Scientia Agricultura Sinica

Gene Cloning and Expression Analysis of Long Empty Glumes Mutants in Rice

ZHU Ling, CHEN Xiao-qiong, DU Kang-xi, HAN Bao-lin, RAN Xiu-hua, ZHANG Hong-yu, XU Pei-zhou, WU Xian-jun

Scientia Agricultura Sinica. 2015, 48(11): 2085-2095. doi:10.3864/j.issn.0578-1752.2015.11.001

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【Objective】 Three long empty glumes mutants were screened from a maintainer line Yixiang 1B(Oryza sativa L. ssp. indica) by ethyl methanesulfonate (EMS) mutagensis. This study is to explore the genetic basis and molecular mechanism for controlling the development of long empty glumes by gene mapping and cloning. And the temporal and spatial expression of related genes were also concerned. 【Method】 Three long empty glumes mutants, Oslg-1, Oslg-2 and Oslg-3, were analyzed by phenotypic observation, allelism identification, gene mapping, bioinformatics analysis, and expression analysis by quantitative PCR. 【Result】The glumes showed no obvious difference at the stage of early floret differentiation between wild type and mutant. However, the epidermal cells of glume presented significant difference during floret maturation. At the heading date, epidermal cells of abaxial glume presented bulge and rough nodules, and formed axially alignment. The epidermal hairs became more, and bulges arranged at a regular intervals, which were similar to the structure of epidermal cells of lemma. Genetic analysis suggested that the mutant Oslg-1 was controlled by one recessive nuclear gene. The Oslg-1gene was mapped between SSR markers RM5344 and RM20934, on the short arm of rice chromosome 7, with genetic distances of 1.11 cM and 0.82 cM, respectively, and with a physical interval of 246.3 kb. By analyzing and sequencing for the candidate genes in this genomic region, it was found that there was a single nucleotide change at 182 base (T→A) in exon of LOC_Os07g04670 gene, which caused a missense mutation at 61 amino acid (Leu to His) in the encoded product. Allelism analysis by inter-crosses showed that Oslg-1, Oslg-2 and Oslg-3 were allelic variation. Detection of the gene OsLG in Oslg-2 and Oslg-3 mutants was made and found that there was a single nucleotide change (316, T→A and 119, T→C) in exon of OsLG gene, which caused a missense mutation (106, Trp to Arg and 40, Leu to Pro) in the encoded product, respectively. These results suggested that the candidate gene may regulate the elongation of rice glume by the homologous gene sequence alignment and phylogenetic analysis. Besides, the relative expression content involved in OsLG gene and another PAP2 genecontrolling glume trait was also analyzed by real-time PCR (qRT-PCR). The results showed that OsLG gene was expressed in roots, leaf sheath and panicle, and presented rather higher expression in panicle than that in root and leaf sheath. By contrast, PAP2 gene was only detected in panicle. Therefore, both of OsLG and PAP2 were tissue-specificaly expressed, and PAP2 was much more higher expressed in panicle than OsLG. It was deduced that these two genes functioned in a synergistic pattern to control the development of long empty glumes as the expression level of OsLG and PAP2 were down in OsLG mutants. 【Conclusion】The Oslgmutants are allelic with G1 reported previously, and the mutation of a single amino acid in functional domain leads to the development of long sterile glume. OsLG and PAP2 are synergistically expressed in panicle.

Safety Assessment of Weediness of Transgenic Drought-Tolerant Wheat (Triticum aestivum L.)

JIANG Qi-yan, LI Xin-hai, HU Zheng, MA You-zhi, ZHANG Hui, XU Zhao-shi

Scientia Agricultura Sinica. 2015, 48(11): 2096-2107. doi:10.3864/j.issn.0578-1752.2015.11.002

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【Objective】 Genetically modified (GM) plants have been widely used in both agriculture and food industry. A scientifically sound environmental risk assessment, including weediness risk, is required for crops derived from genetically modified prior to unrestricted release into the environment. The objective of this study was to assess the potential ecological risk for weediness of transgenic drought-tolerant wheat MGX-L, and to provide scientific data for the formulation of evaluation standard of environmental safety assessment of transgenic wheat in China. 【Method】 In the cultivated lands and abandoned lands, the surviving competition ability of transgenic drought-tolerant wheat MGX-L, recipient variety and conventional wheat varieties were comparatively evaluated by investigating their agricultural traits. The propagation coefficient of wheat planted in abandoned lands was compared and the survival ability in abandoned lands was evaluated. Volunteer possibility, seed shattering and persisting possibility of transgenic drought-tolerant wheat MGX-L were analyzed and compared with the wild type for evaluating the ability of life continuous. The reproducing ability was evaluated by comparing the pollen viability, pollen diameter and sterility of MGX-L with the wild type. The weediness potential of MGX-L was evaluated based on the above results.【Result】There were no significant differences between transgenic wheat MGX-L and the wild type in plant height, spike number per hectare and per spike grain. The 1000-grain weight and yield of the transgenic wheat MGX-L were higher than that of the recipient variety Jimai 22. However, the 1000-grain weight and yield of the transgenic wheat MGX-L were lower than that of conventional wheat varieties or no significant difference. There were no significant differences between transgenic wheat MGX-L and the wild type in survival ability in the cultivated lands. Some wheat seeds could geminate and obtain seeds of next generation in abandoned lands. However, its propagation coefficient was less than 1 and the population size of transgenic wheat declined after the first year as a result of increased competition from native perennial plants. The transgenic wheat population couldn’t persist in abandoned lands like its conventional counterpart. There were no significant differences in seed shattering ability and volunteer plants rate between the transgenic wheat and the wild type. When buried under soil 20 cm, the seeds of transgenic wheat and the wild type were all rotted. When buried under soil 3 cm, the seeds of transgenic wheat and the wild type were rotted or sprouted. There were no intact seeds buried under soil. So there was no significant difference between the transgenic wheat and the wild type in seed persisting ability. 【Conclusion】 It was concluded that the transgenic drought-tolerant wheat MGX-L had the lowest potential weediness like the non-transgenic wheat varieties, based on the assessment of survival ability, seed shattering ability, volunteer plants rate and seed persisting ability.

Analysis of SSR Loci in Nicotina tabacum Genome and Its Two Ancestral Species Genome

TONG Zhi-jun, JIAO Fang-chan, XIAO Bing-guang

Scientia Agricultura Sinica. 2015, 48(11): 2108-2117. doi:10.3864/j.issn.0578-1752.2015.11.003

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【Objective】 The statistical analysis of SSR loci genomic information in Nicotina tabacum (2n = 24Ⅱ= 48 TTSS) and its two ancestral species (N. tomentosiformis 2n = 12Ⅱ= 24 TT and N. sylvestris 2n = 12Ⅱ= 24 SS) are carried out for the genetic analysis of plants of the Nicotiana genus. 【Method】 The data in the above-mentioned three tobacco genomes were downloaded from a public database NCBI (National Center for Biotechnology Information), 50 pairs of random amplified polymorphic SSR were synthesized for each genome, and analyzed by the SSRIT and TRF soft-wares for their SSR-loci distribution. 【Result】 A total of 218 081, 263 478 and 397 432 SSR loci, with their average distance 7.52, 7.78 and 9.06 kb were found in the genomes of N. tomentosiformis, N. sylvestris and N. tabacum, respectively. Almost all the SSR loci are distributed in the intron domains and especially the 5′-UTR domains. The SSR motifs within the genome are dominated by the types of Di-nucleotide and Tri-nucleotide, with more than 2/3 in the total number of SSR motifs, and the type of Di-nucleotide motifs has a most abundant peak, and contains the highest frequency and quantity of A (T)_n motif structure. The most of SSR motifs types have 3-10 repetitions, except mono-nucleotide type. There are 150 pairs of primers synthesized are subjected to PCR for the DNAs from eight tobacco species, all of which can amplify the clearly stable target fragments, with 36 pairs of primers shows polymorphisms. 【Conclusion】 Some distribution characteristcs of SSRs in the genomes of N. tomentosiformis, N. sylvestris and N. tabacum were observed, which indicated a high conservation for the SSR loci among the relatively closed tobacco species.

