Scientia Agricultura Sinica

Study on the Genetic Basis of General Combining Ability with QTL Mapping Strategy

HU Wen-Ming, XU Yang, ZHANG 恩Ying, XU Chen-Wu

Scientia Agricultura Sinica. 2013, 46(16): 3305-3313. doi:10.3864/j.issn.0578-1752.2013.16.001

Abstract ( 510 )

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【Objective】 General combining ability (GCA) is an important indicator for assessing parents. Studying the genetic basis of GCA and the feasibility of mapping QTL related with GCA (QTLGCA) may provide a foundation in breeding. 【Method】 A NCII mating design, where a set of recombinant inbred lines (RIL) derived from two pure lines was used as the tested lines and several randomly selected pure lines were used as testers, and QTL mapping strategy was applied to investigate the genetic component of GCA, the impact factors for QTLGCA mapping and the relationship between QTLGCA and QTL controlling trait per se. 【Result】 Results indicated that when a trait was controlled by one pair of alleles, the GCA effect estimate of RIL and QTLGCA mapping were all associated with additive effect, dominant effect of QTL controlling trait per se and the alleles frequency in testers. When a trait was controlled by two pairs of additive/dominance alleles, the GCA effect estimate of RIL and QTLGCA mapping did not associate with whether the linkage between genes or not, and the impact factors of GCA estimate and QTLGCA mapping were the same as that in one pair of alleles. When a trait was controlled by two pairs of interactive alleles, both GCA estimate and QTLGCA mapping were all associated with the alleles frequency in testers, the major effect of QTL controlling trait and interactive effect between QTL. In addition, GCA estimate was also influenced by the recombination rate between loci. 【Conclusion】 The allele frequency in testers and relative size of effects of QTL controlling trait per se were important factors not only to GCA estimation but also to QTLGCA mapping, and the difference between QTLGCA mapping and QTL mapping also depended on the genetic architecture of quantitative traits and analyzsis method of mapping QTL.

Mining Method of the Upstream Open Reading Frames and Their Translational Regulation in the Plant Genomes

WANG Ting-Zhang, HU Wang-Xiong, XU Jian-Hong, XUE Qing-Zhong

Scientia Agricultura Sinica. 2013, 46(16): 3314-3323. doi:10.3864/j.issn.0578-1752.2013.16.002

Abstract ( 539 )

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Approximately 20% of plant genes have small upstream open reading frame (uORF), which are located in the 5' UTR of a mature mRNA. The mining methods, classification and evolution of the uORF homologous groups, and research progress of their translation regulation to downstream mORF were briefly introduced. Conserved peptide uORF (uORF) are ubiquitous in plants (Arabidopsis and rice) and insects (fruit fly) genomes, and they have similar diversity of sequences, but differ in their average length, genome clustering, and their preferential association with methyltransferases. Plants usually have shorter uORF than that of insects. Comparative analysis of the full-length cDNA sequences between Arabidopsis and rice showed that homology groups of uORF can play the regulatory functions on the downstream mORF, such as transcription factors, signal transduction factors, developmental signal molecules, and translation initiation factor eIF5. The Bayesian phylogenetic analysis of the uORF with the CHCH domain in the eukaryotic genomes showed that the homologous group 8 -like could be divided into five branches of taxa (green algae, arthropods, nematodes, invertebrates and fungi). uORF families could act as trans factors to regulate the expression of major ORF during the growth, development, and physiology in the eukaryotes. Quite large ratio of uORF can regulate the translation of mORF in response to the concentrations of polyamine, sucrose, phosphatidyl choline, and methyl transferase in plant genomes.

Advances and Prospects in Research of Biochar Utilization in Agriculture

CHEN Wen-Fu, ZHANG Wei-Ming, MENG Jun

Scientia Agricultura Sinica. 2013, 46(16): 3324-3333. doi:10.3864/j.issn.0578-1752.2013.16.003

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Biochar has a bright prospect due to its good structure, physicochemical properties and broad raw materials of its production. It has already been a hotspot in the fields of agriculture, energy and environment. The influences of biochar were reviewed comprehensively on soil, crops, agricultural eco-system, and its important roles in food security of China. The application value and industrialization of biochar in agriculture from the low-carbon, recycle and sustainable point of view were discussed. Utilization of biochar would play much more important roles in improving soil obstacles and increasing crop production capacity of soil, which will benefit the sustainable development of agriculture and the national food security of China. At the end, the prospective of biochar industrialization and development in China were proposed, which will provide relevant references for the well development of biohcar industry.

A Method for Correcting the Meteorological Data from Regional Climate Model and Its Application in Crop Simulation

吕Zun-Fu , LIU Xiao-Jun, TANG Liang, LIU Lei-Lei, CAO Wei-Xing, ZHU Yan

Scientia Agricultura Sinica. 2013, 46(16): 3334-3343. doi:10.3864/j.issn.0578-1752.2013.16.004

Abstract ( 437 )

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【Objective】A method for correcting Regional Climate models (RCMs) output was developed in this research, which will lay a foundation for crop model prediction under future climate scenarios. 【Method】 The daily rainfall intensity, rainfall frequency, solar radiation, maximal and minimal temperatures output data of RegCM3 from 1994 to 2010 were corrected based on the historical daily weather records from 1960 to 1993 in Xuzhou, Huai’an, Zhengzhou, Weifang and Shijiazhuang. 【Result】 Compared with the raw meteorological data from RegCM3, the corrected monthly mean meteorological variables, the corrected monthly meteorological variables, and the probability distribution of daily rainfall, temperature and solar radiation from 1994 to 2010 agreed better with the observed values, especially for the extreme high temperature and high rainfall frequency. In addition, dry spell length after correction in five eco-sites was consistent with the observed values. The determination coefficient and normalized root mean square error (NRMSE) between observed and simulated yields from WheatGrow model with daily corrected RegCM3 at above five eco-sites reached 0.72 and 10.5%, which were 0.35 higher and 8.2% lower than those with daily raw RegCM3 outputs, respectively.【Conclusion】Therefore, the correction method in this research could be further used to correct the future meteorological data which generated by RegCM3, and the corrected meteorological data could be taken as the input data of crop model to improve the prediction accuracy of crop model.

