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Table of Content

    01 October 2016, Volume 49 Issue 19
    CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS
    Biological Characteristics of Plant MicroRNAs and Actions in Environmental Stresses
    ZENG You-ling, YANG Rui-rui
    Scientia Agricultura Sinica. 2016, 49(19):  3671-3682.  doi:10.3864/j.issn.0578-1752.2016.19.001
    Abstract ( 1048 )   HTML ( 13 )   PDF (904KB) ( 1450 )   Save
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    MicroRNA (miRNA) is an extensive class of non-coding and small molecular RNA with length about 21 nt. It is encoded by the endogenous gene and transcribed by RNA polymerase II and the precursor miRNA is processed into mature miRNA by Dicer-Like and a series of the protein complexes. miRNA mainly regulates its targets at the level of post-transcription mediated degradation of target mRNA or translation inhibition. miRNA is an important regulator of gene expression in eukaryotic cells. Lin-4 is the first animal miRNA discovered in Caenorhabditis elegans (C. elegans). It is essential for the growth and development of C. elegans by negative controlling the expression level of target gene lin-14 with partial complementarity to lin-14 mRNA in the 3′untranslated region (UTR), causing the degradation of lin-14 gene and thus inhibiting the expression of lin-14 gene. Arabidopsis miR171 is discovered as the first miRNA in plant, which targets mRNA of Scarecrow-like (SCL) family, and thus miR171 can affect plant growth and development by negative regulating target gene expression. Plant partial miRNAs (from miR156 to miR408) are relatively conserved among different species and other miRNAs after miR408 are species-specific. Plants will encounter all kinds of unpredictably environmental stresses (salinity, drought, heavy metal, pest and pathogen infection), because of plant sessile growth, no moving to avoid the adversely environmental effects like animals. Therefore, it is necessary for plants to cope with these stresses with their special mechanisms. Actually, plants grown chronically in the stressed environments have evolved highly complicated and delicate physiological and molecular mechanisms. Studies have showed that miRNA and its target genes are the main regulatory factors in response to various stresses. miRNAs play important roles in regulating the expression of endogenous resistance genes by involving in plant growth and development, signal transduction, protein degradation, nutrient deficiency, preventing pathogen invasion and adapting to high salt- and drought-stressed environments as well. So far, lots of miRNAs are identified and have significantly differential expression by next-generation deep sequencing, advanced bioinformatics and real-time quantitative PCR technologies in response to environmental stresses; and the plants show the resistant or sensitive phenotypes by microRNA-based transformation. The different members of miRNA family responding to environmental stresses are also taken on being species-specific. It is well-known that China’s Xinjiang is a kind of typical continental arid climate with a low annual rainfall and more acid areas. In such harsh environments, some extremely salt- and drought-tolerant halophytes and xerophytes can still survive healthy and strong. It is very essential to study deeply how plant miRNAs play regulatory roles in coping with environmental stresses. This paper mainly summarizes plant miRNA biosynthesis, the modes of actions with target genes and research status of some miRNAs involving in the abiotic and biotic stresses. Some prospects are expected by microRNA-based biotechnology.
    Isolation of Thinopyrum ponticum Genome Specific Repetitive Sequences and Their Application for Effective Detection of Alien Segments in Wheat
    YAO Han, TANG Cai-guo, ZHAO Jing, ZHENG Qi, LI Bin, HAO Chen-yang, LI Zhen-sheng, ZHANG Xue-yong
    Scientia Agricultura Sinica. 2016, 49(19):  3683-3693.  doi:10.3864/j.issn.0578-1752.2016.19.002
    Abstract ( 433 )   HTML ( 3 )   PDF (5348KB) ( 629 )   Save
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    【Objective】As a tertiary gene pool of wheat, Thinopyrum genus has many useful traits, such as tolerance to both abiotic and biotic stresses. DNA repetitive sequence is one major component for genomes of most eukaryotes. Isolation of genome specific repeats is very helpful for understanding of sub-genome component of polyploid species, their evolution and utilization in crop improvement. To identify Th. ponticum chromatin in wheat genetic background more accurately and efficiently, genome specific repetitive sequences were cloned from Th. ponticum.【Method】Screening the plasmid library of Th. ponticum via dot-blot hybridization, combining with fluorescence in situ hybridization (FISH) analysis of the wheat-Th. ponticum partial amphiploids, Th. ponticum genome specific repetitive sequences were isolated and their distribution patterns on chromosomes were characterized by FISH. The Th. ponticum genome specific repeats were analyzed by Repeat Masker, Triticeae Repeat Sequence Database and NCBI. A set of PCR primers was designed for detecting Th. ponticum chromosomal fragments. Wheat-Th. ponticum hybrid derivatives were employed to testify the genome specificity and effectiveness of these repeats in identifying Th. alien fragments in wheat genetic background. 【Result】Seven Th. ponticum genome specific repetitive sequences were obtained. Their FISH signals dispersively covered all chromosomes in Th. ponticum and Th. intermedium. Furthermore, they could discriminate chromosomes of Th. ponticum and Th. intermedium from wheat chromosomes without wheat DNA block by FISH. This was further proved in wheat-Th. ponticum substitution lines and translocation lines. Ten PCR primers were developed for efficient amplification of Th. ponticum segments in wheat. Their detection efficiency ranged from 73.3% to 95% in 109 wheat-Th. ponticum derivatives. 【Conclusion】Th. ponticum genome specific repetitive sequences were isolated, which can be used for detection of wheatgrass fragments in wheat by FISH or PCR amplification.
