Scientia Agricultura Sinica

Theory and Practice on Breeding of Two-Line Hybrid Rice, Liangyoupeijiu

Lü Chuan-gen, ZOU Jiang-shi

Scientia Agricultura Sinica. 2016, 49(9): 1635-1645. doi:10.3864/j.issn.0578-1752.2016.09.001

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Two-line hybrid rice, Liangyoupeijiu, is a landmark for super rice breeding in China National High-tech R&D Program. It has the largest planting area for years after the widely planted three-line hybrid Shanyou 63, and a significant impact in rice breeding area. Breeding practice in Liangyoupeijiu experienced a difficult and tortuous process of scientific research and production applications. Initially, the authors attempted CHA using a typical indica-japonica hybrid, Yayou 2. After failure of the attempt, the technical route was adjusted as: developing hybrid by partial-use of indica-japonica heterosis, and screening a plant ideotype suitable for ecological conditions in Changjiang-Huaihe region. The hybrid should have higher and stable seed setting rate, ultra-high yield, good grain quality, multiple resistances to disease, extensive adaptability, and safety for seed production as well. By analyzing key agronomic traits of hybrids, which used a lot of PTGMS lines as female, different ecological type lines as male, the authors determined the key female of Pei’ai 64S which was 1/4 relatives to javanica, and selected elite indica lines as male which belongs to the advantages cline and can avoid long growth period and higher plant of its F1, and screened the combinations adapting to the climate conditions in ChangJiang-Huaihe rice region. By screening a large number of combinations, the hybrid Liangyoupeijiu was obtained, which achieved genetic, morphological and functional “three excellent”, and the good grain quality, ultra-high yield, multiple resistances are gathered in one. Through studies on the hazardous high and low temperatures to fertility conversion and pollination of Pei’ai 64S, and frequency analysis on such temperatures in the various ecological regions, the authors put forward the suitable area for seed production and corresponding security heading date of parents. On the basis of the study on the plant temperature to Pei’ai 64S fertility conversion, the techniques for safeguarding the seed purity when confronted with low temperature weather in seed production were proposed. Through study on the biological characteristics and appropriate growth conditions of Liangyoupeijiu, the suitable planting areas and corresponding seeding and heading dates were proposed, the problems that may occur in the production were predicted and the corresponding preventive measures and solutions were proposed, and the cultivation techniques for increasing seed setting rate and its stability were developed. Supported by the research and application of the above techniques, Liangyoupeijiu was successfully cultivated in large-scale for years in Southern China. After reviewed the design of the research project, breeding theory and technical route, the tortuous processes for breeding, researching, developing and popularizing the hybrid, the authors discussed the significance of the achievement, put forward a complementary thinking of “fishgig-like” stem distribution for middle and base parts on rice plant ideotype, proposed the availability for selecting rice palatability by the complementation between indica and japonica types. To large-scale successfully cultivate a hybrid rice, prospective studies for the optimum growth conditions, the optimum planting area, the optimal cultivation measures, and a series of planting techniques to exert its advantages and avoid its disadvantages are very important.

Identify QTL Associated with Soybean 100-Seed Weight Using Recombinant Inbred Lines and Determine QTL Diversity Within Nature Population

CHEN Qiang, YAN Long, FENG Yan, DENG Ying-ying, HOU Wen-huan, LIU Qing, LIU Bing-qiang, YANG Chun-yan, ZHANG Meng-chen

Scientia Agricultura Sinica. 2016, 49(9): 1646-1656. doi:10.3864/j.issn.0578-1752.2016.09.002

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【Objective】 Seed weight is a major target of breeding as it is not only a component of seed yield but it also affects quality, so, different uses have different requirements for it. This study is to identify QTL associated with 100-seed weight, obtain the linkage markers and explore its diversity. Eventually provide a scientific basis of genetic improvement of seed weight.【Method】Recombinant inbred lines (RIL) derived from a cross of Jidou12×Heidou were used to detect the QTL associated with 100-seed weight, the composite interval mapping (CIM) method in WinQTL Cartographer software was used for the QTL analysis based on the seed weight observed in two years. A putative QTL was claimed using a genome-wise type I error of P＜0.05 determined by 300 permutations. In addition, a total of 205 soybean landraces and improved cultivars were used to identify phenotype through 3 years, and genotyped by SSR marker that linked with seed weight. Allelic frequency variation is more than 5% (corresponding to the number of resource materials is more than 10) as the efficient allelic variation.【Result】In RIL, the distribution frequency of seed weight was basically in normal, and the narrow-sense heritability was 88.72%. Five QTL for 100-seed weight were identified. These QTL scattered on Chr.02 (D1b), Chr.06 (C2), Chr.08 (A2) and Chr.17 (D2), respectively and could explain 7.68%-12.83% of the phenotype variation. The additive effect of the QTL varied from 0.65 g to 0.84 g. Two QTL were detected in two years. The qSW-6-1 flanked by SSR markers Satt457 and Sat_062 on Chr.06 explaining 12.02% of the phenotype variation, linked marker was Satt281, and the additive effect was -0.81 g. The qSW-17-1 flanked by SSR markers Satt301 to Satt310 on Chr.17 explaining 12.83% of the variation, and the additive effect was -0.84 g. Based on the germplasm, the allele number of the eight SSR loci, associated with five QTL identified via RIL, varied from 2 to 8, and the genetic diversity index varied from 0.34 to 0.82. Six alleles, Satt281-227 bp, Barcsoyssr_2_304-245 bp, Satt301-199 bp, Sat_406-214 bp, Satt119-136 bp and Satt341-218 bp, were correlated with large seed weight. Satt281-227 bp was the novel alleles in RIL and nature population, mainly distributed in the domestic improved cultivars that with big seed weight. Among the 205 accessions, only three accessions (Lü75, Zhongpindaheidou, Zhongye2) contained more than four alleles associated with large seed weight. 【Conclusion】 In this study, five QTL associated with 100-seed weight were identified by linkage analysis in RIL which crossed by improved cultivars Jidou12 and landraces Heidou. One allele of these was additive effect among RIL and nature population. The diversity distribution was made clear among the 205 accessions. The results can be applied to the parental selection and marker-assisted breeding in the process of seed weight improvement.

