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    16 May 2016, Volume 49 Issue 10
    CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS
    Genetic Analysis and Candidate Gene Identification of the Panicle Degradation Mutant spd11 in Rice
    ZHONG Ping, CHEN Pu-rui, WANG Qian, XIAO Fu-liang, ZHANG Kuan, MA Fu-rong, HUANG Mei-ling, WANG Ping-rong, DENG Xiao-jian, SUN Chang-hui
    Scientia Agricultura Sinica. 2016, 49(10):  1835-1843.  doi:10.3864/j.issn.0578-1752.2016.10.001
    Abstract ( 645 )   HTML ( 4 )   PDF (4612KB) ( 720 )   Save
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    【Objective】 The objective of this study was to gain a better understanding of the regulation mechanism of early rice panicle development by genetic analysis and candidate gene identification of panicle degradation mutant spd11. 【Method】 A panicle degradation mutant, spd11, was isolated by ethyl methanesulfonate mutagenesis from the mutant library of japonica rice dense and erect mutant dep2 of Zhonghua 11. Phenotypes and the main agronomic traits of spd11 were analyzed under field conditions in Chengdu, Sichuan. Because spd11 can not seed, we individually harvested each plant of the isolate spd11 mutant strains lines and planted each of the plants as an independent line for genetic analysis. We investigated the segregation of each strain and analyzed the genetic pattern of spd11. The mutant gene of spd11 was mapped using F2 populations by crossing spd11+/spd11-plants with Gang 46B. Then, we conducted DNA sequencing, phylogenetic and alignment analysis of LOG proteins in different species. 【Result】 Compared with the control, spd11 exhibited long flag leaf blade lengths. The lengths of primary branches were shortened along with a 58% decrease in branch numbers. The florets of spd11 almost degenerated to white floc, though incompletely degenerated florets consisting only of a completely closed husk could be found occasionally. Other agronomic traits such as tiller number and flag leaf blade width showed little difference across lines. Genetic analysis showed that some lines were wild-type plants, but other lines isolated mutant plants. The segregation ratios between wild-type and mutant plants were all fitted to 3:1, indicating that spd11 was controlled by a single recessive nuclear gene. Then, the mutant gene was mapped to a region of 43.2 kb between two In/Del markers ch1-2295 and ch1-2299 on the long arm of chromosome 1, and the genetic distance were 0.23 cM and 0.46 cM, respectively, containing 8 predicted genes. Sequencing analysis revealed that a single base was changed (G116A) in the coding region of the OsLOG gene which encodes a cytokinin-activating enzyme, causing a missense mutation (C39Y). Alignment and phylogenetic analysis indicated that the LOG proteins were highly conserved in different species, and the mutation in spd11 was located at a very important amino acid site. Comprehensive analysis of the mutation sites and the phenotypes of reported log allelic mutants hinted that this site (C39Y) may lie within OsLOG. 【Conclusion】 SPD11 may be allelic to the OsLOG gene, and the mutation on a key site of OsLOG could affect cytokinin activation in spd11 mutant, eventually leading to the panicle degradation phenotype.
    Identification and Characterization of the AQP Gene Family in Sesame
    WU Xiang-yang, CHENG Chao-ze, Lü Gao-qiang, WANG Xin-yu
    Scientia Agricultura Sinica. 2016, 49(10):  1844-1858.  doi:10.3864/j.issn.0578-1752.2016.10.002
    Abstract ( 524 )   HTML ( 7 )   PDF (1766KB) ( 774 )   Save
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    【Objective】The present study aimed to identify the AQP gene family in the sesame genome and analyze its phylogenetic relationships in evolution, its linkage mapping, gene structure, transmembrane domain, conservation in amino acid residues and gene expression in response to Ralstonia solanacearum challenge. 【Method】AQP gene sequences were isolated in sesame genome by standard bioinformatic techniques combined with gene annotation information and verified by InterPro. AQP gene sequences from sesame, Arabidopsis and rice were aligned and compared using ClustalW2, among which, XIP members of AQP were compared between sesame and potato. Phylogenetic trees were constructed with MEGA6.0. Linkage mapping and gene structure assay were performed with MapInspect and Gene Structure Display Server 2.0, respectively. ProtParam, WoLF PSORT and TMHMM Server v2.0 were used to analyze the molecular mass and isoelectric point, the subcellular localization and the transmembrane domain respectively; multiple protein sequence alignments of AQP in sesame, Arabidopsis and rice were used to predict the NPA motif, ar/R selective filters and Froger’s Position (P1-P5). AQP gene expressions were analyzed based on the data of transcriptome of sesame upon Ralstonia solanacearum inoculation and further confirmed by qPCR. 【Result】A total of 36 AQP genes were identified in the sesame genome. Sequence comparison and phylogenetic tree analysis suggested that these AQP of sesame can be categorized into 5 subgroups: PIP (13 members), TIP (12 members), NIP (8 members), SIP (2 members) and XIP (only 1 member). 34 AQP genes were mapped onto 12 linkage groups. The gene structure, protein sequence, subcellular location and conservation in amino acids in the same subgroup were similar. Under Ralstonia solanacearum challenge, the expression of some members in PIP and TIP were up or down regulated, but the expression of members in NIP, SIP and XIP had no significant changes. SiPIP1;2, SiPIP1;3, SiPIP2;3, SiPIP2;4 were up-regulated; among them, SiPIP1;3 and SiPIP2;3 retain constant up-regulation, but SiPIP1;2 and SiPIP2;4 exhibited up-regulation at early stage of infection and down-regulation at later stage. SiPIP1;4, SiPIP2;1, SiPIP2;6, SiTIP1;1, SiTIP1;3, SiTIP2;1 and SiTIP2;2 were drastically down-regulated. The expression of these genes were confirmed by qPCR, which showed similar results to that of transcriptome sequencing. 【Conclusion】 Through whole genome analysis, 36 members of AQP genes were found in the sesame genome. These were grouped into five subgroups, and mapped onto 12 linkage groups. Most AQP genes contain 1-4 introns (though SiNIP1;2 has 7 introns). The substrates transported by each AQP group were predicted based on the types of ar/R selective filters and amino acid residues in Froger’s position. Under Ralstonia solanacearum challenge, expressions of some members in PIP and TIP were significantly up or down regulated.