    Comprehensive evaluation and genetic diversity analysis of phenotypic traits of core collection in upland cotton
    DAI Pan-hong, SUN Jun-ling, HE Shou-pu, WANG Li-ru, JIA Yin-hua, PAN Zhao-e, PANG Bao-yin, DU Xiong-ming, WANG Mi
    Scientia Agricultura Sinica. 2016, 49(19):  3694-3708.  doi:10.3864/j.issn.0578-1752.2016.19.003
    Abstract ( 505 )   HTML ( 5 )   PDF (2918KB) ( 537 )   Save
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    【Objective】 The objective of this study is to analyze the genetic diversity and the law of genetic variation of phenotypic traits and explore the comprehensive evaluation techniques for core collection in upland cotton.【Method】The genetic diversity of 419 core collections in upland cotton was analyzed with 17 phenotypic traits. Genetic diversity of phenotypic traits was calculated using Shannon-weaver Information Index. Phenotypic distance was analyzed using Nei’s 1973, and then NTSYS-pc 2.20q software was used for cluster analysis of core collections. With SAS9.2, the best linear unbiased estimate (BLUE) value was gotten from phenotypic trait data, and then the optimum value of phenotypic traits was calculated according to the best linear unbiased estimator. Meanwhile, the principal component, regression and correlation analysis were also used to study the comprehensive evaluation index and method of core collection. 【Result】 Based on the analysis of phenotypic traits of core collection, it was found that the variation of boll number, boll weight, lint percentage, seed index and other traits were greater with coefficient of variation over 10% , while the variation of fiber breaking strength, micronaire level and the upper half of the average length was smaller which their coefficient of variation was below 10%. Variance analysis found that there were significant differences in the phenotypic traits between various locations, between years, and between locations and years. The phenotypic traits of different geographical origin germplasms were quite different. The growing stage elongation, upper half mean length, lint percentage and other traits from the geographical origin of the Yangtze River areas were higher than those of other geographic origins, and the comprehensive properties including the fiber strength, boll weight, uniformity index, plant height, and spinning uniformity index from the Northwest areas were the best. The yield and fiber quality traits from the US germplasms were superior to the sum of the other countries. Genetic diversity indices of phenotypic traits were 0.351-3.796, with an average of 1.715. The result of analysis on the genetic diversity of different geographical origins indicated that the genetic diversity and genetic richness in the Yellow River areas was the highest, but that in south China was the lowest. Cluster analysis showed that the upland cotton lines were overall dispersed with no obvious groups, but the partial accessions with similar characteristics can be clustered into 13 groups. After comprehensive evaluating these core collections, seven principal components were found when the cumulative contribution rate was more than 85%. The comprehensive value (F) of phenotypic traits of core collections averaged 1.740. The highest F value (2.302) was from Australia’s N74-250, and the lowest F value (0.624) was from Liaoyang green cotton. Correlation analysis between 17 phenotypic traits and their F values illuminated that 14 phenotypic traits including boll weight, lint percentage, fiber strength, upper half mean length, etc. were very significantly correlated with the F values, except micronaire, seed index and yellow givers. Finally, the regression equation was constructed for comprehensively evaluating the core collection with 8 phenotypic traits as the independent variables including boll opening stage, boll weight, elongation, flowering, micronaire, plant height, number of fruit branches, and spinning consistent index. 【Conclusion】 The core collection of upland cotton from China has a rich genetic diversity. The difference in genetic variation of the germplasms with different geographical origins is great. The accessions from different ecological zones have unique characteristics.
    TILLAGE & CULTIVATION·PHYSIOLOGY & BIOCHEMISTRY·AGRICULTURE INFORMATION TECHNOLOGY
    Effects of Reduced Chlorophyll Content on Photoinhibition and Photosynthetic Electron Transport in Rice Leaves
    ZHOU Zhen-xiang, LI Zhi-kang, CHEN Ying, WANG Zhi-qin, YANG Jian-chang, GU Jun-fei
    Scientia Agricultura Sinica. 2016, 49(19):  3709-3720.  doi:10.3864/j.issn.0578-1752.2016.19.004
    Abstract ( 558 )   HTML ( 3 )   PDF (5633KB) ( 774 )   Save
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    【Objective】The chlorophyll content of a chlorophyll-deficit rice mutant (YL) is significantly lower than its wild type (WT), but its photosynthetic electron transport rate and net photosynthetic rate are significantly higher than its WT. The aim of this study is to understand the physiological basis, and its potential use in high photosynthetic efficiency breeding was prospected.【Method】A pot experiment in the climate chamber at high light intensity (HL) and low light intensities (LL), and a field experiment were conducted. Chlorophyll fluorescence, the concentration of super oxygen anion and malodialdehyde, superoxide dismutase activity, light fluorescent and electron micrographs, canopy temperature were investigated. 【Result】 The results show that the chlorophyll content was significantly lower in the mutant than its WT, and the light treatment had different effects on chlorophyll content in the mutant and its WT. When compared the HL and LL treatments, the chlorophyll content was increased by 60% in the mutant, but decreased by 20% in the WT. The light response curves showed that the mutant has a higher photosynthetic capacity than its WT. At the irradiance of 1 000 µmol·m-2·s-1, the photosynthetic rate was 46.5% and 9.4% higher in the mutant than its WT, in HL and LL treatments, respectively. The chlorophyll fluorescence measurements indicate that the photosynthetic electron transport rate (ETR), photosystem Ⅱ efficiency (ΦPS), photosynthetic quenching (qP), and maximum efficiency of open photosystem Ⅱ in the light (F′v/F′m) were significantly higher in mutant than its WT. The content of oxygen anion and malondialdehyde were higher in WT than the mutant, especially in HL treatment, indicating photoinhibition in WT. The electron micrographs and light fluorescent micrographs of the mutant and its WT indicated that the chloroplast was to some extent harmed. The inter-vein distance was also found larger in WT than in the mutant, and the area of vascular bundle is smaller in WT than in the mutant, indicating a better water status in mutant than in its WT. The thermal image indicated that the canopy temperature was significantly lower in mutant than in its WT at noon with high irradiance. All the results suggested that the high chlorophyll content in the WT excessively absorbed light energy, causing photoinhibition (high oxygen anion and malondialdehyde content, reduced photosystem Ⅱefficiency and decreased SOD activity), and had a lower photosynthetic rate than the mutant. The excessively absorbed light energy also contributed the higher canopy temperature in WT than in mutant, which is adverse for canopy photosynthesis.【Conclusion】All these results implicate that selecting for moderate chlorophyll content in breeding would help avoiding the generation of reactive oxygen species and alleviating the photoinhibition, improving photosynthesis, especially at noon under high solar radiation.