Nitrogen, Phosphorus and Potassium Requirement and Their Physiological Efficiency for Winter Wheat Affected by Soil Surface Managements in Dryland

HE Gang, WANG Zhao-hui, LI Fu-cui, DAI Jian, LI Qiang, XUE Cheng, CAO Han-bing, WANG Sen, LIU Hui, LUO Lai-chao, HUANG Ming

Scientia Agricultura Sinica. 2016, 49(9): 1657-1671. doi:10.3864/j.issn.0578-1752.2016.09.003

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【Objective】 The objective of the study was to determine the effects of soil surface managements on nitrogen (N), phosphorus (P) and potassium (K) requirements and their physiological efficiencies for the grain yield formation and nutrient accumulation of winter wheat in dryland.【Method】 A field experiment was carried out to study the effects of plastic film mulching (PM), straw mulching (SM), planting green manure (GM), and straw mulching plus planting green manure (SGM) on winter wheat grain yield, grain N, P and K concentrations, their requirements and physiological efficiencies for grain yield formation and nutrient accumulation.【Result】 PM decreased N requirement and increased N physiological efficiency for grain yield formation, and thus increased grain yield by 6%. SM decreased crop N uptake, so it decreased the grain yield by 7%. Grain yield was also decreased by 5% with both the GM and SGM, which could be explained by increased nutrient requirement and decreased physiological efficiency for grain yield formation. Grain N concentration was decreased by 8% with the PM, which was associated with the increased N requirement for grain N accumulation and decreased its physiological efficiency. PM increased crop K uptake, and then increased grain K concentration by 4%. SM decreased grain N concentration by 4%, whereas it increased grain P and K concentration by 6% and 4%, respectively, and this was caused by the decreased P and K requirement, and increased their physiological efficiencies for the grain nutrient accumulation. Grain N concentration was increased by 8% with the GM, which was observed to be linked to the increased N physiological efficiency for grain nutrient accumulation. SGM had no effect on the grain N and P concentration, whereas it increased the grain K concentration by 4% due to the increased K physiological efficiency for grain nutrient accumulation. 【Conclusion】 Overall, PM decreased the N requirement and increased the N physiological efficiency for grain yield formation, thus increased the grain yield. However, PM increased N requirement and decreased its physiological efficiency for grain N accumulation, thus it was an unprofitable measure for increasing grain the N concentration. SM had an adverse impact on crop nutrient uptake, thus inhibited the grain yield formation and its nutrient accumulation. GM and SGM increased the nutrient requirement and decreased the nutrient physiological efficiency for grain yield formation, thus decreased the grain yield. GM was beneficial for increasing the grain N physiological efficiency for grain nutrient accumulation, thus increased the grain N concentration. Therefore, for improving winter wheat grain yield and nutritional quality with the plastic film mulching and straw mulching in dryland, increasing N fertilizer application rate should be a critical strategy. Planting green manure during summer fallow is an effective measure for improving soil fertility, but it has a potential risk of decreasing grain yield. To avoid the yield decrease, annual precipitation and its distribution over seasons should be considered when planting green manure during the fallow season.

Effects of Spraying Chemical Topping Agents on Canopy Structure and Canopy Photosynthetic Production in Cotton

YANG Cheng-xun, ZHANG Wang-feng, XU Shou-zhen, SUI Long-long, LIANG Fu-bin, DONG Heng-yi

Scientia Agricultura Sinica. 2016, 49(9): 1672-1684. doi:10.3864/j.issn.0578-1752.2016.09.004

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【Objective】 The objectives of this two-year field experiment were to study the effects of two chemical topping agents, Flumetralin and Mepiquat chloride, on the characteristics of plant type, canopy structure, canopy photosynthetic production and yield of cotton and to determine how the changes of canopy structure affect canopy photosynthetic production and yield. The results of this study will provide a theoretical basis for more effective use of chemical topping agents.【Method】The study included two cotton cultivars Xinluzao 45 and Zhangmiansuo 50 and one cotton strain 45-21. Conventional manual topping was used as the control treatment. Plant height, plant width, leaf area index (LAI), leaf chlorophyll contents, canopy light transmittance, canopy apparent photosynthetic rate, and yield components were among the variables that were measured. The effects of chemical topping technology on LAI, canopy light transmittance, canopy apparent photosynthetic rate and yield were studied. 【Result】 Plant height was significantly higher in the chemically-topped treatments than in the manually-topped treatment, plant width was significantly lesser in the chemically-topped treatments than in the manually-topped treatment, and lateral growth was significantly inhibited after spraying. The LAI and leaf chlorophyll contents were greater in the chemically-topped treatments than in the manually-topped treatment, the difference in LAI and leaf chlorophyll contents were extremely significant at the early boll opening stage. Light transmittance in the upper and middle canopy layers was greater in the chemically-topped treatments than in the manually-topped treatment. However, late in the growing season, light transmittance in the lower canopy layers was lesser than in the manually-topped treatment. Leaf chlorophyll contents increased to a maximum and then decreased at the end of the growing season. Chemical topping increased leaf chlorophyll content. Canopy apparent photosynthetic rates were significantly greater in the chemically-topped treatments than in the manually-topped treatment. Furthermore, canopy apparent photosynthetic rates remained high for a longer time in the chemically-topped treatments. At the early boll opening stage, canopy apparent photosynthetic rate in the chemically-topped treatments was 16.04 μmol·m^-2·s^-1 which was 14.35%-16.40% greater than those in the manually-topped treatment. Canopy respiration rates were significantly greater in the chemically-topped treatments than in the manually-topped treatment during the first part of the season. After reaching a maximum there was no significant difference among the treatments. The ratio of canopy respiration rate to total apparent photosynthetic rate was higher in the chemically-topped treatment than in the manually topped treatment. Chemical topping increased the number of bolls per plant. Cotton yield in the Flumetraline treatment was greater than that in the manually-topped treatment. 【Conclusion】Chemical topping technology can shape plant type and adjust the formation of cotton canopy structure. Compared with manual topping, chemical topping increased the LAI and the leaf chlorophyll content. The LAI and chlorophyll content both remained high for a relatively longer time in the chemically-topped treatments. Chemical topping improved light distribution in the canopy and increased the photosynthetic area, thus ensuring the chemically-topped treatments had the highest canopy apparent photosynthetic rate and the longest duration of photosynthetic activity.