Effects of Dislodging Subtending Leaf on Grain Filling and Yield of Summer Maize

QIN Ying-ying, DONG Shu-ting, WEI Shan-shan, ZHANG Ji-wang, LIU Peng, ZHAO Bin

Scientia Agricultura Sinica. 2015, 48(11): 2118-2126. doi:10.3864/j.issn.0578-1752.2015.11.004

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【Objective】 A field experiment was conducted to study the effects of dislodging subtending leaf on grain yield, grain volume, dry weight, dehydrating rate and grain filling rate of summer maize.【Method】Three maize cultivars XY335, ZD958 and DH661 were used to analyze the effects of subtending leaf on grain yield and yield components, grain filling (grain volume, dry matter and dehydrating rate), and the time of milk lines disappearance and black layer appearance of summer maize by setting different dislodging subtending leaf treatments such as dislodging 1/4 subtending leaf (S1), dislodging 1/2 subtending leaf (S2), dislodging all subtending leaves (S3), and CK. 【Result】 The results showed that grain yield of summer maize decreased significantly after dislodging subtending leaf. Grain yields of S1, S2 and S3 of XY335 decreased by 9.45%, 13.78% and 27.89% respectively, compared to CK. Yields of ZD958 decreased by 10.72%, 15.07% and 24.75%, respectively, compared to CK. Yields of DH661 decreased by 9.31%, 16.01% and 22.35%, respectively. The damage increased with increasing number of dislodging subtending leaf. Grain weight and grain number per panicle of summer maize decreased significantly after subtending leaves were dislodged. The greatest effect was observed under the S3 treatment. Grain weight and grain number per panicle of S3 decreased by 15.30% and 14.87% in XY335 compared to CK, that of ZD958 decreased by 11.22% and 15.24% compared to CK, and that of DH661 decreased by 15.10% and 8.55% compared to CK. In addition, dislodging subtending leaf significantly affected grain filling characteristics. The damage increased with increasing number of dislodging subtending leaf. Days of reaching maximum grain filling rate (D_max) decreased significantly, and was the most susceptible to damage from dislodging all subtending leaves (S3). That of XY335, ZD958 and DH661 decreased by 3 d, 4 d, and 3.5 d compared to CK. Weight of reaching maximum grain filling rate (W_max) and active grain filling period (P) decreased significantly after the subtending leaves were dislodged, of which the most susceptible to damage was from the S3 treatment. W_max of XY335, DH661, and ZD958 is S3 decreased by 12.75%, 17.78%, and 17.12%, respectively, compared to CK. P of S1, S2, and S3 in DH661 decreased by 6.22%, 9.82%, and 11.78%, respectively, compared to CK. XY335 decreased by 5.39%, 8.39%, and 13.09%. ZD958 decreased by 0.17%, 2.39%, and 5.97%, respectively, compared to CK. Furthermore, dislodging subtending leaf decreased grain volume, grain fresh weight and days of reaching milk lines disappearance and black layer appearance, and increased dehydrating rate. Days of reaching milk lines disappearance of S3 decreased by 10 d, 6 d, and 4 d, respectively, compared to CK in XY335, ZD958, and DH661. Days of black layer appearance decreased by 10 d, 5 d, and 4 d, respectively, compared to CK in XY335, ZD958, and DH661.【Conclusion】Dislodging subtending leaf decreased days of maximum grain filling (D_max), weight of maximum grain filling rate (W_max), maximum grain filling (G_max), resulted in the decline of grain dry matter, affected grain filling characteristics, decreased the time of milk lines disappearance and black layer appearance, leading to the decrease of grain yield of summer maize. The effects decreased with the increase of the number of removed subtending leaves.

Effects of Growth Substances on the Fiber Growth and Development of Color Cotton Ovule Culture in vitro

LIU Song-jiang, GONG Wen-fang, SUN Jun-ling, PANG Bao-yin, DU Xiong-ming

Scientia Agricultura Sinica. 2015, 48(11): 2127-2142. doi:10.3864/j.issn.0578-1752.2015.11.005

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【Objective】 In order to establish and optimize theovule culture system of brown cotton and green cotton, the effects of growth substances (osmotic regulators, plant growth regulators and flavonoids biosynthesis precursors) on the fiber growth and development were studied.【Method】Brown cotton variety Z1-61, green cotton variety CC28 and the white cotton variety RT-White (control sample) were used as plant materials. The conventional ways of cultivation and management were adopted. Flowers were tagged at 0 day post anthesis (DPA), and then the ovules of 3 DPA were used for ovule culture in vitro. Besides the addition of 10.0 μmol·L^-1IAA and 5.0 μmol·L^-1GA3, different concentrations of osmotic regulators (mannitol, NaCl, KCl and sucrose), plant growth regulators (MeJA and BR) and flavonoids biosynthesis precursors (phenylalanine and ferelic acid ) were added to the basic BT medium, respectively. Except investigating the interaction effect between brassinolide and methyl jasmonate, the other experiments were chosen variety and treatment for dual factors test. After being cultured in vitro for 30 days, the fiber color, fiber length, ovule fresh weight, ovule dry weight and fiber dry weight of Z1-61, RT-White, and CC28 were determined. The mean value and standard error were calculated and multiple comparisons were carried out using least significant difference(LSD) or shortest significant ranges(SSR).【Result】Color cotton pigmentation was observed until 15 d after ovule culture in vitro. The best treatment for color cotton ovule growth and fiber development was 5 g·L^-1 sucrose, under which the fiber length, ovule fresh weight, fiber dry weight and ovule dry weight of Z1-61 was significantly increased by 28.35%, 20.69%, 103.70% and 75.84% (P＜0.05), while that of CC28 was significantly increased by 8.10%, 16.07%, 63.14% and 82.76% (P＜0.05), respectively, compared with the controls. In 0.05 μmol·L^-1MeJA treatment, the fiber length of Z1-61 was significantly increased by 22.90% (P＜0.05), while that of CC28 was increased by 4.39%. In 0.5 μmol·L^-1BR treatment, the fiber length of Z1-61 and CC28 was significantly increased by 22.46% and 11.56% (P＜0.05), respectively. In 25 μmol·L^-1 ferelic acid treatment, the fiber length of Z1-61 and CC28 was significantly increased by 8.72% and 8.81%（P＜0.05), respectively. Furthermore, 30 g·L^-1 mannitol, 0.1 mol·L^-1 NaCl, 0.20 mol·L^-1 KCl, 10 g·L^-1 sucrose, 40 μmol·L^-1 methyl jasmonate or 0.10 mmol·L^-1 phenylalanine were conducive to the pigment accumulation of brown cotton fiber. Among them the best treatment was 40 μmol·L^-1 MeJA. However, the growth substances used in this experiment did not cause any significant change in the pigment appearance of green cotton.【Conclusion】Sucrose provided the sugar source and osmotic environment for color cotton ovule growth and fiber development, and the appropriate concentrations of MeJA, BR or ferulic acid benefited the fiber elongation. There was a cross-effect between BR and MeJA. BR was mainly involved in fiber elongation, while MeJA may be involved in the pigment accumulation of brown cotton. Furthermore, certain concentrations of mannitol, NaCl, KCl, sucrose, MeJA or phenylalanine were advantageous to the brown cotton fiber pigment synthesis. However, neither osmotic regulators, plant growth regulators nor flavonoids biosynthesis precursors caused any significant change to the pigment accumulation of green cotton.

