90% of total fats. Packaging methods and storage temperature had a great impact on various fatty acids. In addition to arachidonic acid (C20:0) and docosenoic acid (C22:1), the other kinds of fatty acids were greatly influenced by packaging methods. The storage temperature had a significant relationship with myristic acid (C14:0) and behenic acid (C22:0) while had little effect on the rest of the fatty acids. The changes of fatty acids agreed with the results that the amount of highly unsaturated fatty acids decreased with increasing temperature, these data also demonstrated negative correlations between polyunsaturated fatty acids (stearic acid (C18:0) and oleic acid (C18:1)) and saturated or monounsaturated fatty acids (linoleic acid (C18:2) or linolenic acid (C18:3)). The starch grains of fresh rice exist in a compound grain form with a large shape and a thick film on the surface, neat and orderly arranged with clear radial section in both sides of the sheet between the cells. The structure of endosperm cell cross-section became sloppy and disorderly, while the number of intracellular sections were more than that of intercellular sections. In addition, some “small holes” can be observed in sections with some protein and fat droplets scattered in the amyloplast. The compound starch granule broke into a large number of single granulocytes due to the damage of cell wall. Part of the shape of single starch granules became nearly circular with some blunt edges. And the radial structure became blurred or even disappeared when aging seriously.【Conclusion】The value of fatty acids and total acid showed an upward trend first and then a downward trend, the smoothness of endosperm surface deteriorated, and the number of holes and tilt of surface proteins film increased, as well as the radial structure trend disappeared gradually during storage. The rice quality deterioration seriously with woven and natural hypoxic packaging, it could delay the deterioration process of rice quality with vacuum packaging effectively.

POMC Expression and Localization in Sheep Skin with Different Coat Color

MA Shu-hui, LI Ya-nan, HE Xiao-yan, WANG Hai-dong, YANG Lei, DONG Chang-sheng

Scientia Agricultura Sinica. 2015, 48(9): 1818-1824. doi:10.3864/j.issn.0578-1752.2015.09.15

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【Objective】The objective of this study is to identify the regulation effect of POMC on coat pigment formation via analyzing the differences of POMC expression and location between different coat colors of sheep skin, in order to further enrich the theoretical knowledge about the formation mechanism of animal coat color. 【Method】 Three black adult sheep and three white sheep with matched age were selected, then three pieces of skin tissue from the buttocks of sheep were collected, respectively. One of them was used for immunohistochemistry assay to investigate the expression location of POMC between different coat color sheep after tissue embedded, the others were hold in liquid nitrogen for the extraction of total RNA and protein. The POMC-specific primers were designed by blasting in the NCBI database. After the reverse transcription of total RNA, QRT-PCR assay was proceeded to detect POMC mRNA expression of different coat color sheep. Then the protein expression level of POMC between different color sheep was evaluated by using Western blotting. All data were analyzed by SPSS19.0 to explore the mediate effect of POMC on coat color formation mechanism. 【Result】Immunohistochemistry showed that POMC was expressed in both black and white sheep skin, and the expression sites were located around the keratinocytes of hair follicle root sheath and epidermal. QRT-PCR showed that the relative expression of POMC mRNA in black sheep skin was 2.5004±0.2577^**, while in white was 1.0032±0.0350, the quantity expression of POMC mRNA level of black sheep was 2.50 times as that of white sheep (P＜0.01). Western blotting result showed that the expression of POMC protein in black and white sheep were 1.5253±0.04026^* and 1.0005±0.0180, respectively, the expression of POMC protein in black sheep was 1.55 times as that in white sheep (P＜0.05). 【Conclusion】POMC expressed normally in black and white sheep skin, and its expression was located in consistent position, the expression of POMC in black and white sheep skin have differences, which may involve in formation of coat color.