Molecular Cloning Flanking Sequences of T-DNA Insertion from the Ustilaginoidea virens Mutant Strain B1241

BO Hui-wen, YU Mi-na, YU Jun-jie, YIN Xiao-le, DING Hui, WANG Ya-hui, LIU Yong-feng

Scientia Agricultura Sinica. 2016, 49(9): 1685-1695. doi:10.3864/j.issn.0578-1752.2016.09.005

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【Objective】The objective of this study is to explore the function of pathogenicity-relative gene basing on the phenotypes and T-DNA integration flanking sequence of a mutant strain B1241, and to provide a theory foundation for illustrating the pathogenic mechanism of Ustilaginoidea virens.【Method】 With the wild type strain P1 as the control, biological phenotypes of B1241 were analyzed. Artificial inoculation of mixtures of hyphae broken and conidia was made by injecting at the leaf sheath of the rice plant seven days before booting stage. Number of diseased grain per panicle was counted until false smut balls developed. After five generations of mutant strain B1241 inoculated on the PSA without hygromycin, the stability of T-DNA insertion was detected. Southern blot was used to identify the copy number of T-DNA in B1241. The flanking sequence of T-DNA was obtained by HiTail-PCR and the full length of the flanking gene was cloned by RACE-PCR. The gene expression was detected by quantitative RT-PCR.【Result】There was no significant difference in colony morphology and growth rate between B1241 and P1 culturing on MM, PSA and TB3. A field inoculation trials showed that the pathogenicity of B1241 was significantly declined compared with P1. After five generations of mutant strain B1241 inoculated on the PSA without hygromycin, gene GFP and HPH were still amplified, which indicated that T-DNA had been stably inserted into the genome of B1241. Genomic southern blot analysis confirmed that B1241 is a single T-DNA insertional event. Analysis of flanking sequence indicated that there were 28 bp sequences of U. virens loss in T-DNA insertion site and 37 base pairs were not found in T-DNA sequences and U. virens genome. Sequences analysis indicated that full length of flanking gene was 2 650 bp with a 14 bp of 5′ untranslated regions and a 319 bp of 3′ untranslated regions. The cloned flanking gene homologous with UV8b-7878 in strain UV-8b encodes a glycoside hydrolase family 18 protein which has a conserved domain D××D×D×E. The T-DNA insertion into the promoter region of Uvt-1241 led to lower transcription levels of gene Uvt-1241 in this mutant strain.【Conclusion】The mutant strain B1241 showed great changes on the conidia production and pathogenicity, probably due to inserted T-DNA in the promoter region of the gene. These results implied that gene Uvt-1241 might played an important role during the growth and pathogenic process of U. virens.

Analysis of Detoxification Enzyme Genes in the Multiple Pesticide-Resistant Strain of Tetranychus urticae

ZHOU Xing-long, SONG Li-wen, YANG Shun-yi, LI Jing-jing, WANG Jin-jun, ZHANG Xin-hu, SHEN Hui-min

Scientia Agricultura Sinica. 2016, 49(9): 1696-1704. doi:10.3864/j.issn.0578-1752.2016.09.006

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【Objective】Tetranychus urticae is an important mite in agricultural production, which is easy to produce resistance to pesticide because its small size and rapid propagation. The objective of this study is to discuss the change of mRNA relative transcript level, find out multiple resistant mechanism of T. urticae and provide a theoretical basis for its integrated management (prevention and control). 【Method】SS and Mp-R strain of T. urticae were raised in laboratory. Mp-R strain was continuously selected with median lethal concentration (LC₅₀) of mixture of spirodiclofen, fenpropathrin and abamectin. After spraying insecticide for 4-5 times, biological assay was done referring to leaf-residue method which recommended by FAO, LC₅₀ and resistance ratio were calculated, and a selected cycle was recorded. After 3 cycles, acaricides concentrations were gradually increased, then toxicity equation, LC₅₀, resistance ratio, and chi-square value were calculated by PoloPlus software. The activities of CarEs, GSTs and MFOs were measured by biochemical analysis in egg, larva, nymph and adult of SS and Mp-R strains of T. urticae. Ten ditoxifying genes expression which were related to resistance were measured with RT-qPCR technology using ELFn as reference gene through comparing Ct value method.【Result】After 50 generations of selection, LC₅₀ of T. urticae to abamectin, spirodiclofen and fenpropathrin reached 1 103.55, 5 993.33 and 2 345.62 mg·L^-1, and the resistance ratios were 603.03, 167.65 and 51.77 times, respectively. Activities of MFOs in egg of Mp-Rstrain increased significantly compared with the SS strain, but CarEs and GSTs activities had no significant difference. CarEs and GSTs activities at other stages of Mp-R strain increased significantly compared with the SS strain, but MFOs activity did not increase. In Mp-R strain, CarEs and GSTs activities of female adult mite and MFOs activity in egg increased significantly compared with other stages, and compared with the SS strain, the expression of TuGSTd05, TuGSTd06 and TuGSTd09 genes were significantly up-regulated by 1.8 times at all developmental stages of Mp-Rstrain, and the expression of TuGSTd01 gene was significantly up-regulated by 1.63 times in nymph of Mp-R strain, but at other developmental stages had no significant difference. CYP392E10 gene expression significantly increased by 5.87 times in egg, significantly increased by 2.15 times in larva and 2.09 times in nymph, but in adult non-significant. CYP392A6 gene expression significantly increased by 1.89 times in egg, 1.64 times in larva and 1.59 times in nymph, but in adult non-significant. CYP392A16 gene expression significantly increased by 6.97 times in egg, 8.20 times in larva, 8.88 times in nymph, 7.34 times in male adult and 8.59 times in female adult. CYP392D8 was significantly up-regulated by 2.18 times in egg, 2.00 times in larva, 2.03 times in male adult and 2.41 times in female adult, but in nymph non-significant. TuCCE35 significantly increased by 1.58 times in nymph, 1.86 times in male adult and 2.65 times in female adult. TuCCE36 significantly increased by 1.73 times, 1.89 times in male adult and 2.14 times in female adult.【Conclusion】Ten detoxifying genes expression had various changes at different developmental stages of Mp-R strain in T. urticae compared with SS strain. CYP392A16 might participate the resistance formation of different developmental stages of T. urticae, but other genes’ expression had no obvious change, whether or not involved in the metabolism of the 3 acaricides about these genes need to be further verified.