    Grain Yield and Water Use Efficiency and Soil Water Changes of Dryland Corn with Film Mulching and Close Planting
    FAN Ting-lu, LI Yong-ping, LI Shang-zhong, LIU Shi-xin, WANG Shu-ying, MA Ming-sheng
    Scientia Agricultura Sinica. 2016, 49(19):  3721-3732.  doi:10.3864/j.issn.0578-1752.2016.19.005
    Abstract ( 640 )   HTML ( 1 )   PDF (1574KB) ( 725 )   Save
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    【Objective】Drought and water shortage are the main limiting factors for dryland agricultural production on Loess Plateau of China, and studies of the effects of film mulching and close planting and hybrids on dryland corn yield and water use efficiency (WUE) are of benefit to understand the relationship of future grain yield increase and water environment safety.【Method】A 4-year field experiment with three replications was conducted in Pengyang county, Ningxia, a hilly and gully region on China’s Loess Plateau, in continuous spring corn seasons from the year 2012 to 2015, and a randomized design was used with full film ridge-furrow cover (FPRF) and half film-flat cover (HPFC) , three corn hybrids of XY335 and JX1 with close planting and mid-late mature, and JD4 with spaced planting and early mature, and three plant densities of 4.5×104 (Low), 6.75×104 (Middle) and 9.0×104 (High) plants/hm2. The area of each plot was 6.5 m ´ 3.3 m. Soil water contents were determined at main crop stages in soil depths of a 200 cm with 20 cm depth as a soil layer. Seasonal ET amounts were calculated with growing season precipitation (precipitation received from planting to harvesting) and soil water changes. Grain yields in all plots were determined by hand-harvesting 12 m2. Soil moisture contour maps of 2012-2015 were drawn using SURFER V11 software. 【Result】Experimental factors highly affected dryland film mulched corn yield and water use, but the order of influence on grain yield and WUE was corn density > film mulching pattern > corn variety. With year’s type from dry years to normal and rainy years, grain yield increased from 7.72 and 8.79 t·hm-2 to 11.86 to 11.15 t·hm-2, but the highest WUE value was obtained in normal year not in rainy year. When corn density increased from 4.5 ×104 to 6.75 ×104 plants/hm2, ETs, grain yield and WUE increased by 10.6 mm, 20.0% and 3.45 kg×mm-1×hm-2, and the density improved from 6.75×104 to 9.0×104 plants/hm2, ETs did not increase again and grain yield and WUE increased by 12.0% and 2.97 kg×mm-1×hm-2, respectively. Average grain yield of corn and WUE in FPRF increased by 15.72% and 21.09% compared with that in HPFC. Grain yield and WUE of JX1 and XY335 hybrids, with close planting and middle-late mature, increased by 15.46% -24.45% and 13.35% -15.55% compared with that of JD4 hybrid with low density and early mature. Whatever rainfall in the four years, profile soil water content at 0-200 cm layers during crop growing season was always higher in the FPRF than in the HPFC treatment, particularly the FPRF stored more than 50-90 mm soil water at the 0-200 layers in grain filling period, which played an important role in drought-resistance and WUE increase during serious summer drought. More importantly, the FPRF with significant corn yield increase by close planting did not deplete much soil water, yet not resulted in deep dry soil layers formation whether normal and rainy or dry year, and negative effects on soil water cycle were not observed in the FPRF. However, a dry soil layer with less than 8% of soil water was formed significantly in the HPFC treatment at deep layers in dry years, and dry soil thickness increased and it’s scope extended as corn growth. 【Conclusion】On the basis of film mulching with density of 5.3×104 plants/hm2 currently, the package of full ridge-furrow film mulching and close planting hybrids and density improvement of 1.5×104 plants/hm2 will be a key technique for sustainable grain yield increase of dryland corn and high efficient use of water in dryland area of annual rainfall over 450 mm, and the yield-increasing techniques with close planting did not cause deep dry soil layers formation.
    PLANT PROTECTION
    Acquisition and Biological Characteristics of Fluopicolide-Resistant Isolates in Phytophthora infestans
    LUO Yan-tao, MENG Run-jie, ZHAO Jian-jiang, HAN Xiu-ying, MA Zhi-qiang, WANG Wen-qiao, ZHANG Xiao-feng
    Scientia Agricultura Sinica. 2016, 49(19):  3733-3745.  doi:10.3864/j.issn.0578-1752.2016.19.006
    Abstract ( 458 )   HTML ( 1 )   PDF (541KB) ( 783 )   Save
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    Objective】The objectives of this study are to research the cross resistance between fluopicolide and the other fungicides and assess the resistance risk of Phytophthora infestans to fluopicolide. Method】The fluopicolide-resistant mutants were acquired by UV irradiating sporangia, UV irradiating mycelia and fungicide taming. The mutation frequency was calculated, the resistance levels were measured, whether the resistance could be inherited stably under the condition of no fungicide after 10 generations was studied, the fitness (including mycelial growth rates, sporulation capacity, and comprehensive fitness index) on RSA medium and detached leaves were measured and the competitiveness of those resistant mutants and their parental isolates were studied. Cross resistance between fluopicolide and other fungicides were analyzed. The resistance risk of P. infestans to fluopicolide was assessed by the resistance risk quantitative evaluation criteria which was created by the authors’ lab. Result】Twenty-one fluopicolide-resistant mutants were acquired in this study. The resistance levels of those resistant mutants ranged from 61 to 3 157 and the mutation frequency of UV irradiating sporangia was 2.78×10-7. The fitness of most of the tested resistant mutants showed no significant difference with their respective parental isolates. In the test of competitiveness, among the 18 resistance frequency measurements of 1st, 3rd and 7th generations of two fluopicolide-resistant mutants, 3 were significantly lower than their parental isolates, 4 were significantly higher than their parental isolates, while the other 11 showed no difference. The resistance of all those 21 fluopicolide-resistance mutants could be inherited stably. The resistance of all those 21 fluopicolide-resistance mutants could be inherited stably. The correlation coefficient (r) between the lgEC50 of the fluopicolide-resistant mutants and the fluopicolide-sensitive isolates to fluopicolide and the lgEC50 of those isolates to azoxystrobin, pyraclostrobin, cymoxanil, dimethomorph, mandipropamid, metalaxyl and fluoride ether bacteria amide were 0.104 (p =0.654), 0.311 (p =0.170), 0.228 (p =0.081), 0.376 (p =0.093), 0.214 (p =0.351), 0.122 (p =0.599), 0.963 (p =0.000). The inherent resistance risk value of P. infestans to fluopicolide was 15.Conclusion】The cross-resistance relationship was found between fluopicolide and fluoride ether bacteria amide. No cross-resistance relationship was found between fluopicolide and azoxystrobin, pyraclostrobin, cymoxanil, dimethomorph, mandipropamid and metalaxyl. There could be high inherent risk of P. infestans developing resistance to fluopicolide. The risk management of P. infestans to fuopicolide should be enhanced. It is suggested that the fluopicolide should be used alternately or in mixture with other not cross-resistant fungicides.