Analysis of Biological Phenotypes and Molecular Cloning of T-DNA Integration Flanking Sequences of Ustilaginoidea virens Mutant Strain B-726

HUANG Lei, YU Mi-na, HU Jian-kun, YU Jun-jie, YIN Xiao-le, NIE Ya-feng, CHEN Zhi-yi, LIU Yong-feng

Scientia Agricultura Sinica. 2013, 46(16): 3344-3353. doi:10.3864/j.issn.0578-1752.2013.16.005

Abstract ( 442 )

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【Objective】 The objective of this study is to analyze the phenotypes and T-DNA integration flanking sequence of a mutant strain B-726 and to clone a novel gene which normal expression is disrupted by T-DNA insertional event, understand the role of the gene in U. virens molecular pathogenic process, and to develop a new strategy for controlling rice false smut. 【Method】 Biological phenotypes of B-726 were analyzed by testing growth rate, sporulation ability and pathogenicity. The copy number of T-DNA inserted in B-726 was identified by Southern blot. The flanking sequence of T-DNA was cloned using TAIL-PCR and the completed gene sequence in the flanking was cloned by RACE-PCR. The gene expression was detected by RT-PCR. 【Result】 Phenotypic analysis of B-726 showed that the pathogenicity of B-726 was significant reduced, the sporulation ability and growth rate were declined. Genomic Southern bolt analysis confirmed that B-726 was a single T-DNA insertional event. The T-DNA insertion site was in the 344 bp upstream of a gene named Uvt-726. RT-PCR analysis confirmed that Uvt-726 transcriptions were expressed in B-726 significantly decreased compared to P1. 【Conclusion】 A novel gene maybe associated with pathogenicity of U. virens was cloned, and these results imply that the Uvt-726 might play an important role during the pathogenic process of U. virens.

Evaluation of Resistance to Powdery Mildew in 68 Chinese Major Wheat Cultivars and Postulation of Their Resistance Genes

YANG Li-Jun, ZENG Fan-Song, GONG Shuang-Jun, SHI Wen-Qi, ZHANG Xue-Jiang, WANG Hua, XIANG Li-Bo, YU Da-Zhao

Scientia Agricultura Sinica. 2013, 46(16): 3354-3368. doi:10.3864/j.issn.0578-1752.2013.16.006

Abstract ( 516 )

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【Objective】 Resistance of 68 major wheat cultivars, each of which is planted at least over 70 thousand hectares yearly in China, to powdery mildew was analyzed based on resistance frequencies to mildew population from different provinces/ autonomous regions and whole China in order to evaluate the risks of mildew epidemics. Their resistance genes were also postulated.【Method】1 094 wheat single colony powdery mildew isolates were collected from 12 provinces (autonomous region) which grow wheat in China in the spring of 2011. Resistance frequencies of 68 cultivars were estimated by the mean of inoculating detached leaves segments of each cultivar with 1 094 isolates, respectively. Cluster analysis was carried out with UPGAMA (unweighted pair group arithmetic mean analysis) model of the software NTSYSpc2.10e based on the phenotypic resistance data. Resistance genes of the 68 cultivars were postulated by comparing resistance spectra of the cultivars to 31 differential isolates with those of materials possessing known resistance genes to the isolates. 【Result】 The resistance spectrum of each cultivar differed significantly from that of others. Resistance frequencies of Neimai8, Neimai9 and Mianmai37 were higher than 99%, while those of Jimai22, Yangmai11, Yangmai12, Yangmai13 and Lunxuan987 were between 70% and 90%. Among the 68 cultivars, 54 cultivars counting for 79.4%, had resistance frequencies lower than 40%, suggesting the resistance of these cultivars had been overcome. The resistance frequency of a cultivar to mildew population from the region where the cultivar was grown was lower than that to the mildew population from the region where the cultivar was not grown. Cluster analysis indicated 68 cultivars could be classified into four groups. Group one included 6 cultivars and out of them, resistance frequencies of 5 cultivars to all isolates were between 40% and 70%. Group two included 7 cultivars with resistance frequencies higher than 70%. Group three composed of 54 cultivars with resistance frequencies lower than 40%. The fourth group had only one cultivar with resistance frequency of 46.1%. The cultivars came from the same province and the cultivars with the similar resistance frequency had been clustered to the same group. It showed that they may have a similar resistance genetic background. Results of Pm postulation showed that Neimai8 and Neimai9 contains Pm21, Yanzhan4110, Xinmai208 and Yangmai11 possess Pm4b, Jimai22 possesses Pm2+ta, while the rest cultivars contain other unknown resistance factor(s) which could not be postulated. 【Conclusion】Nearly 80% of the cultivars wildly grown in China have a lower resistance frequency to wheat powdery mildew population. Referring to a cultivar, it was vulnerable to mildew population from the area where the cultivar is grown. There would be a risk of wheat powdery mildew epidemic if weather and other conditions favor for mildew epidemic. Many cultivars from the same region were clustered into the same group, it indicated that the resistance heterogeneity is lower in wheat grown in the region.