Dynamic Differences of Organic Carbon Mineralization in Different Types of Paddy Soil Under Long-Term Located Fertilization

WU Meng, LI Zhong-pei, FENG You-zhi, CHEN Rui-rui, JIANG Chun-yu, LIU Ming

Scientia Agricultura Sinica. 2016, 49(9): 1705-1714. doi:10.3864/j.issn.0578-1752.2016.09.007

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【Objective】The objective of this study is to explore dynamic differences and influence factors of organic carbon mineralization in different types of paddy soil under long-term located fertilization. The results could help us to illuminate the characteristics of organic carbon mineralization in different types of paddy soil and provide guidance for formulating reasonable nutrient management measures.【Method】In this study, the paddy soils in four different places were used, including Changshu (gleyed soils) in Jiangsu province, Yujiang (reddish soil of low fertility) in Jiangxi province, Wangcheng (reddish soil of high fertility) in Hunan province, and Chongqing (purple soil). The soils were collected in long-term fertilization location including three different treatments, as control (no fertilizer, CK), N, P and K fertilizer (NPK), N, P and K fertilizer plus rice straw (NPKS). The organic carbon mineralization laboratory incubation experiment was conducted in 27 days. The cumulative carbon mineralization was measured through alkali absorption method in constant incubator. The Jones first-order kinetic model was used to express the dynamics of cumulative carbon mineralization with respect to incubation time and was fitted to compare the influence of soil type and different applications of long-term fertilization on the dynamics of carbon mineralization.【Result】Long-term fertilization could improve the content of soil total N, P, K and organic carbon but decreased the soil pH. Long-term fertilization could significantly improve the cumulative carbon mineralization (C) except Changshu soil and different fertilizations caused the increase of 11.7%-86.1%. Two parameters in mineralization kinetic equation, potential mineralization of organic carbon (C0) and easy mineralization of organic carbon (C1), could be significantly improved by fertilization in the reddish paddy soil of Yujiang and Wangcheng. In the same fertilization treatment, C, C0 and C1 were bigger in Changshu and Wangcheng while smaller in Chongqing and Yujiang; Ratio of C0 to soil organic carbon (C0/SOC) was bigger in Yujiang. Correlation analysis indicated that soil organic carbon, total nitrogen, available nitrogen and total phosphorus showed a very significant positive correlation with C, C0 and C1 (P＜0.01); pH and total potassium showed a very significant negative correlation with C; C0/SOC showed a significant negative correlation with SOC, pH and all nutrient indexes. The fertilization treatments had no significant effect on mineralization rate constant (k) while the soil type affected k significantly. The k displayed a very significant negative correlation with pH, C/N, total K (P＜0.01) and a significant negative correlation with available K (P＜0.05). The relative influence of different parameters was obtained through aggregated boosted trees and the result showed that available nitrogen and soil organic carbon had a bigger impact on C and C0 with relative influence at 23.8%, 20.2% (for C) and 29.5%, 23.7% (for C0); soil organic carbon and pH had a bigger impact on C1 and C0/SOC with relative influence at 26.7%, 15.9% (for C1) and 34.4%, 23.0% for (C0/SOC); pH and total potassium had a bigger impact on mineralization rate constant (k) with relative influence at 34.2% and 22.1%, respectively.【Conclusion】The type of soil exerted a bigger impact on soil organic carbon mineralization dynamic than long-term located fertilization. The difference of parent material, pH, organic carbon and nutrient content of soil in different long-term fertilization locations account for the dynamic difference of organic carbon mineralization.

Selenium Content of Wheat Grain and Its Regulation in Different Wheat Production Regions of China

LIU Hui, YANG Yue-e, WANG Zhao-hui, LI Fu-cui, LI Ke-yi, YANG Ning, WANG Sen, WANG Hui, HE Gang, DAI Jian

Scientia Agricultura Sinica. 2016, 49(9): 1715-1728. doi:10.3864/j.issn.0578-1752.2016.09.008

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【Objective】 In order to determine wheat grain selenium content in different wheat production regions in China, and to investigate factors that influence grain selenium content and agronomic measures that could increase selenium content, a wheat grain sampling and investigation and then a field experiment of foliar-selenium application on wheat were conducted.【Method】A total of 655 field wheat grain samples including 73 spring wheat and 582 winter wheat samples from different wheat production regions in China were collected from 2008-2009, 2009-2010, and 2010-2011 wheat seasons, and grain yield was investigated and grain selenium content was measured. The field experiment was conducted in 30 comprehensive experimental stations of China Agricultural Research System on Wheat in 14 provinces (municipalities) from 2010 to 2011, including two treatments of foliar applied with water and 0.017% sodium selenite solution at the middle and late jointing stages in a randomized complete block design with 3 replicates, and selenium content of plant before jointing and grain yield and selenium content at harvest were measured. 【Result】Over the 655 wheat grain samples, the average selenium content was 64.6 µg·kg^-1, which was too low to meet the selenium requirement of populations who rely primarily on wheat-derived foods, and grain selenium content ranged from 0 to 821.0 µg·kg^-1, with the average of spring and winter wheat of 67.5 and 64.2 µg·kg^-1. Of these samples, 63%, 19% and 8% had selenium content be deficient, low and enriched, no sample was found to have selenium content reach the toxic level. Selenium of wheat grain in different regions exhibited higher content in northern regions than that in southern regions, while it was higher in western regions than in eastern regions. Results from the field experiments showed that foliar application of sodium selenite did not significantly affect wheat grain yield, with yield for no selenium application and foliar selenium application averaged 6 650 and 6 649 kg·hm^-2. Foliar selenium application significantly increased grain selenium content, when no selenium was applied, grain selenium content averaged 31.0 µg·kg^-1, applying selenium at 116 g·hm^-2increased the grain selenium content to an average of 647.8 µg·kg^-1, which reached selenium-enriched level, but still below toxic level. For 1.0 g Se·hm^-2applied, grain selenium content was increased by an average of 5.3 µg·kg^-1, and applying 51 g Se·hm^-2 could increase the selenium content of wheat grain from the average of 31.0 µg·kg^-1to above 300 µg·kg^-1. Grain selenium content was not influenced by yield, but under no selenium applied it was significantly correlated with the available soil selenium content in 0-20 cm soil layer and with selenium content of plant before jointing. For each 1.0 µg·kg^-1 increment of available soil selenium content from 6.3 to 30.7 µg·kg^-1, the grain selenium content without selenium applied increased by an average of 2.1 µg·kg^-1, and for each 1.0 µg·kg^-1increment of selenium content of plant before jointing from 0 to 147.2 µg·kg^-1, the grain selenium content without selenium applied increased by an average of 0.7 µg·kg^-1. Grain selenium content with selenium applied as well as selenium biofortification index were both significantly correlated with selenium content of plant before jointing. For each 1.0 µg·kg^-1increment of selenium content of plant before jointing, the grain selenium content with selenium applied increased by an average of 5.7 µg·kg^-1, and selenium biofortification index increased by an average of 0.043 μg·kg^-1(g·hm^-2)^-1. 【Conclusion】In agriculture production, applying selenium in soil before wheat jointing to increase the available soil selenium level, or applying foliar selenium fertilizer at the middle or the late jointing stage etc., are all effective measures to make the plant accumulate more selenium and to transfer it into grain at filling stage, and therefore increase the grain selenium content.

