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    20 March 2018, Volume 34 Issue 3
    Advances on Key Gene DXS Involved in the Terpenoid Biosynthesis in Plants
    ZHANG Hao-yu, FAN Jun-miao, WANG Ting, HAN Yuan-huai, DU Fang
    2018, 34(3):  1-8.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0779
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    Terpenoids are a class of natural compounds widely present in nature and have important values in plants,animals and human beings. 1-deoxy-D-xylulose-5-phosphate synthase(DXS)is the first and a rate-limiting enzyme in 2-C-methyl-D-erythritol 4-phosohate(MEP)pathway,and plays a key role in the regulation of terpenoids biosynthesis. It is located in the thylakoids and has a plastid transit peptide sequence and three functional domains:a TPP binding domain,transketolase domain and pyrimidine binding domain. The DXS enzyme activity can be measured by high performance liquid chromatography. At present,DXS genes have been cloned and isolated from 178 plants and can be sorted into three classes based on the homology of protein sequences:DXS1,DXS2,and DXS3. The expression patterns of DXS genes were demonstrated by tissue specificity,circadian rhythm and related to developmental stages of flowers and fruits as well as varieties. Overexpression or inhibition of DXS genes in transgenic plants with exogenous or endogenous DXS genes can influence the variations of photosynthetic pigments(such as chlorophylls and carotenoids),endogenous hormones(such as abscisic acid and gibberellin)and other terpenoids. In this paper,characteristics of DXS protein,the types of DXS gene,and their expressions,regulations and genetic transformations were reviewed prominently in order to provide theoretical and practical references for plant breeding and metabolism regulation.

    Research Advances on Plant Thaumatin-like Protein Family
    LIU Cha, HAN Li-hong, WANG Hai-bo, GAO Yong, TANG Li-zhou
    2018, 34(3):  9-17.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0774
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    Thaumatin-like proteins(TLPs)play an important role in the process of plant resistance to stress. TLP family belongs to multi-gene one,and its molecular weight is small,and it has typical thaumatin domain of highly conserved. Typical TLP is composed of 16 cysteine residues forming eight disulfide bonds,and the three-dimensional structure possesses three conserved domains I,II,and III,forming a “V” shape acidic cleft on the surface,which ensure the catalytic function of TLP. Most species’ TLPs are classified into 10 clusters,and unbalanced amplification occurs in each cluster. TLPs possess antifungal,glucanase,and allergens activities,and can be induced by biotic or abiotic factors,such as hormones,stress signal,thus they play a role in the life process and plant innate immunity. Growingly TLP family genes with antifungal potential have been used to improve plant disease resistance through genetic engineering. In this study,we summarized recent researches on the structure,evolution,biological function and application of TLPs,so as to provide a theoretical basis for further study.
    Research Advances on the Gene for Gibberellin 3-Beta-Dioxygenase in Higher Plants
    YANG Yi-hong, XU Hao, CHEN Duan-fen, GAO Zhi-min
    2018, 34(3):  18-22.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0751
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    As one of key enzymes involved in gibberellin synthesis,gibberellin 3-beta-dioxygenase(GA3ox)is directly acting on the formation of active GAs,and plays a vital role in the growth and development of plants. In this paper,we reviewed the advances on the cloning and expression pattern of GA3ox gene,as well as the functional effects in the study of higher plants. GA3ox gene has been cloned from Arabidopsis,tobacco,sorghum,poplar and other plants. In the different growth stages of plants and in different tissues,the expressions of GA3ox gene varied,which mainly resulted from the genetic factors of a species,and meanwhile,were affected by the environmental factors and exogenous hormones. Studies on mutants of GA3ox revealed that the gene mutations hindered the GA biosynthesis and thus indirectly affected the growth of plant,resulted in the phenotype changes of plant stalk,flowers,seeds,fruit and roots,including plant dwarf and seeding difficulties. Overexpression of GA3ox promoted the production of GA,such as the induction of cell division and differentiation,the radial elongation of root tip,and seed germination. Here studying the relevant functions of GA3ox in different plants and discussing its role in plant growth and development is aimed to provide reference for understanding the regulatory mechanisms of GA3ox on plant growth and development,and to better use the GA3ox in genetic engineering research for directly breeding of new plant varieties in practice.
    The Effects of By-products of Hydrolyzing Lignocellulose on Ethanol Fermentation and Relevant Countermeasures
    LIN Bei, LI Jian-Xiu, LIU Xue-ling
    2018, 34(3):  23-30.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0657
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    The pretreatment of raw materials is necessary in ethanol production from lignocellulose,in which some compounds of inhibiting microbial growth and subsequent fermentation are generated. The inhibitors are usually classified into three major groups:furan aldehydes,phenolics and weak acids. In recent years,significant progress has been earned in the research of inhibitory compounds. Firstly,we introduced the generation and the inhibition mechanisms of the inhibitors,focusing on the researches in recent years. Then,we discussed the countermeasures to inhibitors,including using new pretreatment methods to eliminate the generation of inhibitors,using effective detoxification of the pretreated substrates to reduce the concentration of inhibitors before fermentation,breeding of the inhibitor-tolerant strains and also fermentation control to reduce the toxic effects of the inhibitor. At present,a large number of studies focus on the development of inhibitor-tolerant strains,we summarized these methods of mutagenic breeding,adaption and metabolic engineering,as well as the research progresses and achievements of jointly using these methods. We pointed out that the metabolic engineering of microorganisms is the most promising approach for overcoming the effects of inhibitors on ethanol fermentation,and prospected the future research direction,aiming at providing references for the researchers in this field.