Research on Digitizing Morphological Structure and Growth Process of Grape Tree

WEN Wei-liang, WANG Yong-jian, LI Chao, WANG Chuan-yu, GUO Xin-yu

Scientia Agricultura Sinica. 2015, 48(11): 2143-2151. doi:10.3864/j.issn.0578-1752.2015.11.006

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【Objective】 Describing the morphology structure and growth process of plants in a digital way is an effective means of correctly understanding and expressing the development regulation of plants. In order to accurately describe and quantitatively analyze the grape trees with complex morphological structure, the digital expression and processing technologies of grape trees were studied in depth. 【Method】 The basic structure unit was divided and defined according to the morphological structure and growth process of grape tree, including the truck, perennial canes, new shoot and organs on the shoots. The new shoots include bearing and vegetative shoots. Organs on the shoots include buds, inflorescence, ears, secondary shoots, leaves and tendrils. Referencing the experience, habits, and trend of structure naming strategy at home and abroad, combining the actual field observation and following the temporal and space order of growth sequences of each structure unit, a naming scheme for the above ground structure units of grape trees at an annual rate was proposed. The first letters indicate the type and the figures showing the birth order of structure unit. The naming scheme also contains information of the growth and branching source relationship between the relative structure units. It integrated the shoot change process from the initial new shoot to the cane and then to the perennial cane under the perennial mechanism. The new shoots were constituted using an ordered set way in the scheme. On the basis of the naming scheme, the representation method of compound organs and complex operations was given, including representation method of complex organs such as bearing basal shoots and bearing shoot group; digital description of grape tree structure changes by artificial pruning; growth representation of grape trees at an annual rate; growth process backtracking of perennial grape trees; quantitative statistical analysis method of each structure unit. 【Result】 Taking the short shoot pruned V font shape Cabernet Sauvignon grape tree in Zhengzhou National Grape Germplasm Resources Garden and T font shape Xiangfei grape tree in the experimental greenhouse in Institute of Forestry and Pomology of Beijing Academy of Agricultural and Forestry Sciences as an example, the digital expression of the selected two trees was given respectively. The digital expression realized a clear representation of the two trees with complex structures, including the unique naming of each organ and the connection relationship of each other. On the basis of the structure expression, structure units on a new shoot were counted to make a quantitative statistic. The morphological structure change expression such as secondary shoot and tendril pruning and tree annual growth were illustrated as well. The application of digitized grape trees showed the universality and effectiveness of the method for the description of different forms and different growth periods of grape trees. 【Conclusion】 This study contributes to a more intuitive understanding of grape tree morphological structure and growth mode. It has important significance for constructing the functional-structural model, for realizing precisely describing morphological structure of grape trees, visualization expression and quantitative analysis, etc. The study also has important significance for quantitative calculating and simulating the condition, development and evolution of the internal factors of grape trees, and for further prediction and evaluation of these factors. At last, the structure unit definition and naming scheme have reference values to research the woody plants of similar morphological structures.

Advances in Research on Maize Resistance to Ear Rot

DUAN Can-xing, WANG Xiao-ming, SONG Feng-jing, SUN Su-li, ZHOU Dan-ni, ZHU Zhen-dong

Scientia Agricultura Sinica. 2015, 48(11): 2152-2164. doi:10.3864/j.issn.0578-1752.2015.11.007

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Maize is one of the most important crops in the world. The percentage of maize yields is 38.12% in gross cereal yields of China. Ear rot is a serious disease of maize, which often leads to a decline in yield and quality, and that hazards animals and human health because of the toxins that are produced in the ear rot. The development and utilization of resistant cultivars is the most economical, safe, and effective method for controlling ear rot. So far, many studies on pathogens causing maize ear rot have been reported and more than 40 pathogenic fungi have been isolated and identified. The ear rots caused by Fusarium verticillioides, F. graminearum and Aspergillus flavus are very common and serious diseases. The four methods of resistance identification have been established, i.e. two toothpicks method, silk spraying, silk channel injection and kernel injection. Thousands of maize germplasm have been identified and evaluated for resistance to ear rot, and some accessions with resistance to F. verticillioides, F. graminearum or A. flavus also have been screened out. Resistance inheritance and gene mapping have been conducted for some materials, and more than 60 quantitative trait loci (QTL) associated with resistance to A. flavus infection and aflatoxin accumulation in maize have been mapped on chromosomes 1-8. Furthermore, 55, 29 and 16 QTLs resistance to F. verticillioides, F. graminearum and fumonisin and deoxynivalenol (DON) accumulation have been discovered, respectively. Based on QTL associated with ear rot resistance in maize, the meta-QTL analysis has been projected and some stable QTLs have been detected. Also, more than 10 ear rot resistant accessions have been developed and some new varieties with different levels of resistance have been bred. In addition, physical and biochemical resistance mechanisms were demonstrated, which mainly reflected in anti-infection and anti-diffusion. However, there are few successful cases that the resistance research and disease-resistant breeding results are applied to production practice, resulting in lack of maize cultivars with stable resistance as well as high yield and quality. In this paper, the advances including pathogens, identification of resistant maize germplasm, resistance inheritance, discovery and mapping of resistance genes, resistance mechanism, and breeding resistant varieties in research of maize resistance to ear rot are reviewed, and several important directions for the future researches are prospected. The main aim is to supply useful information so as to promote the development of the research on maize resistance to ear rot.

