Scientia Agricultura Sinica

Subcellular Localization and Verticillium Wilt Resistance Analysis of Cotton GbRvd in Overexpressed Tobacco

YANG Zhan-wu, YANG Jun, ZHANG Yan, WU Jin-hua, LI Zhi-kun, WANG Xing-fen, WU Li-qiang, ZHANG Gui-yin, MA Zhi-ying

Scientia Agricultura Sinica. 2016, 49(21): 4065-4073. doi:10.3864/j.issn.0578-1752.2016.21.001

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【Objective】In order to resolve the mechanism of GbRvd-mediated resistance to Verticillium wilt in cotton, subcellular localization and resistance of GbRvd-overexpressed tobacco were studied.【Method】Expression vector pJCV52 was modified by inserting gfp gene into SpeⅠenzyme site and the resulted vector was named as GPJCV52 which can be used for subcellular localization. Gateway-based constructs, made for subcellular localization and overexpression of GbRvd, were transformed into Agrobacterium strain GV3101 for tobacco transformation. Subcellular localization of GbRvd was predicted and observed by bioinformatics analysis and Agrobacterium-mediated transient expression in tobacco leaf, respectively. Transformation of tobacco was made by Agrobacterium-mediated method, and final transgenic tobacco plants were obtained through tissue culture. PCR and semi-quantitative RT-PCR were used for screening positive transgenic plants and detecting gene expression levels, respectively. Verticillium dahliae conidial suspension was prepared and incubated with T3 generation of transgenic tobacco strains through the method of soil drench. The disease severity for plants was graded from 0 to 4 and then the disease index was calculated. 【Result】ProtComp, an online analysis software, predicted that GbRvd is an extracellular (secreted) protein. Transmembrane prediction showed that GbRvd contains three transmembrane domains. On-line analysis software WoLF PSORT prediction results showed that GbRvd localization in the cell membrane, endoplasmic reticulum and chloroplast scores were 7, 3 and 1, respectively, indicating that GbRvd mainly existed in the plant cell membrane. In order to further confirm the location of GbRvd in the cell, expression of GbRvd fused with GFP was examined. Fluorescence signal of control GFP protein in tobacco cells was observed in the nucleus, cytoplasm and cell membrane, and fluorescence signals of GbRvd fused with GFP were mainly detected in the plasma membrane and cytoplasm of tobacco epidermal cells. The transgenic tobacco plants were produced by Agrobacterium-mediated infection and tissue culture method. PCR detection results showed that the expected size of DNA band could be amplified in positive transgenic tobacco plants, and that could not appear in wild type plants. By semi-quantitative RT-PCR expected band could also be detected in positive transgenic tobacco plants, which indicates that GbRvd successfully integrated to the tobacco genome and can be normally transcribed. Three independent T3 transgenic tobacco lines were selected to analyze Verticillium wilt resistance. The results showed that the disease index of transgenic lines was significantly lower than that of wild-type, indicating that overexpression of GbRvd can effectively improve plant resistance to Verticillium wilt. 【Conclusion】GbRvd mainly exist in plant cell plasma and membrane, and its overexpression can significantly increase the resistance of tobacco to Verticillium wilt.

Screening for Cooking-Processing Potato Lines According to Potato Tuber Qualities and Properties

LIU Juan, LIANG Yan-chao, SUI Jing-hang, YU Bin, WANG Run-run, ZHANG Xiao-wei, CHENG Li-xiang, WANG Yu-ping, ZHANG Feng

Scientia Agricultura Sinica. 2016, 49(21): 4074-4084. doi:10.3864/j.issn.0578-1752.2016.21.002

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【Objective】 Cooking of potato is a main important processing method in China. The cooking quality of potato should be analyzed and classified according to the commercially processing criterion. Screening for cooking-processing potato lines according to potato tuber qualities and properties from varieties and advanced lines are efficient methods under current consumptions of potatoes. As the processing indexes are required for different production purposes, potato breeding schemes have to be improved and changed to suitable for different cooking-processing standards. 【Method】Selection at an early breeding stage, the characteristics with agronomic traits such as yield, tuber size, tuber shape, tuber colour, eye depth, diseases resistance and commodity rate were evaluated based on three different locations in three years. Fourteen potato varieties and lines were selected for cooking qualities test. Samples of potato tuber were cooked in boiling water steamer for 25 min. After cooled for 20 min and 7 days under room temperature, the tuber colour change, the hardness and softening of the tubers from before-cooking to after-cooking were determined and analyzed. The contents of dry matter, starch, total glycoalkaloid, sucrose, reducing sugars, free amino acids, chlorogenic acid, ascorbic acid, citric acid were measured. The effect of change of the above components on tubers umami, colour change and texture were analyzed. The essential cooking-processing indexes were defined. 【Result】Comprehensive analysis and assessment on agronomic characters of potato varieties and lines showed that the first-line for selecting cooking-processing potato is that the agronomic characters of potato should meet the commercial processing criteria. The content of reducing sugars should be below 0.4% because its effect on tuber sweet flavor. There were no significant difference between tuber umami and the content of free amino acids (30-70 µmol) in fourteen varieties and lines. The content of total glycoalkaloid should be below 150 mg·kg^-1, which negatively influences on tuber bitter flavor. The value of ΔE colour between the cooking end and seven day later after-cooking was the important colour index in potato cooking-processing. The tuber softening difference was another important index of potato cooking-processing. 【Conclusion】The present results indicated that the after-cooking tuber umami, colour change and texture are the most important cooking-processing indexes. The varieties Burbank, Shepody and lines 0724-58, 0712-66 are the better softening potato varieties and lines. The potato lines 0744-8 and 0722-26 are the better hard lines.

Genetic Analysis of Traits Related to Cold Resistance in Winter Rapeseed (Brassica rapa L.)

LIU Lin-bo, SUN Wan-cang, LIU Zi-gang, WU Jun-yan, FANG Yan, LI Xue-cai, ZENG Xiu-cun, YANG Gang, DONG yun, CHEN Qi, FANG Yuan, YUAN Jin-hai

Scientia Agricultura Sinica. 2016, 49(21): 4085-4095. doi:10.3864/j.issn.0578-1752.2016.21.003

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【Objective】The objective of this paper is to understand genetic way and to provide a theoretical foundation of cold resistance traits in winter rapeseed (Brassica rapa L.). 【Method】 In this study, six winter rapeseed cultivars with different cold resistances were used as parent materials to make complete diallel cross combinations with the Griffing methodⅠ. The parents and F1 generations were planted to obtain the data of cold resistance traits. By using the additive-dominant model and MINQUE(1) method, the genetic variance, genetic correlation, parent additive effects, combination dominant effects and heritability, and heterosis of over wintering rate and 11 traits in winter rapeseed were analyzed.【Result】Over wintering rate, POD activity, SOD activity, SS content, relative electrolytic leakage, chlorophyll content, transpiration rate and stomatal conductance were mainly controlled by additive effects, and dominant effects were much less important. The dominant effects prevailed in the traits of CAT activity, MDA content, SP content and net photosynthetic rate. Over wintering rate was detected to be significantly and positively correlated with CAT activity, POD activity, SOD activity, SP content, SS content, chlorophyll content, transpiration rate, stomatal conductance and net photosynthetic rate, but negatively correlated with MDA content and relative electrolytic leakage. Longyou 7 and Longyou 8 had positively additive effects in the traits of overwintering rate, CAT activity, POD activity, SOD activity, SP content, SS content, chlorophyll content, transpiration rate, stomatal conductance and net photosynthetic rate, which indicated Longyou 7 and Longyou 8 could be used as parents that improved cold resistance of generations. The results of dominant effects analysis showed that Longyou 7×GY and Longyou 7×Longyou 8 had stronger cold resistance. As for the narrow heritabilities, SOD activity, relative electrolytic leakage, transpiration rate and overwintering rate were 74.63%, 59.06%, 57.49% and 56.87%, respectively, and CAT activity was the lowest (2.83%). CAT activity, SP content and net photosynthetic rate had stronger population mean heterosis and population over-parent heterosis. 【Conclusion】Overwintering rate, SOD activity, relative electrolytic leakage and transpiration rate were used in selection at early generation. Among the 6 elite breeding lines, Longyou 7 was recommended for using as direct parental line due to its higher general combining ability in cold resistance traits.