    Herbicidal Activities of Alkaloids from Cephalotaxus sinensis
    MA Shu-jie, LIU Lin, LU Xiao-peng, MA Zhi-qing, ZHANG Xing
    Scientia Agricultura Sinica. 2016, 49(19):  3746-3753.  doi:10.3864/j.issn.0578-1752.2016.19.007
    Abstract ( 397 )   HTML ( 3 )   PDF (403KB) ( 503 )   Save
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    【Objective】The objective of this study is to evaluate herbicidal activity of alkaloids from Cephalotaxus sinensis against four species of weeds and provide a basis for its further study. 【Method】 C. sinensis extract, total alkaloids, and eight alkaloids (drupacine, 11-hydroxycephalotaxine, cephalancetine A, isocephalotaxine, cephalotaxine β-N-oxide, 4-hydroxycephalotaxine, wilsonine and cephalotaxine) were measured by seed germination method and pot experiments, with Trifolium pretense, Amaranthus retroflexus, Lolium perenne and Sorghum sudanense as indicators.【Result】Seed germination experiment showed that C. sinensis extract, total alkaloids, drupacine, 11-hydroxycephalotaxine, cephalotaxine and cephalotaxine β-N-oxide had strong inhibiting effects on the growth of T. pretense with the EC50 values of 634.35, 690.47, 69.47, 71.21, 174.11 and 243.12 mg·L-1 against roots growth, and 411.79, 413.11, 68.52, 139.25, 237.72 and 413.62 mg·L-1 against stems growth, respectively. C. sinensis extract, total alkaloids, drupacine, 11-hydroxycephalotaxine, cephalotaxine and cephalotaxine β-N-oxide showed strong inhibiting effects on the growth of A. retroflexus with the EC50 values of 176.32, 29.69, 21.01, 48.08, 199.57 and 90.74 mg·L-1 against roots growth, and 257.69, 46.20, 25.51, 47.85, 65.08 and 66.34 mg·L-1 against stems growth, respectively. Only C. sinensis extract, total alkaloids and drupacine showed inhibiting effects on the growth of S. sudanense with the EC50 values of 349.54, 254.27 and 84.71 mg·L-1 against roots growth, and 556.50, 168.66 and 154.34 mg·L-1 against stems growth, respectively. Only C. sinensis extract, cephalotaxine β-N-oxide and drupacine showed inhibiting effects on the growth of L. perenne with the EC50 values of 214.40, 187.89 and 26.52 mg·L-1 against roots growth, and 695.58, 290.15 and 122.26 mg·L-1 against stems growth, respectively. The pot experiments showed that C. sinensis extract, total alkaloids, drupacine, 11-hydroxycephalotaxine and cephalotaxine had strong herbicidal effect in soil treatment against dicotyledon weeds T. pretense and A. retroflexus, and drupacine showed the strongest herbicidal effect with inhibitory rates of 78.89% and 81.11%, but low inhibitory rate of less than 25% against S. sudanense and L. perenne. Spraying treatment showed that the five samples had a weak herbicidal effect against dicotyledon weeds T. pretense and A. retroflexus with an inhibitory rate of less than 30%, and had no effect against S. sudanense and L. perenne.【Conclusion】C. sinensis alkaloids has potential herbicidal activities against dicotyledon weeds, and its main active constituent is drupacine, with an inhibiting effect on young buds growth.
    SOIL & FERTILIZER·WATER-SAVING IRRIGATION·AGROECOLOGY & ENVIRONMENT
    Isolation and Biological Properties of Endophytic Diazotrophs from Rice and Their Influences on Rice Seedling Cd Accumulation
    YUAN Mei, TAN Shi-juan, SUN Jian-guang
    Scientia Agricultura Sinica. 2016, 49(19):  3754-3768.  doi:10.3864/j.issn.0578-1752.2016.19.008
    Abstract ( 475 )   HTML ( 1 )   PDF (1271KB) ( 731 )   Save
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    Objective】The objective of this study is to isolate, identify and analyze phylogenetics of endophytic diazotrophs from rice planted in Hunan province, test the biological characteristics of the isolates, and to explore the influences of diazotroph inoculation on rice seedlings Cd accumulation. Method】Surface sterilization and low nitrogen medium were used to isolate endophytic diazotrophs. nifH detection was conducted based on PCR amplification to confirm the isolates as nitrogen-fixing bacteria. 16S rRNA was amplified with PCR, blasted in EzTaxon after sequencing, and analyzed with Clustalx-MEGA to make phylogenetic tree. Greenhouse trials were conducted to investigate the influence of diazotroph inoculation on rice seedling Cd accumulation. 【Result】Nineteen endophytic diazotrophs were isolated from root, stem and leaf of 8 rice samples. These 19 strains phylogenetically belong to 4 genus 13 species of Bacillus aryabhattai, B. cereus, B. idriensis, B. sindicus, B. licheniformis, B. megaterium, B. methylotrophicus, B. subtilis, B. tequilensis, Brevibacterium. halotolerans, Fictibacillus phosphorivorans, Paenibacillus barcinonensis, P. lautus. Biological tests showed that about 1/3 of the 19 strains produce protease and cellulose, grow well at 48℃, form spores well with percentage 60%-90%, produce alkali with final pH 8.5-9.0. About 1/6 of the 19 strains are antagonistic against plant pathogenic Rhizoctonia solani ACCC36246, Fusarium graminearum ACCC36249 and Fusarium sporotrichioides ACCC37402 with rate of 42%-55%. About 2/3 of the 19 strains showed sensitive to antibiotics and resistant to fungicide. Four representative strains of the 19 could utilize 7 of the 78 carbon sources, sodium lactate, sucrose, dextrose, glycerol, malic acid, alanine and glucuronic acid amide. Greenhouse trials showed that 6 of the 19 strains promoted rice seedling Cd absorption with increase of 6.41%-38.45%, and other 13 strains decreased rice seedling Cd absorption with 2.06%-34.46% compared with control.Conclusion】Nineteen endophytic diazotrophs were isolated from rice planted in Hunan. These 19 strains phylogenetically belong to 4 genus 13 species of Bacillus, Brevibacterium, Fictibacillus and Paenibacillus. Partial strains produce protease and cellulose, grow well at 48℃, form spores well, antagonistic against plant pathogenic Rhizoctonia solani, Fusarium graminearum and Fusarium sporotrichioides, have good prospects of application. Inoculation of diazotroph can significantly affect rice seedling Cd absorption. The results suggest that application of microbial method to control paddy Cd is a very worthwhile pathway.