Effects of Dietary Zinc on Immunity, Antioxidant Capacity and MT-I mRNA Gene Expression Level and Their Factor Correlation Analysis of 5-15 Weeks Old Goose

WANG Bao-wei, CHEN Miao-lu,WANG Bing-han, ZHANG Ming-ai, GE Wen-hua, CHENG Fan-sheng, YUE Bin

Scientia Agricultura Sinica. 2015, 48(9): 1825-1835. doi:10.3864/j.issn.0578-1752.2015.09.16

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【Objective】 This experiment was conducted to determine the required level of zinc in goose feed. The effects of different levels of dietary zinc on immunity, antioxidant capacity and metallothionein (MT-I) mRNA gene expression to 5-15-week-old Qing-Nong-Hui geese were evaluated. 【Method】 Three hundred and sixty 29-day-old Qing-Nong-Hui geese were randomly selected and divided into six treatment groups with six replicates for each group, and each replicate contained ten geese (5♂+5♀). The content of added dietary zinc in each group was 27.46 (GroupⅠ, control), 77.46 (GroupⅡ), 127.46 (Group Ⅲ), 177.46 (Group Ⅳ), 227.46 (Group Ⅴ), and 277.46 (Group Ⅵ) mg·kg^-1, respectively. The geese were fed for 11 weeks. 【Result】The results were as follows: Index of thymus, spleen and bursa of fabricius showed an increase and downward trend with the effect of zinc level in feed. The thymus index obtained the highest (P＜0.05) level when the added zinc in feed was 127.46 mg·kg^-1, while the spleen index and the fabricius bursa index reached the highest level (P＜0.05) when the added zinc in feed was 77.46 mg·kg^-1. The index of immune organs decreased as long as the dietary zinc in feed was out of the range of 77.46-127.46 mg·kg^-1. The antibody titer reached the highest (P＜0.05) after the geese were immunized by goose paramyxovirus vaccine for 14 days when the zinc level in feed was 157.41 mg·kg^-1. The activity level of total antioxidant capacity (T-AOC) for the goose serum and liver reached the highest (P＜0.01) when the zinc level in feed was 153.84 mg·kg^-1. The activity level of both catalase (CAT) and glutathione peroxidase (GSH-Px) for the serum and liver of goose was the highest (P＜0.01) when the zinc level in feed was 148.33 mg·kg^-1. The activity level of superoxide dismutase (CuZn-SOD) was the highest (P＜0.01) in the goose’s serum and liver when the zinc level in feed was 148.33 mg·kg^-1. On the other hand, when the zinc level in feed was 177.46 mg·kg^-1, the malonaldehyde (MDA) in the serum and liver had the lowest level (P＜0.05); With the increase of the zinc level in feed, the expression quantity of MT-I mRNA was significantly increased (P＜0.05). And when the zinc level in the feed was 177.46 mg·kg^-1, the expression quantity of MT-I mRNA of liver was the highest. It was highly significant that the expression quantity of MT-I mRNA in goose liver was positively correlated with the activity level of T-AOC (r= 0.94, P＜0.01) and CuZn-SOD (r= 0.97, P＜0.01). The MT-I mRNA expression level in goose liver was also positively correlated with the activity level of CAT (r=0.85, P＜0.05), GSH-Px (r=0.89, P＜0.05), and negatively correlated with MDA (r=-0.70, P＞0.05). The expression level of MT-I mRNA in goose liver was significantly positively correlated with activity levels of T-AOC (r=0.99, P＜0.01), CAT (r=0.92, P＜0.01) and CuZn-SOD (r=0.94, P＜0.01) in serum. It was also positively correlated with GSH-Px (r=0.81, P＞0.05) and negatively correlated with MDA (r=-0.39, P＞0.05).【Conclusion】 Appropriate additional level of dietary zinc promoted the immune organs development and antioxidant capacity of goose. Dietary zinc also intervened the MT-I mRNA expression level, which was closely related with the body’s antioxidant capacity. Feed zinc levels in the 77-150 mg·kg^-1 range can keep the body in high immunity and antioxidant capacity level of the 5-15 weeks old geese.