Optimization of Fermentation Condition for Laccase Production by Setosphaeria turcica Using the Response Surface Methodology and the Enzymatic Characters

CAO Ke-ke, LIU Ning, MA Shuang-xin, CAO Zhi-yan, LIANG Dong-xu, CHAI Jiang-ting, DONG Jin-gao

Scientia Agricultura Sinica. 2015, 48(11): 2165-2175. doi:10.3864/j.issn.0578-1752.2015.11.008

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【Objective】The objective of this study is to optimize fermentation parameters for the laccase production by Setosphaeria turcica, investigate its enzymatic properties, and to develop the applications of the laccase.【Method】According to the results of single factor experiments to confirm the categories and concentration scales of the carbon source, nitrogen source and Cu²⁺ as single parameters, the response surface experiments of fermentation using S. turcica were preformed which the enzymatic activity was chosen as the response value. The experiments were designed by the central composite design (CCD), and analyzed by Multivariate Quadratic Regression Analysis of Design Expert for three factors and three levels, then the optimal fermentation medium for laccase production by S. turcica was determined. And the properties of laccase isolated from the fermentation culture were characterized using ABTS as substrate, including the optimum temperature and pH, the stability at different pH and temperatures, and the kinetics constants of Km and Vm.【Result】The multivariate quadratic regression model which the enzymatic activity was chosen as response value was significantly different (P=0.0001), which meant the established regressing equation for activity of laccase had an excellent goodness of fit and the laccase fermented by S. turcica could be analyzed by this model. According to the model, the factors influencing the activity of laccase were in the order as follows: Cu²⁺＞glucose＞urea and the interaction of glucose and urea was very significant. The optimum conditions, which were predicted by this model, were glucose 50.05 g?L^-1, KH₂PO₄ 1 g?L^-1, urea 1.46 g?L^-1, MgSO₄ 0.5 g?L^-1, peptone 2 g?L^-1, maize steep liquor 0.5 g?L^-1, CuSO₄ 0.07 g?L^-1 and Tween80 3 mL?L^-1, at 28℃, and 150 r/min. The maximal enzyme activity was (40.00±1.20) U?mL^-1 after 7 d of cultivation. The laccase of S. turcica had a molecular weight of approximately 80 kD analyzed by SDS-PAGE after preliminarily purified. It was demonstrated that the optimum pH and temperature were pH 4.2 and 50℃, respectively, and the laccase of S. turcica had higher activity at higher temperature and weak acidic condition, using ABTS as substrate. It also exhibited high thermal stability and the stability of pH, retaining its activity basically unchanged after incubation at pH 4.2 for 14 h and above 60% at 50℃ for 14 h. The Km was 0.036 mmol?L^-1 and Vm was 28.63 mmol?L^-1·min^-1, at room temperature and pH 4.2. 【Conclusion】The enzymatic properties of the laccase of S. turcica were investigated, and the fermentation medium by response surface methodology was optimized. The laccase of S. turcica has a potential value for further research due to the advantages of high activity, short fermentation period and high stability.

Control Efficacies of Two Preparations of Difenoconazole and Fluazinam by Seed-Coating Against Peanut Soil-Borne Fungal Diseases

GUAN Lei, GUO Bei-bei, WANG Xiao-kun, ZHANG Da-xia, WANG Kai, LIU Feng

Scientia Agricultura Sinica. 2015, 48(11): 2176-2186. doi:10.3864/j.issn.0578-1752.2015.11.009

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【Objective】 In peanut production area of China, disease of crown rot, root rot and Sclerotium blight commonly occurred and caused heavier damage in recent years. The objective of this study is to compare the toxicity of FS (flowable concentrate for seed treatment) and CS (microcapsule suspension) of difenoconazole and fluazinam to three peanut soil-borne fungi and definite the safety, control efficacies and application methods of two preparations of difenoconazole and fluazinam by seed-coating against three peanut soil-borne fungal diseases. 【Method】 Firstly, the toxicity of two preparations of difenoconazole and fluazinam to Aspergillus niger, Fusarium solani and Sclerotium rolfsii were determined. Then the safety of seed-coating of the two preparations of difenoconazole and fluazinam on peanut was evaluated. Finally, field trial was conducted in order to confirm their control efficacies. 【Result】 The EC₅₀ values of 6% difenoconazole FS, 15% difenoconazole CS, 50% fluazinam FS and 12% fluazinam CS in inhibiting A. niger were 0.0503, 0.4959, 5.6141 and 46.5433 mg?L^-1; in inhibiting F. solani were 0.5386, 2.8604, 0.3780 and 28.1665 mg?L^-1; in inhibiting S. rolfsii were 1.1435, 3.2615, 0.0951 and 3.8920 mg?L^-1, respectively. All the treatments exhibited no significant difference compared with control check about peanut emergence rate and time, root length and fresh weight, plant height, fresh weight of stem and leaf (in an incubator at 20℃ and 25℃). In the field trial, all the treatments exhibited no significant difference compared with control check about peanut emergence rate and time. The control efficacies of 6% difenoconazole FS and 15% difenoconazole CS at the rates of 100 and 200 g a.i./100 kg seed by seed-coating to the three peanut soil-borne fungal diseases were all higher than 68.03%. The control efficacies of 12% fluazinam CS at the rate of 160 g a.i./100 kg seed by seed-coating to the three peanut soil-borne fungal diseases were all higher than 74.97%, which were higher than the same dosages of 50% fluazinam FS. Pod yield of all treatments significantly increased compared with control check. The yield growth rate increase of 15% difenoconazole CS at the rate of 200 g a.i./100 kg seed treatment was higher, and ranged from 7.22% to 11.47%.【Conclusion】Difenoconazole 6% FS and 15% CS at the rates 100 and 200 g a.i./100 kg seed, and fluazinam 12% CS at the rate 160 g a.i./100 kg seed were all security to peanut and were high-efficient in controlling peanut crown rot, root rot and sclerotium blight by seed-coating treatment.

Effects of Root Layer Regulation on Nitrogen Utilization and Soil NO3--N Residue of Wheat-Maize System

PENG Ya-jing, WANG Xin-ying, ZHANG Li-juan, HAO Xiao-ran, QIAO Ji-jie,WANG Wei, JI Yan-zhi

Scientia Agricultura Sinica. 2015, 48(11): 2187-2198. doi:10.3864/j.issn.0578-1752.2015.11.010

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【Objective】 Low resource utilization efficiency of water and fertilizer and high soil nitrogen accumulation were common problems of the winter wheat-summer maize system in the North China Plain. This study was conducted to investigate the effects of different root layer regulation measures on nitrogen utilization and soil NO₃^--N residue of crops. 【Method】 The field experiment was conducted in the high-yield farmlands in North China Plain. Traditional water-nitric nitrogen, water-nitric nitrogen regulation, regulation+Agh (soil conditioner), regulation+CRU (controlled-release urea), regulation+GGR (plant growth regulator) were designed as five treatments. Soil, plant and root samples were collected and measured, and the nitrogen utilization effects of different root layer regulation measures were analyzed.【Result】The results indicated that under the premise of controlling water and nitrogen, the wheat-maize yield and nitrogen uptake in Agh and GGR treatments were higher than traditional water and nitrogen treatments. At jointing stage of wheat, roots at 80-100 cm soil layers in GGR treatment distributed much, which shows that GGR could promote the development of roots at lower layer. At bell stage of maize, at 20-50 cm layer, root length density of Agh treatment in Gaocheng and GGR treatment in Daming was significantly higher than those of traditional water and nitrogen treatment. In the first rotation season, in 0-200 cm layer, the soil residual nitrate-N in GGR treatment in Gaocheng and Shenzhou was significantly lower than that of traditional water and nitrogen treatment, especially at 60-100 cm layer soil residue nitrate nitrogen was the lowest. In the second rotation season, the soil residual nitrate-N in Agh treatment in Gaocheng and GGR treatment in Daiming were significantly lower than traditional water and nitrogen treatment. The amount of apparent deficiency of nitrogen of Agh in the first rotation season and GGR (Gaocheng) in the second rotation season was large, which indicate that root regulation could promote crop to absorb soil accumulated nitrogen. Root layer regulation measures were of benefits to economy and ecology, Water Use Efficiency (WUE) and Partial Factor Productivity_Nitrogen (PFP_N) increased by 2.47 kg·m^-3 and 18.08 kg·kg^-1 compared with traditional water and nitrogen treatment, and the average income was 258.43 yuan/667 m². 【Conclusion】 In high yield land in the North China Plain, different root-layer regulation measures of single cropping and annual production of wheat and maize respectively increased by 8.58%, 5.99% and 7.13% in average compared with the traditional water and nitrogen treatment. After two seasons the residue of soil nitrate nitrogen in 0-100 cm layer soil were reduced by 70.73 kg·hm^-2 and 59.44 kg·hm^-2 in average compared with the conventional water and nitrogen treatment, obviously reduced the residue of soil nitrate nitrogen, slowed the leaching loss of nitrogen to deep soil, and promoted root development during critical growing period of wheat-maize. All in all, Agh and GGR were effective measures to improve crops yield, use the soil accumulated nitrogen, achieve cost synergies and improve water and fertilizer use efficiency.