Prokaryotic Expression and Polyclonal Antibody Preparation of Trichothecene 3-O-Acetyltransferase Gene Tri101

DONG Fei, JI Fang, WU Ji-Rong, YIN Xian-Chao, XU Jian-Hong, SHI Jian-Rong

Scientia Agricultura Sinica. 2013, 46(16): 3369-3376. doi:10.3864/j.issn.0578-1752.2013.16.007

Abstract ( 425 )

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【Objective】Tri101 was cloned and the purified protein was obtained by prokaryotic expression. Polyclonal antibody of high titer and high sensitivity was obtained by SPF rabbits immunization, thus providing a basis for the establishment of the transgenic plants and products.【Method】Tri101 was obtained from Fusarium graminearium Schwabe F3210 by PCR and connected to the expression vector pET29a (+), the recombinant vector was transformed into E. coli BL21(DE3) and induced by IPTG. The results indicated that the recombination expression plasmid was constructed and transformed into E. coli BL21 (DE3). The Tri101 protein was induced by IPTG and used as the antigen to immune the SPF rabbits. 【Result】The result of SDS-PAGE showed that the molecular weight of expressed protein was about 50 kD, existing in the form of inclusion body. The expressed protein was purified by HisTrap, and the best imidazole concentration was 200 mmol•L-1. His-tag present in recombinant protein was removed by TEV protease and the digestion product was purified by HisTrap. The purified protein was used to prepare polyclonal antibody. The result of ELISA showed that the titer of rabbit anti-Tri101 antiserum was 1﹕12 000，the detection sensitivity was 0.05×10-6 µg•mL-1. The result of Western blotting demonstrated that polyclonal antibody had a very good specificity.【Conclusion】Through Tri101 cloning and prokaryotic expression, the purified fusion protein was prepared by immunizing SPF rabbits. Polyclonal antibody was obtained and used for the establishment for the transgenic wheat and products.

Resistance of Field Population Aphis gossypii Glover to Carbosulfan and Imidacloprid in Xingtai and Nanjing District

WANG Na, LANG Zhi-Fei, HE Kang, LI Fei

Scientia Agricultura Sinica. 2013, 46(16): 3377-3383. doi:10.3864/j.issn.0578-1752.2013.16.008

Abstract ( 421 )

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【Objective】 The objective of this study is to monitor the insecticide resistance of Aphis gossypii Glover in filed to carbosulfan and imidacloprid, and to provide a better understanding of resistance mechanism. 【Method】The cotton aphids were collected from fields in Nanjiang city of Xinjiang Uygur Autonomous Region (XJ), Xingtai city of Hebei Province (HB), and Nanjing city of Jiangsu Province (JS). The resistances of field cotton aphid to conventionally used insecticides imidacloprid and carbosulfan were investigated. The cotton aphids from Xinjiang were treated as the sensitive strain. The bioassay, enzyme activities and mRNA relative expression levels by qPCR were performed to study the resistance ratios and their potential molecular mechanisms. 【Result】A. gossypii of Xingtai city of Hebei Province (HB) and Nanjing city of Jiangsu Province (JS) were still sensitive to carbosulfan, but their insecticide resistance to imidacloprid reached 4.87 and 8.72 folds. Three field populations of cotton aphids had significantly different activities of oxidases, esterase and glutathione-S-transferase. The activities of esterase in Nanjing city of Jiangsu Province were the highest among three field populations. Compared with XJ strain, quantitative real-time PCR indicated that the relative expression level of carboxylesterase gene was the highest, which was 3.28-fold higher. 【Conclusion】The cotton aphid has developed low-level resistance to imidacloprid. The increased esterase activity confers the low-level resistance to imidacloprid.

Molecular Clone and Its RNA Interference Effect Analysis of Lactate Dehydrogenase Gene in Meloidogyne incognita

MEI Mei, HUANG Yong-hong, MAO Zhen-chuan, LIU Zhi-min, XIE Bing-yan

Scientia Agricultura Sinica. 2013, 46(16): 3384-3391. doi:10.3864/j.issn.0578-1752.2013.16.009

Abstract ( 338 )

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【Objective】The objective of this study is to clone and analyze the lactate dehydrogenase gene in Meloidogyne incognita (mildh), and to determine the inhibitory effect of mildh silencing on the diseases caused by Meloidogyne spp..【Method】In the preliminary studies, the number of RNA interference (RNAi) phenotype genes of nematodes in their genomes were predicted by bioinformatics methods. In this study, using special primers designed based on the predicted nematode ldh, the ldh in M. incognita (mildh) were cloned which was transferred into tomato plantlets by virus-induced gene silence (VIGS) technique after analyzing the sequence characters to investigate the influence of the mildh silencing on the amounts of root knot on the tomato plantlet inoculated with M. incognita.【Result】The cloned mildh contained a complete coding sequence that coded 315 amino acids. The identity between the cloned mildh and the predicted mildh was as high as 94.9% in nucleotide sequence and 95.5% in the deduced amino acid sequence. The silencing vector pTV-mildhi, mediated by Tobacco rattle virus (TRV), was constructed. The tomato plantlets were inoculated with M. incognita after treated by pTV-mildhi. Sixty days later, statistical analysis showed that the amounts of root knot on tomato plantlets of which the mildh was silenced decreased by 48.6% compared to that of the empty vector control, by 48.4% compared to that of the water control. 【Conclusion】The study revealed the mildh silencing had a significant inhibitory effect on root-knot nematode disease caused by M. incognita. It also showed that mildh genes may be involved in the pathogenesis of nematode, which provided a theoretical basis to the research of nematode pathology and to the control of nematode disease.

