Scientia Agricultura Sinica

Cloning and Functional Analysis of a Cotton Nicotinamide Synthetase Gene GbNocotin and Its Promoter

YANG Yu-Wen-1, ZHOU Jian-Wu-1, GAO Yuan-Yuan-2, CHEN Tian-Zi-1, ZHANG Bao-Long-1, NI Wan-Chao-2

Scientia Agricultura Sinica. 2014, 47(6): 1041-1050. doi:10.3864/j.issn.0578-1752.2014.06.001

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【Objective】Cloning and characterizing the expression pattern of a cotton nicotinamide synthase gene and its promoter to analyze it’s application in transgenic crop breeding.【Method】 According to the sequencing results of a clone (78L16) of Maxxa BAC, the promoter of a cotton nicotinamide synthase gene GbNocotin was obtained firstly. The online database PLACE was utilized to predict regulatory elements on the promoter. Its recombinant vector pGbNocotin::GUS was constructed and was transformed into Arabidopsis by flower dip method and the transgenic plants was obtained, which were used for GUS staining analysis at the seedling and mature stages. Then the open reading frame (ORF) of GbNocotin was obtained by RT-PCR. Phylogenetic tree was made by mega5.0 and expression analysis in different organs and by stress treatments was conducted by real time-PCR. 【Result】 A specific Fe-deficiency-related element IRO2OS, several stress-responsive such as dehydration, heavy metal and pathogen related elements and phytohormone responsive elements are present in the 1.8Kb promoter region of GbNocotin. GUS staining analysis of transgenic Arabidopsis plants by pGbNocotin::GUS showed that GUS gene was strongly expressed in the root and hypocotyls at the seedling stage, while it was also detected at the base of pods, inflorescence and petioles as well as root in the flowering Arabidopsis plants. GbNocotin has an ORF with 864 nucleotides and encodes a protein of 287 amino acids, its isoelectric point is 6.76 and the predicted molecular weight is 32.7 kD. Although the conserved NAS domain (PFAM accession number: PF03059) was located at amino acid of 11-286, but the similarity of GbNocotin with homogenous genes from other plant is very low, with the highest similarity of ATNAS3 in Arabidopsis is only 43%. GbNocotin exhibits an organ differently expressed pattern. It was strongly expressed in root and stem, moderately expressed in cotton fibers and flowers, in contrast to weakly expression levels in leaves and ovule. In addition, Fe deficiency induced the expression of GbNocotin significantly, as the expression level increased as 9 fold compared to the control plants after one week with the treatment of Fe deficiency. Although the treatments of CuSO4, PEG, abscisic acid (ABA) and gibberellin (GA) all depressed the expression of GbNocotin, but the content varied largely, as the expression all decreased dominantly at 24h by the 4 treatments but the inhibitory effects of ABA and CuSO4 were most significantly. And after 48 h, the expression level by PEG and GA treatments has been restored, while that of the CuSO4 and ABA treatments was still very low.【Conclusion】GbNocotin was differentially expressed in various tissues and organs. It was induced by Fe deficiency and depressed by CuSO4, PEG, ABA and GA treatments. The GbNocotin gene may participate in the absorption and utilization of Fe in cotton and may have potential applications in breeding.

Molecular Biology Identification of Transgenic Cotton Lines Expressing Exogenous G10aroA Gene

WANG Xia-1, 2 , 4 , MA Yan-Bin-2, WU Xia-2, SHEN Zhi-Cheng-3, LIN Chao-Yang-3, LI Peng-Bo-2, SUN Xuan-2, WANG Xin-Sheng-2, LI Yan-E-2, LI Gui-Quan-1

Scientia Agricultura Sinica. 2014, 47(6): 1051-1057. doi:10.3864/j.issn.0578-1752.2014.06.002

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【Objective】It is of great value to develop glyphosate-resistant cotton in China. To obtain the glyphosate-tolerant transgenic cotton line, a new G10aroA gene encoding a 5-enolpyruvylshikimate- 3-phosphate synthase (EPSPS) was transformed into the cotton by Agrobacterium-mediated transformation method. The glyphosate-tolerant transgenic cotton lines can be obtained by somatic callus tissue culture techniques.【Method】 Firstly, the callus induction efficiency of recipient cotton was tested under different concentrations of glyphosate. Secondly, G10aroA was transformed into the hypocotyl segments of cotton line R15 using Agrobacterium-mediated method. The transgenic cotton plants were regenerated from callus tissue culture under selection of glyphosate. The T1 cotton plant seeds were obtained by self-pollination of T0 plants. The G10aroA gene in the transgenic cotton genome was confirmed by PCR, and the transcripts of the G10aroA gene in the transgenic cotton lines were analyzed by RT-PCR. The expression of EPSPS encoded by G10aroA gene was detected by Western blot. 【Result】 Glyphosate concentration of 2.5 mmol•L-1 was selected by comparative analysis for tissue culture. Under this condition, the G10aroA gene was introduced into R15 cotton and thirty-two positive cotton lines were regenerated. These T0 transgenic lines were strictly self-pollinated. The G10aroA gene was confirmed by PCR detection of G10aroA in twenty-seven transgenic lines of T1 generation. The results suggested that the exogenous gene was stably inherited in all of twenty-seven T1 transgenic plants. Quantitative real-time PCR analysis revealed that the exogenous G10aroA gene was expressed varied among different tissues in transgenic line L12 and L14. The order of relative expression level from high to low was stems, bracts, leaves and flowers. In addition, the EPSPS protein of 46 kD size encoded by G10aroA gene was detected in the leaves of transgenic line L7，L12 and L14. 【Conclusion】 The G10aroA gene has been integrated into genomic DNA of the cotton lines via Agrobacterium-mediated method. The transcripts and protein of G10aroA were both detected in the T1 progeny. Thus the transgenic lines may be used for cotton breeding research of glyphosate-tolerant cotton.

Subcellular Localization and Expression Analysis of Nicotiana sylvestris KUP/HAK/KT Family K+ Transporter Gene NsHAK11

SONG Yu-Feng-1, 2 , DONG Lian-Hong-1, 2 , JIN Yi-Rong-1, 2 , SHI Su-Juan-1, 3 , ZHANG Liang-1, 2 , LIU Chao-Ke-4, FENG Xiang-Guo-4, HU Xiao-Ming-4, WANG Qian-1, LIU Hao-Bao-1

Scientia Agricultura Sinica. 2014, 47(6): 1058-1071. doi:10.3864/j.issn.0578-1752.2014.06.003

