Scientia Agricultura Sinica

Genome-Wide Identification of Glyco-hydro-16 Family in Maize and Differentiation Analysis

LIN Feng, GE Min, ZHOU Ling, ZHAO Han

Scientia Agricultura Sinica. 2016, 49(11): 2039-2048. doi:10.3864/j.issn.0578-1752.2016.11.001

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【Objective】Genome-wide identification was carried out for Glycoside hydrolase family 16 in maize, and their expression profile across tissues and differentiation between heterotic groups were analyzed. 【Method】Based on the maize V3 sequences, genome-wide survey of Glycoside hydrolase family 16 was conducted according to conserved sequences and domains through hmmersearch program. Blast2GO was used for gene annotation and phylogenetic relationships were analyzed through protein sequences. Expression profiles were examined within the whole transcriptome context at different tissues across development stages in B73. In line with the chromosome locations of the family genes, the authors screened the SNP markers and analyzed their genetic differentiation in different heterotic groups. 【Result】Totally 34 genes were identified in maize throughout genome-wide survey and annotated as xyloglucan endotransglucosylase with 3 conserved motifs discovered in all members. According to the phylogenetic relationships and sequence similarity they were divided into 8 subgroups. Glycoside hydrolase family 16 members were conserved in Gramineae, however, most maize members were not closely related with other Gramineae plants except only 3 members (AC210669.3, GRMZM2G413006, and GRMZM2G166944). The family genes in maize distributed on almost all chromosomes except on chromosomes 3 and 6 with several genes clustered on chromosomes 2, 5 and 10. Different expression profiles across tissues indicated their diversity functions. According to their chromosome location, the authors screened the SNP markers and calculated the genetic differentiation coefficient between the heterotic groups SS and NSS. Ten genes in Glycoside hydrolase family 16 differentiated significantly between the two heterotic groups, distributing on chromosomes 1, 2, 4, 5, 7 and 9. The one on chromosome 2 corresponding to GRMZM2G091118 got the highest Fst 0.52, indicating possible role contribute to heterosis. 【Conclusion】Based on the V3 sequences of maize, 34 genes were identified for Glycoside hydrolase family 16 and annotated as xyloglucan endotransglycosylase. Their expression profiles were different across tissues implying diversity functions. Some family members differentiated significantly between heterotic groups SS and NSS.

Analysis of Genetic Diversity and Structure of Tartary Buckwheat Resources from Production Regions

QU Yang, ZHOU Yu, WANG Zhao, WANG Peng-ke, GAO Jin-feng, GAO Xiao-li, FENG Bai-li

Scientia Agricultura Sinica. 2016, 49(11): 2049-2062. doi:10.3864/j.issn.0578-1752.2016.11.002

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【Objective】 The objective of this study was to understand the genetic diversity and population structure of tartary buckwheat resources from different production regions and to provide theoretical foundation of functional gene and resources use. 【Method】 Eight phenotypic traits were evaluated by correlation and principal component analyses. Plants were cultivated in the greenhouse, and 10 fresh leaves were selected in the three leaf stage. DNA of tartary buckwheat was extracted using the CTAB method, and then they were amplified based on the SSR marker, detected by the electrophoresis, and took photos and saved. 0, 1 matrix was structured based on the detection of SSR, and eighty-three materials were used to analyze genetic diversity and genetic structure by PowerMarker3.25 and Structure2.3.4 software. 【Result】 Distribution of the eight phenotypic traits were scattered with most apparently interrelated. The cumulative contribution rate of four principal components formerly reached up to 85.2%, which may show the relative relationships of the plant traits. The variation coefficient and genetic diversity of plant height and main stem diameter contributed the most to this metrics between all plant traits. Among the distinct geographical regions, the mean H′ (1.82) of Tibet population was the richest, and in Sichuan population was the second with a mean H′ of 1.78, just slightly lower. Genetic diversity of plant traits from the distinct regions showed that the H′ of growth stage, plant height, and main stem branch number in Sichuan population were the highest, while the H′ of leaf width was the richest trait in the Shaanxi population, andof 1000-seeds weight H′ in Yunnan population was the richest trait for plants of that region. Principal component analysis of 7 different geographical populations showed plant traits of similar production regions may have a close relation. Genetic diversity of 83 individuals of tartary buckwheat germplasm resources was detected by 13 pairs of SSR primes. A total of 208 loci were detected, among which 200 (96.2%) were polymorphous. The number of amplified fragments and polymorphous fragments per primer combination were 16 and 15.4; the number of alleles varied from 4 to 58, and the frequencies of major alleles varied from 0.02 to 0.86; gene diversity was between 0.38 and 0.98, and polymorphic information index was 0.35-0.98. Genetic diversity of different production regions showed that genetic structure of the northerly populations showed close relationship, and genetic structure of southwesterly population was also so, which showed the relationship between population cluster and production regions. The number of alleles (12.1), genetic diversity index (0.84), and polymorphic information content (0.83) in Shaanxi were the highest among the regions. All resources were divided into three clusters based on modules by genetic structure analysis, and cluster analysis based on genetic distance showed dispersive resources and no regionalization. Genetic distances were close from plant materials of the same production regions among different resources. 【Conclusion】 Polymorphic information contents of tartary buckwheat from main production regions were high, and the genetic diversity among them was rich. Resource interflow and genetic material exchange were observed in northerly and southwesterly production regions.

Al-Induced Hydroxamates Cyclic Secretion from Roots to Resist Al Toxicity in Maize

GAO Xiao-feng, GUO Tian-xiang, TANG Xin-lian, LI Xiao-feng

Scientia Agricultura Sinica. 2016, 49(11): 2063-2071. doi:10.3864/j.issn.0578-1752.2016.11.003

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【Objective】Al-induced secretion of Hydroxamates Cyclic from roots was investigated and we provided a theoretical basis for revealing the mechanism responsible for Al-resistance in Zea Mays L..【Method】The experiment was conduced using a Zhengdan 958 variety and a Taiyu No.11 variety with Al treatment by hydroponics. The Al-resistance and the effect of Al on the secretion of Hydroxamates Cyclic from roots and Hydroxamates Cyclic ameliorated Al toxicity were studied by measuring the root elongation, Al content, callose content, Hydroxamates Cyclic secretion, and relevant indicators of Hydroxamates Cyclic-ameliorated Al toxicity among these two varieties.【Result】With comparison to the control treatment (without AlCl₃), root elongation was inhibited significantly, while Al and callose content of root tips increased significantly in Zhengdan with 10 μmol·L^-1AlCl₃ treatment after 24 h. When root elongation was reduced by 47.77%, Al and callose content of root tips increased by 226.64% and 60.74%. On the contrary, there were no significant effects on the root elongation and callose content of root tips in Taiyu. Although Citrate secretion increased significantly by 41.28% from roots in Taiyu with 20 μmol·L^-1AlCl₃ treatment after 44 h, Citrate secretion did not increase significantly from roots in Taiyu and Zhengdan with AlCl₃ treatment during 24 h with comparison to control treatment (without AlCl₃), meaning Al cannot induce Citrate secretion from roots in maize during 24 h. Al-induced Hydroxamates Cyclic (DIMBOA and MBOA) secretion from roots of Taiyu, the amount of Hydroxamates Cyclic secretion increased significantly with the increasing AlCl₃ concentration (10, 15 and 20 μmol·L^-1) and the duration of AlCl₃ treatment (3, 6, 9, 12 and 24 h), but Hydroxamates Cyclic secretion did not increased significantly from roots in Zhengdan with AlCl₃(10, 15 and 20 μmol·L^-1). And dark condition can significantly improve the secretion of Hydroxamates Cyclic in Taiyu and effectively reduce Al-induced (20 μmol·L^-1AlCl₃) inhibition of root elongation compared to the light condition. DIMBOA and MBOA secretions increased by 40.81% and 72.02%, and root elongation increased by 33.15%. Compared to the sole Al (20 μmol·L^-1AlCl₃) treatment, addition of DIMBOA (3 mg·L^-1) and MBOA (3 mg·L^-1) to Al solution can significantly increase root elongation, reducing Al content of root tips in maize. With the addition of DIMBOA and MBOA to Al solution, root elongation of Zhengdan increased by 14.16% and 13.85%, and Taiyu increased by 16.16% and 11.33%; Al content of root tips of Zhengdan was decreased by 39.94% and 43.54%, Taiyu decreased by 39.92% and 42.10%. On the other hand, 20 μmol·L^-1AlCl₃ treatment increased significantly Hydroxamates Cyclic content of roots and leaves in Taiyu. However, Al treatment decreased significantly Hydroxamates Cyclic content of the leaves in Zhengdan. 【Conclusion】These results suggested that Al-resistance bears on Hydroxamates Cyclic secretion. Al-induced secretion of Hydroxamates Cyclic from roots may be an effective mechanism for maize under Al-stress, Hydroxamates Cyclic content of the leaves may be involved in the secretion in the maize.