Characteristics of Background Emissions and Emission Factors N₂O from Major Upland Fields in China

XU Yu-xiu, GUO Li-ping, XIE Li-yong, YUN An-ping, LI Ying-chun, ZHANG Xuan, ZHAO Xun, DIAO Tian-tian

Scientia Agricultura Sinica. 2016, 49(9): 1729-1743. doi:10.3864/j.issn.0578-1752.2016.09.009

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【Objective】Published literatures regarding to N₂O emissions from upland fields in China were collected to establish a dataset. The established database was used to analyze the N₂O background emissions (emission from soil without fertilization), emission factors (EFs) and associated factors influencing the N₂O emissions from major upland fields (for example wheat fields, maize fields and vegetable fields) in China. The results will provide supports for estimating regional N₂O emission inventory and provide appropriate mitigation strategies.【Method】Subgrouping analysis and regression analysis were used to analyze the factors which influence N₂O background emissions (for example the soil nitrogen content and C﹕N ratio of the soil) and EFs (for example the nitrogen application level and fertilizer types such as nitrification inhibitors and slow release or controlled release fertilizers).【Result】(1) The N₂O background emissions from upland fields ranged between 0.70 and 3.14 kg N₂O-N·hm^-2. Either N₂O background emissions from wheat and summer maize fields or daily N₂O background emissions from vegetable fields increased with increasing soil total N (TN) while decreased with the increasing of soil C﹕N ratio. Irrigation could promote the N₂O background emissions from wheat fields. (2) EFs increased with increasing N application rate and its value was ranked in the following order: vegetable fields (0.56%-0.61%)＞summer maize fields (0.50%-0.68%)＞spring maize fields (0.35%-0.40%)＞wheat fields (0.22%-0.36%). EFs from summer maize fields were two times higher than those from wheat fields. (3) Nitrification inhibitors could decrease N₂O EF by 34%-60% ranking in the order: DCD+HQ (58.9%)＞NBPT+DCD (52.9%)＞DMPP (51.1%)＞NBPT (44.1%)＞Pyridine (39.5%)＞DCD (38.9%). The effects of different nitrification inhibitors on EF decline were varied in different fields ranking in the order: wheat fields (60.0%)＞vegetable fields (50.6%)＞spring fields (39.6%)＞summer maize fields (34.7%). (4) Compared with normal urea, slow release or controlled release fertilizers decreased EF to an extent between 15.9% and 79% ranking in the order: long-effect ammonium bicarbonate (78.9%)＞Polymer-coated urea (59.8%)＞Urea formaldehyde (53.4%)＞Resin-coated urea (44.9%)＞Sulfur-coated urea (30.6%)＞Calcium-magnesium-phosphate-coated urea (15.9%). The decline effects of different slow release or controlled release fertilizers on decreasing EFs varied in different fields ranking in the order: vegetable fields (78.4%) ＞spring maize fields (58.2%)＞winter wheat fields (49.2%)＞summer maize fields. The effect of slow release or controlled release fertilizers on decreasing EFs were lower in summer maize fields than in other croplands.【Conclusion】N₂O emissions are influenced by many factors including soil properties (i.e. soil TN content and C: N ratio), management practices (i.e. irrigation and fertilization) and other factors. In order to reduce N₂O emissions, it is critical to take appropriate practices specific to oriented climate, soil and cropping regimes and combined with appropriate rational nitrogen fertilizer application rate and type.

Present Situation and Prospect of Potato Processing in China

MU Tai-hua, CHEN Jing-wang

Scientia Agricultura Sinica. 2016, 49(9): 1744-1745. doi:10.3864/j.issn.0578-1752.2016.09.010

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Domestic and Abroad Research Progress of Potato Tuber-Specific Storage Protein Patatin

ZHANG Du-qin, MU Tai-hua, SUN Hong-nan

Scientia Agricultura Sinica. 2016, 49(9): 1746-1756. doi:10.3864/j.issn.0578-1752.2016.09.011

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Potatoes are the fourth main crops after wheat, rice, and corn. China is the world’s largest potato production country. Along with the development of the potato processing industry, studies on potatoes are going on gradually. Patatin is the potato tuber-specific storage protein, as well as the main part of the potato protein. In addition to its Lipid acyl hydrolase (LAH) and antioxidant activity, Patatin has excellent physico-chemical and functional properties such as solubility, emulsifying, foaming, and gelling properties, and is one of the most popular vegetable proteins. However, no overviews about the physico-chemical properties, the isolation, extraction and purification methods, the application in the food industry, and the future research hotspots of patatin were found up to now. In this paper, the molecular weight, structural characteristics, and physico-chemical properties of patatin were introduced, the method of isolation, extraction, and purification methods of patatin were summarized, and the advantages and disadvantages of each method were compared in order to provide a theoretical reference for the further application, research, and exploitation of patatin in the food industry. The content of patatin in potato protein ranged from 5.4% to 38.0%, relating to the cultivars and metabolic physiology of potato. The molecular weight of patatin varied from 39 kDa to 45 kDa, depending on the degree of glycosylation. Patatin is a kind of globulin with compact structure, and the primary structure contains more than 360 amino acid residues and remained unaffected by the surrounding elements, while the advanced structures were affected by temperature, pH, and so on. Patatin has excellent physico-chemical and functional properties such as solubility, emulsifying, foaming, and gelling properties, which were affected by surrounding elements. Gel chromatography and ion-exchange chromatography are the most common methods to isolate and purify patatin from potato protein, the reversed-phase chromatography (RPC), immunoaffinity chromatography, and gene expression can produce highly purified patatin. Although not suitable for large-scale production, expanded bed adsorption (EBA) chromatography can prepare large amount of patatin with low purity. Research studies on the application of patatin in the food industry are scarce. Patatin has great potential as food ingredients in the food industry, further research studies on Patatin will continue progress toward simplifying extraction methods, cutting the extraction cost, and developing foods, health products, and drugs with high-functional value.

