Scientia Agricultura Sinica

Morphological Characterization and Candidate Gene Analysis of Zebra Leaf Mutant zebra524 in Rice

LI Yan-Qun, ZHONG Ping, GAO Zhi-Yan, ZHU Bai-Yang, CHEN Dan, SUN Chang-Hui, WANG Ping-Rong, DENG Xiao-Jian

Scientia Agricultura Sinica. 2014, 47(15): 2907-2915. doi:10.3864/j.issn.0578-1752.2014.15.001

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【Objective】 The present study was aimed at morphological characterization and candidate gene analysis of zebra leaf mutant zebra524 in rice, so as to lay the foundation for further functional analysis of this gene and its application in agricultural production. 【Method】 A zebra leaf mutant, designated as zebra524, was isolated from an ethyl methanesulfonate (EMS)-mutagenized population. Morphological characteristics and the agronomic traits of the mutant and its wildtype were contrasted systematically. Using spectrophotometer, photosynthetic pigment contents in leaves of the mutant and its wildtype were measured at seedling and booting stages, and in glumes at grain-filling stage, respectively. After the zebra524 mutant was crossed with normal green varieties, the leaf colour phenotypes of the F1 progenies and the segregation ratio of zebra leaf plants to green leaf plants in the F2 populations were investigated. A total of 575 zebra leaf mutant individuals in the F2 mapping population generated from the cross between the mutant and G46B (Indica) were used for mapping using molecular markers. In addition, putative genes in the mapped region were analyzed, and candidate genes in the mutant and its wild-type were sequenced, respectively. Subsequently, DNA sequencing of β-OsLCY gene and alignment of the deduced amino acid sequences of homologous β-OsLCY proteins were conducted. 【Result】 During seedling and tillering stages, the zebra524 mutant showed alternating transverse pale green and albino sectors on leaves. During booting stage, its albino sectors turned into pale green and hereafter, the newly grown leaves became completely pale green. The zebra524 mutant also showed pale glumes during heading and grain-filling stages, and brown nodes, pink basal internodes, orange-red embryos and basal caryopsises at maturation stage. In addition, plumules of zebra524 showed an orange-red color in the dark. Chlorophyll contents in leaves of the zebra524 mutant decreased by 82.8% and 20.9%, and carotenoid contents decreased by 64.7% and 32.6% at seedling and booting stages, respectively, compared with those of the wild type. In addition, the levels of chlorophylls and carotenoids in glumes of zebra524 mutant were reduced by 38.1% and 42.8%, respectively, at the grain-filling stage. Its plant height, number of spikelets per panicle, seed setting rate and 1000-grain weight were reduced by 12.3%, 9.5%, 13.0% and 5.4%, respectively, at maturation. All F1 plants displayed wild-type phenotype, and the number of normal seedlings versus that of mutant seedlings in the F2 populations was fitted to 3:1, indicating that the zebra524 phenotype was controlled by a single recessive nuclear gene. The mutant gene was mapped to a region of 235 kb between SSR marker RM7082 and InDel marker Y2 on the short arm of chromosome 2. Sequencing analysis of candidate genes between the mutant and its wild-type revealed a single-nucleotide G-to-T mutation was found at position 235 in the coding region of the LOC_Os02g09750 gene for lycopene β-cyclase, which resulted in an amino acid change from Gly at position 79 to Cys in the encoded product. 【Conclusion】 The zebra524 mutant gene was allelic to β-OsLCY gene which was documented previously. The zebra524 mutant phenotype may be attributed to a point mutation of β-OsLCY gene encoding lycopene β-cyclase.

Genetic Contribution of Seedling Root Traits Among Elite Wheat Parent Jing 411 to Its Derivatives

XIAO Yong-Gui-1, LU Ya-Ming-1, WEN Wei-E-1, CHEN Xin-Min-1, XIA Xian-Chun-1, WANG De-Sen-1, LI Si-Min-1, TONG Yi-Ping-2, HE Zhong-Hu-1, 3

Scientia Agricultura Sinica. 2014, 47(15): 2916-2926. doi:10.3864/j.issn.0578-1752.2014.15.002

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【Objective】The breeding program in crop root plays an important role in determining yield potential and broad adaptation. Jing 411 as one of the most important elite parents has been widely used in the Northern Winter Wheat Region. Understanding the principal components and detecting the root traits loci in Jing 411 will provide important information for future genetic improvement by high density SNP markers.【Method】The core parent Jing 411 and its 14 derivative varieties and lines including six derivative and eight varieties in the first and second generations were used in present study. The uniform grains of each variety were selected for germinating in seedling plate for 6 days, and then six healthy and consistent plants were selected to culture 15 days in cultivating plates. Every variety with three replicates in each cultivating plate was planted in nutrient solution. Nine root parameters including maximum length root, length of branch root, length of main root, total length of roots, surface area of branch roots, surface area of main root, surface area of total roots, number of total root tips, and root dry weight were measured after 15 days of culture. The genetic structure and components of Jing 411 and its derivative populations of different generations were compared. The whole-genome association mapping was employed to identify the chromosome region controlling seedling root traits loci using 90K SNP markers. 【Result】The averaged genetic similarity index was 57.9% among Jing 411 and its derivatives, and the clustering results for these genotypes were generally in consistent with their pedigree. Jing 411 had the same allele ratios of 63.9% and 67.9% to its first and second generation derivatives, respectively. Most of Jing 411 derivatives were obtained from the second generation by backcrossing with the elite parent or its derivatives, and the contribution of Jing 411 was higher than theoretical expectation. On A, B and D genomes, the same allele ratios from the elite parent were 62.2%, 61.3% and 74.3%, respectively. Jing 411 exhibited more branch root and total root tips than its derivatives, however, the maximum length root and root dry weight has been improved significantly in the derivatives. Thirty-five loci showed significant association with root traits using stepwise model selection with Proc GLMselect in SAS software. Two new loci for root traits on the long arm of chromosomes 3D and 5B were linked with the SNP markers wsnp_Ex_c1032_1972861 and BS00100708_51, respectively. The gene and its linked SNP marker could be used for selecting root traits in the derivatives of Jing 411. Alleles were positively related with root traits from Jing 411 at 26 loci, and Zhongmai 175 and CA0958 remained more positive root loci delivered from the core parents, accounted for 73.1%. 【Conclusion】Conventional breeding program could effectively collect available genetic components, and promoted the root characters through selected outstanding above-agronomic parameters. A notable example in the improvement of Jing 411 was the variety Zhongmai 175, which carried more loci from the elite parent than other derivatives, and had superior root traits, agronomic traits and wide adaptation than its parent Jing 411.

Analysis of Shade-Tolerance and Determination of Shade-Tolerance Evaluation Indicators in Different Soybean Genotypes

LI Chun-Hong-1, 2 , YAO Xing-Dong-1, JU Bao-Tao-1, ZHU Ming-Yue-1, WANG Hai-Ying-1, ZHANG Hui-Jun-1, AO Xue-1, YU Cui-Mei-1, XIE Fu-ti1, SONG Shu-Hong-2

Scientia Agricultura Sinica. 2014, 47(15): 2927-2939. doi:10.3864/j.issn.0578-1752.2014.15.003

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【Objective】 The main purpose of the present study was to explore the methods of evaluating shade-tolerance, analyze the shade-tolerance of different soybean genotypes, screen suitable evaluation indicators of shade-tolerance, and establish a mathematical evaluation model of shade-tolerance. 【Method】 Thirty soybean cultivars(lines) were treated under shading and natural light conditions in a field experiment. Corn-soybean strip cropping system created the shaded conditions in the field. The corn variety Zhengdan 958 was used in the experiment. At the maize tasseling stage, shading rate was 70%. Soybean single planting was used as control. Main stem height (X1), stem diameter (X2), main stem node number (X3), branch number (X4), lowest pod height (X5), internode length (X6), plant weight balance (X7), lodging (X8), pods per plant (X9), seeds per plant (X10), seeds per pod (X11), 100-seed weight (X12), seed-stem ratio (X13) and yield per plant (X14) were investigated at the mature period. Shade-tolerance coefficient (STC) of all indexes from both treatments was used to comprehensively evaluate the shade-tolerance by principal component analysis, membership function method, hierarchical cluster analysis and stepwise regression analysis. 【Result】 Firstly, the 14 single indexes were transformed into 5 independent comprehensive components through principal component analysis, which represent 87.685% information of the raw data of the traits related to shade-tolerance. The membership function method was employed to calculate comprehensive shade-tolerance value (D) and carry on cluster analysis, and 30 soybean cultivars (lines) were divided into three shade-intolerance types. Class I was the high shade-tolerance type. Class II was the medium shade-tolerance type. Class III was the shade-intolerance type. At last, a mathematical evaluation model for soybean shade-tolerance was established by stepwise regression analysis, D=-0.5495+0.2932X3+0.0592X4+0.1628X6-0.0244X8+0.5943X9+0.1345X12+0.8210X14 (R2 =0.9908), whose accuracy was higher than 93.67%. Based on the model, seven indexes closely related to the shade- tolerance, including main stem nodes, branch number, internode length, lodging resistance, pods per plant, 100-seed weight and yield per plant were screened out. 【Conclusion】 Thirty soybean cultivars (lines) were divided into three shade-tolerance types, which were the high shade-tolerance type, the medium shade-tolerance type and the shade-intolerance type. Main stem nodes, branch number, internode length, lodging resistance, pods per plant, 100-seed weight and yield per plant can be used for identification of soybean varieties to shade-tolerance.