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【Objective】The aim of this study is to try to isolate and clone new potassium transporter KUP/HAK/KT gene in tobacco through bioinformatics method, then to determine its subcellular localization and analyze its expression. Using this method so as to provide reference for study about tobacco KUP/HAK/KT potassium transporter gene family.【Method】Firstly, by searching the genome database of Nicotiana sylvestris and N. tomentosiformis, the KUP/HAK/KT family members were predicted, and then phylogenetic trees of the above two tobaccos were constructed by multiple approaches of bioinformatics. According to the above results, a new N. sylvestris K+ transporter gene, NsHAK11, was found. Secondly, subcellular localization of the protein coded by the new gene was predicted through Wolf PSORT. Then the leaves of N. benthamiana were inoculated by infiltration with culture of Agrobacterium, and the subcellular localization of the protein was observed with laser scanning confocal microscope, which was based on the fluorescence produced by fused fluorescent protein. In this way, the prediction could be verified. Lastly, the expression pattern of NsHAK11 gene was predicted through PLACE website. The fluorescent quantitative RT-PCR method was adopted to detect the expression of NsHAK11 gene in two situations. On the one hand, the differential expression of NsHAK11 gene in different organs of N. sylvestris at flourishing flowering stage was studied. On the other hand, the expression changes of NsHAK11 gene in leaves of N. sylvestris at seedling stage under low temperature, high temperature and low-K+ stress were studied by using the same method, too. Thus, the authenticity of the prediction was verified by the fluorescent quantitative RT-PCR results.【Result】These preliminary results showed that both of N. sylvestris and N. tomentosiformis KUP/HAK/KT families have 19 members, and either of the two families could be divided into 4 clusters evolutionarily. According to the predicted sequence, a new gene of N. sylvestris KUP/HAK/KT family was cloned, which belongs to Cluster III. The new gene was named NsHAK11, because the protein coded by NsHAK11 has the highest similarity with AtKUP11 in Arabidopsis thaliana. However, the similarity of the protein with NrHAK1 and NtHAK1, which were acquired in tobacco, were only 48% and 49%. This showed that NsHAK11 is a new gene in tobacco. The results of Wolf PSORT predicted that the protein NsHAK11 is located mainly on plasma membrane. The subcellular localization experiment showed that the location of NsHAK11 fused with GFP and DsRed2 separately, two reporter proteins, were both on plasma membrane, which were basically in line with the prediction. The analysis of NsHAK11 promoter through PLACE website predicted that NsHAK11 gene can be expressed in the tissues of root, flower and germ, and its expression may be influenced by low temperature, high temperature, drought and phytohormones. The results of fluorescent quantitative RT-PCR matched with the prediction: NsHAK11 was expressed in all tissues of N. sylvestris at full flowering stage but at different levels, while the expression quantity in main roots was the highest, lower in the lateral roots and the lowest in leaves. In another experiment, NsHAK11 in leaves of N. sylvestris at seedling stage expressed different types of up-regulation when treated with low temperature, high temperature and low-K+ stress. The up-regulation of expression of NsHAK11 was maximum and earliest under low temperature stress. The level of up-regulation was lower under high temperature stress, while there was an obvious suppression at early stage. And the expression showed up-regulation but not significant when treated with low-K+ stress.【Conclusion】NsHAK11, a new KUP/HAK/KT family member of tobacco was obtained. And it encodes a potassium transporter protein, which is mainly distributed on plasma membrane. Besides, NsHAK11 is extensively located in the tissues and organs, and it can be involved in response to low temperature, high temperature and low-K+ stress.

Effect of Phosphorus on Reddish Brown Iron Plaque on Root Surface of Rice Seedlings and Their Nutritional Effects

FU You-Qiang, YANG Xu-Jian, WU Dao-Ming, SHEN Hong

Scientia Agricultura Sinica. 2014, 47(6): 1072-1085. doi:10.3864/j.issn.0578-1752.2014.06.004

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【Objective】 Phosphorus (P) deficiency could significantly induce the formation of reddish brown iron plaque on the surface of rice roots. However, it remains unclear how P induces the formation of reddish brown iron plaque on root surface.【Method】 In this study, 24 rice varieties of different origins were used to measure the iron plaque on their root surface. Rice seedlings were firstly cultivated for 21 days hydroponically, and then submitted to P-deficient solution and 0.1 mmol•L-1 Fe2+ solution, respectively. The parameters including root length, root surface area, root and shoot dry weight and content of iron plaque were determined after treatments. Based on the above parameters, a rice variety (Tianyou 998) was screened with higher amount of iron plaque. In the following experiments, effects of different nutrient deficiencies (N deficiency, P deficiency, K deficiency, and their interactive treatments, etc.), root-split treatments (root system was divided into two parts, both two parts were treated with P supply treatments, one part was treated with P-deficient treatment, and the other was with P supply treatment, or both two parts were treated with P-deficient treatment) and different P/Fe treatments (1﹕1, 1﹕3, 1﹕5, 1﹕8) on the formation of iron plaque were examined with Tianyou 998 as materials. After treatments, contents of iron plaque, inorganic P content on root surface, root and shoot P or Fe uptake were determined to investigate the effect of P on iron plaque and their nutritional effects.【Result】Different rice varieties had various content of iron plaque. The content of iron plaque on root surface among 24 varieties ranged from 3.67 to 9.78 mg•g-1. The amount of iron plaque on root surface was correlated with root length, root surface area and rice biomass. Results from nutrient-deficient experiments indicated that iron plaque by DCB method consisted of reddish brown and non reddish brown iron plaque. Only P deficiency could induce the formation of reddish brown iron plaque on root surface, while N or K deficiency could not. Root-split experiment indicated that the formation of reddish brown iron plaque was affected by exogenous P, rather than internal inorganic P. Results from different P/Fe treatments indicated that P/Fe in solution influenced the amount of reddish brown iron plaque significantly. DCB-Fe concentration on root surface was related to Fe level in solution. The maximal value of iron plaque was 36.50 mg•g-1. Reddish brown iron plaque increased with decreasing P/Fe. When P/Fe was less than 1:3, the reddish brown iron plaque could be induced on root surface of rice seedlings obviously. The formation of reddish brown iron plaque contributed to P and Fe uptake in both roots and shoots of rice seedlings.【Conclusion】The formation of reddish brown iron plaque was induced by exogenous P deficiency, rather than endogenous P signal. The formation of reddish brown iron plaque was associated with external P/Fe levels around root system, while DCB-Fe was related to Fe concentration in solution. Reddish brown iron plaque formed on root surface of rice seedlings can be regarded as a nutrient pool, which would contribute to the uptake of P and Fe.

Study on Tobacco Vigorous Seedling Indexes Model

BAI Yan-1, 2 , SHI Wan-Hua-1, XING Xiao-Jun-3, WANG Yong-3, JIN Yi-Rong-1, 2 , ZHANG Liang-1, 2 , SONG Yu-Feng-1, 2 , DONG Lian-Hong-1, 2 , LIU Hao-Bao-1

Scientia Agricultura Sinica. 2014, 47(6): 1086-1098. doi:10.3864/j.issn.0578-1752.2014.06.005

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【Objective】Researches made by the predecessors mainly focused on the indexes of crop seedling selection, less studies were carried out in quantification of seedling index model and validation, especially reports on the study of tobacco seedling index model are rare, so in practical production of tobacco seedling, exploration and development of scientific evaluations of quantitative indicators of tobacco seedling are badly needed. The establishment of a suitable vigorous seedling index model for tobacco intensive seedling in China was established to provide a theoretical basis for the normal raising seedlings and cultivating vigorous seedlings for tobacco production.【Method】Based on traditional vigorous seedling standards, following the principle of dominant, practice and operability, 17 seedling quality indexes including the plant height, stem circumference, above-ground fresh weight, underground part fresh weight, root activity, chlorophyll, and superoxide dismutase (SOD) were determined. Fuzzy comprehensive evaluation analysis was carried, and on the basis of this analysis, correlation analysis was made on each single tobacco quality indicator and the comprehensive evaluation index. Several representative quality indicators were screened, two different types of evaluation indexes were combined, namely the relative index (two single quality indexes ratio) and the composite index (3 or above 3 single quality indexes), and correlation analysis method was used to analyze the seedling quality comprehensive evaluation index and relative index, composite index, respectively, then the typical combination evaluation indexes were screened and the seedling index model was established. Finally, the seedling index model and tobacco quality index reference frequency distribution table were grouped in a reasonable manner to map each quality index and seedling index linear relationship, and the best value range of seedling index model was obtained.【Result】The range of comprehensive evaluation indexes of tobacco seedlings quality is 0.26-0.80. The range can roundly summarize tobacco seedlings quality, quantitatively and objectively evaluating the quality of tobacco seedlings. The tobacco seedling quality is divided into four levels through the total value of frequency curve method, the range of 0.71-0.80 is the optimal level for seedlings, that of 0.62-0.70 is for the sound seedlings, that of 0.41-0.61 is for the secondary seedlings, and that of 0.26-0.40 is for the weak seedlings. Relative index contains less information. Relative index can not roundly summarize tobacco seedlings quality. However, the composite index constituting multiple agronomic characters is stable, and can roundly summarize tobacco seedlings quality, and the correlation coefficient of (stem girth/plant height)×total fresh weight×10 composite index with comprehensive evaluation indexes is very significant, which can be used as the vigorous seedling index model for evaluating tobacco seedlings quality. The relationship analysis of 17 tobacco quality indexes and seedling evaluation index (stem girth/plant height)×total fresh weight×10 showed that when (stem girth/plant height)×total fresh weight×10 is in the range of 28.57-37.05, tobacco seedlings are strong, root develops well. Through field tests and verification, (stem girth/plant height)×total fresh weight×10 is 28.57-37.05, a range of rapid growth and development of tobacco plant, strong growth potential in the field, the root system is active for absorption of soil moisture and nutrients, the photosynthetic reaction is active, adverse resistance is strong, the flue-cured tobacco leaves quality is good, and the economic benefit is high.【Conclusion】The vigorous seedling indexes model (stem girth/plant height)×total fresh weight×10 can scientifically evaluate tobacco seedlings overall quality, comprehensively describe the tobacco seedling quality indexes. It is simple and convenient in application. Field experiments showed that when the numerical range takes 28.57-37.05, the tobacco seedlings quality indexes reached the maximum values, tobacco seedlings survive quickly and the survive rate was high after transplanting, the comprehensive quality of tobacco plants was good in the growth period, the flue-cured leaves have considerable economic values, and the chemical quality is good.