Effect of Shading on Soybean Leaf Photosynthesis and Chlorophyll Fluorescence Characteristics at Different Growth Stages

WANG Yi, ZHANG Xia, YANG Wen-yu, SUN Xin, SU Ben-ying, CUI Liang

Scientia Agricultura Sinica. 2016, 49(11): 2072-2081. doi:10.3864/j.issn.0578-1752.2016.11.004

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【Objective】The present work studied the effect of shading on photosynthesis and chlorophyll fluorescence characteristics, including leaf area, lamina mass per unit area, chlorophyll content, yield characters, photosynthetic and fluorescence parameters of three soybean cultivars at different growth stages, in order to support the development of maize-soybean intercropping and relay-intercropping systems in hilly and mountainous area of southern China.【Method】Three cultivars named Guixia 2, Nandou 12 and C103, whose period and total days for growth were different with each other, were studied. Soybean plants were shaded by shading nets with 50% transmittance during growth stages of emergence to full-bloom (VER2), emergence to pod-fill (VER5), and full-bloom to full-ripe (R2R8), respectively. Plants grown under natural light were used as control (CK). Leaf area, lamina mass per unit area, chlorophyll content, photosynthetic and fluorescence parameters, as well as yield characters were measured and analyzed. 【Result】 Compared to the control (CK), leaf area, chlorophyll (a+b) content and apparent quanta efficiency at the VER2 stage increased by 15.5%, 13.0% and 74.1%, respectively; lamina mass per unit area, light compensation point and maximum photosynthetic rate were decreased by 15.8%, 26.2% and 26.5%, respectively, compared to CK. Leaf area, chlorophyll (a+b) content and apparent quanta efficiency increased by 0.3%, 10.5% and 28.1%, respectively at the R2R8 stage; while lamina mass per unit area, light compensation point and maximum photosynthetic rate decreased by 10.2%, 20.3% and 12.1%, respectively, compared to CK. When soybean plants were re-illuminated at full bloom stage, maximal quantum yield of PSII was decreased by 3.0% and non-photochemical quenching was increased by 26.8%, compared to CK. When plants were re-illuminated at pod-fill stage, maximal quantum yield of PSII was decreased by 8.5% and non-photochemical quenching was increased by 40.8%, compared to CK. In addition, at VER2, VER5 and R2R8 stages for Guixia 2, yields per plant decreased by 40.8%, 48.7% and 59.2%, and 100-seed weight decreased by 23.7%, 39.3% and 26.4%, respectively, compared to CK. For Nandou 12 at these stages, yields per plant decreased by 46.7%, 54.2% and 21.2%, and 100-seed weight were decreased by 3.9%, 19.9% and 26.1%, respectively, compared to CK. For C103, yields per plant decreased by 69.8%, 74.9% and 73.9%, and 100-seed weight decreased compared to CK by 68.8%, 69.6% and 71.6%, respectively.【Conclusion】Shading to the VER2 stage had a greater impact than for R2R8 on the photosynthesis and chlorophyll fluorescence characteristics of soybean. Photosynthetic capacity of soybean leaves made some recovery from shading when re-illuminated at R2. However, high-light stress was observed for plants re-illuminated at R5. Therefore, we suggest that maize-soybean intercropping to be a more advantageous system than relay-intercropping in hilly and mountainous area of southern China, with the perspective of minimizing the negative effects of shading on soybean photosynthesis. We also suggest, it is necessary to re-illuminate the soybean plants before full-bloom stage, in order to avoid an excessively long shading duration, when making decisions about on cultivar use, sowing time, and planting technology. The effect of shading period can influence yield of soybean. Moreover, the longer the shading duration last, the more the expected yield loss.

A Correcting Algorithm of Crop Productive Potentiality Based on the Terrain Factors in National Scale

CHEN Yan-qing, YANG Jian-yu, YUN Wen-ju, DU Meng, DU Zhen-bo

Scientia Agricultura Sinica. 2016, 49(11): 2082-2092. doi:10.3864/j.issn.0578-1752.2016.11.005

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【Objective】 Light temperature/climate productive potentiality as one of the important index for farmland classification, which directly affects the accuracy of the classification results. In theory, light and temperature conditions should vary in different terrain regions, but existing productive potentiality value that one county, one crop just owns one value can’t accurately reflect the differences of productive potentiality when the terrain differences apparent in the county, which leads to the classification results can’t accurately describe the differences of the cultivated land quality. The objective of this study is to solve this problem.【Method】 Based on terrain had serious relationship with the light, temperature and precipitation which were closely related to productive potentiality, this paper proposes to find the relationship between terrain factor and productive potentiality using the relationship to correct the value of productive potentiality. As productive potentiality was calculated based on a national scale data, in order to ensure the comparability of revised productive potentiality value, this paper carried out correction in national scale and used 900 m × 900 m DEM data as data source of calculating terrain factors. Firstly, by SPSS software, regression analysis was done between altitude, gradient, aspect and productive potentiality respectively, then the highest correlation regression model was screened to reflect their relationships. Secondly, the regression equation, county average terrain values, average productive potentiality and the terrain values of correcting area were used to get correction formula for productive potentiality. Finally, the correlation coefficients of different terrain factors and productive potentiality were used as weights to weight the values of each corrected productive potentiality value by single factor to get the comprehensive correction productive potentiality value.【Result】 This paper did regression analysis using the data productive potentiality value at the current farmland classification and the DEM data. There were 3 779 samples participated in correcting light temperature productive potentiality and 2 765 samples participated in correcting climate productive potentiality. Regression analysis results showed that the correlation coefficient between light temperature productive potentiality and gradient was 0.0008 and the correlation coefficient between light temperature productive potentiality and aspect was 0.0002. This proved that when 900 m × 900 m DEM data were used as data source to calculate gradient and aspect in national scale, both gradient and aspect almost had no correlation with productive potentiality. On the other hand, correlation coefficient was 0.835 between altitude and light temperature productive potentiality, and the value of correlation coefficient between climate productive potentiality and altitude was 0.721, which meant there was high correlation between altitude and productive potentiality. According to the regression equation between altitude and productive potentiality, the influence coefficient of altitude to the light temperature productive potentiality was 1.479, to the climate productive potentiality was 1.095. Changning County in Sichuan Province was used as a case example verification. The results showed that the revised production potential value had the same trend as elevation of the trend, which reflected that terrain impacted light and temperature conditions, and the more elevation deviating from average elevation was, the greater the revised productive potentiality was different from average productive potentiality.【Conclusion】In national scale, altitude has an important effect for productive potentiality, and the impact for light temperature productive potentiality is greater than climate productive potentiality. On the contrary, both gradient and aspect have no obvious correlation with productive potentiality in national scale. Based on the data limitation, the purpose of this paper is to focus on discussion of mathematical model method and the fixed thought, there is still a certain distance with the practical application, in the future study, we can use more detailed data to analyze the impacts of gradient and aspect to productive potentiality in local area on the premise using the national control of comparability.