Expression and Identification of the σ3 Gene of Avian Reovirus in Transgenic Tobacco

WANG Sheng, XIE Zhi-xun, HUANG Li, XIE Li-ji, DENG Xian-wen, XIE Zhi-qin, LIU Jia-bo, LUO Si-si, ZENG Ting-ting

Scientia Agricultura Sinica. 2015, 48(9): 1836-1844. doi:10.3864/j.issn.0578-1752.2015.09.17

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【Objective】 An experiment was carried out in laboratory to study on expression of Avian reovirus σ3 gene in tobacco and its genicity reactions of the expression products. 【Method】 A pair of primers was designed according to the major antigen region of the σ3 gene derived from GenBank. A plant vector of pBI121-σ3 constitutively expressed σ3 gene was constructed. The pBI121-σ3 vector was transferred into the Agrobacterium strain EHA105 by thermal activation method and a recombinant Agrobacterium strain EHA105 containing pBI121-σ3 was obtained. After transformation of tobacco plants via Agrobacterium tumefaciens, the resistant plants were selected with kanamycin. The resistant plants were firstly analyzed by PCR, using σ3 gene specific primers, and then real-time fluorescence quantitative PCR was used to further estimate the copy number of the σ3 gene in positive plants, and endogenous RNR2 gene in tobacco was used as a reference gene. With a serial of dilutions, the standard curves of the cycle threshold (C_T) relative to the log of each initial template copy of σ3 and RNR2 genes were obtained. The transgenic copy number was obtained by comparing the initial template copy of σ3 gene with that of RNR2. Western blot analysis was used to examine the σ3 protein expression in transgenic tobacco. 【Result】 The recombinant plasmid was identified by restriction endonuclease analysis and by gene sequencing. It was confirmed by DNA sequencing that the recombinant plasmid pBI121-σ3 contains a complete σ3 gene, a correct insertion site and expected open reading frame (ORF). The plant nuclear vectors expressed the σ3 gene by using CaMV 35S promoter and NOS terminator, and a kanamycin resistance marker (NPT II). The σ3 gene may be expressed as a 61.6 ku green fluorescent fusion protein(GFP) in tobacco. MS medium containing 350 mg cefotaxime·L^-1 inhibited the growth of Agrobacteria and 100 mg kanamycin·L^-1 can provide enough selective pressures. Eight resistant plants containing σ3 gene were obtained by PCR screening. The standard curve of RNR2 gene was expressed as y=-3.5352x+15.143, the standard curve of σ3 gene was expressed as y=-3.5366x+1.8265. Among the five putative transgenic lines, five had four copy numbers, whereas the negative control had none. Western blotting results showed that the 61.6 ku recombinant σ3 protein was successfully expressed, and the protein was specifically recognized by anti ARV positive serum. 【Conclusion】 A plant expression vector of pBI121-σ3 was successfully constructed and the regenerated transgenic σ3 gene tobacco plants were obtained. Recombinant σ3 protein has a good reactivity with anti ARV positive serum. The research findings provide a basis for further analysis on plants as bioreactors development and the production of σ3 oral vaccines.

Screening and Identification of Silkworm Probiotic Bacillus SWL-19 and Its Effect on Intestinal Microflora Diversity

LI Guan-nan, XIA Xue-juan, SENDEGEYA Parfait, HE Shi-bao, GUO Dong-dong, ZHU Yong

Scientia Agricultura Sinica. 2015, 48(9): 1845-1853. doi:10.3864/j.issn.0578-1752.2015.09.18