Characteristics of Mineralization and Nitrification in Neutral Purple Paddy Soil from a Long-Term Fertilization Experiment

QIN Zi-Xian-12, ZHANG Yu-Ting-12, ZHOU Zhi-Feng-12, SHI Xiao-Jun-12, GUO Tao-12

Scientia Agricultura Sinica. 2013, 46(16): 3392-3400. doi:10.3864/j.issn.0578-1752.2013.16.010

Abstract ( 524 )

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【Objective】The objective of the experiment is to reveal the effects of long-term fertilization on nitrogen internal cycle of neutral purple paddy soil ecosystem and the ways maintaining soil nitrogen supplying potentials. 【Method】 A long-term fertilization experiment lasted for 21 years was carried out in Beibei District, Chongqing City. Waterlogging incubation method proposed by Warning was used to investigate N mineralization characteristics. The total number of soil nitrobacteria, the potential nitrification rate and the intensity of soil nitrification were studied.【Result】Nitrogen accumulations and nitrogen mineralization potential (N0) were significantly improved under the chemical fertilizer (except N) or organic fertilizer or combined application of organic manure and inorganic NPK, and combined application of organic manure and inorganic NPK was the highest (164.43 mg•kg-1), nitrogen mineralization potential (N0) improved by 59.29%, as compared to CK. But the increasing extent of combined chloride-containing fertilizer and organic manure was much lower than the fertilization of organic manure and inorganic NPK. Different fertilization in all treatments (except NPK or M) increased the rate of mineralization (k). Compared to CK, the total number of soil nitrobacteria was significantly improved under fertilizers N, especially organic manure and inorganic NPK fertilizer combined application, which was about 74.25 times of CK. The potential nitrification rate and the intensity of soil nitrification showed a consistent trend, which applied the organic manure and inorganic NPK fertilizer had the best effect, and combined chloride-containing fertilizer and organic fertilizer was significantly lower than CK. Soil pH affected significantly the potential nitrification rate and the intensity of soil nitrification, with a correlation coefficient of r =0.925 (P<0.01) and r =0.773 (P<0.05). 【Conclusion】 Long-term combined application of organic manure and inorganic NPK is considered the best way for maintaining the soil fertility, chloride-containing fertilizer inhibits nitrification process and it can be used as nitrification inhibitor.

Effects of Deep Rotary Sub-Soiling Tillage on the Physical Properties and Crop Growth of the Sticky Loamy Soil in North China

YANG Xue-1, PANG Huan-Cheng-1, LI Yi-Bing-1, REN Tian-Zhi-1, DONG Guo-Hao-2, GUO Zhi-Hui-2, WANG Xiang-Jun-2

Scientia Agricultura Sinica. 2013, 46(16): 3401-3412. doi:10.3864/j.issn.0578-1752.2013.16.011

Abstract ( 457 )

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【Objective】This study aims at exploring the feasibility of application of deep rotary-subsoiling tillage on crop growth in North China concerning the problems of shallow plough layer, solid plough pan and low soil productivity resulted from the application of rotary tillage in successive years.【Method】Four treatments including rotary tillage 15 cm, interval sub-soiling 30 cm, deep rotary sub-soiling 30 cm, and deep rotary sub-soiling 50 cm were designed in an experiment carried out in Wuqiao of Hebei province to study the effects of different tillage treatments on soil water, soil bulk density changes and crop yield. 【Result】 The results indicate that different tillage treatments greatly affected soil water storage. Deep rotary sub-soiling treatments benefited the precipitation infiltration and water storage, especially under drought conditions. At the turning green stage of the winter wheat, the soil water storage of deep rotary sub-soiling treatments was significantly higher than that of the rotary tillage. Soil water storage of the deep rotary sub-soiling treatment at 50 cm and at 0-50 cm soil layer was 11.69 mm higher than that of the rotary tillage treatment and 10.56 mm higher than the interval sub-soiling treatment, improved by 12.21% and 10.90%, respectively. Different tillage treatments greatly affected soil bulk density. A year and a half after tillage, the soil bulk density of all deep rotary sub-soiling treatments was significantly lower than that of the rotary tillage and interval sub-soiling treatment at 0-30 cm soil layer. Soil bulk density of deep rotary sub-soiling at 50 cm treatment was significantly lower than other treatments at 30-50 cm soil layer. The figure of deep rotary-subsoiling tillage was lower than that of the rotary tillage treatment before winter wheat seeding while showed no difference afterwards. Different tillage treatments affected soil physical property, which further influenced the crop LAI, matter production, grouting process and yield. In all soil tillage treatments, the crop yield of the deep rotary sub-soiling treatment was significantly higher than that of the rotary tillage and interval sub-soiling. After one and a half year, the summer maize yield of the deep rotary sub-soiling at 50cm reached 13 822.68 kg•hm-2 and 13 127.87 kg•hm-2 of that at 30 cm, which is 38.19% and 31.24% higher than conventional rotary tillage, respectively. 【Conclusion】 Deep rotary sub-soiling tillage at 30cm is the most suitable method in this area considering the ecological and economic factors.