    TILLAGE & CULTIVATION·PHYSIOLOGY & BIOCHEMISTRY
    The Possible Effects of Global Warming on Cropping Systems in China Ⅻ. The Possible Effects of Climate Warming on Geographical Shift in Safe Planting Area of Rice in Cold Areas and the Risk Analysis of Chilling Damage
    WANG Xiao-yu, YANG Xiao-guang, Lü Shuo, CHEN Fu
    Scientia Agricultura Sinica. 2016, 49(10):  1859-1871.  doi:10.3864/j.issn.0578-1752.2016.10.003
    Abstract ( 448 )   HTML ( 4 )   PDF (2148KB) ( 734 )   Save
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    【Objective】The period from 1961 to 2010 was divided into two stages, i.e., 1961-1980 and 1981-2010, combined with the scenarios of different temperature increases, then the geographical shift of rice safe planting areas in Heilongjiang province and the possible risk of chilling damage after the shift influenced by historical and future climate warming were quantitatively analyzed. 【Method】 Using the standard of the accumulated temperature zone and some agro-meteorological indicators, combined with ArcGis method, the geographical shift of accumulated temperature zones and northern limit for rice in cold planting areas were compared and analyzed. Using the risk assessment standard of chilling damage, the change characteristics of delayed chilling damage and sterile chilling damage in sensitive area of rice in cold areas were analyzed. The chilling damage characteristics in sensitive region under the background of climate warming and the risk evolution trends under the scenarios of different temperature increases were also evaluated.【Result】Compared with the results during the period from 1961 to 1980, the accumulated temperature zones moved northward and eastward obviously during 1981-2010, the average of which was northward 1.19°. The regions of maximum geographical shift were the fifth accumulated temperature zone and the sixth accumulated temperature zone. The region of maximum area increasing was the second accumulated temperature zone, which nearly covered Songnen Plain. The accumulated temperature zones under the future climate warming scenarios moved northward obviously. The region of maximum area increasing was the first accumulated temperature zone. Compared with the period 1961-1980, the average geographical shift of safe northern limit for rice in cold areas was northward 121 km and the maximum spatial displacement was in central part of Nenjiang- Wudalianchi- northern part of Xunke. The safe northern limit of rice in cold areas under the scenarios of 1-3℃ temperature increasing moved northward 410.5-545 km and the maximum spatial displacement was in northern part of Huma, Heilongjiang province. Rice could be planted in the whole region except Mohe under the scenarios of 3℃ temperature increasing. Compared rice sensitive planting region with nonsensitive region, the chilling damage risk in the sensitive region was obviously higher than nonsensitive region and the risk of sterile chilling damage was higher than delayed chilling damage. In rice safe planting areas of Heilongjiang province, the frequency of severe chilling damage was the highest, followed by light chilling damage, the frequency of moderate chilling damage was the lowest. The severe and light delayed chilling damage in the sensitive region during 1981-2010 showed an obvious increase. The risk of moderate delayed chilling damage in both sensitive region and nonsensitive region was low. The different grades of sterile chilling damage in sensitive region were higher than that in nonsensitive region. The different grades of chilling damage in sensitive region would be higher than that in nonsensitive region under future temperature increasing scenarios.【Conclusion】Under the background of climate warming as well as scenarios of different temperature increases, the accumulated temperature zones and safe northern limit of rice in cold areas had shown an obvious northward moving. The risk of chilling damage in sensitive region was obviously higher than that in nonsensitive region. The risk of chilling damage would be decreased as temperature increasing, but the defensive measures in sensitive region should be strengthened, such as adopting improved cultivation techniques, planting early-maturing varieties, as well as avoiding blind area and varieties expansion in rice planting areas.
    Effect of Different Ratios of Basal to Tiller Nitrogen on Rice Yield and Nitrogen Utilization Under Different Soil Fertility
    FAN Li-hui, XU Shan-shan, HOU Peng-fu, XUE Li-hong, LI Gang-hua, DING Yan-feng, YANG Lin-zhang
    Scientia Agricultura Sinica. 2016, 49(10):  1872-1884.  doi:10.3864/j.issn.0578-1752.2016.10.004
    Abstract ( 580 )   HTML ( 7 )   PDF (477KB) ( 600 )   Save
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    【Objective】 The objective of the experiment is to study the effect of ratio of basal and tiller fertilizer on yield, nitrogen use efficiency and rice population indices, and explore the optimum split ratio of basal and tiller fertilizer under different soil fertility for high-yielding rice. 【Method】 In this study, a field plot experiment was carried out with five different ratios of basal and tiller N fertilizer (0:10, 3:7, 5:5, 7:3, 10:0) under low and high fertility soil using Wuyunjing 23 as planting material, respectively. The tiller dynamics, dry matter accumulation and translocation, N uptake and utilization, and yield and yield components were measured.【Result】 Under low fertility soil, the maximum tiller number decreased, while the tillering rate, dry matter accumulation, nitrogen utilization and yield showed an inclination first then declination with the increasing ratio of tiller fertilizer, the yield and NUE were significantly higher than other treatments when the basal: tiller ratio of 3:7 and 5:5 with the total N rate of 300 and 240 kg·hm-2, which could reach 13.12 t·hm-2, 13.16 t·hm-2 and 27.00 kg·kg-1, 29.28 kg·kg-1, respectively. Under high soil fertility, with the increasing ratio of tiller fertilizer, the panicle number first increased then declined while grains per panicle appeared increasing, a declination tendency was observed for yield and nitrogen utilization, but the differences among different treatments were not significant. Compared with those under low soil fertility, the tillering rate and maximum tillers were higher and the duration to maximum tillers was shorten under high soil fertility; more dry matter was accumulated after heading under high soil fertility, thus the filling rate and grain weight was higher than that of low soil fertility which resulted in the higher yield under high soil fertility. The tiller number and the maximum tillers were the lowest under no basal fertilizer treatment, and the tillering was delayed in spite of the soil fertility, which proved that basal fertilizer is essential for tillering. With the increase of basal N rate, the time for tillering was shorten, but the tillering rate reduced and the time to effective panicles was prolonged due to the lack of nitrogen supply at tillering stage. Therefore, optimizing the N split ratio of basal to tillering is essential for high rice yield. For this study, 60 kg N·hm-2 of basal N and 1:1 of basal to tillering N was optimum for low fertility soil, and the N rate can be reduced from 300 kg·hm-2 to 240 kg·hm-2 while maintaining the high yield of 13 t·hm-2.【Conclusion】The effect of different ratios of basal and tiller N fertilizer on yield and NUE varied with the soil fertility condition, and also depends on the total N rate. For low fertility soil, rational split ratio of basal and tiller N is very important to achieve the high yield and avoid the excess N input.
    PLANT PROTECTION
    Development of the Agrobacterium tumefaciens Mediated Transformation System of Alternaria alternata Apple Pathotype
    GU Xue-ying, SHI Wen-xiao, WANG Hong-kai, GUO Qing-yuan
    Scientia Agricultura Sinica. 2016, 49(10):  1885-1891.  doi:10.3864/j.issn.0578-1752.2016.10.005
    Abstract ( 420 )   HTML ( 10 )   PDF (1043KB) ( 377 )   Save
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    【Objective】 Alternaria alternata apple pathotype is one of the important pathogens on apple which can infect leaves and fruits seriously. The objective of this study is to establish the Agrobacterium tumefaciens mediated transformation system of Al. alternata apple pathotype, provide a technical basis for study molecular mechanisms of pathogenesis. 【Method】 Vector pKO1-HPH is a shuttle plasmid which is constructed on the backbone of pCAMBIA1300 containing hygromycin phosphotransferase gene (hph) and GFP gene. Fungal transformation was carried out by co-incubation of Ag. tumefaciens strain AGL1 containing plasmid pKO1-HPH with conidia of Al. alternata apple pathotype. Transformants were screened at PDA plates containing hygromycin B. Hph gene expression was detected by incubation of transformants on PDA plates containing 50 μg·mL-1 hygromycin B, GFP was detected using Green fluorescence microscope. Influences of fungal cultural time and pretreatment method of conidia on transformation were evaluated. The stability of transformants was determined by growing on PDA plates containing hygromycin B after five-generation successive growth. PCR assay was used to confirm the existence of the hph gene and GFP gene in transformants. The copy number of inserted T-DNA in transformants was determined by Southern blot. Pathogenicity test of some transformants was performed on apple fruits and leaves. 【Result】 Spores of Al. alternata apple pathotype were formed enough when 100 μL of spore suspension (about 105 spores) was spread on PCA plate and incubated for 36-48 h. The maximum of transformation efficiency was up to 200 transformants/107 conidia when pretreatment of conidia at 4 for 6 h before transformation. The resistance to hygromycin B in transformants was stable after five successive generations. Results of specific PCR showed the exogenous genes (GFP and hygromycin B) were integrated into genome of Al. alternata apple pathotype strain and single copy insertion was detected in genome of majority of the transformants according to southern blot analysis. Green fluorescence was detected in the mycelia and spores under the fluorescent microscopy, indicating GFP was expressed in spores and hyphae of transformants. Results of pathogenicity test on fruits and leaves showed that the pathogenicity of several transformants was reduced.【Conclusion】Ag. tumefaciens mediated transformation system of Al. alternata apple pathotype is developed in this research and useful for further studies on molecular interaction of apple and Al. alternata apple pathotype.