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【Objective】 The objective of this study is to screen the Bacillius probiotic for silkworm, which could be used to improve silkworm intestinal micro-environment and promote the development of sericulture production. 【Method】 Heat treatment was used for primary screening of Bacillus in the gut of silkworm. Further screening were conducted based on extracellular production of protease, lipase, cellulose and amylase, combined with hemolysis test, antibiotic susceptibility assay and abilities of producing digestive enzymes, moreover, colonized in the silkworm gut. Strains’ identification was carried out based on morphological, physiological and biochemical and 16S rDNA sequence analyse. In addition, denaturing gradient gel electrophoresis (DGGE) technology was used to detect the colonization of the strain and its effect on microflora diversity in the gut of silkworm. 【Result】 Two strains termed as SWL-17 and SWL-19 were screened from 57 bacterial strains. Both of them did not cause hemolysis and were able to produce protease, lipase, cellulose and amylase, simultaneously. Kirby-Bauer disc diffusion method was employed to perform antimicrobial susceptibility testing by using 14 kinds of paper disc, which showed that both SWL-17 and SWL-19 were sensitive to most of the drugs. The physiological and biochemical characteristics, like gram’s staining, spore staining, production of gas and acid, citrate utilization, glucose fermentation, nitrate reduction and V-P test etc. of the two strains were the same, except that SWL-19 couldn’t ferment sucrose. The growth rates of SWL-17 and SWL-19 were similar, while the colony morphologies were different. The colony morphology of SWL-17 was light yellow, round, ridgy, humid and tidy edge, while that of SWL-19 was white opaque, flat, dry and jagged edge. The protease, lipase, cellulose and amylase producing abilities of SWL-17 were (1.62±0.04), (2.12±0.11), (1.87±0.03) and (1.43±0.03), respectively; those of SWL-19 were (2.91±0.05), (2.43±0.04), (3.24±0.12) and (3.48±0.10), respectively. The protease, cellulose and amylase producing abilities of SWL-19 were significantly higher (P＜0.05) than that of SWL-17. Through homology analysis of 16S rDNA sequence, strains SWL-17 and SWL-19 were assigned to Bacillus sp. and B. subtilis,respectively. However, genetic background of SWL-19 were more clearly, which indicated that it was more suitable for further study. The dominant microflora of control group was Enterococcus spp., after exogenous adding strain SWL-19, the Bacillus sp. and Enterobacter sp. became the dominant microfloras. These results suggested that strain SWL-19 were able to permanent colonizes in the gut of silkworm and change the diversity of bacteria. 【Conclusion】 Screening of Bacillus probiotic is believed to have an important application value in the development of sericulture compound micro-ecological agents.

Chromosome Analysis of Peanut (Arachis hypogaea L.) Based on Sequential GISH-FISH

DU Pei, LIU Hua, LI Li-na, QIN Li, ZHANG Zhong-xin, HUANG Bing-yan, DONG Wen-zhao, TANG Feng-shou, QI Zeng-jun, ZHANG Xin-you

Scientia Agricultura Sinica. 2015, 48(9): 1854-1863. doi:10.3864/j.issn.0578-1752.2015.09.19

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【Objective】The cytogenetic study of peanut has been hindered mainly by the small chromosomes and limited cytological markers of peanut. In order to reveal the correspondence between cultivated peanut (Arachis hypogaea L., 2n=4x=40, AABB) and wild donor parents chromosomes and to characterize the chromosome structure variants, the objective of this study is to establish a highly distinguishable karyotype of A and B genome chromosomes for improving accuracy of chromosome identification. 【Method】Using the total genomic DNA of the two possible donors (A.duranensis, 2n=2x=20, BBand A.ipaënsis, 2n=2x=20, AA) of cultivated peanut and 5S and 45S rDNA as probes, sequential GISH-FISH combined with DAPI staining were employed to develop the karyotypes of Z5163 and the two donors based on distinguishing A and B genome of A. hypogaea clearly. The new karyotype was then used to analyze more peanut cultivars in order to reveal the characteristics of peanut chromosome constitution.【Result】 GISH indicated that all 20 B genome chromosomes of A. hypogaea produced clear and stable signals after hybridized with the total genomic DNA of A.ipaënsis, while only 18 chromosomes except “A chromosomes” of A genome produced signals using A. duranensis as a probe. Sequential mc-FISH using 5S rDNA and 45S rDNA as probes and combined with DAPI staining revealed that all the signals distributed on A and B genome of A. hypogaea were almost identical to its respective possible donor genome chromosomes of A. duranensis and A.ipaënsis, which suggested that A. duranensis and A.ipaënsis were the donors of A. hypogaea. Furthermore, the present study also found that 14 B genome chromosomes showed centromeric bands after DAPI staining, which were quite more than the previous reports, indicating that it was difficult to distinguish the A and B genome chromosomes of A. hypogaea only by DAPI staining as previous reports. Therefore, based on the above findings, a new GISH-FISH karyotype of the cultivated peanut was developed which could clearly distinguish all the chromosomes of A genome from those of B genome in A. hypogaea. And then the karyotype was used to characterize three peanut varieties, and a spontaneous monosomic substitution line of A. hypogaea, MSB1(A1), was found, indicating the homoeologous relationship between chromosomes B1 and A1.【Conclusion】The two genomes of cultivated peanut were very separately correspondent to its possible donor parents A. duranensis and A. ipaënsis; The new karyotype of the cultivated peanut could not only distinguish most of A and B genome chromosomes but also identify spontaneous chromosome variations produced in the progress of human-selection and polyploidization of peanut, indicating homoeologous relationships between chromosomes of A and B genomes of peanut.