    Research Progress on the Testing Technologies for Composition in Genetically Modified Products
    WANG Hao-qian, CHEN Rui, LI Xia-ying, WANG Meng-yu, LIU Peng-cheng
    2018, 34(3):  31-38.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0814
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    With the large-scale commercialized planting of GM(Genetically Modified)crops and the constant development of GM technologies in recent years,GM products with disease resistance,insect resistance,herbicide tolerance,stress resistant,high yield and fine quality have growingly increased and also widely been applied. GM products brought convenience and economic benefits to people while also brought security issues. To strengthen the management of GM products,the development of testing technologies for GMOs were especially critical. At present,the testing technologies for GMOs and their derived products are mainly classified into two categories:one is based on exogenous nucleic acid,mainly including conventional PCR,quantitative PCR,isothermal amplification technology,gene chip technology and so on;the other is based on exogenous protein,mainly including ELISA,Western blot,test strip technology and so on. Each testing technology has its advantages and disadvantages and the scope of application,in real testing process we should select the most effective technology or combination of them for meeting the purpose of testing,while based on the requirements of testing as well as the types and characteristics of GM products. In this paper,we briefly reviewed the principles,advantages and disadvantages and research progress of each testing technology,meanwhile summarized and prospected the development direction of the testing technologies for GMOs,aiming at promoting testing technologies for GMOs in China to better adapt to the current development of transgenic area.
    Research Progress on Omics Technologies in High-amino-acid Tea Plants(Camellia sinensis)
    MA Lin-long, CAO Dan, LIU Yan-li, JIN Xiao-fang
    2018, 34(3):  39-42.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0901
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    Green tea made from high-amino-acid tea cultivars has favorable economic and social benefits in the market due to its rich aroma,umami flavor,velvety taste,and integrated benefits to both human health and ornamental value,thus studies on it have become the hot spot in the tea science. With the development of life sciences into multi-omics era,transcriptomics,proteomics,and metabolomics have been developing rapidly,and are widely being used in various fields of tea science. Consequently,the multi-omics application becomes an indispensable part of studying high-amino-acid tea cultivars. This review covers recent progress of transcriptomics,proteomics,and metabolomics application on high-amino-acid tea cultivars,and also evaluates shortcomings and urgently-to-be-solved difficult problems while applying omics technologies in the study of high-amino-acid tea cultivars. Moreover,this paper briefly discusses the future study perspectives and application of omics technologies in high-amino-acid tea cultivars,aiming at providing a reference and direction for the further study of high-amino-acid tea cultivars,and also providing theoretical basis for new high-amino-acid tea cultivars breeding and efficient cultivation.
    Application of Metagenomics in Prevention and Control of Infectious Diseases
    LI Pei-han, LI Peng, SONG Hong-bin
    2018, 34(3):  43-52.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0787
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    The outbreak of new infectious diseases poses a serious challenge to global public health prevention and control.Rapid identification of pathogens is the primary challenge in dealing with outbreaks emerging infectious diseases. It is difficult to detect unknown pathogens or pathogens with large mutations using traditional pathogen detection methods. Metagenomics based on high-throughput sequencing has shed light on the pathogen detection and identification. Nucleic acid extraction,high-throughput sequencing,data analysis and other key technologies continue to develop,making metagenomics become an important research direction for the prevention and control of new infectious diseases. Metagenomics can direct sequence many samples of the prevention and control of infectious diseases to obtain high-throughput sequencing data. It can combine the pathogenic nucleic acid database,through sequence alignment,mutation evolution analysis and other bioinformatics methods in to use. In this way,we can monitor the suspicious samples of the epidemic outbreak forecast and warning,identify pathogens and provide medication guidance,determine the phylogenetic relationship,trace the origin of the pathogen and investigate the epidemic. Ultimately,we can realize the target of rapid identification,typing,drug resistance and traceability analysis of the new outbreak of infectious diseases. As a new technology,metagenomics has great potential and development prospects in the field of prevention and control of infectious diseases. This article reviews the application of metagenomics in the pathogen detection,detection and traceability of infectious diseases in order to provide a new perspective for the prevention and control of infectious diseases.
    Estimation of the Copy Number of Exogenous Genes in Genetically Modified Rice by Droplet Digital PCR
    WANG Yong, LAN Qing-kuo, ZHAO Xin, CHEN Rui, SHEN Xiao-ling, LI Wen, ZHANG Yao-zhong, LI Liang, WANG Qin-ying
    2018, 34(3):  53-58.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0756
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    Copy number of inserted exogenous gene is an important index of assessing the safety of genetically modified organisms. However,there are issues such as poor maneuverability in previous methods for identifying the copy number of gene,therefore,a simple-operation and accurate identification method is urgently needed. This study testes the feasibility of this method while droplet digital PCR was applied to identify the copy number of target gene in transgenic rice. The experimental results showed that the Cry1Ab/PLD ratio of four samples was 674/662,516/541,737/759 and 516/541,respectively,with 0.97 in average. In other words,the copy number of target gene was 1 by droplet digital PCR,which was consistent with the results from Southern blotting. Conclusively,as a novel method to identify the copy number of inserted exogenous gene,droplet digital PCR has some advantages such as simple operation,high accuracy,and flexible design.