    Effect of Rain-Shelter Cultivation on Temporal and Spatial Dynamics of Airborne Sporangia of Plasmopara viticola
    YU Shu-yi, LIU Chang-yuan, WANG Hui, LIU Li, GUAN Tian-shu
    Scientia Agricultura Sinica. 2016, 49(10):  1892-1902.  doi:10.3864/j.issn.0578-1752.2016.10.006
    Abstract ( 366 )   HTML ( 1 )   PDF (515KB) ( 632 )   Save
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    【Objective】 Downy mildew, caused by Plasmopara viticola, is the most serious disease in grape. Severe yield losses are caused wherever the weather during the growing season is humid and rainy. The objective of this study is to understand the effect of rain-shelter cultivation on airborne sporangia of P. viticola, define the relationship between airborne sporangia and changes of disease index, reveal the control of rain-shelter on the pathogen source, determine the control of rain-shelter on the pathogen source, analyze the primary infection sourced under the cultivation and provide a basis for formulating comprehensive control of the disease. 【Method】Form July to September during 2013, the diseased leaf and disease index on the same shoots were investigated under rain-shelter and open-field cultivation, respectively. The data were used to fit the regression models of dynamic of grape downy mildew under the two cultivations by regression curve estimation in SPSS19.0, the epidemic phases and rate of the disease were derived based on the data. The sporangia of P. viticola were investigated under rain-shelter and open-field cultivation. The dynamics of airborne sporangia were compared under two different cultivations, the relationship between airborne sporangia and changes of disease index in the key epidemic phase were analyzed. The main source of sporangia in the key epidemic phase and the main influence factors of airborne sporangia were cleared by comparing the effect of rain-shelter on the horizontal and vertical sporangia dispersal. 【Result】The seasonal epidemic curves of grape downy mildew under rain-shelter and open air cultivation were typical single peak S curve. The logistic model could reflect the temporal dynamic of grape downy mildew well by using SPSS19.0. The epidemic phases under the two cultivations, the exponential phases were from July 5th to July 23rd and August 18th to August 30th. The logistic phases were from July 23rd to August 19th and from August 30th to September 17th. The degenerating phases were from August 19th to the end of grape growing season and September 17th to the end of grape growing season. The seasonal airborne sporangia under open-field cultivation were a multi-peak curve. The daily rainfall had significant effects on the concentration of airborne sporangia. The peak periods were July to August and August to September under the two cultivations. Rain-shelter cultivation significantly reduced the number of airborne sporangia, thereby reduced the contact probability of sporangia and grape leaves in shelter facilities, and achieved the goal of reducing basic number of germs. Sporangia were trapped earliest on border row of the shelter facilities. The first trapped time delayed, and the concentration of airborne sporangia diminished with the closer to the center. Grape leaf canopy had significant barrier on airborne sporangia in horizontal direction. The concentration of airborne sporangia diminished with the increase of height under the two cultivations. There was a significant barrier effect of rain-shelter facilities on the airborne sporangia close to the roof. 【Conclusion】The first visibility of the disease and the first trapped time of airborne sporangia were significantly delayed, the epidemic process of the disease and cycle of airborne sporangia were significantly shortened, epidemic degree of the disease and airborne concentration of sporangia were significantly reduced by rain-shelter facilities. There were significantly barrier effects on both rain-shelter facilities and grape canopy. The sporangia matured on the spot under open-field cultivation, discharged in rain and dispersed with airflow, finally provided main inocula for the epidemic of the disease under rain-shelter cultivation.
    SOIL & FERTILIZER·WATER-SAVING IRRIGATION·AGROECOLOGY & ENVIRONMENT
    Effect of Long-Term Fertilization on the P Balance and Critical Value of Soil Olsen-P in Paddy Soil from Yellow Earth
    LIU Yan-ling, LI Yu, ZHANG Ya-rong, ZHANG Wen-an, JIANG Tai-ming
    Scientia Agricultura Sinica. 2016, 49(10):  1903-1912.  doi:10.3864/j.issn.0578-1752.2016.10.007
    Abstract ( 386 )   HTML ( 1 )   PDF (451KB) ( 871 )   Save
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    【Objective】The study examined the relation of soil phosphorus (P) availability with P balance under long-term fertilization and the critical Olsen-P value for crop yield and reasonable application of P fertilizer and provided theoretical basis for the scientific application of P Fertilizer and keep sustainable high production efficiency in paddy soil from yellow earth. 【Method】 The study was based on the long-term fertilization experiment of yellow soil in Guizhou. Soil and plant samples were collected from six treatments: without fertilization (CK), two single chemical fertilizer treatments (NK and NPK), single manure treatment (M), and two organic and inorganic fertilizer combination treatments (1/2 M+1/2 NPK and MNPK). Nineteen years (1995-2013) of paddy soil from yellow earth were analyzed for content of soil Olsen-P and the amount of P uptake by plants. The relationship between soil Olsen-P and soil apparent P balance, the relative yield of maize, and phosphorus application rate were studied by the method of linear regression based on a long-term fertilization experiment. The response relationship of crop yield to soil Olsen-P was used the Mitscherlich equation to examine the critical Olsen-P value for crop yield. 【Result】 The results showed that long term application of P fertilizer could significantly increase the content of soil Olsen-P. The Olsen-P annual growth rate was between 0.72 and 2.47 mg·kg-1·a-1 of the treatments with P fertilizer. Among them, MNPK treatment was the highest, and the NPK treatment was the lowest. Compared with single application of chemical fertilizer treatment (NPK) and single application of organic fertilizer (M), The treatments of combined application of organic fertilizer and chemical fertilizer (1/2 M +1/2 NPK) could promote the absorption of P in crops. The soil P content was depleted each year in the no-P treatments, while in the treatments with P fertilization there was surplus. The accumulative phosphorus amount was between 176-1 200 kg·hm-2, the P surplus with MNPK treatment was the highest. The P balance was significantly (P<0.05) correlated with the Olsen-P. With an average surplus of 100 kg P·hm-2, the soil Olsen-P increased by 4.0 mg·kg-1, 2.0 mg·kg-1, 3.2 mg·kg-1, 2.0 mg·kg-1, in the NPK, M, 1/2 M +1/2 NPK, MNPK treatments, respectively. The critical Olsen-P value for crop yield was 15.8 mg·kg-1 in paddy soil from yellow earth. The P balance and Olsen-P content every year were very significantly (P<0.01) correlated with P application amount. When the average P application amount was 17.4 kg P·hm-2·a-1, the P balance was flat. The critical value of soil Olsen-P corresponding to the amount of fertilizer was 37.2 kg P·hm-2·a-1. 【Conclusion】Soil Olsen-P content is affected by the surplus and deficit of soil P, and also by the P fertilizer input amount. When the average P application amount is 17.4 kg P·hm-2·a-1, the P balance was flat. When the average P application amount is 37.2 kg P·hm-2·a-1, there will be higher crop yield, high production of P fertilizer efficiency, and less accumulation in the soil.When the average P application amount is greater than 37.2 kg P·hm-2·a-1, it will lead to low response in crop yields, which not only results in a large amount of P surplus in soil, but also increases the loss risk of soil P. Under the condition of a certain amount of P surplus, long-term application of chemical P fertilizer resulted in the increased amount of Olsen-P which is higher than treatments with manure.