Screening of ‘Jincheng’ Orange CTV Response Genes Using Subtractive Suppression Hybridization

CHENG Chun-Zhen-12, ZENG Ji-Wu-2, BEI Xue-Jun-12, WU Bo-12, YANG Jia-Wei-3, ZHANG Yong-Yan-12, JIANG Bo-2, ZHU Shi-Ping-1, YAN Shu-Tang-1, ZHONG Yun-2, ZHONG Guang-Yan-2

Scientia Agricultura Sinica. 2013, 46(16): 3413-3423. doi:10.3864/j.issn.0578-1752.2013.16.012

Abstract ( 379 )

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【Objective】The objective of this study is to make clear the molecular mechanism and screen citrus tristeza virus (CTV) response genes in CTV susceptive citrus species ‘Jincheng’ orange [Citrus sinensis (L) Osbeck].【Method】 A suppression subtractive hybridization (SSH) library of ‘Jincheng’ orange was successfully constructed by using the virus-free leaves as Driver and those infected with CTV as Tester. 【Result】After sequencing of the randomly picked 850 clones, 742 clones were successfully sequenced and a total of 692 ESTs were obtained after Vectorscreen and adaptors deletion. All these ESTs were then matched to citrus genome transcripts and a total of 137 genes were identified. BLAST2GO gene ontology and KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis results showed that the basic energy metabolisms of ‘Jincheng’ orange were significantly influenced with differentially expressed genes (DEGs) involving in carbon fixation in photosynthetic organisms, nitrogen metabolism, starch and sucrose metabolism, etc. Moreover, DEGs largely involved in defense and resistance and some plant defensins synthesis related pathways such as carotenoid biosynthesis, phenylpropanoid biosynthesis, and phenylalanine metabolism. 【Conclusion】Results generated in this study suggested that CTV susceptible species ‘Jincheng’ orange reacted severely to CTV infection to maintain its growth and development by elevating energy metabolisms and enhancing the expression of stress resistance related genes.

Effects of Chitosan Treatments on Inhibition of Superficial Scald and Quality of European Pears (Pyrus communis L. cv. Early Red Comice) During Storage

WANG Bao-Gang, HOU Yu-Ru, LI Wen-Sheng, YANG Jun-Jun, SHI Lei, YANG Yuan, FENG Xiao-Yuan

Scientia Agricultura Sinica. 2013, 46(16): 3424-3431. doi:10.3864/j.issn.0578-1752.2013.16.013

Abstract ( 445 )

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【Objective】The control method and development regularity of superficial scald of Early Red Comice during storage were investigated in this paper. 【Method】The effects of water-soluble chitosan (WCTS) or acid-soluble chitosan (ACTS) treatment on the development of superficial scald and quality of European pear (Pyrus communis L. cv. Early Red Comice) were investigated during storage at (0±0.5)℃. 【Result】Superficial scald leads to plasmolysis and ultimately death of fruit skin cell of pear fruit. Afterwards, brown or black lesions appeared on the fruit surface. Compared with WCTS treatment, ACTS could more effectively inhibit the development of superficial scald of pear fruit during storage. Superficial scald incidence in ACTS-treated or WCTS-treated fruit was 63.89% or 44.44% lower than that in control on the 120 d of storage. The accumulation of α-farnesene or conjugated trienes in fruit peel was reduced, membrane lipid peroxidation was weakened by chitosan treatments. The respiration rate of fruit treated with chitosan was lower than that of control. The accumulation of the reducing sugar content was enhanced, but the decline of TA content was inhibited by chitosan treatments in the early days during the storage period. 【Conclusion】Chitosan treatment could inhibit the development of superficial scald of Early Red Comice pear, and ACTS treatment was better than WCTS treatment. Chitosan treatment could be used as an effective and convenient method for control superficial scald development of Early Red Comice pear.

Applications of Solid Sampling Analytical Technologies of Elements for Quality and Safety of Agri-Products

MAO Xue-Fei-1, LIU Ji-Xin-2, WANG Min-1, QIAN Yong-Zhong-1

Scientia Agricultura Sinica. 2013, 46(16): 3432-3443. doi:10.3864/j.issn.0578-1752.2013.16.014

Abstract ( 534 )

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Solid sampling technology without digestion treatments is one vigorous branch of elements analytical methods for agri-products, which is able to reduce the limit of detection (for direct solid sampling method), simplify the pretreatment, avoid loss of trace elements, and being more environmental friendly and safer. This paper covered the current developments of sample pretreatment, sample introduction and instrumental methods of the solid sampling technology, and discussed the key problems of sample homogeneity and matrix interference; the applications of direct solid sampling and slurry sampling techniques, and electrothermal vaporization, laser ablation, direct sample insertion, neutron activation analysis, X-ray fluorescence, etc. for heavy metals and other elements analysis in agri-products were reviewed.