    The Translational Regulation of Gene Expression in Gluconobacter oxydans by Small RNAs
    LI Zhen-zhen, ZHAO Jia-chen, MA Yu-shu
    2018, 34(3):  59-66.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0837
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    This study is aimed to establish an effective tool to control gene expression in Gluconobacter oxydans. Cis- or trans-acting non-coding small RNAs,complementary to ribosomal binding site(RBS),were designed and constructed to modulate gene expression in G. oxydans at translational level. Using green fluorescent protein as reporter gene,in a cis-acting configuration,a cis repressor sequence inserted into the RBS upstream bound with RBS to form a stem-loop structure in mRNA,which repressed the reporter gene expression. Such a stem-loop was disrupted by an anti-repressor sequence that had higher affinity than a cis repressor sequence,thus this allowed the reporter gene to re-initiate translation. In a trans-acting configuration,the complementary double strands of small non-coding RNA transcribed from a separate promoter and a RBS were able to specifically repress the reporter gene expression by blocking the binding of ribosome and mRNA. Based on the above results,a series of trans-acting small RNAs were designed and imported,by which the endogenous gene pstI expression inhibition in G. oxydans was realized. In summary,this study offers an alternative approach to study gene functions and regulate gene expression in G. oxydans other than gene knockout.
    Comparison and Optimization of the Detection Method for Pseudomonas aeruginosa
    ZHANG Fan, LI Shu-yao, ZHANG Zi-hao, CAI Xue-feng, REN Yan, LIU Kai, HOU Cui-yan, YI Xin-xin, GAO Xiu-zhi
    2018, 34(3):  67-74.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0742
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    This work aimed to obtain a simple-operation,more accurate method and requiring low test condition by analyzing and comparing 3 Pseudomonas aeruginosa detection methods. The identification results of VITEK 2 Compact automatic bacterial identification system were used as reference,the test methods of P. aeruginosa in GB 8538.57-2016 and GB/T7918.4-1987 were applied to identify 27 strains. The results showed that the accuracy of the GB 8538.57-2016 method was the lowest,and the false positive rate was 29.6%,while the false positive rate of GB/T7918.4-1987 was 7.4%. In the case of GB 8538.57-2016 P. aeruginosa as an example,three steps were added to the optimized method,the blood agar plate culture,Gram stain and microscopy,and pyocyanin test for all suspicious colonies of the blood agar plate. The 27 test strains were verified by the optimized detection method,and the results were consistent with those of VITEK 2 Compact.
    Quantitative Detection of Microcystin-LR by Fluorescent Nanosphere Immunochromatographic Assay
    ZHANG Yi, DING Xin-liang, ZHANG Jue, ZHOU Bin, GUO Ming-ming, HUANG Biao,
    2018, 34(3):  75-79.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0873
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    This study was designed to develop a rapid quantitative detection strip for MC-LR(microcystin-LR)in water samples using the fluorescent nanosphere immunochromatograhic technology. Based on competitive mode,an immunochromatograhic strip system was developed for detecting MC-LR,and the reagent performance was evaluated for its sensitivity,accuracy,specificity and stability. Several parameters for preparing immunochromatograhic strip,i.e.,the concentration of artificial antigen on the test line,labeling rate of nanosphere and antibody,the concentration of nanospheres on the conjugate pad,were optimized. After the sample added for 15 min,the immunochromatographic assay was finished with a linear working range,0.195 μg/L of sensitivity,99.4% of recovery,below 10% of variable coefficient,little cross reaction with MC-LF,well consistency with commercial kits,and solid stability for 6 days at 37℃. In summary,this fluorescent nanosphere immunochromatographic strip is sensitive,stable,accurate,rapid,quantitative,and easy performed for MC-LR detection.
    Betaine Improves the PCR Amplification of Rice GC-rich DNA Sequence
    WANG Tong-tong, ZHANG Li-ping, YIN Kang-quan
    2018, 34(3):  80-86.  doi:10.13560/j.cnki.biotech.bull.1985.2017-1105
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    As a powerful tool for the amplification of DNA sequences,polymerase chain reaction(PCR)has been commonly used in molecular biology. Rice is one of the most important crops and has been widely studied as a model plant. However,the rice genome is rich in GC content,and the amplification of sequence with high GC content by standard PCR procedures is rather tough,which hinders the functional characterization of rice genome sequences with high GC content. To overcome this problem,several additives have been described to improve amplifications. Here after testing different additives,we found that betaine most significantly improved the amplification of rice GC-rich DNA sequences,compared to dimethyl sulfoxide and glycerol. Then we carried out PCR amplifications of multiple GC-rich rice DNA segments,and we found that the 1 mol/L to 2 mol/L betaine demonstrated remarkable effect in enhancing PCR amplification. In addition,betaine was effective in improving PCR amplifications at multiple DNA polymerases.
    Effects of Exogenous Methyl Jasmonate on the Tolerance of Wheat Seedlings to Low Temperature
    LI Yang-yang, JIAO Zhen
    2018, 34(3):  87-92.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0780
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    The effects of exogenous methyl jasmonate on the physiological and biochemical characteristics and the expression of cold-resistant transcription factor in wheat seedlings under cold acclimation were studied with cultivar “Aikang 58”. The test wheat seedlings were set in two groups:the controlled group was sprayed with double distilled water at low temperature of 4℃,and the treated group was sprayed with 100 μmol/L of methyl jasmonate at 4℃ for a week. The physiological and biochemical results showed that the survival rate of wheat seedling treated with 100 μmol/L of exogenous methyl jasmonate was 91%,significantly increased than that of the controlled group. The SOD、CAT and POD activity of the treated group increased 34%、 50% and 14% than that of the controlled group,respectively,the content of soluble proteins increased 30%,and intracellular free proline content increased 14%,while MDA content decreased by 5%. There was a significant difference between the treated group and the controlled group. The quantitative real-time PCR showed that 100 μmol/L of exogenous methyl jasmonate significantly increased expression of cold responsive transcription factors TaCBFⅣd-B4,TaCBFⅢd-B19,TaCBFⅢc-D3,and TaCBFⅢd-D19,and the expression of TaCBFⅣd-B4 was the highest. The results indicated that spraying 100 μmol/L exogenous methyl jasmonate enhanced the tolerance of wheat seedlings to low temperature stress.