Development of a LAMP Approach for Detection of Ralstonia solanacearum

HUANG Wen, XU Jin, ZHANG Hao, XU Jing-sheng, DING Wei, FENG Jie

Scientia Agricultura Sinica. 2016, 49(11): 2093-2102. doi:10.3864/j.issn.0578-1752.2016.11.006

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【Objective】 Bacterial wilt of plants, caused by Ralstonia solanacearum, is one of the most devastating soil-borne diseases in the worldwide and severely restricts production of economically important crops. Simple and sensitive detection assay is the basis for effective prevention and control. The objective of this study is to establish a rapid and specific method for detection of R. solanacearum using an isothermal method known as loop-mediated isothermal amplification (LAMP), to make it possible for researchers and technical staff achieve simple detection of this pathogen. 【Method】 Four specific LAMP primers were designed to target the lpxC of R. solanacearum using online design software Primer Explorer Version 4.0, the inner primers are FIP (5′-TACGCCGTTTCATCGGCCAGGTACACGGCGCACAAGT-3′) and BIP (5′-ATCGTCACGTTCGACAAGGTGGAATGCCG GCTGCAACTG-3′) , the outer primers are F3 (5′-CCTGTACGTGGTCGGCTAT-3′) and B3 (5′-ACCGCAACACGGGATCA-3′). Single-factor experiments were conducted to optimize the parameters of the reaction system, the reaction temperatures were set ranging from 60 to 65℃, the concentrations of Mg²⁺ were set ranging from 2 to 12 mmol·L^-1, the concentration ratios of inner and outer primers were set ranging from 2﹕1 to 12﹕1. The specificity of LAMP was tested by using 24 strains of R. solanacearum isolated from different hosts and 5 different strains of non R. solanacearum (Ralstonia mannitolilytica, Ralstonia pickettii, Enterobacter sp., Acidovorax citrulli, Burkhoderia cepacia), of which 3 species are closely related to R. solanacearum and the others are common bacteria in nature. The sensitivities of LAMP and PCR for detecting R. solanacearum were compared by using ten-fold serially diluted DNA of GMI1000 as templates (including original DNA, 10¹, 10², 10³, 10⁴, 10⁵, 10⁶, and 10⁷ times of diluent). The LAMP method was used to detect the mixture of potato tissue and strain Po41, mixture of ginger tissue and strain Z-Aq-1, also to detect the wilted tomato plant inoculated with R. solanacearum strain Po82 and the health one. Furthermore, the samples of potatoes which may be infected by R. solanacearum were detected by LAMP method. The results of LAMP could be observed by the magnesium pyrophosphate precipitate produced during the reaction, or by the color changing after adding SYBR Green Ⅰ, the positive samples were green and negative ones were orange. 【Result】 The LAMP assay for rapidly and specifically detecting R. solanacearum was established. In this reaction system, the reacting temperature was determined as 63℃ and the concentrations of Mg²⁺ was 6 mmol·L^-1, the concentration ratios of inner and outer primers was 8﹕1 (1.6﹕0.2 μmol·L^-1). The result of specificity test showed that only the reaction liquids with the DNA of R. solanacearum change green, which indicated this method had a good specificity. Sensitivity experiments indicated that LAMP could detect original DNA, 10¹, 10², 10³, 10⁴, and 10⁵ times of diluent, the sensitivity was 1.42 pg which is 10 times higher than conventional PCR. Also this assay could quickly and accurately detect R. solanacearum from plant tissue suspension, diseased plants as well as infected potato tubers sampled in the field. 【Conclusion】 The LAMP assay established in this study had advantages of high sensitivity, specificity, efficiency and low cost over traditional methods and conventional PCR, the reaction results could be directly observed by naked eyes. All the characteristics of LAMP made it suitable to be widely used in field and grass-roots units.

Induced Resistance by Polysaccharides Isolated from Ganoderma lucidum in Tomato Against Gray Mold

NING Yu-bo, WANG Hong-yan, QIAO Kang, LIU Xiu-mei, WANG Kai-yun

Scientia Agricultura Sinica. 2016, 49(11): 2103-2112. doi:10.3864/j.issn.0578-1752.2016.11.007

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【Objective】The systemic long-lasting and broad-spectrum resistance was induced with natural or synthetic compounds. The objective of this study is to investigate the induced resistant effect of polysaccharide extracted from the fruiting body of Ganoderma lucidum (GLP) in tomato against gray mold.【Method】The 1-2 true leaf of tomato plants were sprayed with polysaccharides (50, 100, 200 and 400 mg·L^-1)for 3 times once every 2 days and inoculated spore suspension of Botrytis cinerea after 2 days later of the last time GLP treatment ((1-2)×10⁶spores/mL), meanwhile, the plants treated with the same volume of distilled water used as control instead of GLP solution and inoculated spore suspension of B. cinerea. A syringe stabbed the stem of tomato plant before inoculated spore suspension. Covering plastic film in order to moisturize 24 h and keeping humidity the 2 days of inoculating spore suspension of B. cinerea, the greenhouse relative humidity was not less than 90% and temperature was (15±5) ℃, normal light was recovered 3 days later. The disease index and disease reduction against gray mold in tomato plants was investigated. Simultaneously, the tomato seedlings were soaked in the concentration (50, 100, 200 and 400 mg·L^-1) of polysaccharides and then cultivated in pots. The germination rate, root length and plant height, shoot weight, fresh weight of tomato seedlings were measured 20 days after treatment with polysaccharides. 【Result】Compared with the clear water control’s disease index 49.25, the GLP treatment groups’ disease index were between with 32.96-43.85, significantly reduced, it was found that the lowest disease index 32.96 at the concentration of 400 mg·L^-1 GLP treatment and the disease reduction was 33.07%. The activity of protective enzyme in leaves showed different dynamic changes after GLP treatment, catalase (CAT) and polyphenoloxidase (PPO) activities remarkably increased, and the activities reached the highest peak after 3 days of induction. The highest CAT (162 U·min^-1·g^-1 FW) and PPO (98 U·min^-1·g^-1FW) were 2.13 and 1.71 times compared with the control group, respectively. Peroxidase (POD) activity reached the highest peak after 4 days of induction then decreased gradually, significantly higher than the control. The highest POD (434 U·min^-1·g^-1 FW) was 3.29 times compared with the control groups. MDA content after application of GLP showed a down trend compared to that of control. When the seed was treated with polysaccharides, the germination rate, root length and plant height, shoot weight, root weight of tomato seedlings all increased to a certain degree. The treatment at the concentration of 200 mg·L^-1 GLP showed the highest germination rate 87.3%, 10.0% higher than the control group germination rate 77.3%. Simultaneously, the treatment at the concentration of 200 mg·L^-1 GLP showed the highest promotion in the tomato plant height and shoot weight, enhanced 12.9% and 33.3% respectively than the control. The tomato seed germinal length and root weight enhanced 0.16 cm and 0.33 g respectively than the control at the concentration of 100 mg·L^-1 GLP.【Conclusion】GLP at appropriate concentration had the ability to induce systematic resistance against gray mold. In addition, GLP promoted the growth of tomato seedlings.