Effects of Different Fertilization Levels on Soil Organic Matter and Dissolved Organic Matter in Two Paddy Soils After Multi-Years’ Rotation of Chinese Milk Vetch and Double-Cropping Rice

ZHOU Guo-peng, CAO Wei-dong, BAI Jin-shun, NIE jun, XU Chang-xu, ZENG Nao-hua, GAO Song-juan, WANG Yan-qiu, Shimizu Katsuyoshi

Scientia Agricultura Sinica. 2016, 49(21): 4096-4106. doi:10.3864/j.issn.0578-1752.2016.21.004

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【Objective】The objective of this study was to evaluate the integrative effects of Chinese milk vetch in corporation and its combination with different rates of fertilizer on soil fertility of paddy field in Southern China. 【Method】 Soil samples were collected from the 6-7 years’ experiments located in Hunan (purple alluvial soil, PA soil) and Jiangxi (yellow clayey soil, YC soil). Seven treatments were designed: winter fallow (CK), 100% fertilizer rate (100F), sole incorporation of milk vetch (MV), and MV combined with different rates of fertilizer (MV+40F, MV+60F, MV+80F, MV+100F). The soil organic matter (OM), dissolved organic matter (DOM) and their spectroscopy characteristics were investigated.【Result】The OM content, dissolved organic carbon (DOC) content and DOM’s fluorescence intensity were significantly higher in the PA soil than those in the YC soil, but the content of dissolved organic nitrogen (DON) and the UV-Vis absorption peaks of DOM appeared opposite trends. The OM content of MV treatment was higher than those in other treatments in both the two soils, except for MV+100F in the YC soil. Compared with 100F, MV+100F reduced the OM content in the PA soil, while increased it in the YC soil. The accumulation of OM tended to decrease with the increase of fertilizer application after the MV being incorporated. The DOC content was increased in MV+80F and MV+100F treated soil (except for MV+100F in YC soil) when compared to 100F, while the MV, MV+40F and MV+60F treatments showed different effects in the two soils. The MV treatment increased the DOC/SOC ratio in two types of soils, and the ratio was significantly lower than 100F’s in YC soil. The DOC/SOC ratio tended to increase with the increase of fertilizer application after the MV being incorporated (except for MV+100F YC soil), it showed that combination with more fertilizer would be more beneficial to activation of the soil carbon pool. The MV treatment had lower DON content than 100F (except for MV+100F YC soil), especially in the PA soil. The UV-Vis spectrometric analysis indicated that the UV-Vis absorption peak of DOM in the treatments of green manure and its combination with 40%-60% fertilizer was lower than that in 100F treatment, and the E250/E365 value decreased with the increase of fertilizer addition, it indicated that DOM had the higher molecular weight and more stable molecular structure with the increase of fertilizer application. The fluorescence spectrometric analysis indicated that the fulvic-like humic was the main component of DOM in the two types of soils. Higher fluorescence intensity of DOM than 100F was found in most treatments with MV except for MV+80F and MV+100F in the YC soil, while the humic index was reduced.【Conclusion】OM content was improved by the green manure incorporation, green manure combination with fertilizer could reduce MV’s contribution to OM. After the green manure incorporated, the activity of soil carbon pool and DOM’s humic index increased with the increase of fertilizer, and these could be the reasons why green manure combination with fertilizer reduced MV’s contribution to OM.

Spatio-Temporal Changes of Cropping Types in Northeast China During 1980-2010

LIU Zhen-huan, TANG Peng-qin, FAN Ling-ling, YANG Peng, WU Wen-bin

Scientia Agricultura Sinica. 2016, 49(21): 4107-4119. doi:10.3864/j.issn.0578-1752.2016.21.005

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【Objective】 The aim of this study is to investigate the spatio-temporal changes of cropping types in Northeast China since 1980. 【Method】 In this study, the SPAM-China model was used to simulate the spatial information of crop distribution by 10 km pixel. A new definition of cropping type was used in this study that is over 30% or rank in top three in pixel per crop. And spatial analysis method was used to analyze the cropping pattern and transfer matrix method was employed to detect the variation of cropping types. 【Result】 The results indicate that (1) priority choice of crop in this region has shifted from corn＞soybean＞wheat＞rice to corn＞soybean＞rice＞wheat since 2000. (2)In the past thirty years, 14 kinds of cropping types were found in Northeast. The proportion of six kinds of rice types increased from 8.30% to 18.64%, which was distributed in Liaohe Plain, Songnen Plain and Sanjiang Plain etc. Seven kinds of maize types accounted for over 60%, which was the most common crop in central and west of NEC. Five kinds of soybean types reduced by 4.2%, which has shifted from a dispersal pattern to a concentrated pattern and was located in Heilongjiang province. The proportion of seven kinds of wheat types dropped from 26.82% to 3.17% and it was the most significant change type in the structure of cropping system. Now, only a few pixels are distributed in Nenjiang, Heilongjiang province. (3) Three changes of cropping types were classified by crop transition, reclaimed land planted with new crops accounted for 20.91%, and the main planted crop is rice; single cropping type changed to two or more crops combination type, accounting for 34.90%, most of the crops are rice and soybean; the combination of cropping types changed to single cropping types accounted for 41.36%, large part from maize planting area. 【Conclusion】 Over the past 30 years, corn and soybean played a dominant role in the spatial variation of cropping types, and rice has become an important type of crop, meanwhile, the planting area of wheat was reduced in many regions and now is only confined in Nenjiang. There is also a phenomenon that soybean shift from south of Northeast to north. Cropping structure adjustment could be firstly started from reducing single corn type and increasing soybean and rice combination, which is a basic way to understand food security and food supply pattern. These findings will supply support for decision-making in agricultural structure adjustment and adaptation to climate change in Northeast China.