    Effect of Simulated Short-day on Spike Development of Spring and Summer Sown Oat by Shading
    YANG Xiao-hong, AN Jiang-hong, HAN Bing, ZHOU Hai-tao, YANG Cai
    2018, 34(3):  93-97.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0741
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    In order to study the effect of short-day on the spike development of spring and summer sown oat in the northern China,for which five photoperiod-sensitive oat cultivars and one photoperiod-insensitive oat cultivar strain were planted,and the growth and development of the six oat cultivars planted in natural short-day in Hainan Base were compared. Results showed that illumination time decreased from 15 h to 11 h under shading condition,the spike differentiation process of photoperiod-sensitive oat cultivars was abnormal,and in morphology the spike development degraded seriously,thus their development was not complete. The photoperiod-insensitive oat cultivars’ spikes differentiated normally,and the whole growth and development completed. The experiments of the natural short-day in Hainan indicated that photoperiod-insensitive oat cultivars of spring and summer sown oats under natural short-day eared and seeded,and their whole growth and development completed;while the photoperiod-sensitive cultivars did not ear during growth period,and there was only vegetative growth but no reproductive growth. In addition,there was no difference in the morphologies by scanning electron microscope of different naked oat cultivars in normal spike differentiation. Under the short day of 11 hours per day,the photoperiod-sensitive oat cultivars only grew in vegetative growth stage,even those in the shortest growth periods did not ear and flower,thus resulted in no yield. There was no correlation between growth period and photoperiod-sensitivity of oat varieties.
    Transcriptome Sequencing Analysis of Sweet Sorghum Leaves in the Critical Period of Sucrose Accumulation
    CHEN Si-bo, ZHAO Zhi-han, MA Ai-jun, ZHANG Ao, WANG Zhi-he, RUAN Yan-ye
    2018, 34(3):  98-104.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0876
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    The aim of this study is to find the genes that differentially express between sweet sorghum and common sorghum in the critical period of sweet sorghum sugar accumulation through transcriptome sequencing. Functional analysis of related genes and associated Pathway metabolic pathways were used to further screen the genes related to sugar accumulation and metabolism,which is for laying a solid foundation in fully exploring the biological potential of sweet sorghum and improving sorghum varieties to increase the sugar contents of their stems. In this study,sweet sorghum Liaotian 1 and grain sorghum Xinsu 2 were used as research materials,and their leaf transcriptome data were sequenced and compared at maturity. The 71.98 M reads and 14.54 G total bases were obtained by sequencing. The Cufflinks software was used to assemble the reads,and 1562 new genes were discovered compared with known genetic model. Through differentiated expression,3 115 expressed differential genes were screened,of which 1499 were up-regulated and 1616 were down-regulated. Through GO,COG classification and KEGG pathway enrichment analysis,all of these differentially expressed genes were classified into 94 metabolism pathways covering starch and sucrose metabolism. The five differential genes of β-fructofuranosidase,sucrose synthase and fructokinase from the starch and sucrose metabolic pathway(Pathway ID:Ko00500)were respectively Sb06g031910,Sb06g023760,Sb01g035890,Sb01g033060,and Sb10g0082805 for real-time fluorescence quantitative analysis. The results showed that the expression of these genes at different growth stages was closely related to the activity of their corresponding enzymes,inferring that these genes play a crucial role in the synthesis and accumulation of sucrose in sweet sorghum.
    Evolution Analysis of P5CR and Expression Analysis of P5CR Genes in Cassava
    DING Ze-hong, FU Li-li, YAN Yan, TIE Wei-wei, HU Wei
    2018, 34(3):  105-112.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0796
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    Pyrroline-5-carboxylate reductase(P5CR),which catalyzes the final reaction of proline biosynthesis pathway,plays an important role in plant stress response. Totally 54 P5CR sequences were identified from 45 genome sequence available species(including cassava)by bioinformatic method,and their distribution and sequence characteristics were analyzed;neighbor-joining phylogenetic trees was constructed by MEGA software;and the expression levels of MeP5CR were evaluated in different tissues and in response to abiotic stresses by quantitative real-time PCR(qRT-PCR)method. The results showed that P5CR had a large difference in intron length,but there was no significant difference in amino acid length,exon number,isoelectric point,and molecular weight. There is only one copy of P5CR in most plants,such as in cassava,indicating that P5CR is evolutionarily conserved and originated from a single gene. However,there are 2-3 copies P5CR genes in some plants such as soybean and apple,indicating that P5CR genes experienced several duplication events during the evolution processes,and totally three different evolutionary patterns were revealed. Expression analysis revealed that MeP5CR expressed the highest in leaf,fibrous root,and storage root,but expressed the lowest in stem and petiole. MeP5CR was significantly induced by cold stress but greatly inhibited by drought stress. These findings suggest that MeP5CR is involved in drought and cold stresses at the transcriptional level,and it can be treated as a candidate to further study its functions in abiotic stress in cassava.
    Cloning and Expression Analysis of PwNAC42 in Picea wilsonii
    YUAN Yi-hang, ZHANG He-hua, YOU Han-li, ZHANG Ling-yun
    2018, 34(3):  113-120.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0924
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    As one of the largest plant transcription factors,NAC is widely involved in growth and development,as well as the responses of plants to abiotic stresses. In this study,PwNAC42 cDNA was obtained by Picea wilsonii transcriptome data,its protein sequence was analyzed by bioinformatics,and the expression of PwNAC42 was detected by real-time qPCR. The results showed that the full-length of PwNAC42 cDNA was 1749 bp,including 1140 bp of ORF that encoded 379 amino acids. The molecular weight of PwNAC42 was 42.92 kD and the pI value was 6.53. PwNAC42 had phosphorylation sites of serine,threonine and tyrosine. It was a hydrophilic protein that had no signal peptide domain and transmembrane domain. RT-qPCR results showed that PwNAC42 mainly expressed in cones. At the same time,the expression of PwNAC42 under ABA,NaCl,drought and low temperature treatment changed significantly. Therefore,P. wilsonii PwNAC42 may play a role in cone development and response to abiotic stress.