Control Efficacy and Action Mechanism of Mineral Nutrition on Tobacco Bacterial Wilt

ZHENG Shi-Yan, DING Wei, DU Gen-Ping, YANG Liang, LIU Xiao-Jiao, ZHANG Yong-Qiang

Scientia Agricultura Sinica. 2014, 47(6): 1099-1110. doi:10.3864/j.issn.0578-1752.2014.06.006

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【Objective】 The objective of this study is to research the effect of four kinds of mineral nutrition (Ca, B, Mg and Mo) on tobacco bacterial wilt and defense enzyme activities, and discuss their mechanisms of action. Furthermore, from the perspective of control efficacy and physiological and biochemical regulation ways, finding out the crucial element related to control tobacco bacterial wilt closely.【Method】 A plot experiment was carried out repeatedly by means of greenhouse experiment combined with field test. Adding application of Ca, B, Mg and Mo nutrition for tobacco plant were individually with spraying. Subsequently, a comparison among control efficacies and defense enzymes activities of different treatments were carried out by investigating, detecting and analyzing the impact of different elements on occurrence of tobacco bacterial wilt, tobacco agronomical trait, direct inhibition and defense enzymes in tobacco plant.【Result】The greenhouse experiment results showed that, being on the basis of guaranteeing the basic nutrition, adding application of Ca, B, Mg and Mo all performed inhibition against tobacco bacterial wilt. However, the control efficacy of Mo treatment was significantly higher than others, followed by Ca nutrition, which were 64.79% and 57.67%, respectively. Besides, the areas under disease progress curve (AUDPC) of Mo and Ca treatments were significantly lower than others, which were 300.00, 244.44 (calculated with disease incidence) and 380.56, 352.78 (based on disease index) lower than control, respectively. Furthermore, there were direct inhibitions for Mo and Ca against Ralstonia solanacearum, the highest rate of which was 35.93% and 33.13% individually after treating 24 h. Additionally, Mo and Ca nutrition dramatically increased POD, CAT, SOD, PPO and PAL activities, and decreased MDA level in infected tobacco plant. Compared with the control treatment, which raised 3.11, 1.10, 0.82, 1.68 and 0.60 times, and 1.26, 0.73, 0.90, 1.00 and 0.32 times in turn, reduced 0.50 and 0.26 times, respectively. Moreover, the field experiments results showed that, supplying with Ca, B, Mg and Mo to tobacco plant, which were cultivated in tobacco-growing soil existing nutrition unbalance, tobacco bacterial wilt was controlled effectively. And the field control effects of Mo and Ca treatments were higher than any others, which were 49.46%-65.52%, 46.80%-57.40% (2011) and 45.28%-62.17%, 42.91%-62.57% (2012), respectively. What’s more, in terms of tobacco agronomical characteristics, Mo and Ca nutrition showed distinctly acceleration in the field, particularly plant height, width and area of maximum leaf and stem diameter.【Conclusion】The condition of tobacco nutrient level is closely related to its resistance. Replenishing tobacco with Mo and Ca nutrition regularly will benefit to enhance the defense ability of tobacco against bacterial wilt, and improve tobacco resistance to bacterial wilt. Besides, there is an obvious control effect against tobacco bacterial wilt. Furthermore, the above results would play an increasingly important role in the construction of nutrition-disease resistance model on plant diseases.

Development and Application of a Multiplex RT-PCR Assay for Detecting Three Sweet Cherry Virus Species

ZONG Xiao-Juan, WANG Wen-Wen, WEI Hai-Rong, WANG Jia-Wei, CHEN Xin, XU Li, LIU Qing-Zhong

Scientia Agricultura Sinica. 2014, 47(6): 1111-1118. doi:10.3864/j.issn.0578-1752.2014.06.007

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【Objective】The objective of this study is to develop a multiplex RT-PCR protocol to detect 3 sweet cherry virus species Prunus necrotic ringspot virus (PNRSV), Prune dwarf virus (PDV) and Little cherry virus-2 (LChV-2) simultaneously. 【Method】 The leaves of the sweet cherry (Prunus avium Lindl.) infected by 3 virus species were selected as the experimental materials. Total RNA was extracted using CTAB extraction buffer. For cDNA synthesis, reverse transcription was carried out using random hexamer primer. Six pairs of primers were designed according to the genome sequences of PNRSV, PDV and LChV-2 which were published in GenBank. Single RT-PCR and multiplex RT-PCR were carried out, respectively, to select the primer groups which could be used in the multiplex virus detection. Annealing temperature and the number of the PCR cycles were evaluated to optimize the multiplex RT-PCR conditions. The specificity of the primers was analyzed by using the single-virus infected samples, complex-virus infected samples and virus-free leaf samples. To analyze the sensitivity of the multiples RT-PCR, the transcript which was prepared from the complex-virus infected samples was diluted to 2-fold series. All of the reactions were carried out in the same reaction buffer and under the same conditions. To confirm the accuracy of the multiplex RT-PCR, each amplified fragment was purified by DNA gel extraction kit, cloned into pMD18-T vector and sequenced. In this paper, the multiplex RT-PCR was also used to detect virus infection in Chinese cherry (P. pseudocerasus Lindl.) trees which are cultivated in the orchards in Taian, Shandong Province. 【Result】 Two primer groups were selected and could be used in the multiplex RT-PCR detection. Group 1 including primers ‘PNRSV-S1/A1, PDV-S2/A2 and LChV2-S1/A1’ amplified the fragments of 733, 467 and 337 bp, respectively. Group 2 including ‘PNRSV-S1/A1, PDV-S3/A3, LChV2-S1/A1’ amplified the fragments of 733, 265 and 337 bp, respectively. The PCR products were consistent with the expected size. It was showed that both of the two primer groups could be used to detect 3 sweet cherry virus species with high specificity. Sensitivity analysis showed that virus fragments could be detected in the 23 dilution of the plant cDNA using both of the two primer groups. However, the intensity of each virus fragments was slightly different. The lowest concentration of the plant total RNA transcripts was 107.9 ng•μL-1. Sequence analysis confirmed that it was reliable and accurate to detect the viruses using these two primer groups. This method was then applied to detect the incidence of virus disease in Chinese cherry trees. The results indicated that all of the samples were infected by 2 virus species at least. Of the 9 detected samples, 5 of them were infected by 3 virus species simultaneously. Two of them were infected by PDV and LChV-2 and another two samples were infected by PNRSV and LChV-2. 【Conclusion】 The multiplex RT-PCR protocol can simultaneously detect PNRSV, PDV and LChV-2 with high stability, accuracy and sensitivity.