    Chemical Structure Characteristics of All Fractionations in Mollisol Organic Matter Under Long-Term Continuous Maize Cropping
    ZHANG Fu-tao, QIAO Yun-fa, MIAO Shu-jie, HAN Xiao-zeng
    Scientia Agricultura Sinica. 2016, 49(10):  1913-1924.  doi:10.3864/j.issn.0578-1752.2016.10.008
    Abstract ( 477 )   HTML ( 3 )   PDF (633KB) ( 636 )   Save
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    【Objective】 The objective of this paper is to reveal the effects of 24-year continuous maize cropping on Mollisol organic matter structure in all classes aggregates and density fractionations through studying infrared spectra characteristics of Mollisol organic matter before and after setting experiments, which can supplement soil organic matter (SOM) chemical structure dynamic theory under long-term continuous cropping.【Method】 Soil samples were obtained under 24-year continuous maize cropping in Hailun Agro-ecology Field Research Station, Chinese Academy of Sciences. SOM were divided according to aggregate class and density, and measured carbon content and infrared spectra of SOM in all aggregates and density fractions by Element Analyzer and Fourier Infrared Spectrometer separately. The spectra of SOM before and after continuous maize cropping were compared and analyzed.【Result】 After 24-year continuous cropping, carbon content in bulk soil decreased by 5.3%, carbon content in 2-0.25 mm aggregates significantly decreased, the carbon content had the decreasing trend in other class aggregates; free light fractionation (LF) carbon content significantly increased by 32.74%, but in occluded light fractionation (OF) significantly decreased by 16.72%, while no significant change in mineral-associated fractionation (MF). The relative intensity of aliphatic -CH, polysaccharide C-O, phenolic alcohol -OH increased, while the relative intensity of aromatic C=C and carboxyl C=O decreased, the ratio of -CH/C=C increased. The ratio of -CH/C=C mainly increased in >2 mm, of which -CH/C=C value of three density fractionations increased, especially LF; while in the density fractionations of other class aggregates had increasing trend. 【Conclusion】 So Mollisol organic matter structure became aliphatic and simple under long-term continuous maize cropping, the protection abilities of aggregates and mineral association decreased under long-term continuous maize cropping, which can’t benefit for the stability and storage of organic matter.
    Quantitative Analysis of Soil Salt and Its Main Ions Based on Visible/Near Infrared Spectroscopy in Estuary Area of Yellow River
    LIU Ya-qiu, CHEN Hong-yan, WANG Rui-yan, CHANG Chun-yan, CHEN Zhe
    Scientia Agricultura Sinica. 2016, 49(10):  1925-1935.  doi:10.3864/j.issn.0578-1752.2016.10.009
    Abstract ( 422 )   HTML ( 2 )   PDF (579KB) ( 620 )   Save
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    【Objective】It is crucial to quantitatively and accurately detect saline soil for the prevention of soil salinization and agricultural sustainable development. The objective of this study is to find the characteristic spectra of soil salt and its main ions, and build a quantitative analysis model of soil salinization which is suitable for the estuary area of Yellow River to improve the accuracy and stability of the quantitative analysis. 【Method】 Kenli County in Shandong Province was selected as the experimental area. Firstly, 96 representative soil samples were collected on October 5th-9th, 2014. After soil sample were dried, the contents of soil salt and ions were analyzed. Then the visible and near infrared reflectance hyperspectra of the soil samples were measured in the laboratory by ASD Fieldspec 3 spectrometer, smoothed and transformed to the first deviation. Secondly, the response spectra of salt and its main ions (Cl-, Na+, Ca2+)were analyzed, first soil salinity and main ions content and the first derivative spectra of reflectance by band correlation analysis, according to the principle of correlation coefficient and significant, their sensitive bands were selected as the characteristic bands, feature bands with maximum correlation coefficient were chosen as the significant feature bands. Then the characteristic spectra which can represent soil salt and main ions (Cl-, Na+, Ca2+) were analyzed synthetically using correlation analysis and identified. Finally, the methods of multiple linear regression (MLR), support vector machine (SVM) and random forest (RF) were used respectively to build quantitative analysis models of soil salinity and ions contents. Result】 The overall shape and trend of the spectra curves of soil salinity and major ions (Cl-, Na+, Ca2+) content in study area were very similar. Soil salinity and major ions (Cl-, Na+, Ca2+) spectra response regions were determined to be 1 320-1 495, 1 790-1 920, 2 120-2 290 nm. On account of the correlation between the first derivative of the reflectance and the soil salinity and its main ions content, the sensitive spectral regions were 1 490-1 520, 1 890-1 930 nm, final integrated spectra analysis and correlation analysis the characteristic bands were  1 493, 1 801, 1 911 and 2 289 nm and the significant characteristic bands were 1 493 and 1 911 nm. The models’ accuracy based on the first deviation of reflectance on the significant characteristic bands matched the models’ accuracy based on the four characteristic bands, which indicates that the significant characteristic spectra were effective for quantitative analysis of soil salt and its main ions. Compared the three modeling methods, the prediction ability of the RF was the best, followed by the SVM, the MLR models’ precision was the lowest. The models using the above-mentioned three methods could be used for quantitative analysis of salt, Cl- and Na+, and had good stability and high precision, however the prediction accuracy of Ca2+ contents was still to be improved. In comprehensive comparison and analysis, among the built models, the RF models based on the first deviation of reflectance on the significant characteristic bands (1 493 and 1 911 nm) had higher accuracy and stability for the quantitative analysis of soil salt, Cl- and Na+, and could be applied to the quantitative estimation of Ca2+.【Conclusion】The significant characteristic spectra (the first deviation of reflectance on 1 493 and 1 911 nm) of soil salt and its main ions were selected synthetically using correlation analysis based on the spectral response, then the quantitative estimation models of salt and its main ions were built using the RF regression method, which is suitable for the effective extraction of soil salinization information in the estuary area of Yellow River.