Calcium Degradation Status of Orchard Soil in Weibei Region, Shaanxi Province, China

WEI Bin-meng, WANG Yi-quan, SHI Zong-lin, LI Peng, SHI Hong-ping, LIANG Hua-xue, WANG Jia-xu

Scientia Agricultura Sinica. 2015, 48(11): 2199-2207. doi:10.3864/j.issn.0578-1752.2015.11.011

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【Objective】 Apple production contributes greatly to the economic development of Shaanxi province. But orchard soil has degraded significantly after years of fruit production, which lead to tree senescence and various diseases such as apple bitter pit, water-core and lenticel blotch pit. As a result, fruit yield and quality decreased sharply. The objective of this study was to investigate the distribution and extent of soil calcium degradation in the orchards of different ages, with the aim to facilitate sustainable development of the yield and quality of apple production. 【Method】 Four replicates of ＜10-, 10-20- and ＞20-year old orchards soil and farmland soil as control were selected for the study in Binxian, semiarid region of Shaanxi. Samples were taken within two-thirds of the radius of the tree canopy. Soil samples were used to measure physical and chemical properties such as soil bulk density, pH, CaCO₃, exchangeable Ca, and water-soluble Ca. 【Result】 The soil CaCO₃ content and storage in 0-50 cm increased as orchard age increased, while decreased as orchard age increased in 50-100 cm. Soil CaCO₃ storage in the orchards older than 10 years were significantly lower than that of farmland in 0-50 cm. The exchangeable Ca and water-soluble Ca in all orchard soils were higher in comparison with the farmland soil. The orchard soil exchangeable Ca decreased with orchard age increasing in layers of 0-40 cm and below 60 cm, while slightly increased with orchard age increasing in 40-60 cm. The water-soluble Ca content and storage in 0-50 cm were higher than that in 50-100 cm, and the soil water-soluble Ca has a peak in 10-30 cm in the orchards older than 10 years.【Conclusion】The storage of soil CaCO₃and exchangeable Ca in the layer of 0-100 cm decreased severely with orchards planting years increased, even in the calcareous soil of loess dry-land in Shannxi Weibei Region. The decrease of soil CaCO₃ and exchangeable Ca has become one of the characteristics of soil degradation. Calcium deficiency should be concerned in the orchard fertilization management in this region.

Expression and Protein Interaction Analysis of MdMYB9 and MdMYB11 in Apple

AN Xiu-hong, ZHANG Xiu-de, CHEN Ke-qin, LIU Xiao-juan, HAO Yu-jin, CHENG Cun-gang

Scientia Agricultura Sinica. 2015, 48(11): 2208-2216. doi:10.3864/j.issn.0578-1752.2015.11.012

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【Objective】 This study is aiming at isolating MdMYB9 and MdMYB11 genes that are homologous to TT2 from Arabidopsis, detecting their expression in different tissues and under light treatment, identifying the interaction of MdMYB9 and MdMYB11 with MdbHLH33, and laying the foundation for further function analysis of this two genes. 【Method】 MdMYB9 the protein molecular weight, isoelectric point and the full length amino acid sequences were analyzed using DNAMAN software. In addition, the phylogenetic tree of the MYB proteins, including MdMYB9, MdMYB11 and the R2R2-MYB family proteins from Arabidopsis, was constructed using the neighbor-joining (NJ) method of MEGA 4.0 software. The transcriptional levels of MdMYB9 and MdMYB11 genes were detected in different tissues, fruit skins and seeds at different developmental stages and under light treatment using qPCR. Finally, the interaction of MdMYB9 and MdMYB11with MdbHLH33 protein was detected by Y2H assay.【Result】Sequence analysis showed that the open reading frame (ORF) of MdMYB9 was 873 bp in length, encoding 290 amino acids and sequence alignment revealed 38.13% identity with TT2 in Arabidopsis. The ORF of MdMYB11 gene was 861 bp in length, encoding 286 amino acids and sequence alignment revealed 32.44% identity with TT2. Both the proteins contained the typical R2R3 domain in the N-terminal. qPCR analysis indicated that MdMYB9 and MdMYB11 were expressed in all organs, including roots, stems, leaves and flowers, but their expressions exhibited different levels in the detected tissues. Both genes expressed at different developmental stages of seeds and skins, and the highest expression appeared at the middle stages; however, the expression of MdMYB9 and MdMYB11 were not obviously different under light treatment. Yeast two-hybrid assay showed that both MdMYB9 and MdMYB11 interacted with MdbHLH33.【Conclusion】MdMYB9 and MdMYB11 were expressed in different tissues, developmental fruit skins and seeds, and interacted with MdbHLH33 protein. and MdMYB11 genes associated with proanthocyanin biosynthesis were isolated using homology-based cloning method, and the primers were designed to amplify these two genes. Subsequently,

Research on Influences of Rootstock on Sugar Accumulation in ‘Shatangju’ Tangerine Fruits

LIU Xiang-yu, LI Juan, HUANG Min, LIANG Chun-hui, CHEN Jie-zhong

Scientia Agricultura Sinica. 2015, 48(11): 2217-2228. doi:10.3864/j.issn.0578-1752.2015.11.013