Immunolocalization and Quantitation of ABA and IAA in the Organs of Wheat (Triticumaestivum L.) Under Drought Stress

ZHANG Wei-1, GAO Wei-2, CAO Zhen-1, HE Li-Shan-1, TAN Gui-Yu-1, WANG Bao-Min-1

Scientia Agricultura Sinica. 2014, 47(15): 2940-2948. doi:10.3864/j.issn.0578-1752.2014.15.004

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【Objective】Drought, a serious problem existed in agricultural production, is one of the most important factors limiting agricultural growth and further expanding the crops acreage. Study of the mechanism of plant drought resistance will provide an important safeguard for the increase of agricultural production and incomes. Phytohormone plays a significant role in plant resistance to water deficiency. Based on this study we may further understand the function of phytohormone in plants in response to drought stress, and provide a theoretical basis for the regulation of plant hormone in the production and cultivation of crop in arid regions.【Method】With wheat variety Lumai No.21 as material, immunohistochemical localization was used to investigate the distribution of endogenous hormones ABA and IAA in wheat organs（root, leaf sheath and leaf blade）under drought-stress. While an indirect competitive enzyme-linked immunosorbent assay (icELISA) was applied to quantitate the content changes of ABA and IAA in wheat organs. The chemical signal intensity of the immunohistochemical localization and the endogenous hormone concentration of the icELISA were compared for confirming the effect of the results.【Result】Four weeks after drought treatment with weighing method, the wheat in control group (CK) grew uprightly, leaves were healthy and green, and the whole plant grew well. Wheat in drought treatment group (ND) grew small, leaves areas were reduced and had different degrees of wilting and yellowing phenomenon. Immunohistochemical localization was used to investigate the distribution of endogenous hormones ABA and IAA in wheat organs. The results showed that, the intensity of the staining of ABA in drought-stress wheat organs was stronger than that in wheat of control group, and that the leaf was most noticeable, especially in stomata guard cells and vascular bundle tissue, then was the leaf sheath. Similar results in IAA were obtained, but it was not as noticeable as ABA. An icELISA was applied to quantitate the content changes of ABA and IAA. The result of immunohistochemical localization above is in accord with the result of icELISA. The ABA content in the root was 61.51 ng•g-1 FW, 2.82 times that of controls (21.8 ng•g-1 FW). The ABA content in leaf sheath was larger, 175.35 ng?g-1 FW, 3.65 times that of controls (48.02 ng•g-1 FW). The largest rise of ABA content (512.12 ng•g-1 FW) was in leaves, 10.16 times significantly higher than controls (50.42 ng•g-1 FW). Variance analysis showed that the ABA contents in wheat seedling root, leaf sheath and leaf were significantly different (P＜0.01) under drought stress and normal water supply. The contents of IAA in wheat seedling root, leaf sheath, leaf were 72.81, 274.46 and 195.75 ng•g-1 FW under the normal water supply, respectively. The contents of IAA under water stress condition increased but not very much, and they were 123.56, 400.48 and 417.30 ng•g-1 FW, respectively. Significant analysis (t test) showed that the difference of IAA content in wheat seedling roots under drought stress and normal water supply condition was not significant (P＞0.05), but the difference of IAA content in leaf and leaf sheath was extremely significant (P＜0.01). 【Conclusion】The contents of ABA and IAA tended to increase in the organs of wheat when subjected to water deficiency, and the level of ABA increased obviously, while that of IAA increased gently compared with ABA. The trend of the coordination between ABA and IAA is to cause stomatal closure, promote root growth and slow the growth rate to effectively protect against drought damage.

Effects of Integrated Agronomic Practices on Leaf Senescence Physiological Characteristics of Summer Maize

ZHU Kun-Lun, JIN Li-Bin, DONG Shu-Ting, ZHAO Bin, LIU Peng, ZHANG Ji-Wang

Scientia Agricultura Sinica. 2014, 47(15): 2949-2959. doi:10.3864/j.issn.0578-1752.2014.15.005

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【Objective】This study is to explore the effects of integrated agronomic practices on leaf senescence physiological characters of summer maize. 【Method】 Zhengdan958 was used as experimental material, integrated agronomic managements experiment (MT), including 4 treatments of T1, T2, T3 and T4, and nitrogen rate experiment (0, 129.0, 184.5, 300.0 kg N hm-2, signed by N0, N1, N2, N3) which was based on T4 treatment were designed to explore the effects of integrated agronomic practices and nitrogen rate on leaf area index (LAI), photosynthetic pigments content, superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) activities and malondialdehyde (MDA) content in ear leaves of summer maize through the optimal combinations of planting methods, sowing and harvest date, as well as nitrogen fertilizer application timing and rate. 【Result】 LAI of T4 was always above 4.4 from V12 to 42 days after tasseling (VT), which was significantly higher than those of T1 and T2, and the LAI reduction of T4 was slower in later growing period. As for nitrogen rate treatments (NT), the LAI was increased with the increasing of nitrogen rate. The LAI of N3 decreased by 14.9% from 28 days after VT to R6 which was higher than those of the other treatments. As for MT and NT, the photosynthetic pigments content got the maximum value on 14 day after VT and then decreased, following the change of unimodal curve. From 14 days after VT to 42 days after VT, the chlorophyll (a+b) content of T1, T2, T3, T4 decreased by 21.1%, 22.0%, 27.2% and 18.4% and the carotenoid content of T1, T2, T3, T4 decreased by 26.6%, 26.9%, 32.2% and 25.0%, respectively. On 42 day after VT, the content of chlorophyll a, chlorophyll b, chlorophyll (a+b) and carotenoid in T4 were increased by 6.0%, 13.0%, 6.9%, 9.3% compared to T1, respectively. As for NT, the content of chlorophyll a, chlorophyll b and chlorophyll (a+b) increased significantly with the increasing of nitrogen rate. The carotenoid content of N3 decreased rapidly in latter growing period, and which was decreased by 4.5% compared to that of N0 on 42 day after VT. In MT and NT, the activities of SOD, CAT and POD got the maximum value on 14 day after VT and then decreased, following the change of unimodal curve. The SOD, CAT and POD activities of T4 always remained at high levels and its decreasing was lower in the latter growing period. In NT, SOD, CAT and POD activities were increased as the increasing of nitrogen rate, the protective enzymes activity of N3 decreased faster than those of the other treatments. In MT and NT, MDA content increased following the growth process, and decreased with the increasing of nitrogen rate. MDA content of T4 always remained at low levels. 【Conclusion】 When the nitrogen rate was higher than 184.5 kg•hm-2, photosynthetic pigments content and protective enzymes activity of leaves couldn’t be increased and MDA content couldn’t be decreased continuously with the increasing of nitrogen rate, which was not helpful to increase nitrogen use efficiency. By increasing plant density, changing relay intercropping to direct seeding, and delaying sowing and harvesting date, as well as optimizing nitrogen fertilizer application timing and rate, photosynthetic pigment content of ear leaves in T4 decreased slower, protective enzymes activity were higher, and MDA content was lower, which was helpful to improve both grain yield and nitrogen use efficiency together.