    HORTICULTURE
    Characterization of Mature Fruit Surface Waxes of Three Cultivated Citrus Species
    WANG Jin-qiu, HE Yi-zhong, XU Kun-yang, LUO Yi, SHENG Ling, LUO Tao, LIU Huan, CHENG Yun-jiang
    Scientia Agricultura Sinica. 2016, 49(10):  1936-1945.  doi:10.3864/j.issn.0578-1752.2016.10.010
    Abstract ( 495 )   HTML ( 12 )   PDF (2333KB) ( 885 )   Save
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    【Objective】This study is aiming at characterizing the mature fruits wax from citrus cultivated species(Citrus reticulata Blanco cv. Ponkan, C. limon cv. Eureka and C. grandis Osbeck cv. Shatian Yu) which are widespread in China, establishing the database of citrus fruits wax components, and providing important information for waxing for the specifical cultivar of citrus fruits. 【Method】Wax crystal structure of three citrus cultivars were observed by scanning electron microscopy, and cuticle thickness observed by transmission electron microscope. Subsequently, the fruits waxes of these cultivars were analyzed by gas chromatography-mass spectrometry (GC-MS). Then, the expression levels of wax associated genes were detected in different cultivars using qPCR. 【Result】 Scanning electron microscopy showed that larger and more wax platelets were deposited on the surface of tight-skin citrus Shatian Pummelo than on the loose-skin citrus Ponkan fruits, while wax platelets on lime fell in between. The total waxes of the three citrus cultivars were mostly composed of aldehydes, alkanes, fatty acids, and primary alcohols, while their proportions and carbon number distribution varied among different cultivars. The epicuticular wax concentrations in fruits of Citrus reticulata Blanco, C. limon cv Eureka and C. grandis Osbeck were 1.1, 2.2 and 9.3 μg·cm-2, respectively. The proportions of aldehydes, fatty acids, alkanes and primary alcohols in the epicuticular wax in Citrus reticulata Blanco were 50%, 16%, 28% and 6%, respectively. Their proportions in C. limon cv Eureka were 42%, 30%, 18% and 11%, respectively, while their proportions in C. grandis Osbeck were 50%, 31%, 12% and 7%, respectively. Hexacosanal (C26) and octacosanoic acid (C28) were the most abundant compounds in the epicuticular wax in Ponkan, while in Lemon, their carbon number distributed evenly. Higher carbon number aliphatic compounds were found in Shatian Pummelo, for example, dotriacontanoic acid, hentriacontane and tritriacontane were dominated. The total wax concentrations in fruits of Ponkan and Lemon were 4.3 and 4.6 μg·cm-2, respectively. The proportions of aldehydes, fatty acids, alkanes and primary alcohols in total wax in Ponkan were 57%, 14%, 23% and 4%, respectively. Their proportions in Lemon were 53%, 15%, 19% and 8%, respectively. The chain length distribution of total wax was similar with that of epicuticular wax in both of the cultivars. In addition, terpenoids showed great differences among these cultivars. The most predominant triterpenoid compounds were friedelin, lupeol, α-amyrin and β-amyrin in C. limon cv Eureka. However, there was no friedelin in total wax in Ponkan. Farnesol and squalene were exclusive in tense-skin citrus. qPCR analysis indicated that expression profile of member of beta-ketoacyl-CoA synthase family KCS6 showed a similar trend with wax amounts of the five cultivars, while member of the BAHD superfamily of acyl-transferases CER2 was expressed highest in Shatian Pummelo, which was consistent with high abundance of constituents with carbon number higher than 30. In this study, the wax of citrus fruits was mostly composed of aldehydes, followed by fatty acids or alkanes. Primary alcohols were the least predominant compounds. By comparing the distribution of wax components, it was found that, within the very-long-chain aliphatic wax components, primary alcohols and very-long-chain fatty acides tend to accumulate to higher percentages in the epicuticular layer, while alkanes occurred at lower percentages in it. With terpenoids, triterpenoids and sterols were only present in the total wax, however, squalene and farnesol were detected in both epicuticular and intracuticular wax.【Conclusion】Wax components were regulated by inheritance and peel anatomical structure synergistically. Therefore, loose-skin citrus and tight-skin citrus showed different characteristics of wax profile, which suggested that different synthetic waxes should be adopted in those two types of citrus fruits, respectively.
    Analysis of Protein Phosphorylation Level at Different Developmental Stages of Strawberry Fruit
    Lü Xiao-su, LI Yu-xuan, MIAO Ying, CHEN Liang-ke, SHEN Yuan-yue, QIN Ling, XING Yu
    Scientia Agricultura Sinica. 2016, 49(10):  1946-1959.  doi:10.3864/j.issn.0578-1752.2016.10.011
    Abstract ( 491 )   HTML ( 2 )   PDF (521KB) ( 568 )   Save
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    【Objective】The changes of protein phosphorylation level during the growth and development of strawberry were analyzed in order to reveal the effect of protein phosphorylation on the growth and development of fruit during ripening and senescence.【Method】In this experiment, the protein phosphorylation level of strawberry fruit (Small green fruit stage, big green fruit stage, mature white stage, soft fruit stage, over-soft fruit stage) was analyzed by using iTRAQ-based (isobaric tags for relative and absolute quantification) quantitative proteomic and LC-MS/MS (liquid chromatography-mass spectrometry) methods. And the biological process classification, subcellular localization classification and molecular functional classification of these proteins were identified. The function of the phosphorylated proteins was predicted by comprehensive analysis of the results of multiple databases.【Result】 The results showed that the phosphorylation proteins at different developmental stages were different. During the period of big green fruit stage to soft fruit stage, the total number of phosphorylated protein and the number of proteins involved in the regulation of growth and development are more than at other developmental stage. Biological process classification results showed that most of these phosphorylated proteins were concentrated in the plant signal transduction pathway and the pathway of glucose metabolism. There were 17 phosphorylated proteins involved in signal transduction pathways, and 8 of them were involved in the process of glucose metabolism. Belonging to the MAPK cascade pathway are 101308592, 470122684, and 596127083. The phosphorylation level of 596127083 was relatively stable at various development stages, and 101308592 gradually increased with the development of phosphorylation, While the phosphorylation level of 470122684 began to decrease sharply from the soft fruit period. The results of subcellular localization showed that most of the phosphorylated proteins were localized in the nucleus and cytoplasm. Molecular functional classification showed that most of the phosphorylated proteins had the function of transcription regulation and phosphorylation. In this study, 24 phosphorylated proteins related to growth and development regulation were identified. Among them, four had the function of transcriptional regulation, six were involved in cell division, and there was also a part of the phosphorylated proteins involved in the regulation of the growth and development of hormone response. In addition, this study also identified 3 phosphorylated proteins related to the ripening and softening of fruit. In addition, a part of the protein has a variety of phosphorylation modification, SNF1 related protein kinase beta subunit has three kinds of phosphorylation modification, and its phosphorylation level is not the same.【Conclusion】There may be more than one kind of phosphorylation modification in the same kind of protein. Different phosphorylation modification methods are not the same. The dominant mode of modification in different periods is not only the same. Phosphorylation is not only involved in the regulation of transcription and cell division and differentiation, but also in response to plant hormones and the accumulation of sugars during fruit development. Even involved in the regulation of fruit ripening and softening.101308592 and 470122684 are likely to be involved in the regulation of fruit growth and development. In short, protein phosphorylation modification plays an important role in the growth and development of strawberry.