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【Objective】 The effects of different rootstocks on sugar accumulation, hormone role, sucrose-related enzyme activities and gene expression of ‘Shatangju’ tangerine fruits were studied. The aim is to make research on the physiological and molecular mechanism of citrus rootstock effects on fruit sugar accumulation, and to establish a theoretical basis for the use of rootstocks in citrus production. 【Method】 ‘Shatangju’ tangerine, grafted on Trifoliate orange and Canton lemon rootstocks, were used in this study. The sugar content and sucrose metabolism enzyme activities were determined in fruits of ‘Shatangju’ tangerine. The expression levels of related genes, including sucrose metabolism enzymes and sucrose transport proteins, were obtained from the fruits. Meanwhile, abscisic acid (ABA) levels and expression patterns of NCED1 gene were also measured in the fruits.【Result】 Results showed that total soluble sugar and sucrose content were significantly higher in both the peel and pulp of fruits on Trifoliate orange rootstocks than those on Canton lemon rootstocks at maturation stage. In the meantime, there were higher activities of sucrose synthase (SS) in the fruits on Trifoliate orange rootstocks. qRT-PCR analysis showed that the expression levels of SS2 gene were higher in the fruits on Trifoliate orange rootstocks than those on Canton lemon rootstocks. On the contrary, CWINV1 expression levels were higher in the fruits of ‘Shatangju’/Canton lemon, and was negatively related to sucrose content (r=-0.648*). Furthermore, SUC3 expression increased along with maturity and showed higher levels in the fruits of ‘Shatangju’/Trifoliate orange. There was a significantly positive correlation between SUC3 expression and total soluble sugar content (r=0.87*). In addition, the ABA concentration gradually increased in the peel during fruit ripening, and the ABA concentration was significantly and positively correlated with SS2 and SUC3 expression and significantly greater in ‘Shatangju’/Trifoliate orange after December 5. In the pulp, the ABA concentration time series followed a flattened curve, and the ABA concentration of the pulp from the ‘Shatangju’/Trifoliate orange was also greater. In the peel, NCED1 expression gradually increased with fruit ripening, and showed higher levels in ‘Shatangju’/Trifoliate orange. NCED1 gene was up-regulated and exhibited significant higher levels in the pulp of ‘Shatangju’/Trifoliate orange at later stage. 【Conclusion】 In short, the choice of rootstock had a significant effect on sugar accumulation of ‘Shatangju’ tangerine fruits, mainly through changing the activities and gene expression patterns of sucrose metabolism enzymes, sucrose transport and ABA levels in the fruits.

Effects of Pressure Varied Static Brining on Pork Quality

GUO Xin, HUANG Feng, ZHANG Chun-jiang, HU Hong-hai, CHEN Wen-bo, ZHANG Hong

Scientia Agricultura Sinica. 2015, 48(11): 2229-2240. doi:10.3864/j.issn.0578-1752.2015.11.014

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【Objective】 The objective of this experiment was to investigate the effect of pressure varied static brining on pork quality, made an approach to the superiority of pressure varied static brining and increase quality of brining meat products. 【Method】 Pressure varied static brining (PVSB-test group), atmospheric pressure brining (APB-control 1), vacuum brining (VB-control 2) and pressure increasing brining (PIB-control 3) were employed to prepare cured meat. Subsequently, salting efficiency (salt content, marinade absorption) and various quality indexes including pH value, eating quality (water content, centrifugation loss, drop loss, color, shear force, TPA), salt-soluble protein content, microstructure of pork myolin and myofibrillar protein degradation were investigated. 【Result】 In terms of marinade absorption, there was no significant difference between test group and control groups before 4 h and for 4 h resulted in higher than control groups (P＜0.05). In comparison with control 1, control 2 and control 3, the salt content of test group increased significantly by 31.9%, 10.20%, and 7.29%, respectively (P＜0.05). In comparison with control, the pH value and eating quality increased. Test group resulted in higher pH value during curing than control and presented significances in every time point (P＜0.05). Water content and water capacity of pork muscle treated with PVSB were increased significantly (P＜0.05). Test group presented a significance in lower centrifugation loss than control group 1 and lower drop loss than control group 1, control group 2. PVSB showed a significant effect on tenderness, TPA and color. The test group had lower shearing force, hardness, springiness, chewiness and gumminess than control groups (P＜0.05). PVSB resulted in higher b* of the external than control groups (P＜0.05), a*, b* of the internal and content of myoglobin had no significant differences among four treatments. PVSB also had a significant effect on meat protein and microstructure of pork myolin. With the curing time extension the soluble protein in pork muscle and marinade was improved. The test group resulted in higher salt soluble protein in pork muscle than control group 1 and control group 3, and lower in marinade than control for 6 h (P＜0.05), respectively. The degree of myofibrillar protein degradation in test group and control groups was inconsistent. There was no apparent bands appeared or disappeared from 1.5 to 4.5 h in test group, while it had 40 kD fragment appeared at 6h, but not apparent. All control groups had 40 kD fragment appeared. It was showed that the test group resulted in lower protein degradation than control groups. PVSB had decreased the degree of damage in muscle microstructure. After curing, the muscle fiber bundles of the control group 2 and control group 3 were damaged more seriously than test group and control group 1, so they presented wise integrity in the microstructure of pork muscle, while the loose in control group 2 and control group 3. 【Conclusion】 As compared with APB, VB and PIB, PVSB increased salting efficiency and improved eating quality significantly. Therefore, this technology should have wider application prospects.

Effect of Heat Treatment on the Intramuscular Lipid of Ira Rabbit During Cold Storage

XUE Shan, HE Zhi-fei, XIAO Xia, LI Hong-jun

Scientia Agricultura Sinica. 2015, 48(11): 2241-2250. doi:10.3864/j.issn.0578-1752.2015.11.015

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【Objective】 This paper discussed the variation of the content and fatty acid composition of intramuscular phospholipid of Ira rabbit processed by three heat treatments during the cold storage, in order to provide a theoretical basis for the quality control of Ira rabbit meat as well as its products. 【Method】 The Longissimus dorsimuscle of Ira rabbit was used as material, and then the Ira rabbit was cooked by stewing, microwaving and Al foil-baking, respectively, in order to find out the variation of the content and fatty acid composition of intramuscular phospholipid by gas chromatography analysis. In addition, the change of fatty acid composition of intramuscular phospholipid was analyzed by the way of partial least squares regression analysis (PLSR), making the eight main design factors (raw material, three kinds of heat treatment, four cold storage times) as X variables, and fatty acid composition as Y variables. 【Result】 During the 0-9 d storage, dry weight (%) of cooked samples all significantly decreased (P＜0.05), especially in the 6-9 d late storage. Compared with the other two methods, the microwaving treatment did less effect on the content of intramuscular phospholipid. Besides, all cooked-samples showed a significant decrease in the proportion of polyunsaturated fatty acids (PUFA) and monounsaturated fatty acids (MUFA), whilst a significant increase in the proportion of saturated fatty acids (SFA) was observed during the cold storage (P＜0.05). At the 6-9 d storage phase, the PUFA profile, especially the long-chain PUFA showed higher degree of decreasing. The Al foil-baking was better to keep the UFA of intramuscular phospholipid from degrading than the other two methods, especially the PUFA proportion. In addition, during the 0-9 d cold storage, the n-6/n-3 value of phospholipid fatty acid of cooked Ira rabbit muscle increased significantly (P＜0.05). In spite of this, the n-6/n-3 value of the cooked samples was still within the recommended range. By comparison, the stewing processing was more conducive to retain the lower value of n-6/n-3. According to the PLSR, when the Ira rabbit muscle was cooked by stewing, microwaving and Al foil-baking, respectively, the cold storage time showed a great impact on the composition of intramuscular phospholipid fatty acid than the method of heat treatment. During the storage, the vast majority of the phospholipid fatty acid changed differently in composition, especially at the 0-3 d storage phase, the percentage of the MUFA (C17:1) and PUFA (C20:2n-6, C20:4n-6, C20:5n-3, C22:5n-3 and C22:6n-3）decreased significantly, while the percentage of the SFA (C16:0 and C18:0) increased significantly (P＜0.05). Compared the three heat treatments, the stewing processing was more effective to keep the stable of the composition of intramuscular phospholipid fatty acid of Ira rabbit during the 0-9 d cold storage. 【Conclusion】 The content and fatty acid composition of intramuscular phospholipid of Ira rabbit can be significantly affected by the method of heat treatment and cold storage time. When cooked by stewing, microwaving and Al foil-baking, the influence of cold storage time on the intramuscular phospholipid fatty acids was more obvious than the influence of heat treatment. From the nutritional & healthy point of view, it was quite necessary to use the gentle heat processing method, and make the cold storage time shorter.