Progresses in Study of Virus-Based Vectors of Fruit Trees

ZHOU Yan

Scientia Agricultura Sinica. 2014, 47(6): 1119-1127. doi:10.3864/j.issn.0578-1752.2014.06.008

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Virus-based vectors are commonplace tools for the production of proteins or induction of RNA silencing in plants. But even if the existing vectors from herbaceous plant viruses could infect fruit trees, the time for systemic infection and analysis of the expressed genes in trees generally exceeds the stability of known virus-based vectors. Now this problem has been solved by using virus-based vectors from fruit trees. Progress in studies on virus-based vectors from fruit trees was summarized. The results obtained in recent years are as following: (1) The transmission, host range, differentiation of pathogenicity, genome organization and regulation of gene expression of Citrus tristeza virus (CTV), Citrus leaf blotch virus (CLBV), Apple latent spherical virus (ALSV), Plum pox virus (PPV), Grapevine virus A (GVA) and Grapevine leafroll-associated virus (GLRaV) have been elucidated. Construction of Agrobacterium-mediated infectious cDNA clones or infectious RNA transcripts derived from the full-length cDNA clones of these fruit crops viruses. And then, a foreign open reading frame (ORF) such as green fluorescence protein (GFP) gene, β-glucuronidase gene (GUS) was inserted between coat protein (CP) gene and the adjacent gene as a reporter. The expression of the foreign gene was driven by a duplicated native CP subgenomic (sg) RNA controller element (CE) or an introduced heterologous CE of other virus. (2) Virus-based vectors from fruit trees have been used to elucidate cell-to-cell as well as long-distance movement, spatial separation, localization within the host cells, the processes of stem pitting induced by CTV in Citrus macrophylla and the mechanism of CTV superinfection exclusion. These vectors also can be used as virus induced gene silencing (VIGS) vectors to elucidate gene function. Furthermore, CTV-RNAi vector has been demonstrated to silence the endogenous genes of Diaphorina citri and Candidatus Liberibacter asiaticus. (3) From the environmental safety standpoint, the virus used as the basis of the vector should not cause diseases of plants and not be transmitted by natural vector. Thus, one should choose the non-vectored mild strains. A second approach is to mutate the viral vector, eliminate the determinants needed for pathogenicity and vector-mediated spread. Some viruses of fruit trees are limited to phloem-associated cells. Although the vectors are not the appropriate vectors for expression of genes in other tissues, phloem-limited viruses have resulted in the most stable vectors. Furthermore, this limitation can be reduced somewhat by producing proteins with secretion signal peptides to export the protein out of the cell into the intercellular space. Finally, the stability of virus-based vectors from fruit trees could be improved by using promoters from different tobamoviruses instead of repeated promoters.

Irrigation Water Requirement Based on Soil Conditions in a Typical Irrigation District in a Marginal Oasis

SU Yong-Zhong-1, YANG Rong, LIU Wen-Jie, YANG Xiao, WANG Min

Scientia Agricultura Sinica. 2014, 47(6): 1128-1139. doi:10.3864/j.issn.0578-1752.2014.06.009

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【Objective】Soil property is a key factor for influencing crop irrigation water productivity (IWP) and irrigation water requirement. In accordance with the quantitative relationships between soil properties and crop IWP and irrigation water requirement, as well as the regional spatial distribution of soil properties, crop IWP, irrigation water requirement and their spatial distributions in a typical oasis irrigation district were determined. The aim of the study is to provide a scientific basis for accurately estimating regional irrigation water requirement and determining rational irrigation water allocation.【Methods】In this study, the Pingchuan irrigation district located in the Linze marginal oasis was selected as the study area. Based on soil sampling and analysis of 118 sites in the study area, and maize irrigation experiment under different soil texture and fertility levels, the spatial distribution characteristics in soil properties and the relationships between maize IWP and soil properties were determined. 【Result】 The results indicated that soil sand content in the 0-20 cm plough layer ranged from 29.4% to 91.9% ,with an average of 53.6%. Soil organic matter concentration varied from 1.37 to 17.7 g•kg-1,with a mean value of 10.9 g•kg-1. The mean sand content in the 20-100 cm depth was 51.3%. The farmlands with sand and loamy sand soils occupied above 50% of the total area of the farmlands. The farmlands that soil organic matter concentration was 10.0 g•kg-1 below occupied 26% of the total area. Soil water-holding capacity was poor and soil properties showed a high spatial variability. The field irrigation experiment showed that maize IWP ranged from 1.11 kg•m-3 (sandy soils) to 2.44 kg•m-3 (loam soils), on average. There was a significant correlation between IWP and silt clay content in the 0-20cm (CS1, %) and 20-100 cm (CS2, %) layers, and SOM concentration (OM, g•kg-1) in the 0-20 cm layer. Based on the spatial distribution in soil properties and the relationships between soil properties and IWP, the IWPs in the farmlands of the Pingchuan irrigation district were calculated and ranged from 0.75 to 3.92 kg•m-3, with a mean value of (2.36±0.77) kg•m-3. The farmlands that the IWP was 2.0 kg•m-3 below had 970 ha and occupied 18.5% of the total area. The averaged irrigation water requirement was 558 mm during the maize growing period and the total amount of irrigation water requirement was 28.4×106 m3 in the Pingchuan irrigation district. 【Conclusion】Soil conditions determine crop irrigation water requirement and IWP. In agricultural water management at the irrigation district scale, reasonable irrigation water allocation should be based on patchy unit of different soil properties. Some effective tillage, fertilization, and irrigation measurements that can improve soil structure and fertility should be addressed to improve sustainable water productivity.

Status and Trends of Agricultural Diffuse Pollution Research Based on Bibliometrics

GAO Mao-Fang-1, QIU Jian-Jun-1, LIU San-Chao-2, LIU Hong-Bin-1, WANG Li-Gang-1, PANG Huan-Cheng-1

Scientia Agricultura Sinica. 2014, 47(6): 1140-1150. doi:10.3864/j.issn.0578-1752.2014.06.010

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【Objective】The current situation of agricultural nonpoint source pollution (ANPS) research both in China and the world was analyzed for the purpose of understanding the frontiers and hot topics at present, and providing useful data for scholars and experts in the field of agricultural environment. 【Method】ISI Web of Science and CNKI database were chosen to analyze the published articles on ANPS study in recent 30 years based on paper number, major journals, and cited times. Issues including status and trends, frontiers, major institutions, and international cooperation were analyzed. 【Result】 A total of 280 articles in English and 1517 articles in Chinese were got. The most popular topics for ANPS study were control methods, investigation and analysis of ANPS status, and impact on the environment. Typical watershed such as Three Gorges, Taihu, and Miyun reservoir were hot regions for ANPS study. Agricultural streams are important for nutrient retention and removal in ANPS control. But the theory of the retention and removal process was not very clear now, which will be critical research area. Simulation with the process-based model was the main method. Field observation and laboratory experiment were important validation methods. Wisconsin University, California University and Iowa University from USA and Lancaster University from UK have done great works on ANPS study. Chinese Academy of Sciences, Beijing Normal University, and Xiamen University were famous for ANPS study in China. Cooperation between China and USA increased very much in recent years. The cooperation could help scholars in China focusing on the most important issues in the world and trying to find methods for resolving agricultural nonpoint source pollution. Most of the high quality articles and journals were from USA and Europe. Research ability, article quality, and famous journals in China need to be further improved. Main problems were lack of original research and important discovery, famous research groups, and important authors with high quality articles. The reasons for this phenomenon included short period of most research projects, no enough systematic observation data, difficult to use local models, no necessary cooperation among research groups, few model and data sharing work, unstable research group and staff. Lots of work should be done to resolve these problems. Firstly, make long-term planning for ANPS study and observations. Secondly, support original research for recent progress. Thirdly, reorganize all the financial support and make stable support for important groups. Finally, improve the evaluation system for institute a and staff, and increase the quality of local journals in English. 【Conclusion】ANPS control in watershed scale and simulation with process-based model are frontiers in the world for ANPS study. Nutrient transport and transformation mechanism in agricultural streams and river network will be the emphases and direction in the future research. Published articles from China increased dramatically in recent years, while articles with high impact and good international journals are few. Journal of Environmental Sciences-China and Scientia Agricultura Sinica are journals published high quality articles on ANPS study in China.