Remote Sensing Monitoring of Maize Lodging Disaster with Multi-Temporal HJ-1B CCD Image

Wang Li-zhi, Gu Xiao-he, Hu Sheng-wu, Yang Gui-jun, Wang Lei, Fan You-bo, Wang Yan-jie

Scientia Agricultura Sinica. 2016, 49(21): 4120-4129. doi:10.3864/j.issn.0578-1752.2016.21.006

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【Objective】The aim of this study is to analyze the variance of a variety of vegetation indices derived from HJ-1B (One of the Small Satellite Constellations for Environment and Disaster Monitoring and Forecasting, HJ-1A/1B/1C) CCD multi-temporal images in pre- and post-lodging, in order to achieve the objective for measuring the area of maize lodging and evaluation of disaster rank. 【Method】This paper took Gaocheng, Hebei Province as a research area where suffered a heavy rainstorm which led to a large-scale maize lodging on August 3rd, 2015. A variety of vegetation indices derived from HJ-1B CCD multi-temporal images in pre- and post-lodging period were adopted to conduct the sensitivity analysis among various vegetation index variations (?VIi, the value of vegetation index before lodging minus that of after lodging) and the lodging characteristic values measured in field (Lodging proportion for research areas) to seek the preferable vegetation indices for maize lodging monitoring. The double threshold partition strategy of normal statistical theory was adopted to determine the thresholds and a remote sensing monitoring model for maize lodging was built based on the analysis of the difference of vegetation indices pre- and post-lodging to evaluate the disaster degree of the maize lodging. The accurate assessment of the model was conducted by comparing the in-field measured data and predicted results from the built model at last. 【Result】Based on the features of HJ–1B CCD2 multispectral reflectance curve of the town, water, lodging and no lodging maize, there was a difference between maize and building and water in special reflectance. In the visible spectrum, spectral reflectance of lodging maize was higher than that of no lodging maize, therefore, it was opposite in near infrared wave. From the analysis of RVI difference image, it nearly conform to normal distribution (skewed normal distribution). The result of correlation analysis suggested that RVI (ratio vegetation index) had the best correlation with the proportion of maize lodging (R=0.9377). It means that the more serious of the lodging, the higher the RVI difference. It showed that the maize growth status was obviously affected by lodging, the more severe of the lodging, and it was more difficult for maize to restore to its normal state before lodging and thus causing bigger values of the RVI differences. Generally, RVI differences for different small areas showed a normal (or skew) distribution in general from the statistical analysis of the image of RVI difference. The data measured infield about lodging proportion and the values predicted from the model were adopted to carry out confusion matrix analysis, it showed that a good overall classification accuracy up to 85.7% while the Kappa coefficient was 0.804. The spatial distribution of maize lodging area and the degree of disaster could be mapped via the constructed maize lodging remote sensing model based on the differences of vegetable index derived from the double threshold partition strategy which based on normal (skew) statistical theory. The result from remote sensing mapping was basically consistent with the monitoring data from the local agricultural technical extension station. 【Conclusion】After occurrence of lodging of maize, there was a big difference between the maize growth and the resumption of maize after disaster. Therefore, it was concluded that the remote sensing monitoring model based on the differences of RVI measured in pre- and post-lodging period of maize is a feasible method for reflecting the information which indicate the changes of canopy groups described by different lodging degrees and can satisfy the need for measuring the area of maize lodging and evaluation of disaster rank.

Analysis and Expression of Laccase Gene Stlac2 in Setosphaeria turcica

MA Shuang-xin, LIU Ning, JIA Hui, DAI Dong-qing, XU Miao-miao, CAO Zhi-yan, DONG Jin-gao

Scientia Agricultura Sinica. 2016, 49(21): 4130-4139. doi:10.3864/j.issn.0578-1752.2016.21.007

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【Objective】The objective of this study is to analyze the bioinformatics and infer the functions of Stlac2 in Setosphaeria turcica, research on the effects of substrates generated in the process of lignin degradation on the relative expression of Stlac2. For further study on the function of Stlac2 in the growth, development, and pathogenicity, the Stlac2 was successfully expressed by prokaryotic expression system. 【Method】The protein sequences of Stlac2 were obtained through NCBI and aligned with the known laccases such as Penicillium marneffei PbrB (PMAA_082060), Aspergillus fumigatus Abr2 (AFUA_2G17530), and Aspergillus nidulans YA (AN6635) by ClustalX for the Cu ion binding sites. The secondary structure and biochemical properties were predicted online by online softwares SOPMA and ProtParam, and the three-dimensional structure was modelled by SWISS-MODEL, and analyzed by SAVES. The effects of lignin degradation substrates on the expression of extracellular laccase were measured by oxidazing ABTS and the effect on the relative expression of specific gene Stlac2 was analyzed by using RT-qPCR. Stlac2 was fused into the pET-30a plasmid and expressed by prokaryotic expression system. The protein Stlac2 expressed by prokaryotic cell was extracted and the laccase activity was detected using ABTS as substrate at 420 nm.【Result】 Stlac2 had typical sites bonding with Cu ion. The proportion of α-helix, extended strand, β-turn and random coil were 18.59%, 25.63%, 11.91% and 43.86%, respectively. The protein molecular weight is 61.64 kD, pI is 5.00, grand average of hydropathicity is -0.37, indicating Stlac2 is a hydrophilic protein. The effect of lignin degradation substrates on the production of extracellular laccase was vanillic acid＞vanillin＞4-hydroxybenzaidehyde＞4-hydroxybenzoic acid＞syringaldehyde＞CK. The relative expression of Stlac2 was increased about 5-8 folds compared with CK under the substrates 4-hydroxybenzoic acid and 4-hydroxybenzaidehyde conditions. Stlac2 protein was successfully expressed by prokaryotic expression system and a specific protein band of 67 kD was induced. The laccase activity of this specific protein is (40.7±0.3) U·L^-1.【Conclusion】The biochemical properties of Stlac2 were analyzed systematically and predicted that Stlac2 is a laccase gene. 4-Hydroxybenzoic acid and 4-hydroxybenzaidehyde could increase the relative expression of Stlac2 significantly, indicating it plays a role in the process of lignin degradation. Stlac2 protein can be expressed by the prokaryotic expression systems of pET-30a and the laccase activity is (40.7±0.3) U·L^-1, thus laid a foundation for further study.

The Heterogenous Expression and Enzymatic Characteristics of β-N-acetylglucosaminidase from Locusta migratoria

SONG Hui-fang, LI Ying-long, MA En-bo, ZHANG Jian-zhen

Scientia Agricultura Sinica. 2016, 49(21): 4140-4148. doi:10.3864/j.issn.0578-1752.2016.21.008