    Eco-Economic Potential Analysis of Chinese Main Crops’ Bio-Energy Utilization Straw Resources
    ZHU Kai-wei, LIU Zhen, HE Liang-ping, LIN Jin-chai
    Scientia Agricultura Sinica. 2016, 49(19):  3769-3785.  doi:10.3864/j.issn.0578-1752.2016.19.009
    Abstract ( 481 )   HTML ( 2 )   PDF (656KB) ( 627 )   Save
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    【Objective】Realizing the reasonable development of straw resource on the basis of protecting environment has important significance to Chinese agricultural bio-energy development planning. And effective development of straw resource is also closely related with cost. Thus, from the perspective of protecting environment and cost, this study analyzed the eco-economic potential of main crops’ energy utilization straw resource in different regions, which can provide reference for the layout of agricultural bio-energy industry, prior suitable regions for straw energy utilization project, prior developing technology and government policy formulation.【Method】Based on the research for the studies on the relationship between straw returning amount and soil erosion, soil organic matter, and the crop yield, the concept of soil ecological reserve amount was put forward and the specific returning amount of different crop straws were concluded. Combining the crop yield, planting structure, and grass grain ratio in benchmark year with the soil ecological reserve amount and other straw usages, the total ecological energy utilization straw resource and straw resource density were calculated by regions. Analyzing the policies and costs of different straw energy utilization projects, four developing scenarios and project costs were designed, and the economic collecting radius in different ratio of return on investment (ROI) was calculated by regions. According to the density and economic collecting radius, straw economic collecting amount was calculated, and then comparing the economic collecting amount with the project’s theory straw demand, to conclude the eco-economic straw collecting amount and space distribution of different projects.【Result】The straws can be used for bio-energy are 86.61 million ton, mainly distributed in Guangxi, Sichuan, Shandong and Henan, and the main straws are those of potato, sugar and rapeseed straws. When ROI is 0, the available straw for 6 MW SDFPG, 25 MW SDFPG, 12 MW SGPG, BMF 5 000 t·a-1 and BMF 10 000 t·a-1, respectively, is 86.85, 86.55, 86.85, 33.15, 86.61 million ton at the current level technology and incentive policy. When ROI is 5%, the available straw for 6 MW SDFPG, 25 MW SDFPG, 12 MW SGPG, BMF 5 000 t·a-1 and BMF 10 000 t·a-1, respectively, is 79.43, 83.47, 83.47, 2.60, and 86.61 million ton. When ROI is 10%, the available straw for 6 MW SDFPG, 25 MW SDFPG, 12 MW SGPG, BMF 5 000 t·a-1 and BMF 10 000 t·a-1, respectively, is 18.53, 70.23, 70.68, 0, and 86.61 million ton. At current level technology and incentive policy, under different scenarios, the available straws for CE projects are always 0 in different ROI.【Conclusion】The existing policies have obvious incentives for power projects, the incentives for BMF 10 000 t·a-1 should be appropriately reduced, and the incentives for power projects should be increased. In straw energy industry layout, at present, as for the BMF 10 000 t·a-1, first priority should be given, then first priority should be given to Guangxi, Hubei, Guangdong, Anhui, and Hainan to build 12 MW SGPS, and then construction of 25 MW SDFPG should be considered in Guangxi, Hubei, Guangdong and Anhui, and finally, construction of 6 MW SDFPG in Guangxi should be considered. Now the cost of CE project is so high that it does not necessarily produce economic benefits in every region.
    HORTICULTURE
    Bioinformatics and Expression of the OVATE Gene Family in Grape
    YUAN Yue, ZHANG Ya-guang, GAO Shi-min, TAO Jian-min
    Scientia Agricultura Sinica. 2016, 49(19):  3786-3797.  doi:10.3864/j.issn.0578-1752.2016.19.010
    Abstract ( 532 )   HTML ( 5 )   PDF (3859KB) ( 675 )   Save
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    【Objective】OVATE is a class of transcriptional repressor that regulates plant growth and development. In this study, the authors analyzed the grape OVATE gene family (VvOFPs) from the aspects of bioinformatics and tissue-specific expression in order to lay a foundation for the functional studies in future. 【Method】Based on the conserved OVATE domain (PF04844), OVATE genes were identified from grape genome. Chromosome location, gene structure, conserved domain and subcellular localization were also analyzed by the bioinformatics methods. A phylogenetic analysis was conducted based on the alignment of the OVATE proteins from grape and Arabidopsis genomes. The expression patterns of VvOFPs were tested via quantitative real-time polymerase chain reaction (qRT-PCR). 【Result】Seventeen putative VvOFPs were identified in the grape genome and they unevenly distributed on the eleven chromosomes. No intron was found in the gene structures of VvOFPs. The VvOFPs encoded hydrophilic proteins, containing 115-444 amino acids, and their isoelectric points ranged from 4.55-9.69. In addition, all the VvOFPs contained the entire OVATE domain and were predicted to be located in the nucleus. According to the topology of phylogenetic tree, OVATE proteins from grape and Arabidopsis were divided into six groups (I-VI). Group II, III and V contained only two members, whilst members of groups I, IV and VI were clustered in the species-specific pattern. VvOFPs and AtOFPs contained 10 uncharacterized motifs. Motifs 1 and 2 were located in the OVATE domain region. Besides, group-specific motifs were also found outside the OVATE domain. qRT-PCR analysis showed that VvOFPs exhibited different tissue-specific expression patterns. Interestingly, twelve VvOFPs were found to be expressed in the detected organs of roots, stems, leaves, flowers and fruits while the others were expressed in the specific organ tissues. Most of the VvOFPs were more highly expressed in roots, stems and flowers, whereas a little gene was detected with low level in leaves and fruits. Meanwhile, VvOFPs expressed differently at different developmental stage. Transcripts in flowers a week before or at anthesis were at a high level, while low in fruits four weeks after anthesis. In addition, one pair of paralogues exhibited the same expression patterns, whereas the other three pairs of paralogues exhibited divergent expression patterns. 【Conclusion】Results of this study showed that grape OVATE domain is conserved. VvOFPs exhibited different expression patterns and may be involved in regulation of plant growth and development.
    Characteristics and Expression of Calmodulin Like B Subunit Interaction Protein VvCIPK10 in Grapevine
    YU Yi-he, LI Xiu-zhen, GUO Da-long, ZHANG Hui-ling, YANG Ying-jun, LI Xue-qiang, ZHANG Guo-hai
    Scientia Agricultura Sinica. 2016, 49(19):  3798-3806.  doi:10.3864/j.issn.0578-1752.2016.19.011
    Abstract ( 328 )   HTML ( 3 )   PDF (820KB) ( 349 )   Save
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    【Objective】The objective of this study is to clone serine/threonine protein kinase VvCIPK10 in grapevine, and analyze the kinase characteristics and its expression pattern under stresses. This study will provide a theoretical basis for further study on the molecular function of VvCIPK10 involved in abiotic stress, and investigate the molecular mechanism of grapevine stress resistance. 【Method】The sequences of VvCIPK10 were obtained by electronic cloning technology, and the specific primers were designed to perform RT-PCR reaction. Open reading frame and conserved structure domain of VvCIPK10 were analyzed. The prokaryotic expression vector was constructed and transformed into the expression cells, and the recombinant bacteria were induced by IPTG. The expression products were collected and the protein sample was prepared. The protein samples were analyzed by SDS-PAGE electrophoresis, and the soluble characteristics of the fusion protein were analyzed. The fusion protein was induced by IPTG. The cells were collected and broken by using ultrasonic. The MBP-VvCIPK10 fusion protein was purified by maltose binding protein purification column and analyzed by SDS-PAGE. The purified fusion protein was incubated with in vitro self-phosphorylation buffer solution. After SDS-PAGE electrophoresis, the phosphor screen was performed and the phosphorylation reaction was detected in vitro. The recombinant transient expression vector pBI221-GFP/VvCIPK10 was constructed. The recombinant expression vector pBI221-GFP/VvCIPK10 was transformed into protoplasts by PEG mediated transient transformation. The recombinant expression vector pBI221-GFP/VvCIPK10 was transformed into onion epidermal cells by gene gun mediated transformation, and the fluorescence signal was detected by laser scanning confocal microscope after 16 h of culture. The relatively consistent and robust grape plants were selected, samples were taken at different times after drought, low temperature and salt stress treatments, at the same time, different tissue samples of grapes were taken in the field, with a kit to extract total RNA. After reverse transcription, expression of VvCIPK10 was detected by real-time quantitative PCR. 【Result】The full-length of VvCIPK10 is 1 357 bp, 5′ end of non-coding region is 30 bp, 3′ end of non-coding region is 156 bp, the open reading frame is 1 171 bp. VvCIPK10 open reading frame encoded 436 amino acids, the theoretical isoelectric point is 8.59, molecular weight is 48.7 kDa. Conserved domain prediction analysis showed that the protein has a kinase domain in 5′ terminal, a PPI domain and a NAF domain in 3′ terminal. BLSATP analysis showed VvCIPK10 consistency with peach CIPK (XP_007205151) is highest (74%). Recombinant expression vector pMAL-C5X/VvCIPK10 transformation in Escherichia coli, expressed the molecular weight of fusion protein is consistent with the predicted molecular weight (43 kDa+48.7 kDa). MBP-VvCIPK10 fusion protein was purified by column. VvCIPK10 autophosphorylation activity was dependent on Mn2+ but not dependent on Mg2+ and Ca2+, and EDTA could inhibit the autophosphorylation activity of the VvCIPK10. Subcellular localization showed VvCIPK10 in the nucleus, cell membrane and cytoplasm. VvCIPK10 expressed in various tissues of grapevine. VvCIPK10 transcripts mainly accumulated in grapevine roots and leaves, but showed low expression levels in grapevine stem, inflorescence, fruit and tendril. After drought, low temperature and salt stress treatments, VvCIPK10 showed the induced expression model. The expression of VvCIPK10 reached peak value at 6 h after low temperature stress, and reached the peak at 2 h after drought and salt stresses. 【Conclusion】It was concluded that grapevine VvCIPK10 as a serine threonine protein kinase is able to respond to drought, low temperature and salt stress, suggesting that VvCIPK10 plays an important role in resistance to abiotic stress.