Relationship of Days After Pollination and Vigor Traits on Maize Seed Maturity in Hexi Seed Production Area in China

FAN Ting-Lu-1, WANG Shu-Ying-1, WANG Jian-Hua-2, YANG Zhen-3

Scientia Agricultura Sinica. 2014, 47(15): 2960-2970. doi:10.3864/j.issn.0578-1752.2014.15.006

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【Objective】The objective of this study was to clarify relationships between seed maturity and vigor, and to determine optimum harvest of maize seed with high vigor so as to provide references for seed enterprises to avoid early autumn frost, in Hexi in China.【Method】Samples for maize seeds were obtained at 3 to 5 days intervals after pollination to test seed moisture, standard germination-SG (7 days at 25℃), and vigor, using cold test-CT (3 days at 6℃) and electrical conductivity-EC (three 50-seed samples, 24 hours inhibition at 25℃), and milk line development was classified by a five-stage system, using three genotypes (hybrid XY335, ZD958 and JX1) during the crop seasons of 2013.【Result】Seed moisture and seed moisture-loss rate and germination and vigor were highly influenced by genotypes and days after pollination (DAP). XY335 seed dehydrated early and quick, and seed moisture content was 10-15 percentage points lower in XY335 than that in ZD958 and JX1 at 32 DAP. During grain filling period from 32 to 72 DAP, seed moisture in ear decreased with time, average moisture-loss rate for XY335 was 0.70% per day that was higher than 0.62% per day in ZD958 and 0.49% per day in JX1, and 100-seed weight increased 0.73 g in XY335 and 0.50 g in ZD958 and 0.29 g in JX1 when seed moisture reduced one percentage point. Seed maturity and vigor at different days after pollination had a great difference, and seed quality values in terms of standard germination increased and electrical conductivity values decreased with seed moisture decreases from 70% to 40%, and the SG values increased by 3.37 percentage points in XY335 and 2.76 percentage points in ZD958 and 1.67 percentage points in JX1 as one percentage point reduction in seed moisture. With grain filling moving forward, the SG tended to increase and the EC to decrease but these changes were different across genotypes and DAP, and the SG ranged from 30% to 59% and the vigor at cold test from 2.5% to 36.9% at 32 DAP, and the seed germination values at standard and cold test for XY335 were comparable at 47 DAP onward, but the seed germination at standard was always lower than that at cold test for ZD958 and JX1, suggesting the order of seed low temperature resistance was XY335, ZD958, and JX1, respectively. Seed moisture during grain filling period was highly correlated with milk line development stage, and milk line moved from the seed’s upper to base as seed moisture decreased. Milk line presenting and disappearing were the earliest for XY335, came second for ZD958, and the latest for JX1. When the milk lines for three hybrids were present at the midpoint of the seed, seed moisture would be 38% to 40%, and the SG and vigor in CT would be close to high values, implying optimum harvesting can be made at half- milking stage, in which 100-seed weight of XY335 and ZD958 accounted for 76.6% and 88.0% of its maximum at physiological maturity. 【Conclusion】 Seed maturity affected greatly on seed quality, and optimum harvest for high germination values and vigor using cold test and EC can be determined at half-milk (stage 3) in which seed moisture was about 40%, and can not be made at physiological maturity of seed weight usual reaching its maximum. Suitable harvesting started from 47 DAP onwards for XY335, and 60 DAP for ZD958 and JX1, 10 to 15 days before usual harvesting, to prevent frost damage on seed quality in Wuwei, Gansu.

Function Analysis of BcKMO Gene in Growth, Development and Pathogenicity of Botrytis cinerea

LI Pei-Fen, ZHAO Fu-Xin, DONG Li-Ping, ZHENG Hui-Xin, ZHAO Bin, ZHANG Jing, SI He-Long, XING Ji-Hong, HAN Jian-Min, DONG Jin-Gao

Scientia Agricultura Sinica. 2014, 47(15): 2971-2979. doi:10.3864/j.issn.0578-1752.2014.15.007

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【Objective】The objective of this study is to investigate the function of BcKMO gene in growth, development and pathogenicity of Botrytis cinerea, to further clarify the molecular mechanism of the BcKMO gene in development and pathogenicity of the fungus and provide a theoretical basis for the gray mould control. 【Method】 Bioinformatics methods were used to analyze the BcKMO gene and its encoding product. The BcKMO gene coding region was amplified and cloned into pBARKS1-eGFP, and pBARKS1-BcKMO-eGFP was constructed. The protoplasts of mutant BCG183 were prepared and transformed with pBARKS1- BcKMO-eGFP by PEG-mediated method. PCR, Southern blotting and real-time PCR were used to identify the transformant. Phenotype including colony morphology, mycelium morphology, growth rate, conidial yield, and so on, and pathogenicity assays of wild-type strain BC22 (WT), mutant BCG183 and complemutant BCG183/BcKMO were performed to verify the function of the BcKMO gene in growth, development and pathogenicity. 【Result】The BcKMO gene encodes kynurenine 3-monooxygenase (KMO) having monooxygenase FAD conserved domain and four Aromatic-ring hydroxylase-like motifs. The BcKMO protein displayed high homology to rossmann-fold NAD(P)(+)-binding protein (gi156049701) of Sclerotinia sclerotiorum and FAD-dependent oxidoreductase (gi512192943) of Ophiostoma piceae. The expression vector of the BcKMO gene, pBARKS1-BcKMO-eGFP, was successfully constructed and transformed into the protoplasts of mutant BCG183. Results of PCR, Southern blotting and Real-time PCR showed that the BcKMO gene complementing mutant (BCG183/BcKMO) was successfully obtained. Phenotype assay showed that the mutant BCG183 growed slowly, did not produce conidia and sclerotia, and hyphae slender, but the phenotypes of the BCG183/BcKMO were all similar to that of WT. Compared with WT and the BCG183/BcKMO, the pathogenicity of the mutant BCG183 on kidney bean leaves and cucumber leaves were dramatically increased.【Conclusion】The BcKMO gene is a negative regulation in growth and development, and a positive regulation in pathogenicity of B. cinerea.

Construction of Knockout Vector of GTP Cyclohydrolase II Gene and Mutant’s Biological Characteristics of Valsa mali

SONG Na, DAI Qing-Qing, SONG Na, HUANG Li-Li, HAN Qing-Mei

Scientia Agricultura Sinica. 2014, 47(15): 2980-2989. doi:10.3864/j.issn.0578-1752.2014.15.008

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【Objective】 The objective of this study is to construct the knockout vector of gene Vmgtp1 encoding GTP cyclohydrolase II using the strategy of reverse genetics, and analyze the gene preliminary function by homologous recombination, so as to lay a foundation for the comprehensive analysis of pathogenic molecular mechanisms in Valsa mali and provide a theoretical basis for the technology and pharmaceutical research of apple rot disease. 【Method】 Based on the analysis of the transcriptome data of V. mali, the gene Vmgtp1 (tentatively named) was obtained. After inoculating for 5 days, the expression in the infected tissue was increased. So it is predicted that Vmgtp1 may be related with pathogenicity in V. mali. Using Double-joint PCR method, the up and downstream fragment of Vmgtp1 and the selectable marker hygromycin-phosphotransferase gene (hph) to one fragment were connected. The PCR product and plasmid pHIG2RHPH2-GFP-GUS were double-digested, connected with T4 ligase and transformed into JM109. Then the positive plasmid was transformed into V. mali through PEG-mediated genetic transformation. Next, the hgromycin-resistant mutant strains which can grow normally on PDA medium containing hygromycin were selected. The selected homologous recombinant mutants were then confirmed by PCR with four pairs of primers and Southern Blot. At last, the phenotypes of wild-type strain 03-8 and mutant were analyzed through observation colony color, colony size and propagulum production, the pathogenicities were tested by observing the lesion sizes on the apple limbs which inoculated with wild-type strain 03-8 and mutant. The significance of differences was analyzed by the software of SPSS.【Result】The knockout vector of gene Vmgtp1 encoded GTP cyclohydrolase II using the above-mentioned method was constructed successfully and 101 mutant strains with PEG-mediated transformation were obtained. Only one homologous recombination mutant ΔVmgtp1-90 was obtained after PCR and Southern Blot testing the 101 mutant strains. Compared with the wild type 03-8, the mutant ΔVmgtp1-90 showed slower colony growth rate on PDA medium, the average growth rate of ΔVmgtp1-90 was 11.33 mm?d-1, and the average growth rate of strain 03-8 was 24.67 mm?d-1. At the same time, ΔVmgtp1-90 had lighter colony color and sparser aerial mycelium, had not produced any propagulum on the PDA medium 40 days later under light conditions, but strain 03-8 could produce propagulum after 15 days and overflow conidium after 30 days cultivation on the PDA medium under light conditions. The result of pathogenicity testing also showed that the pathogenicity of ΔVmgtp1-90 was significantly weakened. At 7 days after inoculation, the average lesion diameter on the branch of Fuji apple tree was 24.8 mm after inoculated with strain 03-8, but the average lesion diameter only was 9.3 mm after inoculated with ΔVmgtp1-90.【Conclusion】A mutant of the gene Vmgtp1 encoding GTP cyclohydrolase II through gene knockout and genetic transformation was obtained. Compared the difference of phenotype and pathogenesis of Vmgtp1 mutant ΔVmgtp1-90 and the wild-type strain 03-8, it could conclude that the gene Vmgtp1 may affect mycelial growth and regulate of sporulation of V. mali, and may play a role in the process of V. mali infection. But further research should be done to know whether it plays a major role or not.