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【Objective】β-N-acetylglucosaminidase (NAG) is a key enzyme involved in degradation of chitin. The objective of this study is to obtain the high purified enzyme, and to analyze the enzymatic characteristics. It will be helpful for the biological function study of LmNAG1 during locust development, and will provide a theoretical and practical basis for developing molecular target of pest control. 【Method】The primers with BamH I, Hind III restriction sites and 6×His tags were designed according to the complete cDNA sequence deposited in GenBank (No. JX888720.1). The target sequence consisting of ORF was amplified by PCR, and then ligated to pFastBacTM-Dual vector after double enzyme digestion. The recombinant plasmid was transformed into E. coli DH10Bac competent cells, and the target gene was transposed to baculovirus genome through Tn7 transposon. The white clone was selected by blue-white selection combined with antibiotics screening and then the recombinant baculovirus plasmid (Bacmid) was verified by bacteria liquid PCR with the pUC/M13 primers. The recombinant Bacmid was transfected into Spodoptera frugiperda ovary cell line Sf9 using transfection reagent. The morphology of cell was observed within 72 h continuously. Once the infected phenomenon occurred, the P1 generation recombinant virions were obtained from the supernatant after cell collection and centrifugation, and then used to infect Sf9 cells again. The proteins obtained after lysing were performed western blot to examine whether the target protein was expressed or not. After that, large amounts of Sf9 cells were infected to extract proteins. The recombinant proteins were purified using Ni-NTA agarose beads and Q ion-exchange chromatography, and the protein concentration was determined according to the method of Bradford. The kinetic parameters of purified enzyme, the optimal pH and temperature were determined using the substrate, 4MU-GlcNAc. 【Result】 The recombinant plasmid pFastBac-LmNAG1 consisting of a full-length cDNA of LmNAG1 (1 845 bp) was verified by double enzyme digestion. Furthermore, it was transformed into DH10Bac competent cells, then combined into the baculovirus genome. The correct recombinant Bacmid selected by PCR was used for Sf9 cell transfection. The signs of transfection including cell enlargement and border irregularity were observed under the microscope after 72 h. The recombinant virions were collected after centrifugation, and used to infect Sf9 cells again. The infected Sf9 cells were collected for protein extraction. From western blot, an obvious band around 67 kD, which was in accordance with the molecular mass of LmNAG1. The fusion protein with 6×His tags was obtained successfully. Large amounts of protein were obtained from the infected cells and purified using Ni-NTA agarose beads followed by Q ion-exchange chromatography after dialysis to collect high purified target proteins. The top protein concentration of E3 fraction was 0.057 µg·µL^-1 determined using Bradford method. The in vitro activity detection showed that LmNAG1 exhibited the maximum activity at pH 8.0, and possessed higher stability when pH at 6.0-8.0. The optimum temperature for LmNAG1 was 40℃, the thermostability was high between 30-40℃. However, the enzyme activity was decreased rapidly when the temperature was higher than 45℃. The Km=(0.28±0.02) mmol·L^-1, Kcat= (902.88±38.15) s^-1, and the results showed that LmNAG1 could efficiently hydrolyze β-1,4 linked chitin oligosaccharide.【Conclusion】 The purified LmNAG1 was obtained in this study, and it has been shown that LmNAG1 is able to degrade β-1,4 linked chitin oligosaccharide. LmNAG1 is involved in chitin degradation, which exhibited the similar biological functions with NAG1 of other insects.

Seasonal Variations of Soil Critical Shear Stress in Typical Farmlands in the Hilly Region of Loess Plateau

YU Yao-chuang, WANG Chang-yan

Scientia Agricultura Sinica. 2016, 49(21): 4149-4159. doi:10.3864/j.issn.0578-1752.2016.21.009

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【Objective】The aim of this study is to explore the seasonal variations of soil critical shear stress under two typical farmlands in the hilly region of Loess Plateau in order to provide a reference for ensuring food security and establishing a soil erosion process model.【Method】Maize and millet lands in the hilly region of Loess Plateau were used as the study plots. The scouring experiments were carried out in a laboratory flume to simulate soil detachment by concentrated flow under six different shear stresses ranging from 5.71 to 17.18 Pa. The soil critical shear stress (τ_c) was estimated by the soil erosion process WEPP model. The seasonal variation of soil critical shear stress were discussed under maize and millet farmlands during the crop growing season.【Result】Results show that soil critical shear stress under typical farmlands in the hilly region of Loess Plateau displayed significantly seasonal variations (P＜0.05), which increased during the whole growing season. During the growing season, soil critical shear stress in maize land displayed a pattern of the seasonal variation following declined firstly, then increased, declined again, and rose. τ_c values varied from 1.51 Pa to 4.89 Pa, with a mean value of 3.0 Pa. The minimum and maximum values of soil critical shear stress for maize appeared at the five-leaf stage and at the harvesting stage, respectively. Soil critical shear stress in millet land displayed a pattern of seasonal variation following declined firstly, then increased. τc values varied from 1.06 Pa to 6.53 Pa, with a mean value of 2.93 Pa. The minimum and maximum values of soil critical shear stress in millet land appeared at the seedling stage and at the maturing stage, respectively. The seasonal variations of the soil critical shear stress of maize land and millet land were influenced by the seasonal change of soil cohesion and initial water content, and crop root growth. There were positive relationships between soil critical shear stress and soil cohesion, initial moisture content, content of water-stable aggregates, and crop root weight density. Seasonal variations of soil critical shear stress of maize and millet farmlands could be well simulated with soil cohesion, initial moisture content, and root weight density (R²＞0.74, NSE＞0.72). 【Conclusion】The growth of crop root system, and the seasonal change of soil cohesion and initial moisture content are the main factors influencing the seasonal variations of soil critical shear stress in typical farmlands in the hilly Loess Plateau. Soil critical shear stress of the two farmlands shows positive relationships with soil cohesion, initial water content and root weight density, which are important parameters to simulate the seasonal variations of soil critical shear stress in typical farmlands in the hilly region of Loess Plateau.

Contributions of Wheat and Corn Residues to Soil Organic Carbon Under Fluvo-Aquic Soil Area—Based on the Modified RothC Model

ZHAO Ya-wen, WANG Jin-zhou, WANG Shi-chao, WU Hong-liang, HUANG Shao-min, LU Chang-ai

Scientia Agricultura Sinica. 2016, 49(21): 4160-4168. doi:10.3864/j.issn.0578-1752.2016.21.010

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【Objective】 In order to explore the effect of straw retention on SOC（soil organic carbon）content, the contributions of wheat and corn residues (root and straw) to SOC under fluvo-aquic soil area were studied, which have a great significance to take technical measures to promote SOC content of winter wheat-summer corn rotation system.【Method】The optimized DPM/RPM values (the ratio of decomposable plant material to resistant plant material) of different residues in Roth C-26.3 model was adjusted on the basis of the remaining rates of different organic materials after their decomposition. The modified model was validated with the data obtained from the short-time decomposition experiment (2012.11-2013.11) and the long-term trial conducted in Zhengzhou (1990-2008). Based on the optimized DPM/RPM parameters of Roth C-26.3 model, the contributions of wheat and corn residues to SOC in winter wheat-summer corn rotation system in northern China under three different fertilizer treatments (no fertilizer CK, chemical fertilizer NPK, chemical fertilizer combined with straw NPKS) were simulated. 【Result】DPM/RPM values of wheat root (WR), wheat straw(WS), corn root (CR), corn straw (CS) were 0.89, 3.04, 4.35 and 3.25, respectively, when the model was in optimal condition. It showed that in CK treatment, the carbon input derived from wheat root and corn root were 50%, respectively, while the contributions of wheat root and corn root to newly-formed soil organic (0-20 cm) were 60% and 40%, the retention coefficients of wheat root and corn root were 15.5% and 10.8%, respectively; in NPK treatment the carbon input derived from wheat root and corn root were 60% and 40%, respectively, while the contributions of wheat root and corn root to newly-formed soil organic (0-20 cm) were 71% and 29%, the retention coefficients of wheat root and corn root were 17.5% and 11.4%, respectively; in NPKS treatment the carbon input derived from wheat root and corn root were 47%, 21% and 32%, respectively, while the contributions of wheat root and corn root to newly-formed soil organic (0-20 cm) were 50%, 22% and 28%, the retention coefficients of wheat root and corn root were 16.9%, 11.2% and 11.4%, respectively. In a word, the contribution of wheat residue (50% -71%) to newly-formed SOC was greater than corn residue (22%-40%) in winter wheat-summer corn rotation system in north China whether no fertilization, balanced fertilization or straw returned. The ratio of SOC derived from wheat to newly-formed SOC was greater than the proportion of the carbon input from wheat to total carbon input, instead of the carbon input of corn and its contribution to newly-formed SOC. The carbon efficiency of wheat root (15.5% -17.5%) was more than the carbon efficiency of corn root and corn straw (10.8% -11.4%).【Conclusion】The modified RothC model can be used to explore the contributions of wheat and corn residues to newly-formed SOC in fluvo-aquic soil area. The contribution of wheat root to SOC was greater than corn root in winter wheat-summer corn rotation system in the North China and the retention coefficient of corn root was greater than the corn straw in NPKS treatment, so the application of root residues (especially wheat roots) could promote the soil organic carbon stock.