    STORAGE·FRESH-KEEPING·PROCESSING
    Influence of Addition of Unsaturated Fatty Acids on Fatty Acid Composition of Saccharomyces cerevisiae and Wine Aroma Compounds
    DUAN Liang-liang, PAN Qiu-hong, WANG Ya-qin, YE Dong-qing, DUAN Chang-qing, YAN Guo-liang
    Scientia Agricultura Sinica. 2016, 49(10):  1960-1978.  doi:10.3864/j.issn.0578-1752.2016.10.012
    Abstract ( 480 )   HTML ( 4 )   PDF (900KB) ( 878 )   Save
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    【Objective】To provide a theoretical basis for improving the quality of wine, the effect of different concentrations of unsaturated fatty acids (UFAs, including oleic acid, linoleic acid, and α-linolenic acid) in grape juice on the intracellular fatty acid composition of yeast and volatile profile in wine were investigated in this study.【Method】 Different concentrations of mixed UFAs were initially added into ‘Merlot’ (Vitis vinifera L.) juice including the control without UFAs addition (CK),low concentration of UFAs addition (LFG) and high concentration of UFAs addition (HFG). The growth of yeast (Saccharomyces cerevisiae EC1118) growth (OD600), intercellular fatty acid composition and wine volatile aroma compounds among the three treatments during alcoholic fermentation were compared.【Result】The increase of UFAs can accelerate yeast growth and promote UFAs absorption by yeast, while suppress yeast saturated fatty acids (C4:0-C24:0) synthesis. The influence of UFAs on wine aroma showed that the increase of UFAs can stimulate the production of higher alcohols and ethyl esters by yeast, such as ethyl hexanoate, ethyl octanoate and ethyl decanoate, but inhibit the generation of medium-chain fatty acids (MCFAs, C4:0-C12:0). The influence on grape-derived aldehydes, monoterpenes and norisoprenoids volatiles were little. 【Conclusion】 Increasing UFAs in grapes can considerably improve yeast growth and the wine fermentation rate, more importantly, it can promote yeast-derived ethyl esters volatiles production, which intensifies the fruity, flowery and sweet odors of wine. Therefore, manipulation of initial UFA contents in grapes or grape juice is a potential way to regulate wine aroma quality.
    Effect of Lactic Acid Bacteria Fermentation on Phenolic Profiles and Antioxidant Activity of Dried Longan Flesh
    LAI Ting, LIU Lei, ZHANG Ming-wei, ZHANG Rui-fen, ZHANG Yan, WEI Zhen-cheng, DENG Yuan-yuan
    Scientia Agricultura Sinica. 2016, 49(10):  1979-1989.  doi:10.3864/j.issn.0578-1752.2016.10.013
    Abstract ( 529 )   HTML ( 2 )   PDF (403KB) ( 948 )   Save
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    【Objective】 The effect of lactic acid bacteria fermentation on free and bound phenolics and flavonoids and on antioxidant activity of dried longan flesh was investigated, with the purpose of giving some technical guidance for the deep processing of dried longan, and the development of new nutrition and health products fermented by lactic acid bacteria. 【Method】The culture medium was longan fruit pulp (the dried longan flesh: water=1:1.5), which then was inoculated by seven strains (Lactobacillus acidophilus GIM 1.731, Leuconostoc mesenteroides GIM 1.473, Lactobacillus casei GIM 1.411, Pediococcus pentosaceus GIM 1.428, Lactobacillus plantarum subsp. plantarum GIM 1.380, Lactobacillus delbrueckii subsp. lactis GIM 1.407, Lactobacillus bulgaricus GIM 1.155, bacteria concentration: 8.0-9.0 lg CFU/mL) with 1% at 37℃ for 48 hours. The contents of free and bound phenolics and flavonoids in dried longan flesh were determined. A high performance liquid chromatography (HPLC) method was used to measure the compositions and contents of phenolics in dried longan flesh. The antioxidant activity was analyzed using the ferric reducing ability of plasma (FRAP) and Oxygen Radical Absorption Capacity (ORAC). 【Result】 Seven lactic acid bacterias can increase, to different extents, the content of free phenolics and flavonoids in dried longan flesh (P<0.05), and reduce the content of bound phenolics and flavonoids (P<0.05). Compared to the no fermentation group, the content of free phenolics increased by 0.4%-11.9%, the content of bound phenolics decreased by 14.4%-41.4%; the content of free flavonoids increased 2.8%-19.6%, and the content of flavonoids decreased by 19.6%-70.6%. Lactobacillus plantarum subsp. plantarum GIM 1.380 had more advantages than other lactic acid bacteria on the ability of releasing bound phenolics, The content of free phenolics increased by 11.9%, the content of bound phenolics decreased by 41.4%, the content of free flavonoids increased by 19.6%, and the content of flavonoids decreased by 70.6%. Different lactic acid bacteria had different influence on phenolics compositions in dried longan flesh (gallic acid, syringic acid, (-)-Epicatechin, 4-methylcatechol, protocatechuic acid, p-coumaric acid, and isoquercetin). Compared to the no fermentation group, the content of free gallic acid and 4-methylcatechol were increased by 8.6%-69.8% and 0.4%-29.5%, and the content of bound gallic acid, vanillic acid and 4-methylcatechol were decreased by 80.0%-88.3% and 45.6%-73.9%. After fermentation, the free FRAP value and ORAC value were obviously increased (P<0.05), compared with the no fermentation group. 【Conclusion】 Lactic acid bacteria fermentation could increase the content of free phenolics in dried longan flesh, and reduce the content of bound phenolics, as well as improve its free antioxidant activity. Different lactic acid bacteria fermentations had differently effects on the total phenolic contents and antioxidant capacity and phenolic compositions in dried longan flesh. The result indicated that Lactobacillus plantarum subsp. plantarum GIM 1.380 had more advantages than the other lactic acid bacteria in the ability of releasing bound phenolics and improving antioxidant ability.
    ANIMAL SCIENCE·VETERINARY SCIENCERE·SOURCE INSECT
    Transcriptional Activity of Goat PRNP Gene Promoter
    ZHOU Rong-yan, WEI Yan-hui, XI Jian-zhong, LI Lan-hui, CHEN Hui, GAO Li-jie, ZHANG Zhen-hong
    Scientia Agricultura Sinica. 2016, 49(10):  1990-1997.  doi:10.3864/j.issn.0578-1752.2016.10.014
    Abstract ( 368 )   HTML ( 1 )   PDF (2144KB) ( 477 )   Save
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    【Objective】In order to screen the critical region or transcription factors regulating the expression level of prion in goat, the active region in the promoter of PRNP gene was analyzed. These results will provide a theoretical reference for elucidating the regulation of expression and thought for reducing the infectivity of prion disease with genetic strategy.【Method】The specific primer was designed based on the reference genomic sequence (GenBank Accession: EU870890). The fragment in a 5′ flanking region was amplified and cloned into the vector pEASY-T3. The positive colonies were identified and sequenced. The putative promoter and transcription factor binding sites were predicted with bioinformatic methods and online program. Eleven fragments with different lengths of promoter regions were amplified and cloned into the vector pEASY-T3. The positive colonies and vector pGL3-Basic were digested with two restriction enzymes Mlu I and Bgl II. The digested mixture were purified and ligated with T4 ligase to get the recombinant containing the luciferase reporter gene. The endo-free plamids were isolated after the positive colonies were obtained and sequenced. The transfection to SH-SY5Y was done with a Liposome reagent. The luciferase activity was measured with the dual-luciferase detection kit after 48 hours transfection.【Result】The length of the fragment in 5′ flanking region of PRNP gene in goat was 2 332bp. The predicted active region in the promoter, conserved motifs and multiple transcription factor binding sites were involved in the cloned fragment in the 5′ flanking region. Eleven different lengths of fragments were obtained and ligated with luciferase reported vector. The ratio of transfection reagent and DNA and Firefly luciferase vector and Renilla luciferase were 1﹕0.5 and 50:1 respectively. The core promoter was involved in 5′ flanking region of PRNP gene in goat and this region was from -519bp to +82bp. There were some positive regulatory elements in the region from -220 to +59. The exon 1 played a critical role in regulating the activity of the promoter. The positive regulating elements binding sites were predicted for four conserved motifs. There were many transcription factors, such as Sp1, AP-2 alpha and AP-1, binding sites in the strong active promoter. The transcription factors Foxp3 and COE1 binding sites were also predicted for motif 3 and 4 respectively. 【Conclusion】The core promoter region from -519bp to +82bp was identified in goat PRNP gene. The function of exon 1 in regulating promoter activity was critical.