Sensitivity of Different Field Populations of Tetranychus urticae Koch (Acari: Tetranychidae) to the Acaricides in Beijing Area

GONG Ya-Jun-1, WANG Ze-Hua-1, SHI Bao-Cai-1, CUI Wen-Xia-1, JIN Gui-Hua-1, SUN Yan-Yan-2, WEI Shu-Jun-1

Scientia Agricultura Sinica. 2014, 47(15): 2990-2997. doi:10.3864/j.issn.0578-1752.2014.15.009

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【Objective】The objectives of this study are to survey the sensitivity of different populations of Tetranychus urticae Koch to the acaricides in Beijing area, as well as the relationship between their sensitivity to the acaracides and the activity of resitance-related enzymes. 【Method】 Bioassays of four frequently used acaricides of bifenazate, abamectin, spirodiclofen and pyridaben to five field populations of the T. urticae, i.e. Fangshan, Huairou, Changping, Yanqing and Pinggu of Beijing area, were conducted by using modified slide-dip method. Bifenazate resistance-related mutations were detected by PCR amplification and sequencing of the CYTB gene from individual specimens. Additionally, the activities of four resistance-related enzymes of cytochrome P450 monooxygenases, acetylcholinesterase (AChE), carboxylesterase (CarE) and glutathione S-transferase (GST) were tested by microplate assay in microplate reader for the five populations.【Result】All of the five field populations of Fangshan, Huairou, Changping, Yanqing and Pinggu were sensitive to bifenazate, with LC50 of 2.4880, 6.4693, 6.2398, 0.7882 and 14.7783 mg•L-1, respectively, followed by the abamectin, with LC50 of 22.4712, 35.4431, 14.5260, 15.4904 and 14.0023 mg•L-1, respectively. T. urticae showed low sensitivity to the spirodiclofen and pyridaben in the five tested populations. In populations of Fangshan, Huairou, Changping, Yanqing and Pinggu, the LC50 of spirodiclofen was 49.6833, 81.8826, 72.9609, 204.4609 and 1 433.5137 mg•L-1, respectively, whereas that of pyridaben was 202.6902, 806.8324, 375.3518, 188.3234 and 2 310.9040 mg•L-1, respectively. The sensitivity of the AChE, CarE and GST of Yanqing population was higher than that of other populations, with a value of 14.9508 U/(mg protein), 0.2271 μmol?mg-1 protein?30 min-1 and 58.2962 U/(mg protein), respectively. The cytochrome P450 monooxygenases of Huairou population was significantly lower than that of other populations with a value of 1.4272 μmol?mg-1 protein?30 min-1. There was no linear relationship between the toxicity of bifenazate, abamectin, spirodiclofen and pyridaben and the activities of cytochrome P450 monooxygenases, AChE, CarE and GST. One individual from Huairou population with one site mutation on the CYTB gene (the first codon position of the 126th amino acid was changed from G to A) was detected in 288 individuals of five populations, which lead the amino acid Gly mutated into Ser. The low level of resistance-related mutation in the detected populations of the T. urticae was consistent with their high sensitivity to the bifenazate. 【Conclusion】 T. urticae in Beijing area showed highest sensitivity to the bifenazate, followed by the abamectin, and lastly to spirodiclofen and pyridaben. The sensitivity of T. urticae to the four tested acaricides is not linearly related to the activity of cytochrome P450 monooxygenases, AChE, CarE and GST. The PCR detection of resistance-related mutation indicated that the Huairou population has high risk to generate resistant individuals to the bifenazate. PCR method could fastly and accurately detect the development of acaracide resistance than the bioassay method. In the controlling practice, the pyridaben should not be used, the spirodiclofen should be avoided, and abamectin and bifenazate could be used alternatively.

Comparison and Application Analysis of Several NDVI Time-Series Reconstruction Methods

ZHANG Han-1, REN Zhi-Yuan-1, 2

Scientia Agricultura Sinica. 2014, 47(15): 2998-3008. doi:10.3864/j.issn.0578-1752.2014.15.010

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【Objective】NDVI time-series can simulate plant growth and reflect its growing status. Several models have been fitted in the past to smooth time-series vegetation index data from different satellite sensors. However, differences between the models and fine tuning of model parameters lead to uncertainty and bias between the results amongst users. The current research assessed three techniques: Whittaker smoother, HANTS, and Savitzky-Golay filter for smoothing multi-temporal satellite sensor observations with the ultimate purpose of estimating phenological parameters and deriving the annual multiple cropping index (MCI) reliably. 【Method】The research used MODIS MOD13Q1 data over the year 2000 to 2012 composed at sixteen day intervals covering the Shaanxi Province, and three models were fitted to reconstruct NDVI time-series. First, samples time-series of different vegetation types in three climate zones of research area were picked, and the fitting effect was contrasted by vision. Second, three evaluation indexes (RMSE, the correlation coefficient, and SNR) were used to compare the quality of each model, and then the differences between different types of vegetation were discussed. Third, three phenological parameters (Start of Season, End of Season, and Length of Season) were estimated by the dynamic threshold method, while the means and standard deviations of phenological parameters of different vegetation types estimated from three models were compared. Finally, the MCIs of each year were derived by quadratic difference algorithm and rules using the NDVI time-series of each model, and then were compared with the MCIs derived from statistics.【Result】Savitzky-Golay performed better than others on RMSE and the correlation coefficient, but has a bigger error in extracting phenological parameters. HANTS performed well in extracting phenological parameters, but which needs more complex parameter settings and has a poor accuracy. Whittaker can effectively reduce the signal to noise ratio of the original image with only one parameter, which also has a good performance in getting phenological parameters and the annual multiple cropping index. RMSE has a negative correlation with the correlation coefficient, but the correlation coefficient is more sensitive.【Conclusion】It was concluded that Whittaker smoother is the best approach in the test, which has outperformance in extracting phenological parameters, but remains to be further studied in terms of extracting multiple cropping index.