Methionine and Lysine Requirement of Campbell Sheldrake Ducks from Forty-Two to Sixty-Two Days of Age

YE Hui, WU Ai-Ji, DENG Yuan-Fan, WANG Wen-Ce, YANG Lin

Scientia Agricultura Sinica. 2013, 46(16): 3444-3451. doi:10.3864/j.issn.0578-1752.2013.16.015

Abstract ( 476 )

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【Objective】 An experiment was conducted to investigate dietary methionine and lysine levels on growth performance, slaughter performance and antioxidant capacity in Campbell Sheldrake ducks from 42 to 62 days, and estimate the optimal dietary methionine and lysine levels for Sheldrake ducks.【Method】A total of 936 42-day-old ducks were randomly designed to 3×3 treatments with 4 replicates of 26 birds each. Ducks were fed with diets containing methionine at 0.28%, 0.38%, and 0.48% and lysine at 0.58%, 0.73%, and 0.88%, respectively, for 21 days.【Result】The interaction of dietary methionine and lysine had no effect on growth performance of meat ducks (P＞0.05), but had a significant effect on carcass rate (P＜0.05). Moreover, methionine and lysine interaction had significant effects on hepatic SOD activity and hepatic GSH-Px activity (P＜0.05), which Met 0.38% and Lys 0.88% group showed the most significant effects. The interactions of dietary methionine and lysine had no effect on hepatic level of MDA (P＞0.05). 【Conclusion】 The dietary methionine recommended for Campbell Sheldrake duck at the period of 42-62 days is 0.38%, and the Lysine recommended is 0.73% (ME 11.49 MJ•kg-1; CP16.44%).

The Influences of Over-Expressing miR-137 on TYRP-1 and TYRP-2 in Melanocytes

MA Shu-hui, XUE Lin-li, XU Gang, HOU Ya-qin, GENG Jian-jun, CAO Jing, HE Xiao-yan, WANG Hai-dong, DONG Chang-sheng

Scientia Agricultura Sinica. 2013, 46(16): 3452-3459. doi:10.3864/j.issn.0578-1752.2013.16.016

Abstract ( 484 )

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【Objective】Coat color is one of the most important economic characters. There are as many as hundreds of regulation genes involved in the formation process of coat color, and the mechanism(s) have not been completely determined. TYRP-1 and TYRP-2 are involved in regulation and control of melanin synthesis, and they are members of tyrosinase family. MITF is inhibited by miR-137 at the translational level, however, TYRP-1 and TYRP-2 were regulated by MITF at the transcriptional level. In this study, the effect of miR-137 on the production of TYRP-1 and TYRP-2 by regulating MITF was studied. 【Method】Over-expression of miR-137 was conducted through the cell transfection technique and the contents of melanin in melanocytes were extracted, and then the level of TYRP-1 and TYRP-2 was detected using RT-PCR and western blot. 【Result】 The results showed that the contents of melanin were significantly reduced. TYRP1 mRNA was significantly reduced to 0.4 times and TYRP-2 mRNA was significantly reduced to 0.6 times. TYRP-1 protein was significantly reduced to 0.61 times and TYRP-2 protein was significantly reduced to 0.73 times. 【Conclusion】 Results of the study suggested that miR-137 indirectly regulates the variation of YRP-1 and TYRP-2 by regulating MITF, and the miR-137 may indirectly mediate the alteration of coat color through regulating the expression of TYRP-1and TYRP-2.

Development of iELISA for Detection and Application of Brucella abortus Virb8

ZHANG Jian, MA Wei-ming, ZHANG Liang, HE Hong-bin, DING Jia-bo, YANG Hong-jun

Scientia Agricultura Sinica. 2013, 46(16): 3460-3469. doi:10.3864/j.issn.0578-1752.2013.16.017

Abstract ( 398 )

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【Objective】 Brucella abortus Virb8 is an important Brucella virulence factor. The aim of this study was to construct a prokaryotic expression vector for Brucella Virb8. Then, an indirect enzyme-linked immunosorbent assay (iELISA) for detecting antibodies to Brucella abortus was established using the purified recombinant protein. 【Method】 Brucella S2 genome sequence was extracted and used as a templet in polymerase chain reaction to amplify Virb8 gene. The PCR product was cloned into the pEASY-T3 vector and subsequently sequenced. Then, the gene was cloned into the expression vector pET-32a to construct the recombinant plasmid pET-32a-Virb8. The fused protein pET-32a-Virb8 was expressed and purified by Ni-NTA Spin Kit. Serum samples (n=235) were simultaneously tested by the iELISA and the rose bengal plate test (RBPT). 【Result】 In this study, Brucella Virb8 was successfully cloned and highly purified protein was obtained. The established iELSIA was stable and sensitive. The coincidence of iELISA with RBPT was 97.02%. 【Conclusion】 The iELISA could be used as a method for serological diagnosis of brucellosis and a foundation for advance study on immunogenicity and protection of brucellosis.