Effect of Successive Fertilization on Dynamics of Basic Soil Productivity and Soil Nutrients in Double Cropping Paddy Soils with Different Fertilities

LU Yan-hong, LIAO Yu-lin, NIE Jun, ZHOU Xing, XIE Jian, YANG Zeng-ping

Scientia Agricultura Sinica. 2016, 49(21): 4169-4178. doi:10.3864/j.issn.0578-1752.2016.21.011

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【Objective】Effects of successive fertilization for 3 years on the dynamic change of basic soil productivity (BSP) yield, contribution rate of basic soil productivity, apparent balance of NPK and soil nutrients in paddy soils with different fertilities under double-cropping rice system were studied to provide reference for basic soil productivity cultivation and soil fertility maintaining and improving in different fertility soils.【Method】A pot experiment based on three different fertility soils collected from a long-term fertilizer experiment including no fertilizer (CK), NPK fertilizer (NPK) and NPK fertilizer plus rice straw (NPKS) was conducted. Two treatments were designed including no fertilizer (NF) and NPK application (CF) for three years for the above soils to monitor the impacts of these treatments on the changing characteristics of rice yield, BSP yield, BSP contribution rate of double-cropping rice, soil NPK nutrients and soil organic matter.【Result】Results showed that the orders both of BSP yield and BSP contribution rate in soils with different fertilities were NPKS treatment soil＞NPK treatment soil＞CK treatment soil during the period of the experiment, and the differences of BSP yield and BSP contribution rate among different fertility soils decreased with the experimental years. The differences of BSP yield and BSP contribution rate among soils with different fertilities were insignificant in the last experimental year. Whether fertilization or no fertilization, the higher fertility was of the initial soil, more nitrogen, phosphate and potassium nutrients were taken away from soil by rice plant in six seasons of three years rice planting. There were greater differences between different fertility soils in nutrient fertility change rule under continuous fertilization or no fertilization, which had a certain relationship with the nutrient input-output balance in rice planting system.【Conclusion】The higher the initial soil fertility, the faster the basic soil productivity declined under successive no fertilization. Therefore, reasonable fertilization in low fertility soil should be attached great importance in order to cultivate and improve soil fertility and basic soil productivity, and scientific and attention also should be paid to reasonable adding nutrients in high fertility soil to maintain relatively higher level of soil fertility and sustainable productivity.

Effect of Furrow Irrigation Pattern and Irrigation Amount on Comprehensive Quality and Yield of Greenhouse Tomato

LI Hong-zheng, CAO Hong-xia, GUO Li-jie, WU Xuan-yi

Scientia Agricultura Sinica. 2016, 49(21): 4179-4191. doi:10.3864/j.issn.0578-1752.2016.21.012

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【Objective】This study aims at exploring the effects of different furrow irrigation patterns and irrigation amounts on the comprehensive quality, yield, irrigation water use efficiency (IWUE) and comprehensive benefit of greenhouse tomato, optimizing the furrow irrigation pattern and irrigation amount of greenhouse tomato in northwest China, so as to provide a scientific basis for sustainable development of greenhouse tomato in the region. 【Method】Common furrow irrigation (CFI) and alternative furrow irrigation (AFI) were set as two furrow irrigation patterns. Based on the cumulative evaporation from a 20 cm diameter pan between two irrigations (E), 0.6E, 0.8E, 1.0E, 1.2E were set as four different levels of irrigation amount. The quality of tomato was evaluated comprehensively by principal component analysis (PCA) and grey correlation analysis (GCA). Principal components scores and grey weighted correlation degrees were regarded as the comprehensive quality of tomato. Weight values of comprehensive quality, yield and IWUE were calculated by the coefficient of variation method. Then, TOPSIS method was used to evaluate the comprehensive benefits of all irrigation treatments. 【Result】Tomato quality was affected by both furrow irrigation pattern and irrigation amount. The evaluation results of PCA and GCA were not completely consistent. However, both PCA and GCA acquired that tomato quality under AFI-0.6E and AFI-0.8E treatments were the best. Yield of AFI-0.6E treatment decreased by 22.56% while it just decreased by 9.42% under AFI-0.8E treatment compared to AFI-1.2E treatment which acquired the most yield. While, the IWUE of AFI-0.6E and AFI-0.8E had no significant difference. The result of TOPSIS showed that AFI-0.8E treatment had the largest comprehensive benefit. 【Conclusion】 AFI coupling with the irrigation amount of 0.8E could improve the quality of tomato and guarantee the yield and IWUE, consequently generate the largest comprehensive benefit. It was the optimal combination of furrow irrigation pattern and irrigation amount.

Expression Analysis of Differential Proteins in Three Kinds of Flower Buds with Sex Differentiation of Asparagus

LIU Meng, NIE Lan-chun, WANG Shan-shan, HU Shu-ming

Scientia Agricultura Sinica. 2016, 49(21): 4192-4202. doi:10.3864/j.issn.0578-1752.2016.21.013

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【Objective】 To investigate the proteins which are related to the sex differentiation, and lay a foundation for revealing the molecular mechanism of sex differentiation in Asparagus officinalis L. 【Method】 Differential proteins were analyzed in female flower buds, male flower buds and hermaphroditic flower buds at hermaphroditic differentiation stage by 2-D electrophoresis, mass spectrometry and bioinformatics method. 【Result】 Compared male and female flower buds, 25 specific protein spots and 5 up-regulated protein spots were detected in male flower buds, 13 specific protein spots and 12 up-regulated protein spots were detected in female flower buds. Compared with male flower buds, 19 specific protein spots and 8 up-regulated protein spots were detected in hermaphroditic flower buds. The proteins were identified by LC-MS-MS and analyzed through bioinformatics. Six specific or up-regulated homologous proteins of Asparagus were identified in male flower buds, including luminal binding protein (BiP) which promoted the synthesis of alpha amylase; beta-amylase, glyceraldehyde-3-phosphate and cytosolic phosphoglycerate kinase which are all involved in the glycolytic pathway, PAP fibrillin which is associated with liposomes,Os02g0634900 which its function is unknown. There were 16 specific or up-regulated homologous proteins of Asparagus were identified in hermaphroditic and female flower buds, including enolase 1 and cytosolic phospho-glycerate kinase which are involved in the glycolytic pathway, actin isoform B and actin which maintained cell structure, ATP synthase CF1 alpha subunit and ATP synthase beta subunit which participated in energy metabolism; small GTP-binding protein and GTP-binding protein which participated in material transportation, ribosome inactivating protein RIPm which depressed protein synthesis, ADP-ribosylation factor which participated in material transportation and signal transduction, nucleoside diphosphate kinase which catalyzed phosphate group transfer, plastid-lipid-associated protein which participated in lipid metabolism, oxygen-evolving enhancer protein 1 which was related to photosynthesis, porin which allowed ions, sugars and amino acids across the outer membrane, intracellular pathogenesis-related protein isoform 4 and hypothetical protein which their functions are unknown.【Conclusion】Beta-amylase, glyceraldehyde-3-phosphate dehydrogenase, cytosolic phosphoglycerate kinase, luminal binding protein (BiP), PAP fibrillin and Os02g0634900 which are homologous proteins of Asparagus are related to the male organ development. Enolase 1, cytosolic phosphoglycerate kinase, actin isoform B, actin, ATP synthase CF1 alpha subunit, small GTP-binding protein, GTP-binding protein, ribosome inactivating protein RIPm, with strong similarity to ADP-ribosylation factor, nucleoside diphosphate kinase, plastid-lipid-associated protein, oxygen-evolving enhancer protein 1, porin and hypothetical protein which are homologous proteins of Asparagus, ATP synthase beta subunit and intracellular pathogenesis-related protein isoform 4 are related to the female organ development. Oxygen-evolving enhancer protein 1 and porin which are homologous proteins of Asparagus might be the key proteins of female organ’s development.