    Transcriptome Sequencing and Analysis of Okra Fruit
    LI He-ping, YAO Yun-fa, LIAN Dong-mei, LAI Zheng-feng, HONG Jian-ji
    2018, 34(3):  121-127.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0765
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    In order to study the expressions of functional genes in the transcriptome of okra fruit,RNA-Seq technology was used to sequence 3 samples of okra fruit. Sequencing results showed that three samples received a total of 77 476 Unigene sequences after assembly,and there were annotations in 4 databases for 61 891 Unigene sequences,accounted for 79.88%. Based on KEGG metabolic pathway database,13 336 Unigene sequences of okra fruit may be classified into 128 metabolic pathways. And 3830 SSR(Simple Sequence Repeats)loci were discovered in okra fruit transcriptome,distributed in 3569 Unigenes,accounting for 4.61% of the transcriptome sequences.
    Preliminary Study on Inducing Axillary Buds of Different Pinus serotina Explants
    YANG Meng-li, GAO Xi-xi, LIU Yuan-qiu, HU Dong-nan, LI Jia-jun, ZHANG Zhi-jian, ZOU Gui-wu, HUANG Guo-xian
    2018, 34(3):  128-135.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0409
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    The varied segments from field-growing 1-year old Pinus serotina spring stem and shoot tips from seed germination were used as explants. The explants were disinfected by 0.1% HgCl2,and those with favorable disinfection were used as sterile materials,and then the combination of medium type and the hormones for the axillary bud induction were optimized. The results showed that 10 min disinfection of middle parts of the spring stems by 0.1% HgCl2 was the optimal,the pollution rate was the lowest(0.8),and the best combination of medium and hormone levels to induce axillary buds was MS medium with 2.5 mg/L 6-BA and 0.2 mg/L NAA. And the optimal disinfected time of seed was 4 min,the pollution rate was 2.4%,and the induction rate was the highest in the medium containing 0.7 mg/L NAA and 1.0 mg/L 6-BA. In summary,an effective method to induce the axillary buds from the spring stem and seeds is established,which may provide theoretical evidences and technical support for the rapid and efficient breeding of Pinus serotina.
    Construction of the pBB-GFP Vector for Labeling Plant Pathogenic Bacteria
    ZHAO Zhi-wen, LI Yan-jiao, HU Xun, FAN Xiao-jing, ZHUO Tao, ZOU Hua-song
    2018, 34(3):  136-141.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0807
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    The promoter region of Ralstonia solanacearum RipAK gene was PCR amplified,and fused with lacZ reporter gene to generate pHM1:PRiPAKLacZ. β-galactosidase(LacZ)activity was successfully detected from R. solanacearum carrying pHM1:PRiPAKLacZ cultured either in nutrient rich or minimal medium,suggesting that RipAK promoter effectively drove lacZ gene expression. To construct a vector for labeling plant pathogenic bacteria,the RipAK promoter and gfp gene were cloned into plasmid pBBR1MCS-5,which made the expression of gfp gene could be driven by the RipAK promoter. The constructed pBB-GFP was able to express green fluorescent protein GFP in Escherichia coli. Thus,R. solanacearum,Pseudomonas syringae pv. tomato,and Xanthomonas citri subsp. citri were effectively labeled using pBB-GFP. The 3 plant pathogenic bacteria under fluorescence microscopy were short-rod shaped,and occasionally R. solanacearum formed a linear structure in which multiple cells were connected in series. Moreover,GFP labeling had no effect on bacterial pathogenicity on host plants. The labeled strains were inoculated on the wounds of host plant leaves,where a strong green fluorescence was observed at 24 hours post inoculation. In summary,the pBB-GFP vector constructed in this study may be used for labeling several plant pathogenic bacteria with green fluorescence,and the green fluorescence was visualized from labeled bacteria either cultured in medium or inoculated on host plants.
    Cloning of KISS-1 Gene in Yak and Its Expression in Reproductive Axis
    YANG Yuan-xiao, ZI Xiang-dong
    2018, 34(3):  142-149.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0968
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    KISS-1 plays a pivotal role in the regulation of animal reproduction,thus this study is aimed to investigate the role of KISS-1 gene in regulating seasonal reproduction of the yak. Samples of hypothalamus,pituitary etc. were collected from five adult female yaks and five yellow cattle. KISS-1 gene sequence and its tissue expression characteristics were studied by RT-pcr and qPCR techniques. The results showed that the coding region length of yak and yellow cattle KISS-1 gene was 408 bp,which encoded 135 amino acids,and there were 7 base mutations between these two species. The amino acid sequence identities of yak KISS-1 gene with yellow cattle,Tibetan goats,sheep,wild pig,human,and sewer rat were 97.1%,85%,65%,55%,and 51.5% respectively. Expression of KISS-1 gene was detected in hypothalamus,pituitary,ovary,fallopian tube,and uterus of yak and cattle. The expression levels in hypothalamus and pituitary were the greatest,but there was no significant difference between the two species(P > 0.05). It is concluded that KISS-1 gene is conservative in animal evolution,and its regulation roles in seasonal reproduction of the yak need to be further studied.