Impacts of Climate Change on Wheat Yield in China Simulated by CMIP5 Multi-Model Ensemble Projections

YANG Xuan-1, TANG Xu-2, CHEN Bao-De-3, TIAN Zhan-4, ZHAO Si-Jian-5

Scientia Agricultura Sinica. 2014, 47(15): 3009-3024. doi:10.3864/j.issn.0578-1752.2014.15.011

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【Objective】 By applying climate projections based on 30 Atmosphere-Ocean General Circulation Models (AOGCMs) under representative concentration pathway (RCP) scenarios in the Coupled Model Inter-comparison Project Phase 5 (CMIP5), the effects of climate change on wheat yield in China were assessed in terms of ensemble method. 【Method】 The impact assessment of climate change on crops is typically based on daily data. However, significant uncertainties exist among the AOGCM outputs, particularly in daily data. In this paper, a pseudo global warming (PGW) method was assumed to be a linear coupling of contemporary weather fields and the difference component of climate perturbation signals by AOGCMs. CERES-Wheat model was employed to stimulate the wheat yield in the future and a probabilistic approach is used to address the uncertainties. 【Result】 Warming is expected in all representative stations during the wheat growth period. Temperature increase under the RCP8.5 scenario is higher than that under the RCP2.6 scenario. The temperature in the representative stations of winter wheat is projected to increase by 2.7-2.9℃, and increase by 3.0-3.3℃ in the representative stations of spring wheat at the end of the 21st century. The precipitation rate is projected to significantly increase in the future. Compared with the baseline, the observation data collected from 1996 to 2005 show that the climate-change-induced wheat yield reduced in all representative stations under irrigation conditions. The reduction probabilities increased with climate change. The irrigated yield reduction in the representative stations of spring wheat was greater than that in the representative stations of winter wheat. By the end of the 21st century, the yield in the representative stations of winter wheat is projected to be decreased by 2% under the RCP 2.6 scenario. The yield reduction will be decreased by approximately 6% under the RCP 4.5 scenario and decreased by 9% under the RCP 8.5 scenario with a probability of 85%. In the representative stations of spring wheat, yield will be decreased by 5% under the RCP 2.6 scenario, by more than 8% under the RCP 4.5 scenario, and by more than 15% under the RCP 8.5 scenario with a probability of 90%. In comparison with the baseline, the rain-fed yield in the representative stations of winter wheat will be increased significantly. By the end of the 21st century, the yield in winter wheat is projected to be increased by more than 21% under the RCP 2.6 scenario, more than 22% under the RCP 4.5 scenario, and more than 25% under the RCP 8.5 scenario with a probability of 90%.【Conclusion】The ensemble of daily data were obtained through the PGW method, which efficiently reserve the contemporary weather information, particularly that of extreme weather events. Effects of climate change on wheat yield under RCP2.6, RCP4.5, and RCP8.5 scenarios were assessed through the ensemble method. The results indicate that, with the increasing greenhouse gas emissions, the climate-change-induced yield-reduction probabilities of irrigated wheat in China gradually increased. Rain-fed wheat yield will be increased in the future, with large uncertainties.

The Tetraploid Induction of Shoot-Tip Plantlets with Colchicine in Garlic

KONG Su-Ping-1, 2 , CAO Qi-Wei-2, SUN Jing-Qiang-2, LIU Bo-2, XU Kun-1

Scientia Agricultura Sinica. 2014, 47(15): 3025-3033. doi:10.3864/j.issn.0578-1752.2014.15.012

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【Objective】Garlic is a vegetative crop. It is difficult to use conventional method to breed new varieties. Chemical mutagenesis plays an important role in germplasm innovation, and the effect is admirable in some crops. This paper mainly discussed the mutagenic effect of colchicine on in vitro shoot-tips of garlic with the aim at finding a theoretical and methodical basis for obtaining new garlic germplasm. 【Method】 Garlic cultivars of Cangshan Puke, Zhengyuezao, and Xinjiang purple were collected from different regions. After dormancy broken, the selected shoot-tips from bulbs of garlic were cultured in different media and proceeded to plant regeneration. The rate of plantlet regeneration and tetraploid in vitro shoot-tips were studied in three garlic varieties induced by colchicine exposure. Correlative model of different colchicine concentrations and time duration was established by applying D-optimal design, so as to determine the optimum scheme of the tetraploid induction of garlic. The plant ploidy was identified by Flow Cytometry Analysis and chromosome counts of the root-tips.【Result】The result showed that the DNA content of tetraploid garlic leaf was twice that of diploid garlic Cangshan Puke by flow cytometry analysis. The diploid chromosome number was 2n=2x=16 and the tetraploid chromosome number was 2n=4x=32 by chromosome counting. In colchicine treatment process, the rate of plantlet regeneration was significantly decreased when treated with colchicine at higher concentration and after longer duration. And the concentration of colchicine had a stronger effect on plantlet regeneration than time duration. The difference among the three garlic varieties was significant. As an outstanding example, the plantlet of Cangshan Puke survived significantly fewer than Zhengyuezao and Xinjiang purple. And the plantlet rates were 41.83%, 48.69% and 46.73%, respectively. The concentration of colchicine had a more significant effect on tetraploid induction than the treatment duration. The difference of tetraploid rate among the three varieties was also significant. The tetraploid rates were 15.69%, 15.69% and 23.53% for Cangshan Puke, Zhengyuezao, and Xinjiang purple, respectively. Among the cultivars, the tetraploid rate of Zhengyuezao was the highest (9.15%), Cangshan Puke took the second place (7.84%), and Xinjiang purple was in third place (6.53%). By applying simulation technology, the results showed that over 50% of plantlets had been regenerated and survived when all the varieties were treated by colchicine at a concentration of 0.268%-0.694% after 61.1-113.8 h. While the tetraploid rate was more than 5% when treated at a concentration of 0.531%-0.580% after 79.8-112.2 h. 【Conclusion】 Garlic varieties showed different reactions when treated at various colchicine concentrations and time durations. The difference of mutagenic effect among these was significant. It showed that immersion concentration affected more apparently than the immersion time did. Mutual effect between the concentration and treatment time of colchicine was found. By applying intersection method analysis, the result showed that when treated with colchicine at concentration of 0.531%-0.580% and after 79.8 h-112.2 h duration, more than 50% of plantlets were regenerated and survived, while the tetraploid rate was more than 5%.

Genetic Diversity of Fruit Phenotypic Traits of Wild Pyrus ussuriensis Maxim

AN Meng-Meng-1, WANG Yan-Ting-1, SONG Yang-2, JI Xiao-Hao-1, LIU Chang-3, WANG Nan-1, WU Yu-Sen-1, LIU Wen-1, 4 , CAO Yu-Fen-2, FENG Shou-Qian-1, CHEN Xue-Sen-1

Scientia Agricultura Sinica. 2014, 47(15): 3034-3043. doi:10.3864/j.issn.0578-1752.2014.15.013

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【Objective】 The genetic variation and diversity of morphological traits, volatile components, sugars and organic acids of wild Pyrus ussueiensisi fruits were investigated to provide a scientific basis for resource protection and utilization. 【Method】 Using 35 seedlings of wild P. ussuriensis collected from the germplasm resources nursery and 3 cultivars (Xiaoxiangshui, Nanguo, Jingbai), the ripened fruit vertical length, transverse length, shape index and weight, and the sugars, organic acids were measured by using the Head space-solid phase microextraction, gas chromatography-mass spectrometry (GC-MS), and high-performance liquid chromatography (HPLC). 【Result】 The variation coefficients (CV) of fruit size, content of total volatile compound, numbers and contents of volatile compounds, the segregation ratios and contents of main volatile components, contents of sugars and organic acids, and contents of total sugars and organic acids were over 17%, which revealed significant differences and extremely abundant genetic diversity in 35 seedlings of wild P. ussueiensisi. The further comparison with three cultivars indicated that fruit vertical length, transverse length and weight of wild P. ussuriensis are smaller than that of the fruit of cultivars but have the similar fruit shape indexes. On average, 41 fragrance components were detected in wild P. ussuriensis, being obviously higher than the amount of fragrance components in the fruits of cultivars which contain 25 fragrant components. The average amount of aromas reached 9.44 μg•g-1, which is significantly higher than that of 3 cultivars (5.71 μg•g-1). The wild P. ussuriensis contains organic acid and hydrocarbon derivatives, the 2 special compounds in wild pears, and it also contains other 111 special components which include esters and were categorized into 9 classes. In all, 16 characteristic fragrant components were detected in wild P. ussuriensis and three cultivars. Among them, 1-hexanol, hexanal and other 8 fragrant components commonly exist in both wild P. ussuriensis and three cultivars, serving as the characteristic aromas for both. And hexyl acetate, pentyl acetate and other 6 compounds were unique fragrant aromas for the fruit of wild P. ussuriensis. Three sugars (fructose, glucose and sucrose) and 6 organic acids (malic acid, tartaric acid, citric acid, oxalic acid, lactic acid and succinic acid) almost could be detected in 35 wild seedlings and 3 cultivars with fructose and malic acid showing the highest amount. The contents of tartaric acid and sucrose in the wild seedlings and the content of total sugars and organic acids in some wild seedlings were significantly higher than those of 3 cultivars. 【Conclusion】 There were extensive genetic variation in fruit morphological traits, aroma components, sugars and organic acids components in wild P. ussueiensisi, and 35 seedlings presented significant differences. The genetic diversity of wild P. ussueiensisi was extremely abundant, 111 volatile compounds were unique components in wild P. ussueiensisi, and acids, hydrocarbon derivates were not found in the cultivars. It indicated that the wild P. ussueiensisi has a large potential for further selection.