Longan Pulp Extracts Ameliorate Scopolamine-Induced Learning and Memory of Impairment Mice

BAI Ya-juan, LIU Lei, ZHANG Rui-fen, DENG Yuan-yuan, HUANG Fei, ZHANG Ming-wei

Scientia Agricultura Sinica. 2016, 49(21): 4203-4213. doi:10.3864/j.issn.0578-1752.2016.21.014

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【Objective】The objective of this experiment is to study the effect of different doses of both ethanol extract and aqueous extract of longan pulp on learning and memory function in dementia mice induced by scopolamine and to clarify the mechanism of longan pulp in improving learning and memory of imparment mice.【Method】The contents of the main active substances in the ethanol extract and water extract of longan pulp were analyzed. SPF male Kunming mice were randomly divided into 6 groups for 28 d administration: normal control group, model group, low/high dose longan pulp ethanol extract group (150 mg?kg^-1/300 mg?kg^-1), low/high dose longan pulp aqueous extract group (150 mg?kg^-1/300 mg?kg^-1). At the end of last time administration, the passive avoidance tests were performed after mice received scopolamine intraperitoneal injection, the indexes of step-through latency and shuttle number were marked as acquisition trial, the passive avoidance tests were performed again after 24 h which was marked as retention trial. Then detection of the antioxidant activities of serum and brain, such as SOD, GSH-Px, MDA, as well as ChAT and AChE of brain tissue were carried out. 【Result】By analyzing the constituents of longan pulp extract, it was found that longan pulp aqueous extract contains mainly polysaccharide and protein, and ethanol extract contains not only carbohydrates, but also abundant phenolic acid, flavonoid and phospholipid substances. Moreover, the content of total phenols, total flavonoids and total phospholipid in ethanol extraction was significantly higher than that of aqueous extract. The shuttle number of model mice in acquisition trial of passive avoidance test in 5 min (2.89) was 6.09 times of normal control (0.46). Meanwhile shuttle numbers of the high dose longan pulp ethanol extract and aqueous extract groups were 0.75 and 0.56, respectively, the numbers had no significant difference with normal control. The step-through latency of model group in acquisition trial of passive avoidance test was decreased to 130 s (normal control group was 300 s, P＜0.05). Compared with model group, each dose of longan pulp ethanol extract and aqueous extract could significantly increase the dementia mice in the step-through latency of passive avoidance test (P＜0.05) with dose-dependent, especially the high dose longan pulp ethanol extract and aqueous extract group (289.18 s, 290.80 s, respectively,) basically reached normal level. Biochemical examinations revealed that each dose of longan pulp ethanol extract and aqueous extract could significantly increase the activity of ChAT in brain of dementia mice and significantly reduce AChE activity (P＜0.05) in a dose-dependent manner. At the same dose level, aqueous extract of longan pulp performed better than ethanol extract. ChAT activity recovered normal in high dose longan pulp ethanol extract group, low/high dose longan pulp aqueous extract groups. In the antioxidation, compared with the model group, longan pulp ethanol extract in high dose group was significantly increased SOD and GSH-Px activities and decreased MDA content significantly in serum and brain of dementia mice (P＜0.05), basically reached normal, the effect was better than the same dose of aqueous extract of longan pulp.【Conclusion】Both ethanol extract and aqueous extract of longan pulp have repairing effects on memory deficits and behavioral disorders produced by scopolamine. It was suggested that ethanol extract and aqueous extract of longan pulp may be bene?cial to the treatment of Alzheimer’s disease, but their action mechanisms might be different: to promote learning and memory, aqueous extract of longan pulp mainly regulates cholinergic nerve systemby polysaccharide or glycoprotein, and ethanol extract is mainly by means of providing antioxidation protection, phenolic acid, flavonoid and phospholipid are principal bioactive substances.

The Function Analysis of Over-Expression of Oar MITF-M in Melanocytes

YANG Yu-jing, ZHANG DAN-Jin, NIE Rui-qiang, XIE Jian-shan, FAN Rui-wen, GAO Wen-jun, DONG Chang-sheng

Scientia Agricultura Sinica. 2016, 49(21): 4214-4221. doi:10.3864/j.issn.0578-1752.2016.21.015

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【Objective】 Microphthalmia associated transcription factor M (MITF-M) plays an important role in the melanin synthesis in animal’s skin and hair. The aim of this paper was to clone microphthalmia-associated transcription factor M, to investigate whether the over-expression of oar MITF-M regulated TYR, TYRP-1 and TYRP-2 expression in melanocytes of sheep, and to explore its influence on the formation of melanin. 【Method】The CDS region in oar MITF-M gene was cloned from melanocytes of sheep by primers and PCR, to build a sheep MITF-M cloning vector and eukaryotic expression vector. Over-expression of oar MITF-M was conducted in melanocytes of 5^th generation sheep through the cell transfection technique and the transfer efficiency was observed by fluorescence microscope. The contents of melanin in melanocytes was detected by spectrophotometer, and the levels of MITF, TYR, TYRP-1 and TYRP-2 were detected using Real-time PCR and the proteins of MITF and TYR were detected using Western blot. 【Result】 Results showed that the 1 242 bp cDNA sequence of oar MITF-M gene was obtained by sequencing and splicing. Eukaryotic expression vector was successfully constructed with a startup report gene of green fluorescent protein and specificity TYRP-2 gene promoter. Under the fluorescence microscope, green fluorescence could be identified in melanocytes of sheep. The contents of melanin in melanocytes were increased by 1.15 times (P＜0.05). Real-time PCR results showed that MITF mRNA was significantly increased (P＜0.001) which indicated the oar MITF-M high transfection efficiency. TYR mRNA was significantly increased (P＜0.001). TYRP-1 mRNA was significantly increased by 5.06 times (P＜0.05). TYRP-2 mRNA was not significantly increased by 1.49 times. MITF protein in MITF-M group was 1.65 times more than in empty vector group (P＜0.001) and TYR protein in MITF-M group was 2.38 times more than in empty vector group (P＜0.001), which was consistent with the real-time PCR results. 【Conclusion】 The 1 242 bp length CDS region of oar MITF-M gene was got by PCR, TA cloning and nucleic acid sequencing technology. Results of the study suggested that oar MITF-M increased the production of TYR, TYRP-1 and had little influence on TYRP-2. Therefore, the oar MITF-M may mediate the alteration of coat color through regulating the expression of TYR and TYRP-1.