    Evaluation on Correlations of Three Kinds of Osmoregulation Substances in Tea Fresh Leaves with Low Temperature During Winter and Spring Respectively and Their Difference Among Cultivars
    BO Xiao-pei1, WANG Meng-xin1, CUI Lin1, WANG Jin-he2, HAN Bao-yu1
    Scientia Agricultura Sinica. 2016, 49(19):  3807-3817.  doi:10.3864/j.issn.0578-1752.2016.19.012
    Abstract ( 369 )   HTML ( 3 )   PDF (455KB) ( 365 )   Save
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    【Objective】 The content dynamics of three types of osmoregulation substances in fresh leaves of two cultivars of tea plants and their correlations with air temperatures during winter and early spring were investigated, to evaluate the differences in their responsiveness to the low temperature between the two tea cultivars. 【Method】 Two early bud tea cultivars, ‘Pingyangtezao’ and ‘Zhongcha 102’, were chosen, and air temperatures around their tea shoots were recorded every day from Oct. 21 to April 5 for two consecutive years. The temperature of the dry and wet bulb thermometer at 8:00, 14:00 and 20:00 was daily recorded, and the average of the three dry bulb thermometers set in the experimental tea plantations three times per day standard for the temperature of tea shoot habitat. During these two testing periods adult tea leaves of each cultivar were sampled on the fifth, fifteenth and twenty-fifth days of each month. The soluble sugar contents in the tea fresh leaves were measured by anthrone colorimetric method, the remaining clean fresh leaves were processed into the steamed green tea samples, whereas the contents of proline and free amino acids were determined by SDE-HPLC method. 【Result】The test phase was divided into two stages, the overwintering period (2013/10/21-2014/3/52014/10/21-2015/3/5) and early spring period (2014/3/6-2014/4/52015/3/6-2015/4/5) according to the developmental threshold temperature and the dormancy temperature of the tea plant. Accompanied with initial gradual drops and then maintaining at low air temperatures during the two winters (2013/10/21-2014/3/5, 2014/10/21-2015/3/5), the soluble sugar contents in fresh tea leaves of the two cultivars increased continuingly, negatively correlated with the 10-day average air temperatures. The proline contents increased first and then kept at a high level, also negatively correlated with the 10-day average air temperatures. The free amino acid contents dropped first and then stayed at a low level, which was positively correlated with the 10-day average air temperatures. When the temperature appeared the lowest value, the content of soluble sugars and the content of proline also correspondingly appeared the highest values subsequently, then the content of free amino acids also correspondingly appeared the lowest value. When the temperature was the highest value, the content of soluble sugar and the content of proline also correspondingly appeared the lowest value later, the content of the free amino acids also correspondingly appeared the highest value soon afterwards. During the two spring seasons (2014/3/6-2014/4/5, 2015/3/6-2015/4/5), as air temperatures increased gradually, the soluble sugar contents decreased, while the proline and the free amino acid contents appeared to increase. The frost-resistance of tea plant closely related with air temperature, and soluble sugar, the contents of proline and free amino acids in tea leaves closely related with the frost-resistance of tea plants. During the two winter seasons, compared with ‘Zhongcha 102’, when suffered from the low temperature stress in winter, the content of each of three kinds of osmoregulation substances of ‘pingyangtezao’ more sensitively response to low temperature than ‘Zhongcha 102’, and the contents of the three kinds of substances in cultivar ‘pingyangtezao’ were slightly high. 【Conclusion】 Tea plants are vulnerable to frost, freeze and the late spring coldness from late autumn to Qingming Festival. As important cold-resisting indices of tea plants, the contents of soluble sugars, proline and the free amino acids varied sensitively in responses to the low temperatures, and showed significant correlations with the 10-day average air temperatures during the winter and early spring seasons. The responsiveness of cultivar ‘Pingyangtezao’ to the low temperatures was greater than that of cultivar ‘Zhongcha 102’.