    Isolation of Peripheral Blood Lymphocytes of Sturgeon and the Optimal Proliferate Response Condition
    DONG Ying, HU Hong-xia, TIAN Zhao-hui, WANG Wei, DONG Tian
    2018, 34(3):  150-155.  doi:10.13560/j.cnki.biotech.bull.1985.2017-1000
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    In this study,sterlet(Acipenser ruthenus)was chosen as the model experimental material,different concentrations of lymphocyte separation solution were used to isolate the peripheral blood lymphocytes of sturgeon and study their optimal proliferate response condition. Phytohemagglutinin(PHA),concanavalin A(ConA)and lipopolysaccharide(LPS)were utilized as lymphocyte proliferation mitogen respectively. According to five factors and five levels orthogonal experimental design L25(56),the conditions of sturgeons peripheral blood lymphocytes proliferate response were optimized using Enhanced Cell Counting Kit-8(enhanced CCK-8 or WST-8). Five factors were selected to explore the optimal response condition,including culture time,culture temperature,cell concentration,fetal bovine serum(FBS)concentration,and mitogen concentration. The results showed that 70% Percoll(1.092 g/mL)used as the sturgeon lymphocyte separation solution had the best separation effect. The optimal conditions for proliferation were 3.625×106 initial cell concentration,20 μg/mL PHA or 50 μg/mL ConA or 10 μg/mL LPS,10% - 20% FBS,20-25℃,and culturing for 2 days.
    Analysis of the Genetic Composition in Saccharina japonica and Undaria pinnatifida Based on SSR Markers
    PENG Jie, CUI Cui-ju, LIANG Guang-jin, LI Yan, WU Rui-na, LIU Yan-ling, TIAN Ping-ping, LI Xiao-jie
    2018, 34(3):  156-162.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0785
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    Saccharina japonica and Undaria pinnatifida varieties with higher purity and better character that could be cultivated by the first generation screening of gametophyte and spore seedlings. Using 11 seedling combinations of S. japonica and u. pinnatifida and based on a number of primers that may distinguish male and female parents of each group,the genetic composition of their parents and progeny were analyzed. Thirteen pairs of SSR markers which were able to distinguish male and female parents of 11 combinations were selected in the experiment. Results showed that among the 2013 seedlings,all progenies were the offspring of their parents in No. 48 combination,but only one of progenies was the offspring of their parents in No. 49 combination. Parents were genetically identical to all progenies in No. 22 combination of 2014 seedlings. Among all hybridized combinations of S. japonica and u. pinnatifida in 2015 seedlings No.36,No.38,No.39,No.41 were hybridized successfully,while other combinations were not so. There were no biological relationships between seven of progenies and their parents in No. 59 combination of 2016 seedlings. Analyzing the genetic composition of all individuals and parents in these 11 combinations revealed that some individuals were progenies of their parents,but the genetic composition of some individuals were inconsistent with their parents.
    Isolation,Screening and Identification of Endophytic Bacteria from Isatis indigotica
    WANG Xia, XUE Lin-gui, ZHANG Xiao-hua, HE Xiao-yan, FAN Tao-tao, SHANG Hai,
    2018, 34(3):  163-169.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0855
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    This work is to screen and identify the endophytic bacteria from medicinal Isatis indigotica in the Hexi Corridor of Gansu Province against Fusarium moniliforme,Alternaria alternate,and Exserohilum turcicum. Endophytic bacteria were isolated from the roots,stems,leaves,petioles and flowers of I. indigotica using conventional separation method,from which the antagonistic bacteria were screened by the synchronous culture method,and the screened active endophytic bacteria were identified based on phenotypical and biochemical properties as well as its 16S rRNA gene sequence. As results,a total of 19 endophytes were isolated,and 10 strains of them had varied inhibition effects on F. moniliforme,accounting for the 52.6% of total isolates;the inhibition effect of strain G2 on F. moniliforme was the strongest with inhibition rates up to 94.63%. All 19 strains of the isolates had varied inhibition effects on A. alternata and E. turcicum,accounting for 100% of the total number of isolates. Among these strains,G2,J1,Y5 and B2 were selected for further study,and their inhibitory rates nearly reached 100%. Combined with the physiological and biochemical characteristics,colony characteristics,cell morphological characteristics and 16S rRNA sequencing results,the strains J1 and Y5 were identified as Microbacterium maritypicum,and the strain G2 and B2 as Kocuria rosea and Brachybacterium ginsengisoli,respectively. In summary,antagonistic bacteria on the plant pathogens were isolated from I. indigotica,especially the strain G2,J1,Y5 and B2 presented the strongest inhibitory effects,and thus their inhibitory activities will be worthwhile for further studies.
    Analysis of Antimicrobial Resistance and Resistance Genes of Salmonella from Swine
    ZHI Wei, MA Hai-yan, QIU Yong-feng, HU Su-juan, WEI Ling-ling, ZHUAi-hua
    2018, 34(3):  170-176.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0743
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    The aim of this study is to analyze the relationship between resistance genes of Salmonella in swine and antimicrobial resistance. The antimicrobial resistances of 60 Salmonella strains from swine to 10 drugs were determined by K-B method,and the resistance genes of Salmonella were detected by PCR. The results showed that the antimicrobial resistance of the isolated Salmonella to ciprofloxacin was the highest(98.3% of them were resistant),and about 75% of the strains were resistant to sulfamethoxazole trimethoprim and amikacin. They were more sensitive to gentamicin,tochloramphenicol,norfloxacin,and spectinomycin,and the rate was over 45%. The results of the resistance gene test demonstrated that the detection rates of 7 resistance genes tetA,parC,gyrA,sulI,sulII,floR,and aadA1 were 53.3%,98.3%,66.7%,46.7%,33.3%,48.3%,and 66.3%,respectively,while the resistance gene tetB,tetC,tetG,cat1 and Aaca(3)-Ia were not even detected. Furthermore,by cloning and analyzing the several common resistance genes in the successfully isolates,it is concluded that the resistance genes of Salmonella may determine the resistance phenotype of a bacterium.