Changes in the Nutrients and Fertility of Black Soil over 26 Years in Northeast China

KANG Ri-feng, REN Yi, WU Hui-jun, ZHANG Shu-xiang

Scientia Agricultura Sinica. 2016, 49(11): 2113-2125. doi:10.3864/j.issn.0578-1752.2016.11.008

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【Objective】Relying on fertile black soil resources, northeastern China has developed the biggest grain bases. Clarifying the characteristic of black soil fertility nutrients is essential for soil fertility improvement and maintenance in Northeast China. A long-term cultivated land quality monitoring experiment established in the 1980s was used to investigate the changes in soil nutrients and fertility in the black soil region.【Method】A temporal variation descriptive statistics analysis was conducted to explore the changes in soil organic matter (SOM), total nitrogen (TN), alkaline-hydrolyzable nitrogen (AN), available phosphorus (AP) and available potassium (AK), for three time intervals, including the first (1988-1997), middle (1998-2003) and final (2004-2013) monitoring period, during the study. The changes in soil carbon, nitrogen and carbon-nitrogen ratio (C/N) were analyzed so as to investigate relationship between the carbon and nitrogen budgets. Finally, principal component analysis was used to determine factors contributing to soil fertility, and the general scores of soil fertility at three monitoring periods were calculated. 【Result】 All soil nutrient concentrations showed increased with time and overall soil fertility improved after 10 to 26 years. The SOM, TN, AN, AP and AK significantly increased between the first (1988-1997) and final (2004-2013) monitoring period by 33.9%, 43.9%, 27.6%, 90.3% and 11.8%, respectively. Further analysis indicated that the AP concentrations of 71.4% monitoring sites maintained at a level of 15.0 to 50.0 mg·kg^-1, which meets crop nutrients requirement and would not be expected cause the ground or surface water pollution. However, the concentrations of AP at 28.6% of the monitoring sites exceeded the environmental threshold and some measures should be taken to reduce P fertilizer inputs in these sites. The ratio of C/N decreased with time from 10.3 in 1988 to 9.6 in 2013, decreased by 6.8%. Principal component analysis indicated that SOM and TN were the main factors contributing to soil fertility of the monitoring sites and the 5 fertility factors displayed an order of SOM＞TN＞AN＞AP＞AK. The average general scores of soil fertility at the first and middle monitoring periods were -1.099 and -0.541, respectively, while the black soil fertility has improved significantly and the score increased to 0.5888 at the final monitoring period.【Conclusion】Current farm fertilizer management improved black soil fertility, however a small proportion (28.6%) required better P fertilizer management so as to decrease the risk of ground or surface water pollution. More organic materials should be applied to the black soil to maintain the balance of soil C/N ratio and improve soil fertility.

Hyperspectral Prediction of Soil Organic Matter Content in the Reclamation Cropland of Coal Mining Areas in the Loess Plateau

NAN Feng, ZHU Hong-fen, BI Ru-tian

Scientia Agricultura Sinica. 2016, 49(11): 2126-2135. doi:10.3864/j.issn.0578-1752.2016.11.009

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【Objective】 In terms of the problems in the Loess Plateau, such as many hills, complex topography, low soil organic matter content (SOMC), sampling difficulties, large areas of land damage caused by mining activities and so on, the object of this study is to provide an alternative method for the rapidly quantitative monitoring and evaluation of the SOMC in the process of land reclamation and comprehensive renovation. 【Method】 Taking the cropland soil in the coal mining areas in Xiangyuan County, Shanxi Province was picked as research object, 152 soil samples were collected from the intermediate strip area of land destruction region in a north to south direction. The physical and chemical properties of the soil samples were analyzed. At the same time, the raw hyperspectral reflectance (R) of the soil samples was measured by the standard procedure with an ASD FieldSpec 3 instrument equipped with a high intensity contact probe under the laboratory conditions. The raw spectral reflectance (R) were pretreated by the smoothing or denoising methods of multiplication scatter correction (MSC), baseline offset correction (BOC) and Savitzky-Golay filter in the ParLes 3.1 software. And the raw spectral reflectance (R) was transformed into two types of spectra, which were first order differential reflectance (D (R)) and inverse-log reflectance (lg (1/R)), to analyze the correlation coefficients between the three spectra and their SOMC. Then the significant bands were extracted by the significant correlation coefficients (P=0.01) of the three spectra with the SOMC. Finally, based on the full bands (400-2 400 nm) and significant bands of the three spectra, the hyperspectral predicting models of the SOMC were established by the method of partial least squares regression (PLSR). The optimal models were determined by the assessing indices of predicting accuracies, including coefficient of determination (R²), root mean square error (RMSE), and residual prediction deviation (RPD). 【Result】 The spectra in the bands of 400-1 800 and 1 880-2 400 nm for the raw spectral reflectance (R), 420-790, 1 020-1 040, and 2 150-2 200 nm for D (R), and 400-1 830 and 1 860-2 400 nm for lg (1/R), were significantly correlated with SOMC (P=0.01). And the maximum correction coefficients between the three spectra and their SOMC were 800 nm of the raw spectral reflectance (R), 600 nm of D (R), and 760 nm of lg (1/R). After the transformation of D (R), there were prominent differences among the absorption peaks of the spectral curves in different soil samples, and their correlation coefficients were improved from the value of 0.72 to that of 0.82 in the range of visible bands (400-800 nm). The models of significant bands could obtain better predicting accuracies compared with that of full bands by the method of PLSR. Among the three spectra, the predicting accuracy of lg (1/R) was the best, and R², RMSE of the calibration dataset were 0.95 and 7.64, while R², RMSE, and RPD of the validation dataset were 0.85, 3.00, and 2.56, respectively. For the models of R-PLSR and lg (1/R)-PLSR of full bands, the predicting abilities were good. The R², RMSE, and RPD of R-PLSR were 0.79, 3.64, and 2.10, respectively. And the coefficient of R², RMSE and RPD of lg (1/R)-PLSR were 0.79, 3.53, and 2.17, respectively. However, for the model of D (R)-PLSR, the predicting SOMC were only roughly estimated, and the indices of the predicting accuracies were not satisfying. R², RMSE and PRD of the D (R)-PLSR were 0.61, 5.43, and 1.41, respectively. Finally, by analyzing the predicting accuracies of the three spectra in both full bands and significant bands, it was found that the models of R-PLSR, D (R)-PLSR and lg (1/R)-PLSR in significant bands achieved desirable predicting effect. 【Conclusion】 In the study area, soil spectral reflectance has a high correlation with SOMC, and PLSR is a good method to establish the predicting model of SOMC.