Bioinformatics of the MADS-Box Transcription Factor and Their Expression in Different Apple Tissues

DONG Qing-Long, JI Zhi-Rui, CHI Fu-Mei, TIAN Yi, AN Xiu-Hong, XU Cheng-Nan, ZHOU Zong-Shan

Scientia Agricultura Sinica. 2014, 47(6): 1151-1161. doi:10.3864/j.issn.0578-1752.2014.06.011

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【Objective】The study aimed to analyze the basic information of MADS-box genes of apple (Malus × domestica), and study their expression levels in different apple tissues.【Method】Apple MADS-box genes were obtained by the NCBI database query, and the amino acid sequences were analyzed with CLC Combined Workbench version 6, WebLogo 3, MEGA4.1, MapInspect and MEME bioinformatics software, their expression levels in different tissues were further studied with RT-PCR.【Result】Totally 26 apple MADS-box genes were found in NCBI database. The results of domain analysis indicated that amino acid 10(I), 16-19 (RQVT), 22-23 (KR), 29-31 (KKA), 33 (E), 37, 39 (LCD), 42 (V) and 48 (S) of apple MADS-box domain were conserved. Conserved motif analysis showed that the conserved motifs 1 and 3, which specify the MADS domain and motifs 2 and 4, which specify the K domain, were observed in all apple MADS-box proteins (except for MdMADS9). The result of phylogenetic analysis revealed that apple MADS-box genes were divided into 5 subclasses. MdMADS1, 3, 4, 6, 7, 8, 11 and 18 belong to SEP subclass. MdMADS2, 5 and 12 are AP1 subclass. MdMADS10, 14, 15, 19, 22 and MdAGL belong to AG subclass. MdMADS16, 17, 21, MdSOC1, MdSOC1a and MdSOC1c are SOC1 subclass. MdMADS13, 23 and MdPI belong to AP3 subclass. MdMADS20 belong to SVP subclass. Chromosome mapping analysis showed that the largest number of apple MADS-box genes were found on chromosome 08 (four genes), followed by chromosomes 02, 14 and 17 (three genes each). Only one gene is located on chromosomes 01, 05, 06, 07, 11 and 16, respectively. No gene is located on chromosomes 03, 04, 12 and 15. RT-PCR results indicated that MdMADS genes were highly expressed in flowers and mature fruits. Similar expression patterns were observed within the SEP and AGL subclasses. AP1 subclass was not only expressed in flowers and mature fruits, but also expressed in other tissues. 【Conclusion】 MdMADS genes are highly and structurally conserved and involved in regulation of apple flower and fruit development processes.

Evaluation of the Mastication and Comparison of Fruit Quality with Different Bearing Habits in Nanfeng Tangerine (Citrus reticulata Blanco cv. Kinokuni)

WEI Qing-Jiang-1, WANG Miao-Qiu-1, ZENG Zhi-Fu-2, YANG Cheng-Quan-1, PENG Shu-Ang-1, LIU Yong-Zhong-1

Scientia Agricultura Sinica. 2014, 47(6): 1162-1170. doi:10.3864/j.issn.0578-1752.2014.06.012

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【Objective】The aims of this study are to establish quantitative indices of mastication grade of Nanfeng Tangerine (Citrus reticulata Blanco cv. Kinokuni), and to provide clues for the cultivation measures to improve the mastication of Nanfeng Tangerine based on comparing the quality of fruits with different bearing habits. 【Method】 Nanfeng Tangerine was used in this study, and mastication grades of the fruits were judged by sensory evaluations. Then quantitative indices of fruits with different mastication quality such as thickness of segment membrane, and shear force, hardness and chewiness of segment of fruits were determined. Moreover, the correlation between mastication quality and these indices was analyzed. The mastication quality and the common quality (vitamin C, total soluble solution and titratable acid) of the fruits with different bearing habits were also determined.【Result】Two indices, thickness of segment membrane and shear force of segment, could be used for quantitatively evaluating and classifying the mastication quality of fruits. Based on the statistics of sensory evaluations, thickness of segment membrane and shear force of segment, the mastication of Nanfeng Tangerine could be classified into three grades: the superior mastication fruits with segment membrane thickness ranging from 0.09 to 0.12 cm and the segment shear force ranging from 1.15 to 1.64 kg; the medium mastication fruits with segment membrane thickness ranging from 0.12 to 0.14 cm and the segment shear force ranging 1.64 to 2.63 kg; and the inferior mastication fruits with segment membrane thickness ranging from 0.14 to 0.17 cm and the segment shear force ranging from 2.63 to 5.70 kg. The results of comparing quality of fruits with different bearing habits showed as follow: the big fruits had superior mastication quality than the medium fruits and small fruits，and the medium fruits had superior common quality than the small fruits and the big fruits; the fruits from short branch had superior mastication quality than the fruits from long branches, but they showed no difference in common quality; the fruits from high loads branches had superior mastication quality than the fruits from low loads branches but they also showed no difference in common quality; the outer fruits had superior quality than the inner fruits; and the fruits from middle layers had superior mastication quality than the fruits from upper and lower layers, and the fruits from middle and upper layers had superior common quality than the fruits from lower layers.【Conclusion】Mastication is an important quality index of Nanfeng Tangerine, which can be quantitatively evaluated by the thickness of segment membrane and the shear force of segment. There are different qualities of fruits with different bearing habits. Moreover, it was found that there is no obvious correlation between common quality and mastication quality, but fruits with better mastication quality usually have superior common quality.

Heat Stress Mitigation by Exogenous Nitric Oxide Application Involves Polyamine Metabolism and PSII Physiological Strategies in Ginger Leaves

LI Xiu, GONG Biao, WANG Yun, XU Kun

Scientia Agricultura Sinica. 2014, 47(6): 1171-1179. doi:10.3864/j.issn.0578-1752.2014.06.013

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【Objective】 Ginger is a thermophilic vegetable crop with the characteristics of not tolerating high temperature, which is easily damaged by high temperature. To investigate the relationship between exogenous nitric oxide and polyamine metabolism, as well as the regulation effect on PSII, mitigation effect of heat stress on ginger by nitric oxide was studied. 【Method】‘Laiwu Big Ginger’ was sandy cultured in a climate chamber under 12h/12h photoperiod, 28℃/18℃ (normal) and 38℃/28℃ (heat stress) conditions. Ginger root was treated with 0.1 mmol•L-1 sodium nitroprussiate (an nitric oxide donor) and sodium ferricyanide (reactant of SNP after releasing NO). Relative water content, chlorophyll concentration, electrolyte leakage, chlorophyll fluorescence parameters and polyamine metabolism of ginger leaves were investigated on 0, 5, 10, 15 and 20 d after treatment. 【Result】Electrolyte leakage significantly increased with prolonging stressed time, while relative water content and chlorophyll concentration significantly decreased. Chlorophyll fluorescence parameters including Fv/Fm, ФPSII, Fv′/Fm′ qP and P decreased, and NPQ, β/α-1 and D increased. Main pattern of PSII was shown that its photochemical activity of PSII was decreased for the energy metabolism pathway shifting from photochemical to non-photochemical activity. Under heat stress, free and conjugated polyamines were significantly accumulated and then decreased in different treatment times. Insoluble bound polyamine and Put/PAs ratio kept an increasing trend with prolonging stressed time. Relative water content, chlorophyll concentration, electrolyte leakage of ginger leaves was recovered by exogenous application of nitric oxide, which regulated chlorophyll fluorescence parameters to normalization. Also nitric oxide application improved polyamine metabolism to reduce Put/PAs ratio. 【Conclusion】 Disordered polyamine metabolism, damaged ginger leaves and PSII were shown under 38℃/28℃ conditions. Significant heat stress mitigation by exogenous nitric oxide application was shown in this research involving polyamine metabolism and PSII physiological strategies to improve heat stress tolerance in ginger.