Development and Identification of ELISA Kit for Norfloxacin Determination

XING Guang-Xu-12, WANG Guo-Dong-3, HU Xiao-Fei-2, WANG Fang-Yu-2, DENG Rui-Guang-2, ZHANG Gai-Ping-2

Scientia Agricultura Sinica. 2013, 46(16): 3470-3477. doi:10.3864/j.issn.0578-1752.2013.16.018

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【Objective】The objective of this experiment is to develop an ELISA kit for norfloxacin measurement. 【Method】Based on the prepared norfloxacin monoclonal antibody, the best work concentration of monoclonal antibody and HRP labeled antibody were selected by matrix method to develop an indirect competition ELISA kit for norfloxacin detection, and the properties of which also were examined and conformed. 【Result】The kit has average IC50 of 5.18 μg•L-1, a sensitivity of 0.31 μg•L-1, and the detection limit of 1.00 μg•L-1. The kit has different cross-reactivity with peifloxacin, ofloxacin, ciprofloxacin, lomefloxacin, oxilinic acid. The recoveries of norfloxacin in milk was 103.23%, and the CV was 9.33%. Among the detection results by three different batch kits, the inter-assay and intra-assay coefficient variation were all below 10%. The kit could remain effective for more than six months when stored at 4℃. There was no significant difference in the tested value of norfloxacin in milk between with NEO-kit and with LC-MS/ MS (P≥0.05). Furthermore, the tested value of norfloxacin in practice milk was no difference between with NEO-kit and with LC-MS/MS (P≥0.05). 【Conclusion】 A Nor-kit with high sensitivity, specificity and accuracy was developed in this experiment.

Cloning, Evolution and Expression Features of MAPK1 Gene Family from Brassica Species (B. napus, B. oleracea, B. rapa)

LU Jun-Xing, LU Kun, ZHU Bin, PENG Qian, LU Qi-Feng, QU Cun-Min, YIN Jia-Ming, LI Jia-Na, LIANG Ying, CHAI You-Rong

Scientia Agricultura Sinica. 2013, 46(16): 3478-3487. doi:10.3864/j.issn.0578-1752.2013.16.019

Abstract ( 547 )

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【Objective】The objective of this study is to isolate MAPK1 gene family from Brassica napus, Brassica oleracea and Brassica rapa and analyze the transcriptional expression profiles of MAPK1 in various organs of these three species.【Method】On the basis of in silico cloning, the full-length cDNA and gDNA of MAPK1 genes were isolated from the three species using RACE (Rapid Amplification of cDNA Ends) technology. Organ specificity of MAPK1 in various organs of the three species was detected by quantitative real-time PCR (qRT-PCR).【Result】In this study, four MAPK1 genes including BnMAPK1-1, BnMAPK1-2, BoMAPK1 and BrMAPK1 were isolated from B. napus, B. oleracea and B. rapa. Their gDNA sequences were 2 512-2 525 bp, containing three exons and two introns. Standard mRNA lengths of these genes were 1 599-1 620 bp, all with an ORF of 1 100 bp. Phylogenetic tree showed that BnMAPK1 gene family was originated from the assemblage of B. oleracea and B. rapa MAPK1 genes, BnMAPK1-1 and BnMAPK1-2 corresponding to BoMAPK1 and BrMAPK1, respectively. qRT-PCR showed that MAPK1 genes expressed in all tested organs, but organ specificity showed significant differences among species implying quick evolution.【Conclusion】 In this study, the full-length cDNA and gDNA of MAPK1 genes were isolated from B. napus, B. oleracea and B. rapa. It supports the assumption that B. napus is a heterotetraploid species of B. oleracea and B. rapa by natural interspecific hybridization. Brassica MAPK1 genes show much conservation in gene and protein sequences and structures, but the transcription organ specificity evolves very fast and show significant differences among closely related species.

Determination of Rice Amylopectin Chain-Length Distribution by Using High Performance Size Exclusion Chromatography

YANG Xiao-Yu-12, LIU Zheng-Hui-1, LI Gang-Hua-1, WANG Qiang-Sheng-1, WANG Shao-Hua-1, DING Yan-Feng-1

Scientia Agricultura Sinica. 2013, 46(16): 3488-3495. doi:10.3864/j.issn.0578-1752.2013.16.020

Abstract ( 559 )

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【Objective】The aim of this study is to establish a sensitive high performance size exclusion chromatography with differential refractive index detector (HPSEC-RI) methods for measuring molecular weight distribution of rice amylopectin. 【Method】The proposed method includes three parts: Firstly, separation of amylopectin by butanol precipitation method, Secondly, hydrolysis of rice amylopectin by isoamylase, and Thirdly, qualification of the molecular weight distribution by HPSEC-RI. The proposed parameters of HPSEC-RI are as follows: columns of Shodex SB-G, Shodex SB-803(1 000-100 000) and Shodex SB-802.5(300-10 000) being connected in tandem, column temperature being 40℃, the mobile phase being 0.1 M Tris+0.1 M NaCl (pH=7.40) with a rate of 0.5 mL•min-1, detector temperature being 37℃, sample concentration being 5 mg.mL-1 and the input volume being 20 μL.【Result】The rice amylopectin purified in this study is of high-purity, with the starch content being 99.4%, protein residue being 0.02%, and the peak of rice amylopectin-iodine complex absorption spectrum being 517 nm. The tested rice sample amylopectin chain-length distribution is in the range of DP 2-120. 【Conclusion】The precision and repeatability verification test showed that the proposed method performed well in these aspects including good reproducibility, simple sample preparation, and high efficiency.