Effects of Straw Returning to Field on Soil Respiration and Soil Water Heat in Winter Wheat - Summer Maize Rotation System Under No Tillage

WANG Wei-yu, QIAO Bo, Kashif AKHTAR, YUAN Shuai, REN Guang-xin, FENG Yong-zhong

Scientia Agricultura Sinica. 2016, 49(11): 2136-2152. doi:10.3864/j.issn.0578-1752.2016.11.010

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【Objective】The purpose of the study was to investigate the effects of straw mulching on soil respiration, soil temperature and soil moisture of winter wheat and summer maize rotation system in no tillage.【Method】From October 2011 to September 2014, a long-term location test of 6 different tillage treatments was set in Yangling, Shaanxi. The treatments used were total amount of straw returning to field+fertilization (S1F1), total amount of straw returning to field+no fertilization(S1F0), half the amount of straw to field+fertilization (S1/2F1), half the amount of straw to field+no fertilization(S1/2F0), without straw+fertilization (S0F1), without straw+no fertilization(S0F0), then the differences in soil respiration, soil temperature, soil moisture, crop yield and soil organic carbon of the soil plow layer under different treatments were measured and analyzed from 2011 to 2014. 【Result】During the growth period of winter wheat, soil respiration rate in each treatment showed an increase after the first downward trend and then a decrease. During the growth period of summer maize, the respiration rate in each treatment showed a trend of first increase and then decrease. During the same growth period, the following was S1F1＞S1/2F1＞S1/2F0＞S0F1＞S1F0＞CK in the average rate and cumulative soil respiration. During different growth stages of the same crop, the cumulative soil respiration in different treatments showed a decreasing trend year by year. In the whole research term, the variation trend of soil temperature was similar with the trend of the monthly average temperature. The soil temperature variation trends of different treatments during the same growth period were similar. Simultaneously, the average soil temperatures during the same period of different treatments were decreased with the increase of soil depth. In winter, the soil temperature of different treatments was higher than that of CK, but the average soil temperature was lower than that of CK in a same growth period. The soil moisture decreased with the increase of soil depth, but the soil moisture was affected by rainfall, the change of soil moisture in different rotation cycles was larger, and the average water moisture of each treatment showed a trend of S1F0＞S1F1＞S1/2F0＞S1/2F1＞CK＞S0F1 in a same growth period. Meanwhile, the soil moisture in different straw mulching treatments was significantly different from that of CK, respectively (P＜0.05); 32.5%-60.4% variations in soil respiration rate could be recorded due to the soil temperature, and 38.4%-82.5% variations in it could be recorded due to the soil moisture. In different depths of soil, the correlation between the soil temperature and the soil respiration in 5 cm depth was the strongest, and the correlation between the soil moisture and the soil respiration at 10-20 cm was the highest. In the same year, the yields of winter wheat and summer maize in different treatments, respectively, showed a trend of S1F1＞S1F0＞S1/2F1＞S0F1＞S1/2F0＞CK. In the research cycle, the yield of wheat increased continuously in three years, and the yield of maize increased in the previous two years of three, but the yield in the third year significantly decreased because of the influence of extremely hot weather. After the harvest of single crop, soil organic carbon in different treatments at the same soil depth, respectively, demonstrated a trend of S1F1＞S1/2F1＞S1F0＞S1/2F0＞S0F1＞CK. Meanwhile, the soil organic carbon in different treatments of straw mulching was increased continuously in the three years.【Conclusion】Long-term no tillage with straw returning can effectively reduce soil carbon emissions, improve soil water use efficiency and winter soil temperature, and increase crop yield and soil organic carbon. The effect of S1F0 treatment is the best among different treatments.

Effects of Fusarium Oxysporum f. sp. melonis on Lignin, Activities of Lingin-Related Enzymes and Genes Expressions of CmCADs in Oriental Melon (Cucumis melo var. makuwa Makino)

LIU He-juan, LI Yue-peng, LIU Wei, SHAO Qi, QI Hong-yan

Scientia Agricultura Sinica. 2016, 49(11): 2153-2163. doi:10.3864/j.issn.0578-1752.2016.11.011

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【Objective】The purpose of this study is to investigate the effects of Fusarium oxysporum on symptom, lignin content, activities of lignin-related enzymes and real-time fluorescent quantitative PCR analysis of CmCADs gene expression of CmCADs in oriental melon seedlings and to find out the gene family members of CmCADs that can respond to the fusarium oxysporum. 【Method】The oriental melon ‘Caihong 7’ which is resistant to fusarium oxysporum was used as experimental materials. Then the melon seedlings were inoculated with F. oxysporum at 3 true leaves stage. The control plants were dealt with equivalent sterile water. The changes of phenotype were observed and lignin content, activities of lingin-related enzymes (PAL, POD and CAD) and genes expressions of CmCADs in the vegetative organs of oriental melon seedlings were determined at 0, 1, 3, 5, 7 and 9 days after inoculation.【Result】The symptom of oriental melon seedlings showed obvious changes. The leaves began to wilt slightly 5 days after inoculation and wilt badly 9 days after inoculation. The results showed that compared with the control, the lignin content and the activities of lignin-related enzymes were all increased to a certain extent. The change trend of lignin content in roots, stems and leaves was basically identical with keeping increasing and higher than the control treatment. The activities of PAL which increased first and then decreased in roots and stems while exhibited a global increased trend in the leaves were obviously higher than the control treatment. The activities of POD presented different changes with higher activities than control in each period. The activities of CAD presented a trend of earlier increase and later decrease in roots, stems and leaves. In the study, CmCADs showed tissue-specific expression after inoculation. CmCAD1, CmCAD 2, CmCAD 3 and CmCAD 5 were strongly induced in roots 7 days after inoculation, except CmCAD4, while only CmCAD2 and CmCAD5 in leaves were induced strongly 5 days after inoculation. No CmCADs expressions were observed in stems of melon. 【Conclusion】 It was found that the increase of the lignin content, activities of lignin-related enzymes (PAL, POD, CAD) caused by F. oxysporum suggested that the lignin biosynthesis pathway may play an important role in defending the oriental melon from the F. oxysporum. It was also inferred that CmCAD2 and CmCAD5 which were both induced by F. oxysporum may the members of CmCADs which can response to the F. oxysporum and play a role in resistance to F. oxysporum in oriental melon.

Coupling Effects of Water and Fertilizer on Melon in Plastic Greenhouse of Gravel-Mulched Field Under Drip Fertigation

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

Scientia Agricultura Sinica. 2016, 49(11): 2164-2173. doi:10.3864/j.issn.0578-1752.2016.11.012

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【Objective】The traditional management patterns of water and fertilizer is a limiting factor for the development of melons in a plastic greenhouse of a gravel-mulched field. So the study of the coupling effect of water and fertilizer on melons under drip fertigation has attached great importance to the sustainable development of the greenhouse melon industry in gravel-mulched fields. 【Method】The water and fertilizer effective equations about yield and quality were set up by adopting a “311-B” D optimized saturation design method under the condition of drip fertigation, and the optimal schemes of water and fertilizer were established by means of an analytical model. 【Result】According to the data obtained, mathematical regression models were set up based on the yield and quality respectively by quadratic regression analysis, in which water, N, and K were independent variables and the melon yield and quality parameters were dependent variables. The model analysis shows that water, N, and K significantly influence the melon yield and quality parameters, the influenced order of different factors on the melon yield as N＞water＞K and on the quality as K＞N＞water, the yield and quality increased and then decreased as water, N, and K rates increased. The interaction effects among these factors were positive on the melon yield and quality, with the influenced order as water and N＞N and K＞water and K. The interaction effects of water and N was the biggest positive influence on the melon yield but negative on quality. The single factor is high or low and is not conducive to the formation of melon production, and due to the interaction effect, the combined application of water, N, and K has a stronger role in promotion of the yield and quality. 【Conclusion】According to the optimal effect function model recommendation, the optimum combinations of water, N, and K₂O rates ranged from 786 to796 m³·hm^-2,170 to 227 kg·hm^-2, and 227 to 246 kg·hm^-2, in which melon yield was over 35 000 k·hm^-2 and the quality grade was larger than 85 under the conditions of this experiment.