A Study on the Effect of Subclinical Hypocalcemia in Dairy Cows on Productivity and Manure Emission Characters

ZHANG Zi-yang, SHEN Tai-yu, YU Hong-jiang, GAO San-si, ZHU Kui-ling, HUANG Bao-yin, XU Chuang, YANG Wei

Scientia Agricultura Sinica. 2016, 49(21): 4222-4230. doi:10.3864/j.issn.0578-1752.2016.21.016

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【Objective】The objective of this paper is to study the feed intake, lactation, manure emissions of subclinical hypocalcaemia cows and sewage generated gas pollution emission characteristics. 【Method】 This study was carried out in a Heilongjiang intensive dairy farm. Twelve dairy cows which were similar in postnatal 7-14 days of age, body conditions and parity were selected and divided into subclinical hypocalcaemia group and healthy group, each group had 6 cows, each cow was individually housed, and they were continuously fed for 4 days. Blood samples were taken for measurement of blood Ca, BHBA, NEFA, CLU, P, Mg content indicators every day. Daily milk yield, feed intake, feces and urine of each cow were recorded. Simple dynamic chamber method was used to mix cow manure in the laboratory and NH₃, CO₂ and CH₄ emissions in the mixture were detected and analyzed. 【Result】Cows serum Ca, P and Mg contents in subclinical hypocalcemia symptom group were significantly lower than healthy group (P＜0.01), CLU was significantly lower than that of healthy group (P＜0.05) and BHBA concentration was significantly higher than that of healthy group (P＜0.05) and NEFA concentration was significantly higher than that of healthy cows in healthy group (P＜0.01); milk yield in subclinical hypocalcaemia group was significantly higher than that in healthy group (P＜0.01), 4% energy corrected milk (ECM) was significantly higher than that of healthy cows (P＜0.01) in healthy group, fecal excretion was significantly higher than that of healthy group (P＜0.05); dry matter digestibility and urine volume had no significant difference but a rising trend. Milk yield of cows which were fed 1 kg dry matter was increased significantly (P＜0.01), feces of cows which were fed 1 kg dry matter amount were significantly higher (P＜0.05) in subclinical hypocalcemia calcium group. CH₄ emission curve in healthy group and subclinical hypocalcemia symptom group cows had no significant difference, two groups of gas production trend was basically the same, peak of gas production was on the 52 h, then falling. CO₂ emissions had no significant changes in the trend, no obvious rules, emission peak was appeared in the healthy group and subclinical hypocalcemia symptoms group, respectively, at 36 h and 48 h, the peak in subclinical hypocalcaemia group was appeared earlier than healthy group, and then decreased immediately without regularity and floated up and down. Subclinical hypocalcaemia group of CO₂ cumulative emissions with the passage of time was lower than that of the healthy group; The peak of cows NH₃ emission concentration in healthy group was at 24 hours, then decreased, peaked again at 45 h, emission concentration decreased gradually. The peak of cows NH₃ emission concentration in subclinical hypocalcemia group was appeared at 21 h, then the emission concentration decreased, two groups the test line trend of the two groups were basically the same, all trend were that the peak concentration decreased and floated up and down. The cumulative emissions of NH₃ in subclinical hypocalcemia group were lower than healthy group. 【Conclusion】 During the subclinical hypocalcemia in dairy cows with syndrome of disease due to the feed intake of nutrients can not satisfy the lactation demand in the state of energy and calcium negative balance. At the same time digestive tract absorption rate increased to satisfy the demand of energy lactation; under the same quality of mixed excrements of detection of subclinical hypocalcaemia would not impact on CH₄ emissions, but NH₃ and carbon CO₂ emissions in subclinical hypocalcemia cows manure were lower than that of healthy cows. However, whether reducing greenhouse gas emissions caused the increase of intestinal absorption rate promoted the feed energy uptake still needs to be further studied.

Differences of Methyl Donor S-adenosylmethionine Synthesis and Metabolism Between Apis mellifera ligustica Queen Bee Larvae and Worker Bee Larvae

WANG Li, WANG Hong-fang, XU Bao-hua

Scientia Agricultura Sinica. 2016, 49(21): 4231-4238. doi:10.3864/j.issn.0578-1752.2016.21.017

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【Objective】 The objective of this study is to research the differences of synthesis and metabolism of the methyl donor S-adenosylmethionine (SAM) between Apis mellifera queen bee and worker bee larvae, so as to provide a theoretical basis for the study of DNA methylation and caste differentiation. 【Method】 A total of 890 1-day-old female bee larvae selected from 5 colonies of sister queen group, respectively. Among the 890 larvae, 445 larvae were bred to the king (89 in each colony) and the other 445 larvae were put in the original group so that they can develop toward the worker caste naturally (89 in each colony). The 3-, 4- and 5-day-old queen bee and worker bee larvae were taken to measure the differences of gene expression and enzyme activities of the key enzymes of SAM biosynthesis and metabolism in vivo.【Result】The content of SAM in queen bee larvae didn’t change significantly with the increased days of age (P＞0.05). The SAM content of worker bee larvae increased with the increase of days (P＜0.05). The gene expression of SAMS in queen bee larvae showed a gradient descent trend with increasing age (P＜0.01); but the gene expression of SAMS in worker bee larvae did not change significantly with the increase of days of age (P＞0.05). The gene expression of SAMS in queen bee larva was higher than worker bee larvae at 3- and 4-day-old obviously (P＜0.05), but the gene expression of SAMS in worker bee larva was higher than queen bee larvae obviously at 5-day-old (P＜0.05). The gene expression of Dnmt1a and Dnmt3 was not different significantly between the two types (P＞0.05). The gene expression of Dnmt1a in queen bee larvae showed no significant change with the increase of days of age (P＞0.05), but the enzyme activity of Dnmt1 in queen bee larvae decreased with the increase of days of age (P＜0.05). The gene expression of Dnmt1a was decreased with the increase of days of age in worker bee larvae (P＞0.05), but the enzyme activity of Dnmt1 showed a rising trend (P＜0.01). The enzyme activity of Dnmt1 in queen bee larvae was higher than the worker bee larvae significantly at 3-, 4-day-old (P＜0.05), but the enzyme activity of Dnmt1 in worker bee larvae was higher than the queen bee larvae significantly at 5-day-old (P＜0.05). The gene expression of Dnmt3 in queen bee larvae showed a decreasing trend with the increased days of age (P＜0.05), the gene expression of Dnmt3 in worker bee larvae showed no significant change with the increased days of age (P＞0.05). The enzyme activity of Dnmt3 changed in queen bee larva was not significant with the increased days of age (P＞0.05), the enzyme activity of Dnmt3 changed significantly in worker bee larvae with the increased days of age (P＜0.05), but the enzyme activity of Dnmt3 in queen bee larvae were higher significantly than that of worker bee larvae at 3-, 4- and 5-day-old (P＜0.01). 【Conclusion】There were some differences of the methyl donor (SAM) synthesis and metabolism between queen bee larvae and worker bee larvae at 3-, 4- and 5-day-old. Synthesis of SAM in queen bee larvae was more active than the worker bee larvae before 4-day-old and close to worker bee larvae after 4-day-old. The metabolism process of SAM participating in DNA maintenance methylation in queen bee larvae were more active than worker bee larvae before 4-day-old, but lower than worker bee larvae after 4-day-old. The metabolism process of SAM participating in de novo methylation of DNA in queen bee larva was not lower than worker bee larvae from 3- to 5-day-old.