    Cloning, Sequence Analysis and Tissue Expression of IGFBP-2 Gene in Goat (Capra hirus)
    ZHAN Si-yuan, DONG Yao, LI Li, ZHONG Tao, WANG Lin-jie, ZHANG Hong-ping
    Scientia Agricultura Sinica. 2016, 49(10):  1998-2007.  doi:10.3864/j.issn.0578-1752.2016.10.015
    Abstract ( 363 )   HTML ( 1 )   PDF (3881KB) ( 483 )   Save
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    Objective】The objectives of this study were to clone goat IGFBP-2 (Insulin-like growth factor binding protein -2) coding sequence (CDS), to analyze its sequence characteristics, mRNA and protein expression profiles. It could provide data for further investigation on the function and expression regulation of IGFBP-2 gene in the growth and development of postnatal goat. MethodThe IGFBP-2 mRNA sequences of Ovis aries (NM_001009436) and Bos taurus (NM_174555) were downloaded from GenBank database, and the primers were designed according to the conserved regions by using Primer Premier 6.0 software after sequences alignment, then PCR amplification and the positive clones were obtained using TA clone technology. A complete coding sequence of IGFBP-2 gene was obtained from sequencing. The CDS and coding amino acid sequences were analyzed by using EditSeq, DNAMAN 6.0, MEGA 6.0 and ExPASy online software. The real-time quantitative PCR (Q-PCR) primers were designed using the acquired CDS sequence of the IGFBP-2 gene. The expression level of the IGFBP-2 gene and protein in postnatal Nanjiang brown goat’s different tissues (heart, liver, lung, longissimus dorsi muscle, semimembranosus muscle and triceps brachii muscle) and development stages (3 d, 30 d, 60 d, 90 d and 120 d) were detected using Q-PCR and western blotting. ResultA full-length CDS of 954bp was cloned for goat IGFBP-2, and encodes a protein of 217 amino acid. The GC and AT content is 69.39% and 30.61%. The molecular weight of goat IGFBP-2 protein is 33.8808 kD, and its theoretical isoelectric point is 7.82. The secondary structure of the protein contains random coil (68.14%), α-helix (18.30%) and extended strand (13.56%). Protein structure domain analysis showed that its amino acid sequence contains conserved IB (IGFBP homologues) and TY (Thyroglobulin type Ι repeats) domains, IB domain in the 37-125 amino acids and TY domain in the 250-302 amino acids. Phosphorylation sites prediction indicated that there are five Ser phosphorylation sites and seven Thr phosphorylation sites exists in IGFBP-2 protein sequence. Glycosylation sites prediction found that there are ten N-glycosylation sites and two O-glycosylation sitesexists in IGFBP-2 protein sequence. Sequence analysis indicated that the IGFBP-2 CDS sequence of the Nanjiang brown goat is similar with those of the sheep (98.99%), cattle (97.73%), pig (87.12%), human (78.33%) and mouse (76.26%). Sequence analysis revealed that the IGFBP-2 amino acid sequence of the Nanjiang brown goat is similar with those of the sheep (99.24%), cattle (98.10%), pig (87.07%), human (70.27%) and mouse (73.00%). Furthermore, goat IGFBP-2has the closest phylogenetic relationship to the sheep and cattle IGFBP-2 in amino acids sequences. The mRNA and protein levels of IGFBP-2 in the liver were significantly higher than that in other tissues (P<0.01), and LD were the second (P<0.01); in the liver, the mRNA and protein levels of IGFBP-2 were upregulated from 3 day to 120 day. In addition, the mRNA level of IGFBP-2 showed an “up-down-up” expression pattern during postnatal longissimus dorsi muscle development. 【Conclusion】IGFBP-2 was cloned and its tissue expression patterns were investigated.The sequence characteristics of IGFBP-2 is conserved in species, and liver is the main expression tissue of goat IGFBP-2 mRNA and protein. The expression of IGFBP-2 mRNA and protein occurred with some regularity and indicated that IGFBP-2 may play an important role in early growth and development of postnatal goat.
    Signal Pathway of Interferon-β Induced by Porcine Circovirus Type 2 in PK-15 Cells
    ZHANG Xin-chen, ZHAO Qi-ling, CHEN Meng-meng, SUN Jia-rui, BAO En-dong, ZHANG Shu-xia, Lü Ying-jun
    Scientia Agricultura Sinica. 2016, 49(10):  2008-2016.  doi:10.3864/j.issn.0578-1752.2016.10.016
    Abstract ( 431 )   HTML ( 11 )   PDF (587KB) ( 435 )   Save
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    【Objective】 The objective of this study is to investigate the mechanism of IFN-β production induced by PCV2 in PK-15 cells and provide a theoretical foundation for the pathogenesis and prevention of porcine circovirus disease.【Method】 The PK-15 cells were divided into five groups randomly: Control group, PCV2 group, BX795 (inhibitor of TBK1/IKKε) group, and BAY11-7082 (inhibitor of NF-κB) group, BX795+BAY 11-7082(mixed inhibitors) group. BX795 group, BAY 11-7082 group and BX795+BAY 11-7082 group were pretreated with 0.5µmol BX795, 5µmol BAY 11-7082 and 0.5µmol BX795+5µmol BAY 11-7082 for one hour, respectively. Then the PK-15 cells were infected with PCV2 for 1 h except the control group. The cells were collected at 3, 12, 24, 48 and 72h after infection. The RNA was extracted from the five groups and then reverse-transcribed into cDNA. The mRNA levels of IFN-β, pattern recognition receptors (TLR3, TLR9, RIG-1, MDA-5 and DAI) and adaptor proteins (TRIF, MyD88, Sting, MAVS and IRF3) were detected by real time PCR.【Result】The mRNA levels of IFN-β in PCV2-infected groups were significantly higher than the control at 48h and 72h (P<0.01), indicating that IFN-β can be induced by PCV2 in PK-15 cells. The mRNA levels of TLR9 in PCV2-infected groups were higher than the control at 48h and 72h (P<0.01), the mRNA levels of TLR3 increased obviously at 48h (P<0.01), and the mRNA levels of TRIF and MyD88 which were adaptor proteins of TLR9 and TLR3 were also up-regulated at 48h (P<0.01), demonstrating that NF-κB signal pathway mediated by TLRs is activated by PCV2. The mRNA levels of MDA-5 and RIG-1 were significantly higher than the control at 72h (P<0.01), the expression levels of IRF3, MAVS, Sting which were adaptor proteins of RIG-1 and MDA-5 significantly increased at 48h (P<0.01), indicating that IRF3 signal pathway mediated by RLRs is activated by PCV2. The mRNA levels of DAI were higher at 48h and 72h (P<0.01), also indicating that DNA recognition receptor is involved in the production of IFN-β. BX795 and BAY 11-7082 inhibitors were used to inhibit the signal pathway mediated by IRF3 and NF-κB, the result showed that the mRNA levels of IFN-β in BX795-treated group was obviously lower than the control group (P<0.01), while no significant difference was found between BAY 11-7028-treated group and the control group, demonstrating that the up-regulation of IFN-β induced by PCV2 is mainly relative to the IRF3 signal pathway. 【Conclusion】The PCV2 could induce production of IFN-β in PK-15 cells and IRF3 signal pathway played an important role during this process.