Investigation and Analysis of Main Antinutritional Factors in Soybean and Extruded Soybean

YAO Yi-sha, GU Xu, SHANG Fang-fang, QIU Jing, LI Jun-guo1, LI Jun

Scientia Agricultura Sinica. 2016, 49(11): 2174-2182. doi:10.3864/j.issn.0578-1752.2016.11.013

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【Objective】Soybean is an important food material as well as feed material for its abundant nutriments, but some antinutritional factors (ANFs) in soybean limit its application in the food and feed industries. As a widely used processing technique, the extrusion process can reduce the side-effect of ANFs in soybean to human and animals causing the content of ANFs to decrease while the content of nutrient composition remains about the same. This research is aimed at investigating and analyzing the difference of main antinutritional factors in soybean and extruded soybean and discussing the effect of the extrusion process to these ANFs in soybean, so as to provide a reference for expanded enterprises to choose high quality raw materials and optimize the processing technique in the actual production, and provide some guidance to the design of animal feed formula. 【Method】In this research, 20 batches of soybean and 19 batches of extruded soybean from different areas of the manufacturer were collected, and the content or activity of the main anti-nutritional factors, including trypsin inhibitor, antigenic protein (glycinin and β-conglycinin), oligosaccharide (raffinose and stachyose) and urease, were detected. And these results were compared with 2 batches of soybean and 8 batches of extruded soybean that were produced in one manufacturer. The concentrations of trypsin inhibitor and antigenic protein were analyzed using ELISA. The concentrations of oligosaccharide were determined by HPLC with a differential detector. At the same time the extraction methods of oligosaccharides were optimized. Single factor optimize experiments were the way of extraction, the dosage of activated carbon, volume percentage of ethanol, and solid-liquid ratio. The solution was detected by HPLC after through filter membrane. Through comprehensive analysis of the data, the effect of extrusion technology on the content or activity of main antinutritional factors in soybean was studied. 【Result】 The extraction method is as follows: adding 70% ethanol solution to the samples (solid-liquid ratio: 1﹕25), and then centrifuging and concentrating the extracting solution after using microwave assisted extraction, diluting the concentrated solution with water to 25 mL, centrifuging the solution (2 mL) after blending. In comparison the content of the main antinutritional factors in soybean (22 batches) with the content in extruded soybean (27 batches), the content of trypsin inhibitor and antigenic protein and urease activity in soybean is significantly lower than those in extruded soybean while the content of oligosaccharides has no significant difference between them. The urease activity of most of extruded soybeans is near zero and lower by 99% than the activity of soybeans. The content of trypsin inhibitor, glycinin and β-conglycinin decreased respectively in 66%, 67% and 90%, while the content of raffinose and stachyose remained largely unchanged. From this research the content or activity of trypsin inhibitor, glycinin, β-conglycinin, raffinose, stachyose and urease in the soybean is respectively 32.5-89.6 mg·g^-1, 91.0-143.1 mg·g^-1, 161.1-268.7 mg·g^-1, 3.3-8.78 mg·g^-1, 21.4-34.16 mg·g^-1 and 3.6-9.42 U·g^-1, in the extruded soybean is respectively 10.7-31.1 mg·g^-1, 17.7-64.5 mg·g^-1, 9.3-57.5 mg·g^-1, 4.25-10.21 mg·g^-1, 17.68-34.15 mg·g^-1 and 0.00-0.02 U·g^-1. 【Conclusion】 The extrusion process can significantly reduce the content of the main antinutritional factors in soybean and side effects caused by these factors and can improve the utilization rate of nutrition. This study presents the reference range of the main ANFs in content or activity in soybean and extruded soybean.

Chemical Compositions and Physicochemical Properties of Milk Fat and Fractions Obtained by Short-Path Distillation

GAO Xi-xi, ZHANG Shu-wen, LU Jing, LIU Lu, PANG Xiao-yang, YUE Xi-qing, Lü Jia-ping

Scientia Agricultura Sinica. 2016, 49(11): 2183-2193. doi:10.3864/j.issn.0578-1752.2016.11.014

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【Objective】Milk fat is not suitable for some food applications and has limited functionality because of its complex compositions, structures and processing properties. The objective of this study is to provide a technical method and theoretical reference for fractionation of milk fat through studying the chemical compositions and physicochemical properties and its correlation of milk fat and fractions obtained by short-path distillation in different distillation temperatures.【Method】Commercial milk fat was fractionated by short-path distillation at 150℃, 165℃, 180℃, 190℃, 200℃, 210℃, 225℃, and 240℃, respectively, and the relationship of distillation temperature and yield of distillate was analyzed. Fatty acid composition was determined by gas chromatography to study the effect of distillation temperature on the composition and properties of milk fat and its fractions. Acid value, iodine value, peroxide value, slipping melting point, DSC thermograms of melting and crystallization and polarized light micrographswere also measured.【Result】The lowest temperature that milk fat could be fractionated was 165℃. Distillate yield increased from 1.19% to 36.89% with the distillation temperature increased from 165℃ to 240℃. Acid and peroxide value of distillate and retentate obtained by short-path distillation had different changes with no regularity, while the iodine value of retentate was generally higher than that of distillate. Short-path distillation did not change the kinds of fatty acids in the fractions, but the concentration of certain fatty acid presented substantial differences: the distillate was enriched in short- and medium-chain fatty acids (40%-60%), while the retentate was enriched in long-chain fatty acids (65%-75%) and unsaturated fatty acids (about 30%), which was consistent with the result of iodine value. Distillation temperature had a significant effect on the content of fatty acids in the fractions and the concentration of all kinds of fatty acids showed a trend of approaching to milk fat with the increase of distillation temperature. Relative to milk fat, the slipping melting point of the distillate was depressed and that of the retentate was augmented, while both of them showed an increasing trend as the distillation temperature increased. There was a significant positive correlation (P＜0.01) between the concentration of long-chain fatty acids in the fractions and their slipping melting point, r=0.977. DSC results showed that the melting and crystallization peaks of fractions were offset to the high temperature area as the increase of distillation temperature which indicated that the melting and crystallization properties of fractions were different but changed regularly. Polarized light micrographs showed that the distillate and retentate had different fat crystals, the crystals formed on cooling at 5℃ for 24 h corresponded to spherulites for the retentate whereas the solid fat phase of the distillate corresponded to needle-shape crystals. 【Conclusion】 Milk fat could be fractionated effectively by short-path distillation and distillation temperature had a significant effect on the resulting fractions. Fractionation of milk fat by short-path distillation at different distillation temperatures is able to change the chemical compositions, physicochemical properties and processing properties of the fractions.

Effects of SNPs in the 3’ Untranslated Regions of FADS2 on the Composition of Fatty Acids in Milk of Chinese Holstein

XU Chen-xi, WANG Meng-qi, ZHU Xiao-rui, ZHANG Yu-feng, XIA Hai-lei, LIU Xian-hui, WANG Xiao-long, ZHANG Hui-min, YANG Zhang-ping, MAO Yong-jiang

Scientia Agricultura Sinica. 2016, 49(11): 2194-2202. doi:10.3864/j.issn.0578-1752.2016.11.015