    STORAGE·FRESH-KEEPING·PROCESSING
    Separation and Purification of Polyphenols in Rice Bran by Macroporous Resins
    TONG Xin, ZHANG Rui-fen, DENG Yuan-yuan, XIAO Juan, LIU Lei, ZHANG Yan, WEI Zhen-cheng, ZHANG Ming-wei
    Scientia Agricultura Sinica. 2016, 49(19):  3818-3830.  doi:10.3864/j.issn.0578-1752.2016.19.013
    Abstract ( 376 )   HTML ( 1 )   PDF (712KB) ( 525 )   Save
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    【Objective】The objective of this study is to screen a resin with a good adsorption and desorption performance to rice bran polyphenols (RBP) and optimize the purification technique, to compare the total phenols, total flavonoids content and antioxidant activity before and after purification, thus providing a theoretical basis for the deep processing and utilization of rice bran.【Method】The behavior of static adsorption and desorption, dynamic adsorption and desorption of 9 macroporous resins (HPD-100, HPD-200A, HPD-300, HPD-700, D101, HPD-722, AB-8, ADS17, and HPD-826) with different polarities to RBP was determined. Kinetic characteristics of static adsorption of the preferred resin were analyzed. The purification technique of macroporous resin to RBP was developed through the dynamic adsorption and desorption curves under different loading and elution conditions, including phenolic concentrations in loading samples, sampling rates, sampling volumes and ethanol concentrations in eluent. The phenolic profiles in RBP before and after resin purification were analyzed by HPLC analysis. Their total phenolic and flavonoid contents, oxygen radical absorption capacity (ORAC) and cellular antioxidant activity (CAA) were also estimated. 【Result】HPD-300 exhibited the best capability of adsorption and desorption to RBP among the 9 selected macroporous resins. The saturated absorption quantity of HPD-300 resin to RBP was 9.04 mg GAE·g-1, and the desorption rate was 82.62%. It took 6 h for HPD-300 resin to reach absorption equilibrium. The optimized purification parameters were as follows: the phenolic concentration of loading sample is 1 mg GAE·mL-1, sampling rate is 3.0 BV·h-1, sampling volume is 3.5 BV, eluent is 70% ethanol, flow velocity is 3.0 B V·h-1. After HPD-300 resin purification, the total phenolic content increased by 2.6 times from 88.07 µg GAE·mg-1 to 320.72 µg GAE·mg-1, and the total flavonoids content increased by 3.4 times from 30.04 µg CE·mg-1 to 133.67 µg CE·mg-1. Ten phenolic compounds were identified from rice bran phenolic extract, which were gallic acid, protocatechuic acid, vanillic acid, p-hydroxy benzoic acid, caffeic acid, clove acid, epicatechin, catechins, vanillic aldehyde, p-coumaric acid and ferulic acid by HPLC analysis. The phenolic profiles of the extract were not changed after HPD-300 resin purification, but the concentrations of 10 phenolic compounds increased by 0.4-2.4 times. The ORAC and CAA antioxidant activity of the extract changed from 1 327.51 µmol TE·g-1 and 29.19 µmol QE·g-1 before purification to 3 248.21 µmol TE·g-1 and 95.24 µmol QE·g-1 after purification, and increased by 1.4 and 2.2 times, respectively.【Conclusion】The total phenolic and flavonoid contents and antioxidant activity increased by 1-3 times after HPD-300 resin purification. The individual phenolic compounds in the extract were not lost significantly during purification. Therefore, HPD-300 macroporous resin was suitable to purify rice bran phenolic extract.
    Effect of Fermentation on the Polysaccharides Processing Characteristics in Apple Pomace
    JIA Feng,GUO Yu-rong, LIU Dong, YANG Xi, DENG Hong, MENG Yong-hong
    Scientia Agricultura Sinica. 2016, 49(19):  3831-3844.  doi:10.3864/j.issn.0578-1752.2016.19.014
    Abstract ( 405 )   HTML ( 2 )   PDF (1192KB) ( 428 )   Save
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    【Objective】The output of apple in China accounts for over 50% of the world annually. Among those apples, there are about 20% of apple fruits are used as raw materials to produce deep-processed products. As a result, apple pomace of thousands of tons are produced each year, which has caused the tremendous waste of apple recourse because these pomace are generally sold as a cheap fertilizer or are discarded directly as junks. The difference of physical and rheological property, viscosity-average molecular weight, and basic structure of polysaccharides between raw pomace and fermented apple pomace was studied in order to provide innovative thoughts and a theoretical basis for the exploitation of apple polysaccharides.【Method】Apple pomace (AP), wine fermented apple pomace polysaccharides (WFP), vinegar fermented apple pomace polysaccharides (VFP) were used as experimental materials, the paper investigated overall the processing properties of polysaccharides in fermented apple pomace through comparing with the extract ratio, solubility, hygroscopicity, moisture retention, and emulsifiability, foaming capacity and stability among AP, WFP and VFP. Besides, the rheological properties, microstructure, viscosity-average molecular weight were determined by using rheometer, desktop scanning electron microscope (DSEM), ubbelohde viscometer, respectively.【Result】The extraction ratio of AP was 5.68%, while that of polysaccharide in fermented apple pomace was 6%-7%, both of which had a significant difference (P<0.05). The solubility of VFP and WFP were, respectively, 0.1405 g·mL-1 and 0.0771 g·mL-1, and significantly higher (P<0.05) than AP (0.0283 g·mL-1). Compared to AP, the stability of foaming, hygroscopicity, moistureretention of polysaccharides, fermented apple pomace were significantly increased (P<0.05), while oil absorption, emulsifiability and its stability obviously decreased (P<0.05). According to analysis on rheological property, AP, WFP, VFP could be attributed to typical non-Newtonian fluid, and the apparent viscosity showed obvious dependent-characteristic of concentration. Besides, VFP and WFP had great resistance against temperature change. The findings of analysis of viscosity-average molecular weight and DESM indicated that the viscosity-average molecular weight of AP, WFP, VFP all distributed in the range of 15-130 kDa, and AP>WFP>VFP. Additionally, the microstructure analysis showed that AP consisted of band, bar and flake, twining with each other and forming firm network. WFP contained dentation, bar and flake, so its cross-linking was comparatively lower. But VFP mainly consisted of flake, overlapping with one another, and cross-linking was the lowest.【Conclusion】WFP, VFP were greatly improved in physicaland rheological properties, microstructure and viscosity-average molecular weight, indicating the processing property of polysaccharides in fermented apple pomace was superior to AP.