Optimization and Functional Assessment of Oligosaccharides Compound Prepared by Sweet Potato Residue

DONG Xiang-Yan-1, LI Jing-Mei-2, SHI Bo-1, PENG Qing-1, QIAO Yu-1, OjokohEromosele1 , ZHANG Mi-Min-1

Scientia Agricultura Sinica. 2014, 47(15): 3044-3057. doi:10.3864/j.issn.0578-1752.2014.15.014

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【Objective】The aim of this study was to explore the optimum conditions of sweet potato residue degraded into oligosaccharides by commercialized β-glucanase and polygalacturonse and to investigate glyceollins accumulation in soybean elicited by oligosaccharides, thus providing a scientific basis for the industrial production and application of oligosaccharides.【Method】 Sweet potato residue was hydrolyzed by commercialized β-glucanase and polygalacturonse separately in single factor experiments for optimal conditions such as temperature, pH, substrate concentration, additive amount of enzyme and the reaction time of hydrolysis. Product obtained was determined by thin-layer-chromatography (TLC) and high performance liquid chromatography (HPLC). The optimal conditions for hydrolysis of sweet potato residue were obtained through analyzing the yield of cellobiose and the yield of digalacturonic acid and trigalacturonic acid. Then using the combi-enzyme hydrolyze the sweet potato residue to prepare oligosaccharides compound which could elicit glyceollins accumulation in soybean.【Result】The enzyme reaction conditions for the solubilization were optimized to be addition of 6.9×103 U β-glucanase by weight of sweet potato residue dietary fiber, 7 h treatment at pH3.5, temperature at 40℃, and substrate concentration at 1%. Under these conditions, cello-oligosaccharides were mainly cellobiose, the yield of cellobiose was 100.6 mg•g-1 sweet potato residue dietary fiber, and the conversion rate of cellobiose was 22.37%. The enzyme reaction conditions for the solubilization were optimized to be addition of 1.42×104 U polygalacturonse by weight of sweet potato residue dietary fiber, 4 h treatment at pH2.5, and temperature at 40℃. Using the optimum conditions, the yield of pectic oligosaccharides mainly with a degree of polymerization (DP) of 2 and 3, the yield of digalacturonic acid and trigalacturonic acid was 17.43 mg•g-1, the conversion rate of digalacturonic acid and trigalacturonic acid was 29.9%. The optimum conditions for producing oligosaccharides compound were as follows: temperature at 40℃, pH2.5, concentration of sweet potato residue at 1%, the additive amount of β-glucanase was 6.9×103 U•g-1 and the additive amount of polygalacturonse was 1.42×104 U•g-1, and the reaction time was 7 h. Using the optimum conditions, the yield of oligosaccharides compound mainly with a DP of 2 and 3, and the yield of cellobiose was 136.97 mg•g-1, the conversion rate of cellobiose was 33.57%, the yield of digalacturonic acid and trigalacturonic acid was 25.95 mg•g-1, the conversion rate of digalacturonic acid and trigalacturonic acid was 44.53%. Oligosaccharides compound was used to induce glyceollins synthesis in soybean seeds. The optimal conditions of elicitation were as follows: concentration of oligosaccharides compound 1%, presoaked in the sterile water for 5 h, stored at a controlled temperature of 25℃ and humidity of 50% in dark for 4 d, the yield of glyceollins elicited by oligosaccharides compound was 1.21 mg•g-1 dry weight soybeans. The yield of glyceollins elicited by cello-oligosaccharides with the same concentration of cellobiose was 0.80 mg•g-1 dry weight soybean. The yield of glyceollins elicited by pectic oligosaccharides under the same condition was 0.46 mg•g-1 dry weight soybeans.【Conclusion】 Sweet potato residue, as a cheap reaction substrate, could be used as the substrate for the production of oligosaccharides compound. The optimum conditions for the production of oligosaccharides compound were obtained, and the result showed that oligosaccharides compound has good effect on the generation and accumulation of glyceollins.

Effect of N-acetylmuramidase Knockout on Autolysis and Morphology of Lactobacillus bulgaricus LJJ

CUI Wen-Ming, PANG Xiao-Yang, ZHANG Shu-Wen, LIU Lu, LI Hong-Juan, lv Jia-Ping

Scientia Agricultura Sinica. 2014, 47(15): 3058-3068. doi:10.3864/j.issn.0578-1752.2014.15.015

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【Objective】 Autolysis of lactic acid bacteria exists widely in the production of fermented dairy products. The rate and extent of autolysis not only play an important role in the whole fermentation process, but also have a vital effect on the quality, flavor and production cycle of the product. As main component of peptidoglycan hydrolase, N- acetylmuramidase can destroy the cell wall integrity in lactic acid bacteria autolysis process. It plays a crucial role in the autolysis of lactic acid bacteria. This paper mainly studied the influence of N-acetylmuramidase deletion on autolysis and morphology of Lactobacillus bulgaricus LJJ.【Method】 The primers of upstream homologous arm m-up, downstream homologous arm m-down of N-acetylmuramidase and erythromycin resistance genes were designed based on the sequences of L. bulgaricus genome and pMG36e vector. Then m-up, m-down and erythromycin resistance gene were amplified by PCR and verified by gene sequence analysis. Then erythromycin resistance gene and pUC19 were degested with Kpn Ι，Xba Ι and connected together. The recombinant vector pUC:Emr was verified by double-enzyme cleavage method. Next, m-down and recombinant vector pUC: Emr were digested with Kpn Ι, Sac Ι and connected together. The recombinant vector pUC:Emr:m-down was confirmed by double-enzyme cleavage method. Finally, the m-up and recombinant plasmid pUC:Emr:m-down were digested with Pst Ι, Xba Ι and connected together. The recombinant vector pUC:m-up:Emr:m-down was verified by double-enzyme cleavage method and gene sequence analysis. The recombinant vector pUC:m-up:Emr:m-down as the homologous recombinant N-acetylmuramidase knockout component, was transformed into L. bulgaricus under the electroporation conditions of electric voltage, resistence and capacity of 1.5 kV, 400 Ω and 25 μF, respectively. N-acetylmuramidase gene knockout mutant strains were screened on MRS agar containing erythromycin and verified by PCR. Then the autolysis rate of the gene deletion mutant and the wild type strain was detected and the morphological changes of gene deletion mutant and wild-type strain were observed by scanning electron microscope. 【Result】 N-acetylmuramidase knockout components with erythromycin resistance as selection marker was constructed and used to knock N-acetylmuramidase gene of L. bulgaricus out. N-acetylmuramidase knockout L. bulgaricus mutant strain with erythromycin resistance gene was constructed successfully. Compared with the wild-type strain, the characteristics and morphology of the mutant strain changed significantly. The autolysis rate also decreased significantly. The autolysis rate of the mutant strain was about 35% while the autolysis rate of the wild type strain was about 73% after 24 hours incubation at 37℃. The single cell length of the wild type strain was about 10 μm while that of the mutant strain was approximately 30-40 μm. Compared with the wild-type strain, the single cell length of the mutant strain increased by 3-4 times.【Conclusion】N-acetylmuramidase of L. bulgaricus plays an important role in autolysis and cell division. The deletion of N- acetylmuramidase from L. bulgaricus will result in loss of cell autolysis and the cell length will be increased by about 3-4 times.