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【Objective】D-6-fatty acid desaturase 2 (FADS2) is one of the key limiting enzymes in the conversion of dietary essential 18 carbon PUFAs (18C-PUFAs) such as linoleic acid (LNA,C18:2n-6) to γ-linolenic acid (GLA, 18:3n6), eicosapentaenoic acid (EPA, C20:5n3) and docosahexaenoic acid (DHA, C22:6n3). The objective of this study was to investigate the effects of SNPs in the 3’ untranslated regions of FADS2 gene on the compositions of fatty acids in milk of Chinese Holstein.【Method】In this study, 20 Chinese Holstein cows were selected randomly for PCR amplification and sequencing of the 3’ untranslated regions of FADS2 gene used for SNP discovery. Then the Chinese Holstein cows (n =551) were genotyped using Sequenom MassARRAY (Sequenom Inc., San Diego, CA) based on the previous SNP information in this study, and the associations between SNPs or haplotypes and compositions of fatty acids, unsaturated indexes of fatty acids in milk were analyzed by the least squares method in the GLM procedure of SPSS.【Result】Three SNPs (c.1571 A＞G, c.2743 A＞G and c.2776 A＞G) were identified in the 3’ untranslated regions of FADS2 gene. The genotype GG was the dominant genotype, which the frequencies were 0.800 for c.1571 A＞G, allele G was dominant allele with the frequency of 0.887. The genotype AA was the dominant genotype, which the frequencies were 0.673 for c. 2776 A＞G. The allele A was dominant allele with the frequency of 0.819. The coefficient of disequilibrium was 0.028 between c.1571 A＞G and c.2776 A＞G, which was not significant at P＜0.05. There were 3 haplotypes for FADS2 c.1571 A＞G and c.2776 A＞G, which the frequencies were 0.705, 0.181 and 0.114 for GA, GG and AA, respectively. χ² test indicated that the c.2776 A＞G fitted with Hardy-Weinberg equilibrium in the population (P＞0.05), and the c.1571 A＞G deviated from Hardy-Weinberg equilibrium (P＜0.05). The SNP c.1571 A＞G showed a very significant association with C14:1, unsaturated indexes of C14 and C18 (P＜0.01), and significant association with C18:0, SFA and MUFA (P＜0.05). The individuals with genotype GG had higher C14:1, unsaturated indexes of C14 and C18 than genotypes AG (P＜0.05). The SNP c.2776 A＞G showed a very significant association with C16:1, unsaturated indexes of C16 and C20 (P＜0.01), and significant association with C14:1 (P＜0.05). The individuals with genotype GG had higher C16:1, unsaturated indexes of C16 and C20 than genotypes AG and AA (P＜0.05). In the meantime, the haplotype of FADS2 1571-2776 showed a significant association with C16:1, unsaturated indexes of C20 (P＜0.05). The individuals with genotype GG had higher C16:1, unsaturated indexes of C20 than genotypes GA and AA (P＜0.05). 【Conclusion】The SNPs in the 3’ untranslated regions of FADS2 gene have significant genetic effects on composition of fatty acids in milk, but further investigation will be required to elucidate the biological and practical relevance of these SNPs.

Study on the Temporal and Spatial Expression and Correlation Analysis of Smads and YAP1 Gene in the Hippo Pathway in Sheep Muscle Tissue

BAO Jian-jun, SU Rui, WANG Qing-zeng, Lü Xiao-yang, GAO Wen, YU Jia-rui, WANG Li-hong, CHEN Ling, WU Wen-zhong, SHENG Shui-xing, ZHOU Hong, SUN Wei, DAI Guo-jun

Scientia Agricultura Sinica. 2016, 49(11): 2203-2213. doi:10.3864/j.issn.0578-1752.2016.11.016

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【Objective】 In order to explore the expression pattern and inner connection between the different genes, the mRNA expression level was used to detect the spatial and temporal expression of genes in TGF-β/Smad signaling pathway of sheep muscle tissue. 【Method】 In this study, q-PCR was used to detect relative expression of Smads genes and Yes-associated protein 1(YAP1) in 2 different skeletal muscles (i.e., gastrocnemius muscle, extensor digitorum longus) and 3 different growth stages (i.e., 2-day-old, 2 and 6-month-old) of Hu Sheep, and analyze the relative expression between Smads(Smad2, Smad3, Smad4 and Smad7) and YAP1. 【Result】The results of the spatial and temporal expression of Smads in TGF-β/Smad signaling pathway showed that the expression of Smads in extensor digitorum longus were lower than in gastrocnemius muscle, which may be related to two different parts of the skeletal muscle. The expression of Smad2, Smad3 and Smad4 genes in the 2-day-old were higher than other growth stages, and Smad7 gene expression in the 2-day-old was lower than 6-month-old, and the expression in 2-month-old was the lowest. The expressions of Smads gene of ram in the 2-day-old was higher than ewe, and the expressions of Smad2, Smad4, and Smad7 genes of ram in 2-month-old and 6-month-old were lower than the ewe in 2-day-old, the expression of Smad3 gene of ram in 3 growth stages were higher than ewe. The correlation expression of Smads gene and YAP1 gene in TGF-β/smad signaling of sheep muscle was found that there was no significant correlation in 2-day-old of gastrocnemius muscle between YAP1 and smads (P＞0.05) . The expression between YAP1 and Smad2 had a significant positive correlation (P＜0.05) and the correlation of the expression between YAP1 and other Smads was not significant in 2-month-old of gastrocnemius muscle (P＞0.05). There was a significant negative correlation between YAP1 and Smad3 in different growth stages of gastrocnemius muscle(P＜0.01). YAP1 expression exhibited a high positive correlation with Smad3 and the expression of YAP1 with the others was significantiy correlated in extensor digitorum longus of 2-month-old (P＜0.05). There was no significant correlation between YAP1 and Smads in 2-month-old of extensor digitorum longus (P＞0.05). The expression between YAP1 and Smad7 had a high significant positive correlation (P＜0.01) and the correlation of expression between YAP1 and other Smads was not significant in 6-month-old of extensor digitorum longus (P＞0.05). There were highly significant negative correlations between YAP1 and Smads in different growth stages of extensor digitorum longus (P＜0.01). 【Conclusion】 It was suggested that different tissues, growth stages and gender could affect the expression of Smads in muscle. YAP1 of Hippo pathway may participate in regulating the TGF-β/smad pathway and the process of muscle proliferation and differentiation in gastrocnemius muscle and extensor digitorum longus of Hu sheep.

The Influences of Over-Expressing Pax6^10Neu on MITF and TYR in Melanocytes

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

Scientia Agricultura Sinica. 2016, 49(11): 2214-2221. doi:10.3864/j.issn.0578-1752.2016.11.017

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【Objective】 MITF and β-catenin are important regulatory genes of the melanin system. Pax6 regulates the production of melanin indirectly, through regulates MITF and TYR. In order to explore the function of Pax6^10Neu and the mechanism of Pax6 with its target genes, we explored the function of Pax6^10Neu, which only included 102 N-terminalanimo acids of the paird domain. 【Method】 According to the sequence of Pax6^10Neu in NCBI, the primers of Pax6^10Neu were designed and synthesized by BGI. The coding sequences of Pax6^10Neu were PCR amplified and then confirmed by sequencing. Sal I and Xba I restriction sites were selected by analyzing the sequences of Pax6^10Neu and the mammalian expression vector. The Pax6^10Neu was cloned into the T-Vector, meanwhile, confirmed by sequencing. The fragment was then subcloned into a mammalian expression vector, resulting in a construction that contained a specific mouse tyrp2 promoter driving the expression of the green fluorescent protein (GFP) and the target fragment with Sal I and Xba I restriction sites. The plasmid vector was confirmed by sequencing. The expression vector was amplified by competent cells and obtained without endotoxin by the plasmid midiprep system. Then, the sheep melanocytes were transfected with the vector using Liposome 2000. There were three methods used in the results test which were quantitatively real-time PCR, western blot, and melanin content measurement. 【Result】 The results showed that the MITF mRNA, MITF and TYR protein, melanin content were significantly increased. MITF mRNA was significantly increased to 3.2 times (P＜0.05) and TYR mRNA was increased to 1.31 times, compared with control group, MITF protein was significantly increased to 8.24 times (P＜0.001) and TYR protein was significantly increased to 2.09 times (P＜0.001). Meanwhile, the melanin content was significantly increased to 1.22 times (P＜0.05). 【Conclusion】 We demonstrated that the Pax6^10Neustill interacts with MITF, and then regulated TYR indirectly, meanwhile changing the production of melanin of melanocytes.