    ANIMAL SCIENCE·VETERINARY SCIENCERE·SOURCE INSECT
    Genetic Analysis of Huoyan Trait Based on F2 Resource Population in Huoyan Goose
    YU Jin-cheng, LI Zhe, YU Ning, ZHAO Hui
    Scientia Agricultura Sinica. 2016, 49(19):  3845-3851.  doi:10.3864/j.issn.0578-1752.2016.19.015
    Abstract ( 575 )   HTML ( 1 )   PDF (374KB) ( 480 )   Save
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    【Objective】The Huoyan breed of geese which possesses a good laying performance, is a precious native poultry resource of China. As the breed characteristics of Huoyan goose, they need to be studied about the inheritance patterns of Huoyan trait. The purpose of this study was, by establishing the goose-F2 resource group with Huoyan trait records, combined with the phenotypic analysis, to test the hypothesis that the allele that determines Huoyan trait demonstrates recessive inheritance, so as to understand the genetic mechanism of Huoyan trait. Our findings of the study will lay a theoretical basis for utilization of Huoyan goose genetic resource.【Method】Twenty males and 100 females of Huoyan geese were chosen to generate the F1 generation of Huoyan goose pure line by random mating, and Huoyan appearance and segregation ratio in F1 were observed. By F2-design of outbreed populations, the resource population with Huoyan trait records was established, in which the 3 males and 15 females of Huoyan geese were crossed reciprocally with the 3 males and 15 females of Sichuan White geese, and inter se matings in F1 were carried out to generate the F2 population without inbreeding. Huoyan appearance and segregation ratio in F1 and F2 were observed.【Result】① Huoyan and normal eyelid phenotype in F1 generation of Huoyan goose pure line by random mating was 89% (n=444) and 11% (n=444), respectively. The real ratio of Huoyan vs normal eyelid phenotype of males was 7﹕1(n=238), which was no significant with the theoretic value (χ2=2.09<χ20.05(1)=3.84), and the real ratio of Huoyan vs normal eyelid phenotype of females was 10﹕1(n=206), which was no significant with the theoretic value (χ2=0.06<χ20.05(1)=3.84). It indicated that the hypothesis of the allele that determines Huoyan trait demonstrates recessive inheritance is correct, and that Huoyan trait may be determined by two loci, one influences the formation of eyelid, and another modifies the penetrance of the Huoyan phenotype. ②All of the males and females in reciprocal cross F1 populations of female Huoyan goose crossed with Sichuan White goose ganders were normal eyelid. It indicated that the Huoyan trait exhibits recessive heredity to normal eyelid trait. ③ In F1 populations of Huoyan goose gander crossed with female Sichuan White goose, 100% (n=71) of the males were normal eyelid, as well as the females with Huoyan and with normal eyelid phenotype were 83% (n=76), 17% (n=76), respectively. The real ratio of Huoyan vs normal eyelid phenotype of females was 5﹕1(n=76), which was of no significance with the theoretic value by the χ2 test (χ2=3.51<χ20.05(1)=3.84). It indicated that the Huoyan trait demonstrates sex-linked inheritance. ④ In F2 populations of Huoyan goose ganders crossed with female Sichuan White goose, the real ratio of Huoyan vs normal eyelid phenotype of males and females were 5﹕8 (n=102), 2﹕3 (n=94), respectively, by the χ2 test, and they were of no significance with the theoretic value (χ2=0.36, 0.02<χ20.05(1)=3.84). Meanwhile, in F2 populations of reciprocal cross, the real ratio of Huoyan vs normal eyelid phenotype of males and females were 0﹕1(n=61), 5﹕7(n=60), respectively, by the χ2 test, they were of no significance with the theoretic value (χ2=0.02<χ20.05(1)=3.84). The separation results of Huoyan phenotype in F2 populations reciprocally further proved that the Huoyan trait demonstrates recessive sex-linked inheritance【Conclusion】The Huoyan trait exhibits recessive heredity to normal eyelid trait, and demonstrates sex-linked inheritance. The Huoyan trait is determined by two loci, one gene is on the Z chromosome, and another modifier gene exists on the autosome.
    Cloning and Expression Analysis of Odorant Binding Protein Gene AcerOBP14 from Apis cerana cerana
    DU Ya-li, ZHANG Zhong-yin, PAN Jian-fang, WANG Shu-jie, YANG Shuang, ZHAO Hui-ting, JIANG Yu-suo
    Scientia Agricultura Sinica. 2016, 49(19):  3852-3862.  doi:10.3864/j.issn.0578-1752.2016.19.016
    Abstract ( 471 )   HTML ( 12 )   PDF (3259KB) ( 874 )   Save
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    【Objective】The objective of this study is to clone the cDNA sequence of AcerOBP14 from Apis cerana cerana, predict the protein structure, explore its expression profiles in different tissues and at different developmental stages of A. c. cerana, and to provide a fundamental evidence for the future study of the physiological function of this gene.【Method】 The full-length cDNA sequence of AcerOBP14 was cloned from the entire tissues of A. c. cerana by RT-PCR and submitted to the GenBank database. Physiochemical properties and structure characteristics of the deduced amino acid sequence were analyzed by multiple bioinformatics methods. Phylogenetic tree between AcerOBP14 and its homologous OBPs from Hymenoptera insects was constructed using neighbor-joining of MEGA5.2. The expression levels of AcerOBP14 mRNA in different tissues (antenna, head, thorax, abdomen, legs and wings) at 1, 5, 10, 15, 20, 25 and 30 days were detected by real-time PCR.【Result】The full-length cDNA sequence of AcerOBP14 was obtained (GenBank accession number is KT24648). The open reading frame (ORF) of AcerOBP14 is 408 bp, encoding a protein of 135 aa with an estimated molecular weight of 15.08 kD and pI of 5.44. The encoding protein has a signal peptide and no transmembrance structure, possesses a PBP-GOBP superfamily domain between 18-127 residues, exists multiple obviously hydrophobic regions binding liposoluble odorant and contains four conserved cysteins, suggesting that it belongs to the Minus-C OBP subfamily. Subcellular localization results showed that AcerOBP14 was located on the secretory pathway about endoplasmic reticulum, golgi and plasmalemma which belongs to the secretory protein. AcerOBP14 possesses 7 α-helixs, among which the α1-α6 belong to the core helixs of classical OBPs and α7 is exposed to the surface of protein at the C-terminus. The results of amino acid sequence alignment showed that AcerOBP14 was homologous to AmelOBP14 with 86% amino acid sequence identity, and then had 55% sequence identity with AdorGOBP56a-like. Phylogenetic analysis revealed that AcerOBP14, AmelOBP14, AdorGOBP56a-like, AcerOBP21 and AfloGOBP56d-like belonging to the Apis were clustered into one group, MrotGOBP56a-like (Meqachile), MdemGOBP83a-like (Microplitis), OcorOBP3 (Osmia), BterGOBP56d-like (Bombus) and BimpGOBP56d-like (Bombus) were clustered into the other group. Besides, the results of real-time PCR showed that the AcerOBP14 transcripts were differentially expressed in the antenna, head, thorax, abdomen, legs and wings of the adult workers. The thorax of 1-day-old workers had the highest expression level of AcerOBP14, the antenna had the second highest expression level, and both of them were significantly higher than the head, abdomen, legs and wings (P<0.01). Moreover, the expression profiles in worker bees at other days were significantly higher in antenna than that in other tissues (P<0.01), of which the antenna of 20-day-old workers had the highest expression level. According to the expression profiles of AcerOBP14 at different developmental stages, the antenna of workers had the lowest expression level at 1-day-old which was significantly lower than other days (P<0.01), increased gradually at 5-day-old and 10-day-old, then decreased suddenly at 15-day-old. The expression level at 20-day-old was significantly higher than other days (P<0.01), afterwards, the expression level gradually reduced. Generally, the expression profiles of the head, thorax, abdomen, legs and wings appeared a downward trend with the days of age increasing, and maintained a lower level after decreasing remarkably at 5-day-old.【Conclusion】AcerOBP14 belongs to the Minus-C OBP subfamily with a PBP-GOBP superfamily domain. The results of expression profiling suggested that AcerOBP14 belongs to general odorant binding protein (GOBP) and is involved in not only the olfactory recognition, but also other physiological functions.