Tissue Expression Profile and Key Target Genes Analysis of Porcine miR-192 and miR-215

WU Zheng-chang, YIN Xue-mei, SUN Li, XIA Ri-wei, HUO Yong-jiu, WU Sheng-long, BAO Wen-bin

Scientia Agricultura Sinica. 2015, 48(11): 2251-2261. doi:10.3864/j.issn.0578-1752.2015.11.016

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【Objective】The previous study made by the authors’ group had screened out two microRNAs with extremely significantly increased expression for E.coli F18 infection: miR-192 and miR-215, using Illumina Solexa high-throughput sequencing technology combined with fluorescence quantitative determination. The objective of this study is to understand the miR-192/ miR-215 expression and further screen out the key target genes of miR-192 and miR-215 regulating E.coli F18 infection, which will lay a foundation for probing into the molecular mechanisms of regulating E.coli F18 infection in weaned piglets. 【Method】Real-time PCR was used to detect the miR-192/miR-215 expression in different tissues of Meishan piglets at 35 days of age, meanwhile conservative sequence was analyzed using MEGA 5.0 software. The algorithms TargetScan was used to predict miR-192/miR-215 putative target genes, whose enrichment of Gene Ontology and Pathway was analyzed, respectively. Then the functional classification of target protein was further conducted using DAVID, finally, the intersection between predicted target genes and regulatory genes was taken for the resistance to E. coli F18 infection of weaned piglets which were screened out by the previous study. 【Result】 The results indicated that miR-192/miR-215 were highly expressed in the duodenum and jejunum tissues of Meishan piglets at 35 days of age. The mature sequences of miR-192 and miR-215 are highly conservative in vertebrates. A total of 156 target genes of miR-192/miR-215 were exactly the same, which were significant enrichment in Axon guidance pathway and in 25 GO function (P＜0.05). Target proteins were divided into 12 categories according to the function, and most of the target proteins belonged to phosphoprotein with signal transduction function and DNA binding protein. Through intersection between target genes and regulatory genes (Gene Expression Omnibus, Accession number: GSE26854) for the resistance to E. coli F18 infection, five important target genes such as ALCAM【Conclusion】The results of this study revealed that miR-192 and miR-215 were two important factors of participating in maintaining the intestines function and resisting the E.coli F18 infection, DLG5 was likely to be the important target gene of miR-192 and miR-215 regulating E.coli F18 infection, and further analyses should be conducted to testify whether the DLG5 could be an effective genetic marker for the resistance to E.coli F18 infection in Chinese native pig breeds., DLG5, FRMD4B, MIPOL1 and ZFHX3 were attained. From the point of target genes function, DLG5 was an important factor for maintaining epithelial cells structural integrity.

Effects of Pro-Biotic Microbes on Living Water and Laying Performance in Layer Ducks and Molecular Mechanisms

YING Shi-jia, XU Ai-hong, DAI Zi-chun, XI Zheng-lin, HE Zong-liang, KUANG Wei, ZHAO Wei, WU Yun-liang, SHI Zhen-dan

Scientia Agricultura Sinica. 2015, 48(11): 2262-2269. doi:10.3864/j.issn.0578-1752.2015.11.017

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【Objective】 This study aims to investigate the effects of supplementation of Bacillus subtilis spores in the feed and addition of photosynthetic bacteria (PSB) on living water and laying performance of laying ducks and their mechanisms.【Method】A total of 240 laying ducks were randomly and equally allocated into 24 “slate floor bed + water pool” individual pens using a 2×2 factorial design with 6 replications per treatment. After 7-d pre-feeding, the ducks were received 2 main treatments: supplementation of Bacillus subtilis (viable count 8 × 10⁷ cfu/kg) in the feed, and addition of PSB (viable count 1.8 × 10¹² cfu/kg) to the living water. Neither supplementation of B. subtilis nor addition of PSB into living water was used as the control group (C group), supplementation of B. subtilis was used as the B. subtilis group (B group), addition of PSB into living water was used as the PSB group (P group), and both supplementation of B. subtilis and addition of PSB into living water was used as the combined group (BP group). In the 120-d experimental period, the average laying rate, average feed intake and average feed to egg ratio were calculated every10 days. The living water samples were collected every 10 days for measuring counts of E. coli and Salmonella + Shigella. The living water and blood samples were collected every 15 days for measuring LPS concentration. At the end of the experiment, the fresh feces were collected for measuring counts of E. coli and Salmonella + Shigella. The mucosa and tissues of duodenum, jejunum and ileum were collected for measuring LPS concentrations and mRNA expression of inflammation/apoptosis - related genes, respectively. During the experiment, the animals were maintained under natural photoperiod. Living water and feed were available to the ducks ad libitum. The linear mixed model of SPSS, for the analysis of repeated measurements, was used with comparisons using the LSD correction. The data with repeated measurements included LPS concentrations in living water and plasma, feed intake, laying rate, feed to egg ratio, and counts of E. coli and Salmonella + Shigella in living water. The one-way ANOVA was used for data including counts of E. coli and Salmonella + Shigella in feces, LPS concentrations of intestinal mucosa, and tissue gene expression. Post-hoc differences between treatment groups were further examined using Duncan test.【Result】Both Bacillus subtilis and PSB treatments did not significantly affect feed intake (P＞0.05), Caspase-3 and Bcl-2 mRNA expression in jejunum (P＞0.05), Bcl-2 mRNA expression in ileum (P＞0.05), and IL-1β mRNA expression in spleen, however, they significantly increased laying rate (P＜0.001), and decreased feed to egg ratio (P＜0.001), LPS concentrations of living water (P＜0.001), duodenum (P＜0.01) and ileum (P＜0.01), counts of E. coli in feces (P＜0.001), and mRNA expression of TLR4 in jejunum (P＜0.05) and Caspase-3 in ileum (P＜0.05). There was significant effect of time on feed intake, laying rate, feed to egg ratio, LPS concentration and counts of E. coli and Salmonella + Shigella in living water (P＜0.01). Compared with B and BP groups, LPS concentration in plasma and counts of Salmonella + Shigella in feces in C and P groups were increased (P＜0.05), and LPS concentration in jejunum, counts of Salmonella + Shigella in living water and TNF-α mRNA expression in spleen were also increased (P＜0.05). The count of E. coli in living water in BP group was lower than in C, B and P groups (P＜0.05). Only in BP group was TLR4 mRNA expression in ileum lower (P＜0.05), and IL-10 mRNA expression higher than in C group (P＜0.05).【Conclusion】The results indicate that B. subtilis and PSB treatments improved laying performance of laying ducks by decreasing the counts of Gram-negative bacteria and level of LPS in cultivated water, and decreasing the level of LPS in intestinal mucosa and plasma and inflammatory response.