Effect of Sulphur Availability on Nitrate Accumulation and Expression of Nitrogen and Sulphur Assimilation Related Genes in Non-Heading Chinese Cabbage

XU Yao, MU Jian-mei, ZHANG Guo-qin, MA Jia-jia, XU Jun, LI Jun, LIU Feng-jun, SHE Xu-dong

Scientia Agricultura Sinica. 2016, 49(11): 2222-2233. doi:10.3864/j.issn.0578-1752.2016.11.018

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【Objective】The objective of this paper is to identify the best sulfate to reduce nitrate accumulation in non-heading Chinese cabbage, and analyze the key genes involved in nitrogen and sulphur assimilation. The results of this study will provide new insights into sulphur fertilization and investigation of the molecular mechanisms of nitrate accumulation with the goal of molecular breeding.【Method】Nitrate content of non-heading Chinese cabbage leaves and petioles were measured under treatments of 4 forms of sulphur with 3 concentrations. The expression of 30 nitrogen and sulphur assimilation related genes were analyzed by semi-quantitative RT-PCR. 【Result】Sulphur treatments were significantly increased the aboveground biomass of non-heading Chinese cabbage, and the treatments of Na₂SO₄ had the best effect, and 30 mg·kg^-1Na₂SO₄ was the most effective treatment as the aboveground biomass increased by 49.76% compared with the control. Na₂SO₄and Na₂S₂O₃ reduced nitrate content of non-heading Chinese cabbage more significantly than others. Na₂SO₄decreased nitrate content by 12.23%-23.55% in leaves and by 33.08%-41.98% in petioles compared with the control, and it was also found a positive correlation between the reduction and concentration of Na₂SO₄, and 30 mg·kg^-1Na₂SO₄ also had the best effect. Na₂S₂O₃ decreased nitrate content by 15.34%-33.08% compared with the control in leaves and by 11.95%-19.68% in petioles. Sulphur promoted nitrogen assimilation, and the expression of NR-1, NADH-GOGAT-1, NADH-GOGAT-2, Cytoplasm-GS-4, Cytoplasm-GS-5, and GDH-3 were higher than the control in leaves, and the expression of NR-1, NADH-GOGAT-2, Cytoplasm-GS-1, and GDH-2 were higher than the control in petioles. The expression of NADH-GOGAT-2 was correlated with nitrate levels. Sulphur treatments also had an effect on sulphur assimilation genes, and the expressions of ATPS-2, ATPS-3, ATPS-4, APSR-3, SIR, SAT1.1, and SAT2.1 were higher than the control in leaves, and only the expressions of SIR and OASTL-A were higher than the control in petioles. 【Conclusion】 Na₂SO₄ had the best effect in reducing nitrate content and increasing yield of non-heading Chinese cabbage, and 30 mg·kg^-1 Na₂SO₄was the most effective treatment. The expression of NADH-GOGAT-2 was correlated with nitrate levels. The data suggested that NADH-GOGAT-2 may be the key gene in nitrogen assimilation.

Effect of Six Chinese Traditional Medicine Components on Inhibiting Swine Transmissible Gastroenteritis Virus in vitro

JIN Xiu-zhe, HE Lei, CHENG Xiang-chao, ZHANG Chun-jie, YU Zu-hua, HAN Hai-feng, YANG Dan-fang, MEI Jing-jing, LI Yin-ju

Scientia Agricultura Sinica. 2016, 49(11): 2234-2244. doi:10.3864/j.issn.0578-1752.2016.11.019

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【Objective】To evaluate the antiviral activity mechanism action of phillyrin and other Chinese traditional medicine components to swine transmissible gastroenteritis virus (TGEV), to confirm the antiviral effect of the drug components in vitro, and therefore to provide scientific basis for screening drugs of antiviral activity to TGEV.【Method】Animal cell culture techniques, MTT detection and cytopathic effect (CPE) methods were used to determine the toxic effect of six Chinese traditional medicine components, including phillyrin, forsythiaside, chlorogenic acid, caffeic acid, eugenol and paeonol, on Swine testis (ST) cells. The maximum safe concentrations of the drugs on ST cells were determined by observing CPE of cells, virus TCID₅₀ was determined at the same time, and 100·TCID₅₀ with cell culture maintenance medium was prepared. The original solutions of the 6 drug components were diluted within the maximum safe concentration scope, and their antiviral effects for cell growth inhibition in vitro were measured with three arrangement routes: TGEV before drugs adding, TGEV after drugs adding and TGEV-and-drugs at the same time. Normal cell control and virus control were arranged for each testing group, with three replications for all treatments. The OD₆₃₀ was determined by using Eliasa approach, the rate of inhibition of six Chinese traditional medicine components to TGEV in different ways was determined, and the inhibition of the best Chinese traditional medicine component on the replication of TGEV was screened, the best antiviral effect concentration of each drug component was recorded. After the six Chinese traditional medicine components reacted with the TGEV, the TCID₅₀, RT-PCR was used to evaluate the viral titer in the supernatant. Furthermore, the proliferation inhibition of six Chinese traditional medicine components on TGEV in ST cell was determined.【Result】The results showed that the highest safe concentrations of Phillyrin, Forsythiaside, Chlorogenic acid, Caffeic acid, Eugenol, Paeonol were 320, 200, 80, 125, 100, 200 μmol·L^-1, respectively; the best antiviral effect concentration were 160, 100, 20, 62.5, 25, 100 μmol·L^-1 , respectively. By using Karber method, the TCID₅₀ of initial TGEV was estimated to be 10^-6.25/0.1 mL. All the six Chinese traditional medicine components had inhibition effects on TGEV in ST cells in vitro. In particular, caffeic acid at 62.5 μmol·L^-1 mixed with TGEV at 100·TCID₅₀ showed the best inhibition effect. With this caffeic acid treatment, 72 h after the test started, the tested cells could still keep smooth, without pycnosis, complete, and profile clear between cells, with only a few cells fell off, or death. At that moment, the measured TGEV TCID₅₀ was 10^-3.75/0.1 mL in the supernatants. The results showed that the virus in treatment with caffeic acid was significantly different, compared to the virus control group with 10^-6.45/0.1 mL (P＜0.01). The inhibition rate reached 84.4% according to the OD₆₃₀ value in the direct inactivation experiment. For other tested components, the TGEV TCID₅₀ of phillyrin, forsythiaside, chlorogenic acid, paeonol and eugenol were 10^-4.75, 10^-5.55, 10^-5.55, 10^-5.65, 10^-5.75/0.1 mL, respectively, with no significant difference, compared to the virus control group, with most of the rate of inhibition below 50%. In addition, all of the six Chinese traditional medicine components had proliferation inhibition on TGEV in ST cells, directly killing effect was the most affective one, followed by adsorpt blocking effect, with replicate blocking effect behind. RT-PCR was used to evaluate the viral titer in the supernatant, the result showed that caffeic acid group was dark compared with the virus control group, the virus titer was low, inhibition effect was remarkable to virus. The next were Phillyrin, Forsythiaside, Chlorogenic acid, Paeonol and Eugenol.【Conclusion】All the six Chinese traditional medicine components had proliferation inhibition on TGEV in ST cells, and caffeic acid showed the best inhibition effect. It has the potential to develop into the antiviral drugs.

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