Establishment of a Viscosity Reduction Process by Enzymatic Pretreatment and Its Application in Ethanol Fermentation from Fresh Sweet Potato Without Adding Water

YUE Rui-xue, NIU Fu-xiang, SUN Jian, XU Fei, WANG Hong-yun, ZHU Hong, ZHANG Yi

Scientia Agricultura Sinica. 2016, 49(9): 1757-1766. doi:10.3864/j.issn.0578-1752.2016.09.012

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【Objective】 The objective of this research was to establish a viscosity reduction process for fresh sweet potato (FSP) mash by enzymatic pretreatment before liquification and use it for direct ethanol fermentation from FSP without adding additional water. 【Method】 A total of 12 kinds of starch-type sweet potatoes (SP), widely cultured in China, were selected for this research. The main components of SP, such as dry matter, starch, protein, soluble sugars, pectin, hemicellulose, cellulose, and lignin, were detected and the correlation between these components and viscosity of FSP were analyzed. Based on the results, proper enzymes, cellulase and pectinase, were selected for viscosity reduction. Effects of some key factors of enzymatic pretreatment on viscosity reduction were investigated, including the ratio of substrate to water (1:0, 2:1, 3:2, 5:4, and 1:1), enzymatic pretreatment time (1, 2, 3, 4, and 5 h), enzymes doses (cellulase: 1.0, 1.5, 2.0, 2.5, and 3.0 GCU·g^-1 and pectinase: 1.0, 1.5, 2.0, 2.5, and 3.0 U·g^-1), and enzymatic pretreatment temperatures (30℃, 40℃, 50℃, 60℃, and 70℃). The optimum enzymatic pretreatment conditions were determined by orthogonal experiment design and the effects of enzymatic pretreatment on the following ethanol fermentation performance from FSP were also investigated. 【Result】 For 12 kinds of tested starch-type sweet potato varieties, except for hemicellulose (r=-0.239) and lignin (r=-0.069), all the components were positively correlated to the viscosity (dry matter content, r=0.356; starch, r=0.211, soluble sugar, r=0.307; protein, r=0.266; pectin, r=0.526; cellulose, r=0.600). Wherein the pectin (P＜0.01) and cellulose (P＜0.05) were significantly positively correlated to the viscosity. According to the range analysis, the factors affecting enzymatic treatment effects from high to low were enzyme dosage, the ratio of substrate to water, enzymatic treatment time, and temperature. Based on the experiment results and the consideration on the industrial application, the optimum enzymatic pretreatment conditions were determined as: the ratio of substrate to water 1﹕0, cellulase 1.5 GCU·g^-1, pectinase 1.5 U·g^-1, and 50℃ for 3 h. Besides Wansu 178, mash viscosity from other varieties after enzyme treatment was lower than 3 000 cp, and the viscosity reduction rate was 95.07%-99.31%. At the end of fermentation, the ethanol concentration from 11 kinds of SP varieties was higher than 12% (v/v), and the fermentation efficiency of 9 varieties reached more than 90%. 【Conclusion】 Pectin and cellulose are the main factors affecting the viscosity of SP mash. Fresh SP mash can be directly used for ethanol fermentation after enzymatic pretreatment without adding water.

Lactic Acid Fermentation by Lactobacillus rhamnosus from Sweet Potato Residue

LIU Yu-ting, WU Ming-yang, JIN Yan-ling, SHEN Wei-liang, FANG Yang, ZHAO Hai

Scientia Agricultura Sinica. 2016, 49(9): 1767-1777. doi:10.3864/j.issn.0578-1752.2016.09.013

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【Objective】Lactic acid fermentation process by Lactobacillus rhamnosus using sweet potato residue was studied in this paper in order to provide new technical strategy for utilization of sweet potato dregs.【Method】Firstly, the major elements of sweet potato starch residue were measured by elemental analyzer, meanwhile, there were four fermentation groups carried out with four different time points during the logarithmic growth phase of L. rhamnosus which was identified through OD value of bacteria, the optimal seed age was confirmed taking the fermentation efficiency of lactic acid production using liquid glucose culture as index of investigation. Then on this basis, the sweet potato dregs were used by L. rhamnosus through solid state fermentation (SSF) in the following research: firstly, the effects of inoculation amount,temperature, CaCO₃quantity, nitrogen sources on lactic acid production were investigated through single factor experiments. In addition, the effects of nitrogen sources on viable bacteria counts in fermenting mash after fermentation were studied also. Then the optimal combination was established by further orthogonal design.【Result】Element analysis results showed that sweet potato starch residue was full of C and H, with weight percentage content of 40.34% and 6.16%, respectively. The content of N was only 0.32%. Although C was quite rich in sweet potato starch residue, N was deficient comparatively, it was necessary to add N source to promote growth and metabolism of L. rhamnosus furthermore. The growth curve of L. rhamnosus identified by ODvalue showed that the OD value of L. rhamnosus increased rapidly after inoculated for 2 h, which means that the logarithmic growth phase began. The OD value wasn’t change approximately after 8 h, which means that the stationary growth stage began. It was confirmed that the logarithmic growth phase of L. rhamnosus was 2 h to 8 h. The fermentation efficiency was the highest, reached 92.39%, when the seed age got to 4 h, and the content of residual glucose was the lowest, reached 0.59 g·L^-1, it was confirmed that the optimal seed age of lactic acid fermentation was 4 h. The results of batch single factor experiment through SSF showed successively: The highest fermentation efficiency of 83.87% reached at 10%. The highest fermentation efficiency of 85.55% reached at 37℃, when CaCO₃quantity ranged from 1% to 7%, the highest fermentation efficiency of 90.24% reached at 5%; the fermentation efficiency of urea group reached 91.01% among 4 kinds of inorganic nitrogen source; when urea quantity ranged from 0.4% to 1.6%, the highest fermentation efficiency of 94.13% reached at 0.8%, the viable bacteria counts were 4.32×10⁸cfu/g. Based on the results of single factor experiments above, the optimal CaCO₃quantity of 5% was suitable for neutralizing the lactic acid produced by initial sugar in fermentation system of 10%, so it was unnecessary to change the optimal CaCO₃quantity, and it wasn’t investigated in the next orthogonal design. Besides, adding cellulase as investigation factor, designing the orthogonal experiment with four factors and three levels. Then, the fermentation optimum conditions were obtained, which were: the inoculation size 10%, urea amount 0.8%, cellulose amount 0.4%, temperature 35℃, CaCO₃quantity 5%, fermentation efficiency and viable bacteria counts, respectively, reached (96.55±0.866)% and 3.04×10⁸cfu/g under these conditions. 【Conclusion】A low-cost, simple-process, high-efficiency process condition was established which was not only suitable for lactate industry but also beneficial to sweet potato starch processing farmers.