Relationship Between Color Parameters and Free Amino Acids and Biogenic Amines of Sliced Cooked Cured Beef Ham

SUN Wei-Qing-1, ZHOU Guang-Hong-2, XU Xing-Lian-2

Scientia Agricultura Sinica. 2014, 47(6): 1180-1189. doi:10.3864/j.issn.0578-1752.2014.06.014

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【Objective】Discoloration during storage is a key problem that limits the shelf-life of cooked cured meats. The purpose of the experiment is to study the relationship of color parameters and amino acids and biogenic amines of sliced cooked cured beef ham (SCCBH), to reveal the effects of the nitrogen compounds in meat products on the stability of the heme pigment and apparent color, in the hope of providing theoretical guidances for color protection of this type of products. 【Method】Color parameters, free amino acids and biological amines of SCCH during storage were examined and factor analysis was used to explain the effect of free amino acids and biogenic amines on the color parameters. Principal component analysis (PCA) was used to extract the principal factor. Varimax orthogonal rotation with Kaiser normalization method was adopted in data analysis. 【Result】Eight kinds of biogenic amines and 16 kinds of free amino acids were detected in this experiment. Biological amines showed no significant changes during storage, while, threonine (Thr), valine (Val), methionine (Met), lysine (Lys) and arginine (Arg) changed significantly. The factor analysis results showed that the ?rst principal factor (F1) explained 45.72% of the total variation, and the second (F2) explained 21.52%. F1 was a concentrated reflection of color changes of SCCBH during storage, especially of the redness (a*), Chroma (C) and pigment NO-Heme (NH), and nitrogen compounds directly affecting the apparent color. F2 was mainly expressed the information of the hue and of the nitrogen compounds indirectly affecting apparent redness through the hue (H) or yellowness (b*), these nitrogen compounds mainly contained acidic and alkaline free amino acids. Tyramine (TYR) and spermine (SPM) significantly affected the stability of heme pigment and apparent color of SCCBH, which were shown by a high factor loading value (0.98) in F1. Further, TYR and SPM negatively correlated with the color parameters of SCCBH. Tryptamine (TRY) could keep the NH stable and improve the redness of SCCBH due to the higher factor loading (0.78) in F1 and a significant positive correlation with redness and NH. Threonine and histidine (His) were significantly positively related with redness of SCCBH. Valine, tyrosine (Tyr), methionine and leucine (Leu) had a significant negative correlation with the redness. In addition, glycine (Gly) also had a negative correlation with the redness, but its effect on the apparent color was relatively weak due to its low factor loading in F1. Proline (Pro) had a higher loading (0.78) in F1 and notably related with all of the color parameters, so it affected the color stability apparently. The effect of Pro on the lightness (L*) and yellowness was greater than on the redness just because the correlation coefficients with lightness and yellowness were much higher than it was with redness. Phenylalanine (Phe), lysine and arginine indirectly affected the redness through the lightness and yellowness on the basis of the facts that the three amino acids having high factor loadings in F2 (the factor loadings of Phe, Lys and Arg in F2 were 0.90, 0.94, and 0.97, respectively) significantly related with lightness and hue. The factor loading of glutamate (Glu) in F1 was relatively higher (0.61), which was significantly related to the yellowness and lightness but no effect on redness.【Conclusion】The content and form of the free amino acids and biogenic amines in the meat matrix significantly affected the color stability. This effect might be influenced by matrix pH changing, the combination and competition with porphyrin iron.

Genome-wide Detection of Selection Signature on Sunite Sheep

WANG Guang-Kai-1, ZENG Tao-1, 2 , WANG Hui-Hua-1, ZHANG Shu-Zhen-1, ZHANG Li-1, WEI Cai-Hong-1, ZHAO Fu-Ping-1, DU Li-Xin-1

Scientia Agricultura Sinica. 2014, 47(6): 1190-1199. doi:10.3864/j.issn.0578-1752.2014.06.015

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【Objective】Selection signatures are the selective footprints across the genome because of the effects of selection in the process of species under natural and artificial selection. It could not only reflect the effect of selection in new breed cultivation but also used as a method for QTL mapping which is correlated with economically important traits. Sunite sheep is one of the excellent indigenous sheep breeds distributed in Inner Mongolia through long time artificial selection-, which could adapt to the harsh Gobi natural environment condition. Selection signature detection could be used to search candidate genes due to positive selection and reveal genetic mechanism of economically important traits. Moreover, it could provide genetic evidences for traits undergoing positive selection for a long time in the breed formation process of Sunite sheep. 【Method】 The integrated haplotype score (iHS) method was used to detect genomic selection signatures in the Sunite sheep population based on the Illumina Ovine SNP50K BeadChip data. After quality control, 42616 SNP markers were retained for linkage disequilibrium (LD) analysis and haplotype construction. According to ancestral allelic information, 30537 SNP makers were left to calculate the iHS values. All the iHS values within one window with 500kb length, which was split the whole genome into non-overlapping segments, were averaged. After significant test, the genomic regions with selection signals were annotated. To investigate the biological function of candidate genes, gene ontology enrichment analysis was carried out. 【Result】 The LD decay map of Sunite sheep was constructed. Linkage disequilibrium analysis suggested that LD decreased with marker distances increase, but few pair markers with long distance had high LD levels. There were 204 genomic regions with selection signatures harboring 845 candidate genes were detected. For example, RXFP2 confers the absence of horns in sheep and ASIP could regulate a series of alleles for black and white coat color. HTR4 and SOX10 involved on nervous system development, SOX10 was closely related to neural crest development in embryonic period. E2F2 had an important effect in skeletal development which could activate transcription factor-2 in skeletal growth control. PLA2G6 could affect growth of skeleton and muscle. RPL7 promoted synthesis of ribosomal protein and POL was linked with Ovine Pulmonary Carcinoma. MATR3 could regulate innate and adaptive immune response. Furthermore, candidate genes also included ZWINT, PPP1R1B, GPR98, LUC7L3, CAPZA1 and MYT1L connected with the development of nervous system and disease traits. Bioinformatics analysis found 24 GO items in biological process, 4 GO items in molecular function and 2 GO items in cellular component, respectively. These GO items were mainly related to protein synthesis, macromolecular substances metabolic degradation, targeted transportation of protein, molecular activity, ribosomal components and ribosomal subunits. 【Conclusion】 The first LD map and genome-wide selection signatures map on Chinese sheep breeds were constructed. Many candidate genes related with economically important traits were found through genome-wide selection signatures detection. Some of them have very important significance in the process of sheep domestication. This study provided important reference for further understanding the process of artificial selection on sheep and also provided a theoretical basis for the breeding of Chinese sheep.