    Epidemiological Survey and Molecular Phylogenetic Analysis of Chronic bee paralysis virus in China from 2014 to 2015
    JIA Hui-ru, WU Yan-yan, WANG Qiang, DAI Ping-li, ZHOU Ting
    Scientia Agricultura Sinica. 2016, 49(10):  2017-2026.  doi:10.3864/j.issn.0578-1752.2016.10.017
    Abstract ( 308 )   HTML ( 1 )   PDF (3116KB) ( 311 )   Save
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    【Objective】 The presence of several common honey bee viruses in colonies located in Beijing were investigated in the past years. The Chronic bee paralysis virus (CBPV) was found that its occurrence in diseased colonies was significantly higher than in healthy ones. CBPV was supposed to be investigated in diseased colonies. Since the data on CBPV are little, the objective of this study is to investigate the occurrence and variation of CBPV from China so as to provide a theoretical basis for the prevention of CBPV disease. 【Method】 A total of 136 samples from diseased colonies in Sichuan, Anhui, Zhejiang, Henan, Shandong, Shanxi, Heilongjiang and Beijing in 2014-2015 were collected and detected for CBPV by RT-PCR. Ten heads of bees were randomly collected from each sample. Total RNA was extracted using the TRIZOL method. RNA quality was qualitatively and quantitatively determined by electrophoresis and NanodropND-2000. The first chain of cDNA was synthesized using total RNA as template. The specific fragments of CBPV were amplified using cDNA as template. All amplified bands were performed by agarose gel electrophoresis. Parts of the positive PCR products were sequenced and analyzed. The occurrence of CBPV from each sample was confirmed by comparing the amplified bands with the positive ones. To analyze the phylogeny of Chinese CBPV isolates, RdRP genes of all CBPV positive samples were amplified using cDNA as template, then sequenced and analyzed. The gene sequences were submitted to GenBank for accession numbers. RdRP genes obtained from this study were analyzed and compared with those available on GenBank. All reference sequences and the sequences from this study were aligned using the software of CLUSTAL W. The phylogenetic tree of CBPV was conducted using the neighbor-joining method of MEGA 6.0. 【Result】 RNA integrity from honey bee heads of all samples was relatively preserved. The RNA yield was (980.5±37.1)ng·mL-1 and A260/A280 was 1.92-2.24. A total of 120 samples out of 136 were positive for CBPV and the positive rate was 88.2%. A target band was amplified from each positive sample. The nucleotide sequences of positive PCR products shared >99% sequence identity with target sequences. RdRP sequence analysis revealed that in total 8 CBPV isolates were obtained in this study, and the nucleotide sequences shared >97% sequence identity with those from other countries. Accession numbers for Chinese isolates of CBPV were KT374042-KT374049. There are 5 branches for the worldwide CBPV isolates including two European clades (European clade 1 and European clade 2), two Chinese-Japanese clades (Chinese-Japanese clade 1 and Chinese-Japanese clade 2), and one US clade. All 8 CBPV isolates in this study belongs to the same branch, and had been grouped with 4 Japanese isolates and 6 previous Jiangsu isolates into two mixed Chinese-Japanese clades. 【Conclusion】 CBPV is probably widely spreaded in diseased Apis mellifera colonies in China, and the genetic relationship of prevalent CBPV isolates in China exist no obvious geographical characteristics, seem to be closely related to Japan strains. Intensive CBPV surveillance should be urgently carried out, and put more attention to its function of early warning.
    Interaction of bHLH Transcription Factor Bmdimm and Bmchip in Bombyx mori
    ZHAO Peng, WANG Ye-jing, WEI Shu-guang, LIU Li-na, LI Zhen-zhen, ZHAO Ping, HE Hua-wei
    Scientia Agricultura Sinica. 2016, 49(10):  2027-2038.  doi:10.3864/j.issn.0578-1752.2016.10.018
    Abstract ( 346 )   HTML ( 2 )   PDF (3828KB) ( 630 )   Save
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    【Objective】 Bmdimm is a member of the bHLH transcription factors family, which is one of key transcription factors that regulate the expression of fibroin heavy chain gene in the late stage of the fifth instar of Bombyx mori. Bmdimm was predicted to have a potential ubiquitination site of lysine residue K43, and therefore it is presumed that Bmdimm may be modified by ubiquitin and thus degraded. Bmchip belongs to the U-box subfamily of the B. mori ubiquitin ligase E3 family, which is expressed in the posterior silk gland during the fifth instar of B. mori. Studying on the interaction of Bmdimm and Bmchip will promote us to better understand the ubiquitylation of Bmdimm in vivo and its biological significance on the transcriptional regulation of fibroin heavy chain gene. 【Method】 The expression profiles of Bmdimm and Bmchip in the posterior silk gland of B. mori were analyzed based on the B. mori genome microarray database. The CDS sequences of Bmdimm and Bmchip were obtained by PCR cloning and gene synthesis, respectively. Multiple expression vectors were constructed and then transformed into E. coli to express the target proteins to screen the best expression conditions. The fused Bmdimm and Bmchip were purified using Ni2+ affinity chromatography, and then digested with protease. The fused tag and target proteins were separated via Ni2+ affinity chromatography again. The oligo states of Bmdimm and Bmchip in solution were assessed by gel filtration chromatography. The secondary structures of Bmdimm and Bmchip were studied by circular dichroism spectroscopy. The interaction of Bmdimm and Bmchip in vitro were investigated using Far-western blotting and Pull-down. The affinity of Bmdimm and Bmchip were determined by microscale thermophoresis.【Result】 The B. mori genome microarray expression profiles indicate both Bmdimm and Bmchip were expressed in the posterior silk gland during the fifth instar of B. mori. Multiple expression vectors of Bmdimm and Bmchip were constructed, and it was found that ppSUMO-Bmdimm and pET-32M·3C-Bmchip, 0.3 mmol·L-1 IPTG and 16 were the best expression conditions. Then the recombinant Bmdimm and Bmchip was expressed and purified. Gel filtration analysis suggested both Bmdimm and Bmchip existed mainly as a dimer in solution. Circular dichroism spectroscopy showed both Bmdimm and Bmchip had α-helix structures. Far-western blotting and Pull-down assay suggested Bmdimm could interact with Bmchip in vitro. The dissociation constant KD of Bmdimm and Bmchip was calculated to be (750±28.6) μmol·L-1 using microscale thermophoresis.【Conclusion】 The bHLH transcription factor Bmdimm and B. mori ubiquitin ligase Bmchip are expressed in the posterior silk gland during the fifth instar of B. mori. Bmdimm and Bmchip have transient interaction in vitro, implying Bmdimm may be modified by ubiquitin via the interaction with Bmchip and thus down-regulated the transcription of fibroin heavy chain gene.