Effects of NaCl Concentration and pH Value on the Emulsifying Properties of Sweet Potato Peptides

CUI Shan-shan, MU Tai-hua, SUN Hong-nan, ZHANG Miao, CHEN Jing-wang

Scientia Agricultura Sinica. 2016, 49(9): 1778-1786. doi:10.3864/j.issn.0578-1752.2016.09.014

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【Objective】 The study aims to clear the effects of different NaCl concentrations and pH values on the emulsifying properties of sweet potato peptides and provide a theoretical basis for the application of sweet potato peptides in the food industry. 【Method】Microstructure, average particle size (d_4,3), emulsifying activity, emulsifying stability, rheological property of emulsion and hydrophobicity of sweet potato peptides with different NaCl concentrations (0.2, 0.4, 0.6, 0.8 and 1.0 mol·L^-1) and different pH values (3, 5, 7 and 9) were measured. 【Result】Compared with the sweet potato peptide emulsion without NaCl, when 0.2 mol·L^-1 NaCl was added into the sweet potato peptide emulsion, d_4,3 was increased from 54.19 μm to 59.70 μm, the emulsifying activity index was decreased significantly from 86.29 m²·g^-1 to 56.35 m²·g^-1 (P＜0.05), and the emulsion stability index was decreased from 14.84 min to 13.19 min. However, with the increase of NaCl concentration, the uniformity of sweet potato peptide emulsion was decreased, d_4,3 was further increased from 59.70 μm to 69.72 μm, the emulsifying activity was decreased from 56.35 m²·g^-1 to 32.32 m²·g^-1, emulsifying stability was firstly increased and then decreased, reached a maximum of 15.55 min at 0.04 mol·L^-1, the initial apparent viscosity was increased and exhibited shear-thinning phenomenon, and surface hydrophobicity activity was decreased. In addition, with the increase of pH value, the uniformity of emulsion was increased, d_4,3 was decreased from 64.45 μm at pH 3 to 51.21 μm at pH 9, the emulsifying activity was increased from 3.99 m²·g^-1 at pH 3 to 120.47 m²·g^-1 at pH 9, the emulsifying stability was firstly increased and then decreased, reached a maximum of 29.13 min at pH 3, initial apparent viscosity was reduced and exhibited shear-thinning phenomenon, and the activity of surface hydrophobicity was increased. 【Conclusion】 Emulsifying properties of sweet potato peptides are closely related to the NaCl concentration and pH value. Addition of NaCl (≥0.2 mol·L^-1) reduced the emulsifying activity and stability of sweet potato peptides, while the increase of pH value could effectively improve the emulsifying activity of sweet potato peptides.

Identification of Purple Sweet Potato Color of Cultivar Ningzi No. 1 by HPLC-QTOF/MS and Its Effect on Preventing Obesity in High-Fat-Diet-Treated Rats

ZHANG Yi, WANG Hong-yun, NIU Fu-xiang, SUN Jian, XU Fei, ZHU Hong, YUE Rui-xue

Scientia Agricultura Sinica. 2016, 49(9): 1787-1802. doi:10.3864/j.issn.0578-1752.2016.09.015

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【Objective】Purple sweet potato are the main source for natural antioxidants. This study aims to identify the components of purple sweet potato color of cultivar Ningzi No. 1, and investigate the effectiveness of PSPC for the prevention of obesity and the regulatory mechanism of PSPC in obesity prevention.【Method】 Purple sweet potato color was analyzed using an Agilent 1290 series high-performance liquid chromatography connected to an Accurate-Mass Quadrupole Time-of-Flight/MS (HPLC-QTOF/MS). A pH-differential method was used to determine the total PSPC content. Eight-week-old male Sprague Dawley strain rats were fed on either a normal diet containing 11.4% fat, or a HFD containing 40% fat and intragastrically administered PSPC once a day. Body weights of the rats were measured every week after 6 h of fasting. Activities of serum glucose, triglyceride, and total cholesterol were determined using commercial kits. Serum leptin levels were measured using enzyme-linked immunosorbent assay kits. Expression of leptin and leptin receptor mRNAs in the hypothalamus were detected using Real-time Quantitative PCR. Expression of leptin and its downstream proteins in the hypothalamus were analyzed using Western blotting.【Result】Eleven constituents were separated. Based on previous reports, ten constituents were identified and Peak 1 was newly reported herein. The cyanidin 3-glucoside content of PSPC was 17.4%. After 6 weeks, we first examined the changes in body weight. Our data showed that the effect of middle-dose PSPC (371.6±16.3) g was better than low-dose PSPC (384.0±7.2) g, and the effect of high-dose PSPC (358.2±20.1) g was best. Obesity was prevented and glucose, triglyceride, and total cholesterol induced by the HFD were restored to near-normal levels. Compared with control group rats (2.29±0.28) ng·mL^-1, the levels of serum leptin in HFD group rats significantly increased by 65.50%. Compared with HFD group rats (3.79±0.24) ng·mL^-1, the levels of serum leptin in low (3.13±0.05) ng·mL^-1, middle (2.84±0.12) ng·mL^-1, and high-dose (2.64±0.06) ng·mL^-1 PSPC group rats were significantly decreased. Both of leptin and leptin receptor mRNAs expression in the hypothalamus was upregulated in the different-dose PSPC group compared with that in the HFD group. High-dose PSPC preserved the leptin signaling capability, leading to a decrease in hypothalamic AMPK α activity.【Conclusion】The anti-obesity effects of high-dose PSPC were mediated via a regulation of leptin/AMPKα signaling in the hypothalamus.