Progresses in Research of the Mechanisms of Skeletal Muscle Fiber Formation

LI Bo-Jiang, LI Ping-Hua, WU Wang-Jun, LI Qi-Fa, HUANG Rui-Hua, LIU Hong-Lin

Scientia Agricultura Sinica. 2014, 47(6): 1200-1207. doi:10.3864/j.issn.0578-1752.2014.06.016

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Skeletal muscle, the most important component of animal body, constitutes 40% of the animal body, while muscle fibers as the primary part of muscle, its differences is one of the critical factors affecting meat quality. Therefore, the growth, development of the skeletal muscles and their physiological and biochemical characteristics will directly affect the meat quantity and quality. Generally, the number of the muscle fibers is fixed during the embryonic development, while the increase of the muscle mass is due to the hypertrophy of skeletal muscle fibers after birth. Moreover, the skeletal muscle fibers types are not completely fixed, the change will be occurred according the metabolism and the functional demand during the growth and development of the skeletal muscle. The biological processes of the skeletal muscle growth and development, and the formation of muscle fibers are quite complex, involving in many genes and signal pathway. With the development of molecular biological technologies, many methods are applied to elucidate the regulatory mechanisms of the formation of many complex biological phenomena. To date, a great progress has been made in study of the molecular mechanisms related to the skeletal muscle development, and many key factors involved in the skeletal muscle growth and development have been identified. In the early study, the researches on the skeletal muscle fibers were focused on the identification of muscle fibers types and their physiological and biochemical characteristics, whereas the studies on the molecular mechanisms are relatively rare. Recently, a breakthrough has been made in the molecular regulatory mechanisms involved in the formation of skeletal muscle fibers. So, it is necessary to completely review the types and properties of muscle fiber, molecular regulatory mechanisms and the relationship between the muscle fibers types and meat quality. In this review, the types and properties of muscle fiber are summarized firstly. Then, the research progress in molecular regulatory mechanisms of the slow and fast muscle fibers formation was presented, respectively. Finally, the relationship of muscle fibers and meat quality was discussed. In general, this review will be helpful for further understanding the genetic mechanisms of the formation of skeletal muscle fiber types, and could be a reference for further studying on the formation of muscle fibers in the future. Meanwhile, the review is able to provide useful information for revealing the molecular mechanisms of meat quality, and provide molecular theoretical evidences for cultivating new breeds or lines using biotechnology in the future.

Construction of SSR Genetic Linkage Map and Analysis of QTLs Related to β-glucan Content of Naked Oat (Avena nuda L.)

WU Bin, ZHANG Qian, SONG Gao-Yuan, CHEN Xin, ZHANG Zong-Wen

Scientia Agricultura Sinica. 2014, 47(6): 1208-1215. doi:10.3864/j.issn.0578-1752.2014.06.017

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【Objective】A molecular genetic linkage map for cultivated naked oat based on SSR markers was developed and Quantitative Trait Loci influencing β-glucan content were identified, in order to facilitate the utilization of high β-glucan content oat germplasm resources and provide a theoretical basis for oat molecular marker-assisted selection.【Method】 Taken a segregation population of 215 F2:3 lines which originated from the cross with the high β-glucan content local cultivar Xiayoumai was used as the male parent and improved cultivar Chi38 Youmai as the female as mapping population, a molecular genetic linkage map of oats with SSR markers was developed. The β-glucan content of segregation population was determined by the standard β-glucan measure method (AACC Method 32-23) which was published by American Association of Cereal Chemists and the QTLs for β-glucan content of oat were analyzed and identified by Composite Interval Mapping method. 【Result】 After detection of F2 progenies with 231 pairs of SSR primers, a total of 261 polymorphic markers were obtained. The polymorphic markers mentioned above were analyzed for their genetic linkage relationship by JoinMap 4.0 and finally the oat genetic linkage map was constructed. The map included 26 linkage group, 182 SSR markers and covers 1869.7cM of the whole genome. The average space between markers was 10.6 cM. The number of markers in each linkage group varied from 2 to 14 and the length of the linkage group varied from 10.6 to 235.1 cM. The results of the measurement of the parents and segregation population’s β-glucan content showed that β-glucan content in the offspring groups presented as significant separation and continuous variation with variation coefficient of 18.72%, which indicated that β-glucan content traits are controlled by multiple genes of quantitative traits and the segregation population meets the requirements of QTL mapping. The SSR data were analyzed by QTL analysis software WinQTL Cart 2.5 and whole genome were scanned by Composite Interval Mapping method to identify the possible QTLs for β-glucan content and estimate QTL effect with the LOD=5 as a threshold. Four QTLs correlative to β-glucan content were found. qBG-1 was on the twentieth linkage group and explained 10.9% of phenotype variation with the additive genetic effects value is 0.21 and the nearest marker AM591 showed a genetic distance of 10.0 cM to qBG-1. qBG-2 and qBG-3 were on the twenty-third linkage group and explained 3.2% and 2.7% of phenotype variations with the additive genetic effects values of -0.23 and -0.22, respectively, and the nearest marker AM1832 showed a genetic distance of 4.6cM to qBG-2 and 1.9 cM to qBG-3 by AM641, qBG-4 was on the twenty-fifth linkage group and explained 27.6% of phenotype variation with the additive genetic effects value of 0.84 and the nearest marker AM302 showed a genetic distance of 6.8 cM to qBG-4. The two main QTLs, qBG-1 and qBG-4, all originated from the high β-glucan content male parent Xiayoumai. 【Conclusion】A genetic linkage map was constructed，and four QTLs which affect β-glucan content of naked Oat were identified, thus providing a scientific basis for molecular marker-assisted selection in oat breeding.

Cloning and Expression Analysis of PAL Gene in Seed Coat of Cucurbita pepo

LIU Jia-1, 2 , 3 , XU Bing-Liang-1, 2 , 3 , XUE Ying-Yu-1, 2 , 3 , ZHANG Shu-Wu-1, 2 , 3 , CHEN Rong-Xian-4

Scientia Agricultura Sinica. 2014, 47(6): 1216-1226. doi:10.3864/j.issn.0578-1752.2014.06.018

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【Objective】The aim of this study was to clone full-length cDNA of phenylalanine ammonia-lyase (PAL) gene (CP-PAL) in seed coat of the hulled Cucurbita pepo, analyze its expressions in seed coat development of the hulled and hull-less C. pepo, thus providing a theoretical support for revealing the mechanism of seed development and function of accumulation of lignin in seed coat development. 【Method】 The full sequence of CP-PAL was cloned by RT-PCR and RACE techniques. The bioinformatics method was used to analyze cDNA sequence and deduced amino acid sequence, and the real-time PCR and 2-△△Ct method were used to analyze the expression profile of PAL gene in the whole period of seed coat development. 【Result】 The full-length sequence of CP-PAL consists of 1 720 bp with an intact open reading frame of 1 359 bp, 5′UTR of 114 bp, 3′UTR of 236 bp, polyA of 11 bp, encoding a polypeptide of 452 amino acids. The molecular weight of CP-PAL was 48.86 kD, pI was 6.5, total number of atoms was 6 885 and the formula was C2158H3449N607O657S14. Homology analysis showed that the CP-PAL nucleotide sequences and deduced amino acids were highly homologous to that of PAL gene in Cucumis sativus. CP-PAL contained three functional domains of PAL-HAL, PLN02457, phe_am_lyase and the typical PAL enzyme active site sequence (GTITASGDLVPLSYIA), and it was one member of Lyase_I_Like superfamily. CP-PAL was most likely to be located in the cytoplasm and endoplasmic reticulum, without signal peptide and leader peptide. And it was non-transmembrane and soluble protein. Besides, CP-PAL included casein kinase II phosphorylation site for four, protein kinase C phosphorylation site for six, N-myristoylation site for twelve and N-glycosylation site for two. In addition, CP-PAL had eighteen serine phosphorylation sites, six threonine phosphorylation sites and five tyrosine phosphorylation sites. Random coil was the maximum structural part in the protein secondary structure of CP-PAL, alpha helix and extended strand dispersed in whole protein, N-terminal region was presented mainly in the form of random coil, and C-terminal domain was existed in the form of extended strand. The multiple sequence alignment based on the deduced amino acid sequences of CP-PAL and other PALs of 14 plants showed the functional region was conserved, differences in the N-end. Phylogenetic tree analysis indicated that CP-PAL was very closely related to PAL of C. sativus. The main structural element in CP-PAL protein tertiary structure was α-Helix, less in β-Turn and random coil. Real-time PCR analysis revealed that PAL gene in hull-less C. pepo showed an opposite tendency to hulled C. pepo. The expression of PAL gene in hulled C. pepo was increased, but the expression in hull-less C. pepo was decreased after 20d of self-pollination. The expression of PAL gene in hull-less C. pepo was lower than that in hulled C. pepo. 【Conclusion】The gene CP-PAL which related to lignin synthesis was firstly cloned and characterized in seed coat of hulled C. pepo. The results of study indicated that it may affect seed coat development of the naked kernel variety from C. pepo by regulating the synthesis of lignin.