New Occurrence Regions and Field Infection Regularity of Cereal Cyst Nematode on Wheat in Shaanxi Province

ZHAO Jie, ZHANG Guan-qu, KANG Zhen-sheng

Scientia Agricultura Sinica. 2013, 46(16): 3496-3503. doi:10.3864/j.issn.0578-1752.2013.16.021

Abstract ( 422 )

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【Objective】The objective of this study is to determine current status of cereal cyst nematode (CCN) on wheat and their field infection regularity in Northern Shaanxi Province and northern areas of Weihe River, and to provide a scientific basis for integrated control of CCN in this area. 【Method】 Field surveys of CCN on wheat were performed by sampling with five-point method in field at the booting to milking stages of wheat, isolating CCN cysts with Fenwick flotation method, and identifying CCN species using morphological method. A periodic sampling method in wheat field was used to acquire that the amount and life stage of juvenile (J2) of Heterodera avenae from roots stained by sodium hypochlorite-acid fuchsin solution using a microscope.【Result】Pathogenic CCN species attacking wheat in twenty-four counties in the two areas was H. avenae. Linyou, Yongshou, Liquan, and Wugong counties were first time to be reported the occurrence of CCN on wheat. On the average, the amount of CCN was the highest with 32.0 cysts/100 g air-dried soil in Linyou County, and the lowest with 5.5 cysts/100 g air-dried soil in Yongshou County. No CCN were found in the investigated wheat fields in the Northern Shaanxi Province. No CCN infection was found in the investigated wheat filed in the Northern Shaanxi Province. Second-stage juvenile (J2) of H. avenae hatching from survival cysts in the soil were detected within young roots of autumn-sown winter wheat. Following the next year, J2 attacking the roots of seedling plants could be observed when wheat started regreening. A peak of J2 infection on wheat emerged at time-span from late March to early April. The third-stage juvenile (J3) generated in middle April and started swelling in late April. Obvious white cysts of H. avenae could be seen on the outside of roots of wheat plants by naked-eyes in early May.【Conclusion】Linyou, Yongshou, Liquan, and Wugong counties were new occurrence areas of CCN on wheat. Two infection periods of CCN on wheat occur after autumn-sown winter wheat and regreening in the following year. One generation of H. avenae took place in growing season of wheat in a year in Shaanxi Province.

Hyperspectral Monitoring of the Canopy Chlorophyll Content at Apple Tree Prosperous Fruit Stage

FANG Xian-Yi, ZHU Xi-Cun, WANG Ling, ZHAO Geng-Xing

Scientia Agricultura Sinica. 2013, 46(16): 3504-3513. doi:10.3864/j.issn.0578-1752.2013.16.022

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【Objective】 The aims of the study are to promote the application of hyperspectral techniques in the real-time non-destructive detection of the apple precise fertilization and the diagnosis of the growth of the apple tree by building the quantitative relationship models between the apple canopy chlorophyll content and the characteristics of the canopy spectrum . 【Method】 Taking the apple trees in the Mengyin area as the experimental materials, the spectral reflectance and the apple canopy chlorophyll Chl(a+b) content were measured for two years continuously, the correlation coefficient between them was analyzed, and the RVI, DVI, NDVI and RDVI were calculated by combining any two bands from 400 nm to 1 000 nm. The relationship between them was analyzed and stepwise regression method was used to establish the apple canopy chlorophyll content monitoring model. 【Result】 The results showed that the best spectral index to estimate the content of the chlorophyll is NDVI(975,742), the correlation coefficient is 0.5093. The best apple canopy chlorophyll content monitoring model is Y=-0.56(log1/R)771-0.48 (log1/R)1978+0.20(log1/R)2407-0.10(log1/R)2440+4.749. 【Conclusion】 The model established by using stepwise regression can predict the apple canopy chlorophyll content better, thus it has provided a theoretical basis for reflecting apple growth conditions by using hyperspectral technology.

Molecular Cloning and Expression Analysis of Heat-Shock-Protein90 (LsHsp90) Gene from Leaf Lettuce (Lactuca sativa L.) Under Heat Shock

REN Yue, HAN Ying-Yan, LI Ting, HAO Jing-Hong, FAN Shuang-Xi

Scientia Agricultura Sinica. 2013, 46(16): 3514-3522. doi:10.3864/j.issn.0578-1752.2013.16.023

Abstract ( 378 )

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【Objective】 LsHsp90 gene was cloned and used to analyze the expression profiles under heat shock stresses, in order to provide a foundation for investigating heat tolerant mechanisms of molecular regulation in stress tolerance of Lactuca sativa L.【Method】 With homology cloning approaches coupling with RACE techniques, the full-length cDNA of LsHsp90 was cloned from total RNA of the leaves of L. sativa L. Real-time quantitative PCR (qRT-PCR) was used to analyze the expression patterns of this gene in leaves after heat shock unter 37℃ and 42℃.【Result】The full cDNA sequence of LsHsp90 is 2 330 bp，containing a 2 097 bp open reading flame (ORF) and encoding 698 amino acid residues with a predicted molecular mass of 79.8 kD．The LsHsp90 protein contains a predicted ATPase site and a Hsp90 conservative structure domain, which is highly conserved in plants and especially similar to Hsp90 from Ageratina adenophora (EU269070.1) and Arabidopsis thaliana (AY081302.1). Phylogenetic analysis showed that LsHsp90 and Hsp90 from Ageratina adenophora (EU269070.1) were clustered into one group. Real-time PCR analysis showed that, the expression of LsHsp90 in leaves of two varieties was upregulated under 37℃. LsHsp90 was induced down regulation in heat-sensitive variety S106 under 42℃, while the expression of LsHsp90 in heat-tolerant variety Z36 was upregulated.【Conclusion】LsHsp90 gene was firstly isolated and characterized from L. sativa L. This gene has genetic characteristics similar with all species and the induced expression profiling of LsHsp90 showed different patterns under heat stress. The results indicate that the LsHsp90 gene may play an important role in response to heat shock stress.

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