Establishment and Application of Nested-PCR Method for Detection of Paranosema locustae

HU Hong-xia, MA Yu-xuan, WANG Yan-hong, LIN Jun, LI Zhan-wu, JI Rong

Scientia Agricultura Sinica. 2016, 49(21): 4239-4245. doi:10.3864/j.issn.0578-1752.2016.21.018

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【Objective】The objective of this study is to develop a rapid, sensitive and specific nested-PCR method for detection of Paranosema locustae and provide effective means for the monitoring of locusts and biological control. 【Method】 According to the sequence of small subunit ribosomal RNA genes of microsporidian published in GenBank, two pairs of primers were designed and synthesized (P.L.-F1/P.L.-R1 and P.L.-F2/P.L.-R2), and a series of assays (involving concentrations of primer pairs, Tm value and the number of cycling) were conducted to optimize the nested-PCR. P. locustae treated with 0.2 mol·L^-1 KOH were used as template for nested-PCR. P.L.-F1 and P.L.-R1 were employed as the initial primers for the first amplification, which generated a 298 bp product, and P.L.-F2 and P.L.-R2 as the secondary primers for the final amplification, which generated a 242 bp fragment. Analytical sensitivity and reproducibility were assessed, respectively. The nested-PCR method was used to detect a series of dilution of P. locustae suspension, and to test the P. locustae suspension derived from Barkol Bei mountain in Hami region. At the same time, the P. locustae suspension was extracted and detected with the traditional microscope, and then the sensitivity of two methods was compared. 【Result】The nested PCR was established. The optimal parameters for the nested PCR reaction were performed in standard mixtures of 20 μL containing 2.0 μL of Buffer (10×), 0.3 μL of dNTPs (10 mmol·L^-1), 0.3 μL of Taq DNA polymerase (250 U), 0.3 μL of each primer (10 μmol·L^-1), 1 μL of DNA template (or the diluent of the first-round product was then diluted 10-fold with water) and 15.8 μL of double distilled water. Each PCR run was carried out in a thermal cycler. The conditions of the amplification were as follows: 1 cycle at 94℃ for 3 min, 35 cycles at 94℃ for 30 s, 60℃ (62℃ at secondary PCR) for 30 s, and 72℃ for 20 s and then 1 cycle at 72℃ for 10 min, with a final hold at 10℃. The result of two rounds PCR showed that the nested PCR assay could detect 12.2 spores/µL (1.07 pg DNA). A total of 240 locust samples from Bei mountain in Hami region were analyzed by nested PCR and microscope testing, respectively, the results showed that the positive detection rate was 23.3% and 3.8%, respectively. 【Conclusion】 The nested PCR mothed established in this study is more sensitivity than traditional microscope detection method. As well as, this assay can avoid human error, and more suitable for rapid and large-scale infection intensity survey.

Effect of Canopy Types on the Cluster Micro-Environment and Fruit Quality of the ‘Moldova’ Grapes

LIU Xiao-hong, SUN Yong-jiang, SUN Hong, ZHAI Heng

Scientia Agricultura Sinica. 2016, 49(21): 4246-4254. doi:10.3864/j.issn.0578-1752.2016.21.019

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【Objective】The purpose of this study is to investigate the effects of vertical canopy and horizontal canopy on the cluster micro-environment and fruit quality of ‘Moldova’, and to provide a reference for the establishment of a reasonable type of grape cultivation.【Method】In order to compare the two canopy types, parts of vertical trellis systems (vertical canopy) were changed to pergola (horizontal canopy) under the condition of the same of other cultivation and management techniques in 2013. The temperature, relative humidity around the clusters of the two canopy types were recorded by temperature and humidity recorder. The photosynthetic active radiation above and under the horizontal canopy and both sides of the vertical canopy were also recorded by the photosynthetic active radiation monitor from June 1st. The grapes quality was measured every 2 weeks from 9 weeks after blossom to maturity stage.【Result】The results showed that compared with the vertical canopy, horizontal canopy decreased the temperature around the cluster and the ratio of temperatures above 35℃ significantly. In July, the ratio of temperatures above 35℃ around the horizontal canopy cluster was decreased by 68.97% compared with that of vertical canopy. In August, there no temperature above 35℃ occurred around the horizontal canopy cluster, however, the highest temperature around the vertical canopy cluster reached 41.6℃. At the same time, the horizontal canopy increased the humidity of the cluster microenvironment, reduced the humidity fluctuations, and the photosynthetic active radiation above it in 10:00 decreased by 25.8% and in 14:00 decreased by 5.95% compared with the eastern and western sides of the vertical canopy, respectively, only in 12:00, it showed a higher level than vertical canopy, but the photosynthetic active radiation under horizontal canopy was the lowest all the day. In terms of fruit quality, the horizontal canopy which increased the fruit secondary growth and expanding rate resulted the increase of vertical diameter, cross diameter, fruit shape index and berry weight, especially increased the contents of sugar, titratable acid, total phenols and skin anthocyanin in mature fruits by 2.40%, 4.35%, 5.55% and 7.04%, respectively, compared with that of the vertical canopy in 2015. Besides, the results showed that the horizontal canopy increased the skin lightness significantly, deepened the red and blue chroma which improved the skin color.【Conclusion】Compared with vertical canopy, horizontal canopy can improve the cluster micro-environment and fruit quality.

Plasma Metabolic Profiling Analysis of Dairy Cows with Laminitis Based on GC-MS

LI Ya-juan, WANG Dong-sheng, ZHANG Shi-dong, YAN Zuo-ting, YANG Zhi-qiang, DU Yu-lan, DONG Shu-wei, HE Bao-xiang

Scientia Agricultura Sinica. 2016, 49(21): 4255-4264. doi:10.3864/j.issn.0578-1752.2016.21.020

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【Objective】In order to discover the difference between the cows suffered with laminitis and healthy cows, metabonomics analysis based on gas chromatograph tandem mass spectrometer technology (GC-MS) was utilized in the study.【Method】Twenty plasma samples were collected from cows with laminitis and healthy groups. Plasma metabolic profiling was obtained by GC-MS after derivatization. Multivariate statistics including principal component analysis (PCA) and supervised partial least squares-discriminant analysis (PLS-DA) were conducted to screen the changed metabolites. Bioinformatics was analyzed to identify the significantly changed metabolic pathways.【Result】A total of 242 variables were detected and 37 compounds were significantly different between healthy and laminitis-suffered cows of which 3 were up-regulated including aminooxyacetic acid, lactose and oleic acid and 34 were down-regulated in laminitis group involved amino acid and fatty acid. The main changed metabolic pathways were fatty acid biosynthesis, Gly, Ser, Thr metabolism, biosynthesis of unsaturated fatty acids, purine metabolism, methane metabolism, cyanoamino acid metabolism and phenylalanine metabolism. Results of the the experiment of validation showed that the indexes related to fatty acid metabolism and antioxidation were significantly different between healthy cows and sick cows. 【Conclusion】 Based on GC-MS, this study revealed the changed regularity of metabolism in plasma with laminitis, and the discovered different metabolites would be potential biomarkers in occurrence and development process of laminitis, which can lay a theoretical foundation for further understanding of the pathogenesis, early diagnosis and therapy of laminitis in dairy cows.

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