Expression Analysis of Key Enzyme Genes in Lignin Synthesis of Culm Among Different Lodging Resistances of Common Buckwheat (Fagopyrum esculentum Moench)

HU Dan, LIU Xing-bei, WANG Can, YANG Hao, LI He-xin, RUAN Ren-wu, YUAN Xiao-hui, YI Ze-lin

Scientia Agricultura Sinica. 2015, 48(9): 1864-1872. doi:10.3864/j.issn.0578-1752.2015.09.20

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【Objective】The objective of this study is to explore the critical period and key enzyme genes in lignin synthesis of stem in common buckwheat and to provide a theoretical basis for common lodging resistance breeding cultivation by determining the lignin content and related genes expression characteristics at different stages of different lodging resistance cultivars. 【Method】Degenerate primers were designed to amplify part of the CDS sequences of CAD, CCR, F5H, COMT and CCOAOMT genes and blast was conducted in the NCBI database. The lignin content at the 2nd internode from the bottom were analyzed at branching stage, full bloom stage and milk ripe stage among four different lodging resistances of common buckwheat by UV spectrophotometry. Quantitative real-time polymerase chain reaction was used to analyze the expression characteristics of nine related genes (PAL, 4CL, C4H, C3H, CAD, CCR, F5H, COMT, and CCOAOMT), and the relative expression level was defined as 2^-^△△CT. Microsoft Excel 2003 and SPSS 19 were used for sorting data, drawing and analysis of variance, correlation analysis, respectively.【Result】Amplified CAD, CCR, F5H, COMT and CCOAOMT genes sequences were used to design the subsequent qRT-PCR primers. Lignin content of stem gradually increased from branching stage to milk ripe stage among different lodging resistances of common buckwheat, and lodging resistance cultivar was significantly higher than that of lodging susceptible cultivar. The expression levels of four genes (PAL, 4CL, C4H, CCR) varied significantly or extremely significantly among different cultivars and stages, CCOAOMT, F5H varied significantly among stages and cultivars, respectively. The remaining three genes (C3H, CAD, COMT) showed no significant difference among the cultivars and stages. The expressions of genes PAL, 4CL, C4H, CAD, CCOAOMT, F5H and CCR increased and then decreased, and the maximum appeared at full bloom stage, and lodging resistance cultivar was significantly higher than that of lodging susceptible cultivar. For COMT, the expression level decreased gradually from branching stage to full bloom stage, compared with the front seven genes, it showed an opposite expression pattern between lodging resistance and lodging susceptible cultivars at the first two stages. There was no obvious changing rule in gene C3H from branching stage to milk ripe stage. The lignin content was significantly and positively correlated with expressions of genes PAL, 4CL, C4H, CCOAOMT, CAD, and CCR, negatively correlated with C3H, and negatively correlated at branching stage and full bloom stage with COMT, while positively correlated at milk ripe stage with COMT.【Conclusion】The common buckwheat cultivar with lodging resistance showed high lignin content in the stem. The PAL, 4CL, C4H, CAD, CCOAOMT and CCR genes play a crucial role in the pathway of lignin synthesis. Full bloom stage is the critical period in lignin synthesis of stem in common buckwheat.

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