    Biological Identification of Dgl5 in Deinococcus gobiensis I-0
    ZHANG Heng, LIU Ying-ying, CHEN Yun, PING Shu-zhen, WANG Jin
    2018, 34(3):  177-184.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0875
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    Proteins in LEA5C(Group 5C Late Embryogenesis Abundant)family could involve in the responses to abiotic stresses and protect bio-macromolecules in cells from damages under abiotic stresses, due to their functions as molecular chaperones. The Deinococcus gobiensis I-0 isolated from Xinjiang Gobi Desert possessed highly strong resistance to ionizing radiation,oxidation,and desiccation. The genomic annotation of D. gobiensis I-0 indicated that Dgo_CA1605 encoding protein belonged to LEA5C family,named as Dgl5. Accordingly,the function of Dgl5 was investigated in this article. Recombinant expression strain Dgl5 was constructed by homologous recombination. The abiotic stress results revealed that Dgl5 significantly enhanced high-salt resistance of expression strain BL21 and maximally protected the host from the damages in freeze-thaw cycles. Likewise,the physiological and biochemical experiments in vitro demonstrated that the enzymatic activities of malate dehydrogenase and lactate dehydrogenase were effectively maintained due to Dg15 under freeze-thaw cycles. Therefore,we speculated that the improved resistance of the host adapting to harsh environment such as frozen and high-salt was achieved by Dg15 protecting cellular enzyme activities.
    Extracellular Expression of Laccase Gene from Bacillus pumilus LC01 in Pichia pastoris and Characterization of the Recombinant Laccase
    LI Cheng-ming, WANG Jia-yi, LU Lei
    2018, 34(3):  185-193.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0839
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    To obtain high-yield bacterial laccase with fine stability and efficient decolorization ability,the laccase gene from Bacillus pumilus LC01 was amplified by PCR and cloned into the expression vector pPICZαA. The constructed vector pPICZαA-lac was transformed into Pichia pastoris SMD1168H. The positive P. pastoris strain was induced by methanol to produce the recombinant laccase. Then the recombinant laccase was purified and characterized. The highest laccase activity reached 1 390 U/L at 7 days after cultivation. The purified recombinant laccase had a molecular weight of 65 kD. The optimal pH and temperature for syringaldazine oxidation were 6.8 and 70℃,respectively. The initial enzyme activity was totally retained after 10 d storing at pH 9.0 and 36% of activity remained upon 10 h incubation at 70℃. The purified laccase was found to be totally inhibited by Al3+,Fe3+ and Mn2+. The purified laccase efficiently decolorized remazol brilliant blue R,reactive black 5 and indigo carmine in the presence of acetosyringone. More than 90% of the tested dyes were decolorized within 6 h at pH 9.0,indicating the recombinant laccase is an ideal candidate for the decolorization of dye effluents.
    The Promotion of Proper Carbon Nitrogen Ratio in the Synthesis of Extracellular Polysaccharide by Nitrogen-fixing Strains WN-F
    DENG Chao, DU Xiu-juan, HUANG Tao, GUO Ying, LI Bing-xue, BU Ning
    2018, 34(3):  194-199.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0583
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    The aim of this study is to isolate a nitrogen-fixing strain of producing high-yield polysaccharide from the cultivated maize soil samples around the Long-term Positioning Experiment Station of Brown Soil in Shenyang Agricultural University,and to study the strain identification,the optimization of culture conditions,and the relationship between nitrogen fixation and sugar production. The identification of the strain was carried out by morphological observation and molecular biology. The growth and culture conditions of the strain were determined by analyzing microbial biomass via enzyme mark instrument. The relationship between nitrogen fixation and sugar production was analyzed by measuring OD600 and polysaccharide yield. Results showed that the strain was Bacillus aryabhattai and named as WN-F. WN-F strain reached its highest growth at 37℃ and 180 r/min for 12 h. The medium was optimized as the formula(L-1):sucrose 20 g,beef extract 2 g,KH2PO4 0.4 g,MgSO4 · 7H2O 0.4 g,NaCl 0.4 g,CaSO4 · 2H2O 0.4 g and CaCO3 2 g. The polysaccharide yield of strain WN-F showed a parabolic relationship with the increase of initial nitrogen concentration,and the proper carbon nitrogen ratio promoted the synthesis of extracellular polysaccharide. B. aryabhattai WN-F is a dominant strain in maize soil for long-term positioning experiment. High-yield extracellular polysaccharide has a nitrogen fixation effect,thus,it is a candidate strain for applying combined nitrogen fixation of maize and reducing fertilizer application.
    Fusion of Phytase YiAPPA with the Raw-starch Binding Domain and Characterization of the Fusion Enzyme
    YUAN Lin, HUANG Zhao, ZENG Jing, GUO Jian-jun, ZHANG Ting, Lü Jun
    2018, 34(3):  200-207.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0795
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    This work aims to obtain a fusion enzyme with improved enzymatic properties comprised of phytase YiAPPA and raw-starch binding domain SBD. The raw-starch binding domain(SBD)of thermoacidophilic α-amylase GTamy was introduced into the C-terminal end of Yersinia intermedia phytase(YiAPPA)to generate a fusion enzyme YiAPPA-SBD. The fusion of YiAPPA with SBD resulted in improvements both in terms of thermal activity and stability. Besides,YiAPPA-SBD could absorb raw corn starch. Specifically,YiAPPA-SBD exhibited higher relative activity than YiAPPA within 55-90℃.The half-life of YiAPPA-SBD increased from 15 min to 30 min when incubated at 80℃. The adsorption efficiency of YiAPPA-SBD was 80% when the raw corn starch concentration was over 8%. Moreover,YiAPPA-SBD showed minor changes in its specific activity(3 900 U/mg)at optimal pH(pH 4.5),indicating that YiAPPA-SBD has solid pH stability and resistance to protease when compared to YiAPPA.