Cloning and Prokaryotic Expression of Porcine cGAS Gene

DU Li-li, FAN Shuang-shuang, LI Sai-sai, CHEN Pei-ge, CHEN Lei, SUN Shi-ping, FAN Wen-jie, WANG Jiang, WANG Yue-ying, ZHONG Kai

Scientia Agricultura Sinica. 2016, 49(9): 1803-1809. doi:10.3864/j.issn.0578-1752.2016.09.016

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【Objective】cGAS (cyclic guanosine monophosphate-adenosine monophosphate synthase), as a new type of nucleic transferase was found in mammalian cells recently. It can identify cytoplasmic DNA and catalyse ATP and GTP to generate the second messenger cGAMP, cGAMP binds to STING by dependent way, leading to activate transcription factor IRF3. Then the inherent immunity was starting. This experiment was conducted to construct a prokaryotic expression plasmid, pBbB3a-His6-NusA- cGAS, containing porcine cGAS gene. The gene expression was induced in E. coli, and the cGAS protein was obtained. Then a foundation of the research on the synthesis of cGAMP in vitro and its function in innate immune progress was made.【Method】 The open reading frame (ORF) of cGAS was amplified from the cDNA of porcine spleen, and cloned it into propionate inducible plasmid pBbB3a-His6-NusA-LIC by ligation-independent cloning (LIC) technology. Identification of individual clone was performed by bacteria liquid PCR followed by DNA sequencing. Plasmids were extracted from the confirmed bacteria and transformed into E.coli BL21 (DE3). When the bacteria grew to logarithmic phase, sodium propionate was used to induce the expression of His6-NusA- cGAS fusion protein, 20 mmol·L^-1 sodium propionate at 20℃, 180 r/min were induced by 2 h, 4 h, 6 h, 8 h, 10 h and 0 h, then the optimal induction time was determined. Sodium propionate at 0, 5, 10, 15, 20, 25, 30, 35, 40 and 45 mmol·L^-1, respectively, and at 20℃, 180 r/min induced for 6 h to determine the best induction concentration of sodium propionate. Under the conditions of 20, 30 and 37℃, 20 mmol·L^-1 sodium propionate was used at 180 r/min to cultivate for 6 h to determine the best temperature induction. The His6-NusA-cGAS fusion protein was induced by sodium propionate and identified by SDS-PAGE and Western Blot. 【Result】 (1) Porcine cGAS gene’ORF, which is 1 494 bp in length, was successfully cloned; (2) The propionate inducible plasmid pBbB3a-His6-NusA-cGAS was constructed; (3) At 37℃, 20 mmol·L^-1 sodium propionate to induce for 6 h, the His6-NusA-cGAS fusion protein’s expression amount was the highest. (4) His6-NusA-cGAS fusion protein was efficiently expressed in soluble form with a molecular weight of about 111.87 kD.【Conclusion】 These results indicate that cGAS fusion protein was successfully expressed in E.coli BL21 (DE3) and this will provide technology and methods for cGAS’s fusion protein expression in vitro.

Clone and Activity Analysis of Keratin 10(K10) Promoter in Mouse

ZHANG Jun-zhen, LIU Yu, JI Kai-yuan, YANG Shan-shan, HU Shuai-peng, LIU Xue-xian, FAN Rui-wen

Scientia Agricultura Sinica. 2016, 49(9): 1810-1817. doi:10.3864/j.issn.0578-1752.2016.09.017

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【Objective】 Keratin 10 (K10) is one of the molecular markers of melanosome transfer from melanocytes to the around keratinocytes, which can be used as the specific promoter for the research on the interactions between melanocytes and keratinocytes. Here, the strongest promoter of K10 will be screened to supply the evidence and basis for the research of the function of K10 and other relative genes. 【Method】 Genomic DNA was extracted from a mouse tail. Six fragments of K10 were amplified and subcloned into pMD18-T vector and sequenced. They were then cloned into pGL0 vector to construct pGL0-F1-F6, was transfected into 293T cells by liposome. The luciferase activity were used to identify the strongest promoter from the 6 fragments of K10 promoter (F1-F6), which was measured by luciferase reporter (GFP) from the cell lysis and the prediction of transcript factor binding sites by bioinformatics of the sequence. After the strongest promoter of K10 was identified, it was used as a specific promoter, which recombined with pGL0 (without CMV) to construct pGL0-F-CDK5. pGL0-F-CDK5 was transfected into keratinocytes of mouse by liposome and then Cyclin-dependent Kinase 5 (CDK5) expression was measured by Immunofluorescence chemistry, luciferase reporter (GFP) and quantitative real-time PCR from the cells, cell lysis and total RNA, respectively. 【Result】 Six fragments (F1-F6) of K10 were amplified, cloned and sequenced with the size of 1 201, 908, 664, 787, 790 and 656 bp, respectively. The luciferase activity of F4 with the size of 787 bp was the strongest as measured by the luciferase reporter gene in 293T cell, which suggested that F4 would be used as a specific promoter. But the luciferase activity of F1-F6 was weaker than that of CMV. In the F4 sequence, the basic conserved region (TATAAAA) and the transcript factor binding sites such as C/EBPβ, GATA, HSF and CAP were found by Promoter Scan software, which promotes the K10 expression in keratinocytes. After transfection, the result of luciferase activity measurement showed that pGL0-F4-CDK5 made GFP expression stronger than pGL0-basic-CDK5 with a significant difference（P＜0.01), and made CDK5 mRNA expression higher than pGL0-basic-CDK5 with a significant difference (P＜0.01), which suggested that F4 was the corn region of K10 promoter with the strongest activity. 【Conclusion】 F4 was identified as the corn region of K10 promoter to have the strongest activity in promoting CDK5 expression in keratinocyte, which suggested that F4 would be used as a specific promoter for the gene function research during the interaction between melanocyte and keratinocyte.

Development of Indirect ELISA for Antibody of Brucella abortus

WANG Fang, FENG Yu, ZHANG Ge, JIANG Hui, ZHU Liang-quan, DING Jia-bo

Scientia Agricultura Sinica. 2016, 49(9): 1818-1825. doi:10.3864/j.issn.0578-1752.2016.09.018

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