Study on Differential Expression of Ovine RARG in Ovary at Different Estrous Cycles

WANG Wei-Min, HU Ting-Xi, LI Fa-Di, MA You-Ji, FAN Hong-Ying, PAN Xiang-Yu

Scientia Agricultura Sinica. 2014, 47(15): 3069-3076. doi:10.3864/j.issn.0578-1752.2014.15.016

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【Objective】The objective of this study is to identify ovine retinoic acid receptor gamma (RARG) gene and to discover the differential expression of ovine RARG in ovary at different estrous cycles. 【Method】 Eight one-year old Gansu alpine fine-wool sheep were chosen for estrus synchronization treatment and then divided into 2 groups based on the ovarian physiological situation: one is corpus luteum group and the other one is estrous group. RNA were isolated from the ovarian tissue and RT-PCR was used to clone the ovine RARG gene, bioinformation method was used to construct ovine RARG gene phylogenetic tree as well as the prediction of protein structure and potential function. Q-PCR was applied to discover the differential expression of ovine RARG in ovary at different estrous cycles. 【Result】 A 1 588 bp full-length ovine RARG cDNA was identified and contained a 1 377 bp predicted ORF (GenBank accession No: KF019682), which encoded 458 amino acids with a calculated molecular weight of 50.96 kDa and an estimated isoelectric point (pI) of 7.45. Phylogenetic tree analysis showed that the ovine RARG gene has a close relationship with Bos taurus’, and the gene sequence among these species contained high homologous. Protein structure prediction indicated that the ovine RARG protein contains two main domains: c4 zinc finger in nuclear hormone receptors(ZnF_C4), and ligand binding domain of hormone receptors (HOLI). Q-PCR result demonstrated that the expression of ovine RARG mRNA in estrous situation is higher than that in corpus luteum stage. 【Conclusion】 The ovine RARG gene may work as an important transcriptional factor involved in female ovine estrous cycle control.

Prokaryotic Expression and Protective Efficacy of TPO18 Gene from Taenia pisiformis

HAN Jin-Huan, GOU Hui-Tian, SHANG Qing-Yan, SUN Xiao-Lin

Scientia Agricultura Sinica. 2014, 47(15): 3077-3084. doi:10.3864/j.issn.0578-1752.2014.15.017

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【Objective】The study aimed to investigate the protective efficacy of the recombinant antigen of TPO18 (pGEX-TPO18) from Taenia pisiformis. 【Method】 Total RNA was extracted from oncosphere of T. pisiformis. A pair of primers was designed based on the conserved region of other Taeniidae 18 kD gene. The EcoR I and Xho I restriction sites were introduced into 5′ end of primers. After RT-PCR, amplification product was cloned into pMD18-T, sequenced and the sequence analysis was made with bioinformatics. The products were ligated into the pGEX-4T-1 vector after digestion with EcoRⅠ＋XhoⅠ and purified. The recombinant pGEX-TPO18 plasmid was transformed into E. coli BL21 and spots were picked after shaking bacteria. The correctly identified recombinant plasmid, identified by PCR and sequenced, was named pGEX-TPO18. The recombinant plasmids were induced for expression with IPTG, cells were collected by SDS-PAGE analysis and purified using GST agarose resin. Then, TPO18 protein was detected by Western blotting. Rabbits were immunized with the purified recombinant protein emulsified with Freund’s adjuvant, 206 adjuvant, Al(OH)3 adjuvant, respectively, and rabbit anti-TPO18 serum was prepared. Each rabbit was injected with 50 μg recombinant protein for 3 times. On the 34th day after final inoculation, each rabbit was challenged by 1 500 eggs of T. pisiformis. On the 58th day after infection, rabbits were sacrificed and the number of cysts was counted. Before and after 7 days of every immunization, serum was separated. And serum antibody levels were detected using ELISA assay with 40 µg•mL-1 recombinant protein-coated microtiter plates, 1﹕200 dilution of serum and detection of the OD492nm value of serum samples was made.【Result】 The product of RT-PCR was 339 bp and agree with expectations; The results of sequencing showed no mutation. So, the construction of recombinant plasmid pGEX-TPO18 was successful. The pGEX-TPO18 was transformed into E. coli BL21. SDS-PAGE showed that the 38.6 kD fusion protein was expressed at a high level in E. coli. Western blotting analysis showed that a significant response band at 38.6 kD was displayed between rabbit against Cysticercus pisiformis positive serum and recombinant protein. This result indicated that the recombinant protein has reactogenicity and recognized by sera. On the 43th day after final immunization, the levels of antibody from tested group showed a peak. The reduction rate of cysts in immunized sheep (Freund’s adjuvant, 206 adjuvant and Al(OH)3 adjuvant) was 79.13%, 65.66%, and 50.43% respectively. 【Conclusion】Freund’s adjuvant emulsified recombinant antigens showed high protective activity, thus providing valuable information for the development of highly efficient recombinant vaccines against cysticercoids.

Functional Characterization of BmFAF as an Immune Negative Regulatory Molecule in Silkworm (Bombyx mori)

MA Xiao-Juan, LI Ya-Ming, HU Cui-Mei, WANG Fei, XIA Qing-You

Scientia Agricultura Sinica. 2014, 47(15): 3085-3093. doi:10.3864/j.issn.0578-1752.2014.15.018

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【Objective】The objective of this study is to characterize BmFAF as an immune negative regulatory molecule in silkworm cell line as well as in silkworm larvae and provide a basis for further studies of immune homeostasis in insects.【Method】Domain prediction and analysis were performed after cloning of BmFAF by RT-PCR. Then fluorescence quantitative PCR was used to investigate the spatial-temporal expression profile of BmFAF in different developmental stages and in different tissues extracted from the 5th-instar day-3 larvae, as well as the microbe-induced expression pattern after infection. The plasmid for expressing BmFAF in cells was constructed, and dsRNA for RNAi was also synthesized. After transfection of BmE cells with the constructs or dsRNA, Western blotting or quantitative PCR was used to confirm the over-expression or knock-down of BmFAF in cells. Meanwhile, the change in expression of anti-microbial peptides was detected. Finally, BmFAF was partially silenced by dsRNA injected into the silkworm larvae, and the effect of BmFAF on expression of anti-microbial peptides and survival rate of silkworm larvae after bacterial infection was studied.【Result】BmFAF is highly homologous to FAF family members from other species, the similarity between BmFAF and human HsUBXD8, HsFAF1 or Drosophila DmCaspar is 60%, 42% or 47%, respectively. It also contains a conserved UBX domain. Spatio-temporal expression profile showed that except in gonads, the level of BmFAF was slightly higher in immune tissues than in other tissues, and in molting larvae BmFAF level was significantly higher than in newly exuviated ones, particularly in the 2nd molt and 3rd molt stage. In addition, BmFAF expression level was relatively low in the 5th instar, but increased in pre-pupa stage. BmFAF expression level in fat body drastically decreased 0.5 h post infection of Bacillus bombyseptieus or Serratia arcescens, and did not recover within 12 h, whereas BmCecropinA1 was continuously up-regulated and reached the highest degree at 12 h. Over-expression of BmFAF in BmE cells led to a remarkable decrease of anti-microbial peptides, including BmCecropinA1, BmAttacin and BmMoricin. Moreover, the expression level of BmFAF could be efficiently knocked down to 50%-60% by dsRNA, at the same time, the expression level of BmCecropinA1 and BmMoricin was significantly up-regulated. Injection of dsFAF into the 5th-instar day-2 larvae resulted in a decrease of BmFAF to 45% of the level in larvae injected with dsEGFP at 24 h post injection, and the expression level of BmCecropinA1, BmAttacin and BmMoricin was significantly increased. Statistical analysis showed that the survival rate of silkworm larvae injected with dsFAF was higher than the control at 24 h post infection of S. arcescens, at 72 h 40% dsFAF-injected larvae were still alive but no control survived. 【Conclusion】 Domain analysis, expression features and functional studies in cells as well as in silkworm larvae all indicated that BmFAF acts as an immune negative regulatory molecule by suppressing the expression of anti-microbial peptides, and down-regulation of BmFAF resulted in an increase of anti-microbial peptides which would potentially increase the resistance of silkworm larvae to bacterial infection.