Analysis of P Fertilizer Efficiency Under Rice-Rapeseed Rotation System

BU Rong-Yan-1, REN Tao-1, LU Jian-Wei-1, LI Xiao-Kun-1, CONG Ri-Huan-1, LI Yun-Chun-1, WANG Yang-1, LU Jun-Ming-2

Scientia Agricultura Sinica. 2014, 47(6): 1227-1234. doi:10.3864/j.issn.0578-1752.2014.06.019

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【Objective】A split field experiment was carried out to study the effects of different P fertilizer application rates on crop yield, P uptake, P recovery efficiency and residual efficiency under annual rice-rapeseed rotation. The effects of residual P in rice season on rapeseed yields and crop P uptakes were evaluated to explore the distribution of P fertilizer in annual rice-rapeseed rotation for the purpose of optimization of P fertilization strategy in rice-rapeseed rotation.【Method】An annual rice-rapeseed field experiment was conducted in Honghu County, Hubei province from May, 2010 to May, 2011. In rice season, there were four different P application rates treatment (P0, 0 P2O5, P30, 30 kg P2O5•hm-2, P60, 60 kg P2O5•hm-2 and P90, 90 kg P2O5•hm-2), while in later rapeseed season, the original P application rate treatments were split into two sub-treatments (with P fertilization treatment, 60 kg P2O5•hm-2, and without P fertilization treatment, 0 kg P2O5•hm-2). Besides crop yield, crop P uptake, P recovery efficiency and residual P utilization efficiency under different P treatments were analyzed, the concept of “the substitute rate of P fertilizer” was adopted to estimate the residual effects of P fertilizer applied in rice season on the rapeseed yield. 【Result】 Reasonable P fertilization substantially increased the yield of the rice and rapeseed. Crop yield and P recovery efficiency in rice season were the highest in P60 treatment, with the average of 9 694 kg•hm-2 and 19.2%, respectively. Insufficient or excessive P fertilization decreased rice yield and P recovery efficiency. Compared with without P fertilization treatment in rapeseed season, rapeseed dry matter increased significantly, ranging from 756 to 1 195 kg•hm-2 in P fertilization treatment; while seed yields were also improved, ranging from 427 to 503 kg•hm-2. P fertilizer applied in rice season significantly affected the seed yield and crop nutrient uptake of the following rapeseed. In contrast to the plots without P fertilization in rice season, rapeseed dry matter of the plots with P fertilization in rice season dramatically increased, varying from 212 to 816 kg•hm-2, and the yield of rapeseed varying from 136 to 409 kg•hm-2, and the P uptake by rapeseed increased from 0.4 to 4.9 kg•hm-2. The P fertilizer applied in rice season could be utilized by rapeseed which consequently increased the agronomic efficiencies and the contribution rate of P fertilizer applied in rapeseed season. The recovery efficiency of P applied in rice season ranged from 16.3% to 19.2%, the residual utilization efficiency ranged from 5.4% to 7.3% and the annual P fertilizer accumulate efficiency was 21.8% - 25.6%. P fertilizer applied in rice season had a significant residual effect, which showed a positive correlation with P fertilizer application rate. The residual effects of P fertilizer applied in rice season were equivalent to 2-9 kg P2O5•hm-2 used in the rapeseed season. Furthermore, the residual effects of P fertilizer applied in rice season were also positively affected by the P fertilizer application rate in rapeseed season. The residual effects of P fertilizer in rapeseed season with P fertilization were higher than those without P fertilization. 【Conclusion】 Reasonable P fertilization could substantially increase crop yield, P uptake and P use efficiency under rice-rapeseed rotation system. The P fertilizer application in rice season had a significant residual effect on increase rapeseed yield and P, which showed a positive correlation with P fertilizer application rate. Considering “more P fertilization used in upland season and less P fertilization used in paddy season” in P fertilization management strategy under paddy-upland rotation, P fertilization management in rapeseed season thus should be optimized taking the residual effect of P application in rice season into consideration.

Cloning and Eukaryotic Expression of DXS Gene from Babesia bovis

WANG Jing, LI Bing, LIU Cui-Cui, XIA Ji-Peng, ZHANG Ji-Yu

Scientia Agricultura Sinica. 2014, 47(6): 1235-1242. doi:10.3864/j.issn.0578-1752.2014.06.020

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【Objective】The apicomplexa parasites, including Plasmodium, Babesia, commonly contain a relic, non-photosynthetic plastid-like organelle that has been named the apicoplast and is vital for their survival. The apicoplast harbors several metabolic pathways that supply essential biosynthetic precursors to the parasite. The synthesis of fatty acids and isoprenoid precursors are its most prominent functions, and the apicoplast has been shown to be essential for the survival of P. falciparum and T. gondii specifically. The isoprenoid mevalonate independent biosynthesis of parasitic protozoa in the apicoplast is a promising chemotherapeutic target, because this pathway is different from the mevalonate pathway in mammals and is essential to such parasites. The 1-deoxy-D-xylulose-5-phosphate synthase (DXS) catalyzes the first rate limiting step in the mevalonate independent pathway. To obtain the activive 1-deoxy-D-xylulose-5-phosphate synthase from Babesia bovis Shannxian, dxs gene was cloned and expressed in eukaryotic system. And it is the foundation for further research of the design of anti-babesia agents that can optimally target the active sites of the DXS enzyme.【Method】A full length cDNA of dxs gene from B. bovis Shannxian was obtained using reverse transcription polymerase chain reaction amplification of total RNA extracted. The complete open reading frames (ORF) of dxs was further inserted into T-easy vetctor and the eukaryotic expression vector, then sequenced and bioinformation was analyzed. The positive recombinant plasmids were transfected into the Hela cells by lipofection transfection and detected using inverted fluorescence microscope and Western blot. To obtain the positive monoclonal cells, the limited diluent with G418 was used, and the enzyme activity of the expression product was detected by liquid mass spectrometry in vitro.【Result】Results showed that the full length of DXS gene was 2061bp, encoding 686 amino acid, the similarity with B.bovis T2Bo in GenBank was 98.0%. According to the bioinformation analysis, the enzyme has three domains, namely thiamine pyrophosphate binding region, pyrimidine ring of thiamine pyrophosphate binding region, and transketolase C-terminal region. The enzyme belongs to the common TPP dependent protease family. The positive recombinant plasmids PEGFP-DXS were transfected into Hela cells by lipofection transfection. The inverted fluorescence microscope and Western blot were used to detect the expression of recombinant plasmids. The Hela cells which transfected successfully showed green fluorescence under the inverted fluorescence microscope. And the Western blot results showed a band about 102 kD in accordance with DXS fusion enhanced green fluorescence protein. The positive monoclonal cells were obtained by combining G418 and limited dilution method, the crude cell extracts from the reaction mixture had a product which has the same molecular of product DOXP, the cationic mode m/z=237 by LC-MS/MS, which indicated that the expression product had catalytic activity.【Conclusion】The fusion protein was expressed correctly in Hela cell，and the expression product had enzyme activity. The study is very important for the study of DXS as targets for antiprotozoal drugs.

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