    Synergetic Effects of pH and Ca2+ on Yeast Metabolism and Cell Membrane Function
    XU Yan-jun, LI Jing-yuan
    2018, 34(3):  208-216.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0817
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    This work is to explore the metabolic mechanism of Saccharomyces cerevisiae and the change of the cell membrane function while adding different concentrations of Ca2+ under different pH. Using S. cerevisiae as research target,the metabolic characteristics of yeast cells were studied by adjusting pH and the concentration of Ca2+ in model grape juice medium,and also the metabolic mechanisms were investigated from the perspective of permeability and integrity of the cell membrane. The fermentation was conducted under the condition of temperature was 28℃,rotation speed was 120 r/min,and inoculation was 1×106 mol/L. Results showed that the rate of metabolism was accelerated,and the permeability and integrity of cell membrane were enhanced with the increase of Ca2+ concentration when the pH was 3.0,indicating that the inhibiting effect caused by low pH was eliminated after adding Ca2+. The effect of Ca2+ addition was not significant when the pH was 5.5. The above results indicated that Ca2+ alleviated the physiologically toxic effects of low pH on S. cerevisiae.
    Effects of Raising pCO2 and Osmolality on the Growth,Metabolism and Productivity of CHO Cells at Maintenance Phase
    WANG Chen, WANG Jia-qi, ZHAO Liang, FAN Li, LIU Xu-ping, CHEN Min, ZHANG Li-xiang, TAN Wen-song
    2018, 34(3):  217-224.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0749
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    The aim of this study is to understand the effects of pCO2 and osmolality on the monoclonal antibody(mAb)production during the maintenance phase of Chinese hamster ovary(CHO)cell cultures. Cell maintenance,metabolism,antibody quantity and quality at different pCO2 and osmolality levels were investigated during cell maintenance. It was found that the raise of pCO2 and osmolality brought negative impacts on final antibody production. The increase of osmolality damaged the cell maintenance,while the increase of pCO2 down-regulated the productivity of CHO cells. Through comparing the extracellular and intracellular metabolites,the increase of pCO2 and osmolality resulted in the decrease of carbon source utilization. Besides,on the antibody quality,the increase of osmolality decreased the glycosylation but raised the ratio of neutral charge. Moreover,the increase of pCO2 mainly enhanced the charge heterogeneity but there were no changes on the sugar type distribution of the products. Conclusively,the increase of pCO2 and osmolality during the phase of antibody expression had unfavorable effects on the cell maintenance,both material and energy metabolism,and the expression of product antibody;thus the accumulation of CO2 and osmolality elevation should be avoided as much as possible in order to ensure the normal proceeding of large-scale culture.
    Resveratrol Extraction from Grape Seed and Its Effect on LO2 Cell Oxidative Damage Protection and Anti-aging
    GAO Yuan, WANG Fu-ling, XU Bei-lei, LUO Jiang-han, YAN Li-jun, ZHAO Ying-ying, WANG Yue
    2018, 34(3):  225-229.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0820
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    Resveratrol was extracted from grape seed by ultrasonic assisted two-phase aqueous method. The oxidative damage model was constructed by inducing LO2 cell with alcohol(100 mmol/L),and aging mice model was built via D-galactose. The superoxide dismutase(SOD),glutathione peroxidase(GSH-px)activities and malondialdehyde(MDA)content in the cells and mice liver homogenate were measured after intervention by resveratrol extracts. Resveratrol extraction yield was 2.408±0.088 mg/g when liquid ratio was 1∶10,leaching time was 40 min,ultrasonic time,power and temperature were 300 W,10 min and 50℃,and aqueous two-phase system was ethanol:ammonium sulphate = 1∶1. Resveratrol extracts affecting LO2 cell reduced cells MDA content and increased the activity of SOD and GSH-px induced by ethanol. Moreover,resveratrol extracts presented a certain inhibitory effects on liver MDA production,and increased the activities of SOD and GSH-px in aging model mice.
    Preliminary Study on the Function of Fv-Afe1 Gene in Flammulina velutipes
    JIANG Si-yuan, CHOU Tian-sheng, LI Xiao, HUANG Rong-mei, XIE Bao-gui
    2018, 34(3):  230-234.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0019
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    According to genome data of a Flammulina velutipes strain L11,we selected Fv-Afe1 belonging to adenylate-forming enzymes superfamily. Transcriptome of F. velutipes was used to analyze gene structure,bioinformatics software to predict the amino acid sequence of Fv-Afe1,the predicted results and amino acid sequences of other species in NCBI were combined to construct phylogenetic tree,and qRT-PCR and DGE(digital gene expression profiling)data to analyze the gene expression in different growth stage. The results showed Fv-Afe1 gene contained 967 bp,including 4 exons and 3 introns. The results of bioinformatics revealed that the gene had two domains at tertiary structure of amino acids. Besides,the product of Fv-Afe1 was intracellular enzyme and no transmembrane structure,Fv-Afe1 belonging to the adenylate-forming enzymes superfamily was confirmed by analyzing phylogenetic tree,expression test showed that Fv-Afe1 had the highest expression in stipe-elongation stage and the secondly highest in stipe-maturation.
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    2018, 34(3):  300. 
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    2018, 34(3):  400. 
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    2018, 34(3):  500. 
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