Construction of Multi-Transposon Vectors, and Comparative Study of Transposon Characteristics

SHEN Dan, XIE Yu-xiu, LI Qing-ping, XUE Song-lei, SHI Yun-qiang, WANG Sai-sai, CHEN Cai, QIAN Yue, GAO Bo, CUI Heng-mi, SONG Cheng-yi

Scientia Agricultura Sinica. 2015, 48(11): 2270-2278. doi:10.3864/j.issn.0578-1752.2015.11.018

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【Objective】 Transposons are the basic unit of chromosomes which can autonomously replicate and shift within the whole genome. Sleeping beauty (SB), piggyBac(PB) and Tol2, found from salmonid, cabbage looper moth Trichoplusia ni and Oryzias latipes, respectively, are most active transposons in the vertebrates today. This paper compares the transfection, insertion and cutting efficiencies of the 3 different transposons in 3T3 cells, then obtained the best transposon system at the cellular level.【Method】 The 3′ and 5′ terminal transposable elements were cloned from SB, PB and Tol2 transposon vectors using the high-fidelity PCR in this experiment, ensured the accuracy through sequencing, then the transposable elements were subcloned one-by-one into the pT2-HB carrier frame, thus constructed the multi-transposon vectors, pT3-PST, which included all 3 transposons. Green fluorescent protein expression cassette (pCAG-GFP) and neomycin expression cassette (NEO) genes were cloned into pT3-PST carrier, resulting in two expression vectors, pT3-PST-CAG-GFP and pT3-PGK-NEO. These two expression vectors, with the transposase expression plasmid pCMV-SB100X, were mixed with pCMV-HAhyPBase and pCMV-Tol2 at a mass ratio of 1:1, respectively. They were then wrapped by polycation PEI, and co-transfected into the mouse embrynic fibrilasts (3T3). At the same time, a negative control group was set up using the inactive transposase vector SB△DD. After 36 h post-transfection of GFP, detection of GFP expression was done by fluorescence microscopy, and real-time fluorescent quantitative PCR, after cell genome extraction and using Amp as an internal reference. Primers were designed according to the upstream and downstream sequences, then the cutting efficiency of the 3 transposons was determined by real-time fluorescent quantitative PCR. After 48 h post-transfection of NEO, cells were filtered by using 500 µg·mL^-1 G418 resistance screening until the normal cells were almost dead (10 d). The cells were stained with Giemsa stain, and the number of drug-resistant cells was counted, and finally different transposon activities of different groups were compared.【Result】Multi-transposon vectors, PT3-PST, pT3-PST-CAG-GFP,and pT3-PGK-NEO were successfully constructed. The transfection efficiency of cells in the experimental groups was more than 50%, and the difference was not significant (P＞0.05). The GFP relative copy number of SB group was higher than Tol2 and PB groups, but the difference was not significant (P＞0.05), though it was significantly higher than the control group (P＜0.05). The cutting efficiency of SB group and PB groups was significantly higher than Tol2 group(P＜0.05), though SB and PB group had no significant difference (P＞0.05). pT3-PGK-NEO and transposase co-transfected 3T3 cells, and the results of G418 selection showed that the number of drug-resistant cells of SB group was significantly higher than PB and Tol2 groups (P＜0.05), but the difference between PB and Tol2 groups was not significant (P＞0.05). However, all the three experiment groups were significantly higher than that of the control group (P＜0.01).【Conclusion】The SB transposon system efficiency, at the cellular level, was better than PB and Tol2, which could provide an effective tool for cellular transgenetic research.

Cloning and Expression of Chitinase PbChiIV Genes from Pyrus bretschneideri ‘Yali’

LI Peng-peng, LIU Ning, ZHANG Yu-xing, PENG Jian-ying, DONG Jin-gao

Scientia Agricultura Sinica. 2015, 48(11): 2279-2286. doi:10.3864/j.issn.0578-1752.2015.11.019

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【Objective】This study aimed to clone a full length cDNA of PbChiIV，fruit and the expression after treatment with the salicylic acid(SA) and Botryosphaeria berengeriana de. Not. f. sp. piricola (Nose) Koganecawa et. Sokwwa.【Method】The full lengthof PbChiIV was obtained from Pyrus bretschneideri ‘Yali’ using specific primers. The similarity of the nucleotide sequence and deduced amino acid sequence was analyzed by using BLAST on NCBI. BiotEdit software was used to compare the amino acid sequence. Phylogenetic tree was constructed by using MEGA6.0. Quantitative real-time PCR (qRT-PCR) was performed to determine the expression pattern of PbChiIV in different tissues and the expression of this gene after treatment with SA and B.berengeriana. f. sp. piricola.【Result】The cDNA lengths of PbChiIV genes was 819 bp (GenBank accession number: KJ872676), encoding 272 amino acids. The deduced amino acid sequence shares 100%, 79%, 73%, 73%, 72%, 72%, 72%, 69%, 68%, 67%, 67%, 65%, 67% and 62% identity with P. pyrifolia ACM45716.1, Populus trichocarpa XP_006376418.1, Vitis vinifera NP_001268173.1, Arabidopsis thaliana CAA74930.1, Medicago sativa ACL36992.1, Medicago truncatula AAR87869.1, Vigna unguiculata CAA61281.1, Corylus heterophylla AEM97876.1, Galega orientalis AAP03085.1, Vitis vinifera NP_001268075.1, Vitis pseudoreticulata ABY66958.1, Vitis vinifera AAB65777.1, Nicotiana tabacum BAF44533.1 and Gossypium barbadense AER29902.1. This gene belongs to IV chitinase gene. Expression analysis showed that the expression of PbChiIV in root was the highest, which was 4.32 and 2.96 times compared with stem and leaf, respectively. Followed by the expression in fruit, which was 2.48 and 1.70 times compared to that of stem and leaf, respectively. In the young fruit and mature fruit, the expression of the gene could be induced by SA and B.berengeriana. f. sp. piricola. The highest expression of the gene was 2.83 and 3.80 times compared with controls after treatment with SA, respectively. The highest expression of the gene was 1.82 and 1.66 times as compared with controls after treatment with pathogen, respectively. The highest expression of the gene was 2.49 and 3.43 times as compared with controls after treatment with SA and pathogen. In addition, the expression was up to the peak at 72, 24 and 72 h after treatment with SA and pathogen, respectively.【Conclusion】PbChiIV may be involved in SA-mediated basic signaling pathway of plant defense responses and disease resistance signal transduction network. It is speculated that PbChiIV is involved in defense responses mediated by B.berengeriana. f. sp. piricola. It might play an important role in disease resistance. from Pyrus bretschneideri ‘Yali’ and detect the expression characteristics of PbChiIV in root, stem, leaf

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