PMRP Expression Characteristics and Analysis of the Function in Arabidopsis

ZHANG Hao, YOU Shi-Dong, GAO Jing, ZHANG Hai-Li, LI Sheng-Hui, XING Ji-Hong, WANG Feng-Ru, DONG Jin-Gao

Scientia Agricultura Sinica. 2014, 47(15): 3094-3102. doi:10.3864/j.issn.0578-1752.2014.15.019

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【Objective】The objective of this study is to analyze the expression characteristics of PMRP (putative membrane related protein)gene (the biological funcion was unknown )in Arabidopsis, and make clear of the regulation function of PMRP in Arabidopsis development process.【Method】Bioinformatics method was used to look for the genes which contain the same domain with PMRP in Arabidopsis and draw the phylogenetic tree. Real-time PCR technique was used to analyze the expression level of PMRP in root and stem tissues of 8 and 21 days Arabidopsis, compare the expression of PMRP gene in the 1st, 2nd, 3rd and the 4th pairs of rosette leaves and cauline leaves in 21 days Arabidopsis, analyze the expression level of PMRP in flower organs (such as sepals, stamens and petals) and seeds of Arabidopsis. The gain-of-function mutant of PMRP were obtained by constructing a vector of PMRP gene driven by the 35S promoter of the cauliflower mosaic virus, and introduced it into wild type Arabidopsis Columbia, then tested the expression level of PMRP by RT-PCR technique. The regulating effect of PMRP gene on the growth sites of leaves and stems was analyzed by observing the phenotype of the 35S:PMRP transgenic plants. The function of PMRP gene in differentiation of vascular xylem and phloem was observed using the paraffin sections of the stem transverse section in the PMRP over-expressing transgenic Arabidopsis. The effect of PMRP on the growth and development of Arabidopsis floral organ was studied through observation of the floral organ anatomy. The effect of PMRP on fertility in Arabidopsis thaliana was analyzed by observing the pod formation in the PMRP over-expressing transgenic Arabidopsis. 【Result】PMRP is a 411 amino acids protein which containing a START domain and have transmenbrane segments, there are 35 START proteins in Arabidopsis. Real-time PCR analysis results showed that the expression level of PMRP was higher in cauline leaves ( the relative expression of PMRP was about 2 935) and then the rosette leaves, the growth time of rosette leaves was longer, the more expression of PMRP was tested (the relative expression of PMRP in the 1st, 2nd, 3rd and 4th pairs of the rosette leaves were 1 650, 1 113, 734, and 507, respectively) , then the sepals floral tissues (PMRP relative expression quantity is about 937), PMRP had distribution in the stem, root and seed , but the relative expression amount was less than 270. The relative expression level of PMRP in stamens was the lowest (about 64), far below the expression quantity of PMRP in sepals (937). The expression level of PMRP gene was increased with the increase of growth time, the relative expression of PMRP were 154 and 222, respectively, in 8 and 21 d roots, and 200 and 264, respectively, in 8 and 21 d stems. The rosette leaves were emerged from the branches, stalk lodging susceptible, vascular had no obvious cambium and the xylem and phloem arranged in disorder, the filaments became shorter in floral organs, the number of pod decreased, and the fertility reduced.【Conclusion】The START domain is extremely conservative in function. PMRP was expressed in different organs of A. thaliana, with the extension of time, the expression level of PMRP was also increased. The level of PMRP in the flower stamens was the lowest, once PMRP was over-expressed in Arabidopsis floral organs, stamens dysplasia, resulting in reduced fertility. PMRP had important function in regulating the origination of leaves, the vascular differentiation and development of floral organs.

Temporal Incubation Dynamic of Apple Leaf Brown Spot Caused by Diplocarpon mali

YONG Dao-Jing, LI Bao-Hua, ZHANG Yan-An, WANG Cai-Xia, DONG Xiang-Li, GUO Chang-Fei

Scientia Agricultura Sinica. 2014, 47(15): 3103-3111. doi:10.3864/j.issn.0578-1752.2014.15.020

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【Objective】 Leaf brown spot caused by Diplocarpon mali is the main disease causing defoliation of apple tree in early season. In order to understand the epidemics and design programs to control the disease, temporal incubation dynamics of the disease were examined. 【Method】 Fuji apple leaves at different ages were inoculated with conidia of D. mali for 4-6 times from June to September in 2011 and 2012. Disease development on each inoculated leaf was examined every 3 days after inoculation. Duration from the day of inoculation to the day that the first disease lesion appeared on each leaf, which was defined the incubation period of the leaves, was recorded. Average incubation period, the shortest incubation period and lesions development duration of the disease were calculated from the data. Temperature, rainfall and relative humidity near inoculated trees were recorded at 30 min intervals during the experiment period with automatic meteorological station. 【Result】 Average incubation period of the disease was 23.8 d, the shortest incubation period was 8-20 d with the mean of 13.6 d and the symptom development duration, from the first lesion was observed to last inoculated leaf showing symptoms, was 12-54 d with mean of 31.3 d. The shortest incubation period and the symptom development duration of the disease on the leaves inoculated in June and July were longer than those inoculated in August, as well as on the leaves incubated in indoor were much longer than those incubated in outdoor. Incubation period of the disease was shorter on aged leaves than on young leaves of head at vigorous physiological metabolic stage. The incubation period of the disease on leaves incubated at the early stage of growth (from June to July) was much longer than those incubated at the later stage of growth (from August to September). 【Conclusion】 Symptoms development of apple leaf brown spot was a dynamic process. The incubation period and the disease development duration were relatively longer than other diseases on apple leaves. Incubation period and symptom development duration depended mainly upon the combination of pathogen and the host and largely upon the leaf resistance, which affected by age, degree of senescence and vigorousness of metabolism of the leaf. During the main epidemic stages of the diseased, which occurred from June to September, temperature did not show significant impact on the incubation period of apple leaf brown spot. However, the rain and wetted environment could promote the pathogen sporulation and latent lesions to develop to visible lesions. According to the results, 15 days, even 20 days, after infected by pathogen, systemic fungicides could effectively prevent most infected pathogen developed to disease lesions.

Analysis on the Barriers of Interspecific Hybridization Between Rosa rugosa and Rosa hybrid

YU Xiao-Yan-1, XING Shu-Tang-2, ZHAO Lan-Yong-1

Scientia Agricultura Sinica. 2014, 47(15): 3112-3120. doi:10.3864/j.issn.0578-1752.2014.15.021

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【Objective】 The aim of this study is to reveal the reasons for barriers of interspecific hybridization between R. rugosa and R. hybrid. It is meaningful to improve the theory and techniques of breeding ornamental R. rugosa. 【Method】 A wild R. rugosa from Muping, a cultivar of R. rugosa ‘Tanghong’ , two cultivars of R. hybrid ‘Salvia’ and ‘Red Cap’ were used as plant materials. The fruit setting rate, seed number per fruit, seed length, percentage of seeds with embryo, germination rate of seeds with embryo and survived seedling rate of interspecific hybridization (MP×‘Salvia’, MP×‘Red Cap’) and intervarietal hybridization (MP×‘Tanghong’) were investigated. The growth of pollen tubes in the styles was observed with fluorescent microscope, Fertilization and development of embryos were observed by conventional paraffin sectioning. 【Result】 The fruit setting rate and seed number per fruit of MP×‘Salvia’ were significantly lower than those of intervarietal hybridization. No seed was obtained from MP ×‘Red Cap’. The seeds of MP×‘Salvia’ were of uneven size. The length of 76.67% of small seeds with no embryo and endosperm was simply (0.24±0.07) cm, these small seeds could not germinate. The length of 23.33% of big seeds with embryo and endosperm was (0.61±0.05) cm. But the germination rate and survived seedling rate of these big seeds were significantly lower than those of intervarietal hybridization. Two hours after pollination, the pollens of interspecific hybridization and intervarietal hybridization both germinated on the chapiters, but a great deal of callose deposited at the apex of pollen tubes of interspecific hybridization. Twenty to twenty-four hours after pollination, pollen tubes of intervarietal hybridization grew into ovary, but all the pollen tubes of MP×‘Red Cap’ and most of the pollen tubes of MP×‘Salvia’ were arrested at the middle of the styles. Little callose accumulated in the canal cells and spaces of the style of intervarietal hybridization, but a great deal of callose accumulated in the canal cells and spaces of the style of interspecific hybridization. A few pollen tubes of MP×‘Salvia’ grew into the ovary and fertilization was accomplished. But 79.42% of the embryos of interspecific hybridization aborted at the stage of heart-shaped embryo which formed on the 15th day after pollination, and 20.58% of the embryos of interspecific hybridization continued to develop.【Conclusion】The barriers of MP ×‘Red Cap’ happened before fertilization, and the dual barriers of MP ×‘Salvia’ happened before and after fertilization. The pre-fertilization barriers were caused by the pollen tubes stopping growth at the middle of the styles which may be connected with excessive callose accumulating at the apexes of the pollen tubes and in the canal cells and spaces of the styles. The post-fertilization barriers was due to hybrid embryo abortion at the stage of heart-shaped embryo.

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