Table of Content

26 August 2018, Volume 34 Issue 8

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Research Progress on Plant AP2/ERF Transcription Factor Family

ZHANG Qi, CHEN Jing, LI Li, ZHAO Ming-zhu, ZHANG Mei-ping, WANG Yi

2018, 34(8):  1-7.  doi:10.13560/j.cnki.biotech.bull.1985.2017-1142

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As one of the largest transcription factor family,AP2/ERF transcription factors are common in plants,and have attracted much attention because of their important role in gene breeding. The AP2/ERF transcription factor family contains at least one AP2 conservative domain with about 60 amino acids. According to the number of domains and the sequence of recognition,AP2/ERF transcription factor family can be classified into 5 subfamilies of AP2,ERF,DREB,RAV,and Soloist;and the number of AP2/ERF transcription factor and their subfamily members are variable in different plants,such as Arabidopsis,rice,maize,tomato,etc. Through responses to the regulation of ethylene,cytokinin and auxin,AP2/ERF transcription factors are reported to participate directly or indirectly in multiple processes of plant development including seed development,flower and fruit organ formation. Except for primary metabolism,AP2/ERF transcription factors affect significantly the regulation of plant secondary metabolism,particularly in improving the accumulation of active ingredients of medicinal plants（such as artemisinin,paclitaxel and lignin）. Meanwhile,the AP2/ERF gene of Arabidopsis are also reported to have a positive regulatory function of resistance to Botrytis cinerea,while some AP2/ERFgenes play a vital role in response to drought,high salt,low temperature,hypoxia and other abiotic stresses. In addition,the AP2/ERF transcription factors also participate in the non-biological signal transduction pathways mediated by ethylene. In this study,the structure and classification characteristics of AP2/ERFtranscription factors,their quantity distribution in different plants will be introduced,and the roles of AP2/ERF transcription factors in plant development,secondary metabolism,biotic or abiotic stress and signal transduction will be expounded. Overall,this review is aimed to provide theory basis for cultivating transgenic crops with high-yielding and stress tolerances in the future.

Research Advances in Gene Editing and Plant-virus Interaction and Their Application in Breeding Virus-resistant Crops

YANG Yi-zhou, LI Wei, YI Tu-yong, LI Feng

2018, 34(8):  8-16.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0462

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Viral diseases impose serious threat to crop production and the best management strategy is to breed virus-resistant crop varieties for production. Research on plant-virus interaction has revealed mechanistic insights on the interactions between viral and plant genes which are essential for viruses to complete their life cycle. Once a virus enters its host plant cells, its particle disassembly, gene expression, genome replication, and ribonucleoprotein complex movement through plasmodesmata and phloem tissue are all dependent on interactions between viral products and specific plant genes. The recessive mutation of those plant genes can confer viral resistance to plants. Such mutation exists in plant natural population and can be directly employed in breeding viral resistant crops. CRISPR/Cas-mediated gene editing enables targeted gene mutagenesis in both plants and animals in vivo and there has been an explosion in the development of this technology in the past five years. It allows precise mutation and editing of the plant genes required for virus infection, thus to effectively create virus-resistant crop. In this review, we aim to summarize recent advancements in gene editing research and current knowledge on plant-virus interaction and provide guidelines for creating viral resistant crops using gene editing technology.

Research Progress on microRNA in Landscape Plants

LUO Xiao-ning, ZHAI Li-juan, LI Xiang, SHI Qian-qian, ZHANG Yan-long

2018, 34(8):  17-26.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0002

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MicroRNA（miRNA）is one of the main type of non-coding small RNA with 19-24 nucleotides（nt）. miRNA can negatively regulate target gene expression levels at post-transcription levels through three patterns of cutting,translation inhibition and DNA methylation. They are widely existing in organisms and playing important regulatory roles in plant growth and development,as well as responses to environmental stimuli. Few studies on the miRNA in landscape plants have been carried out,but some achievements have been made. Previous studies show that miRNAs can not only participate in the embryonic development,leaf development,branch development,flower development,fruit development and developmental timing change of landscape plants,but also regulate their secondary metabolisms and signal transductions. In addition,many miRNAs in landscape plants play key roles in the sense and adaptation to biotic and abiotic stresses. This paper reviews the biosynthesis and function mechanism of miRNA in plants,focusing on the biological functions of miRNA in development,secondary metabolism and signal transduction,stress in landscape plants. This is aimed to provide the reference for the further research of miRNA in landscape plants.

Research Progress of Biobutanol Fermentation

GAO Yue, GUO Xiao-peng, YANG Yang, ZHANG Miao-miao, LI Wen-jian, LU Dong

2018, 34(8):  27-34.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0033

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With the depletion of fossil fuels and the worsening of environmental pollution,clean and renewable energies such as biofuels have become a hot topic for research all over the world. Biobutanol becomes a promising one among the new generation of biofuels because of its high energy density,high combustion value and light pollution. Nevertheless,the high production cost of traditional Acetone-Butanol-Ethanol（ABE）fermentation and the low production rate limited the commercial production of biobutanol. In order to reduce the cost of feedstock and to achieve the effective industrial transformation of cheap biomass,many researches focus on economic fermentation technology based on biomass resources. By exogenous adding technology,strain phenotype and fermentation performance were revealed rapidly in fermentation system,which contributed to the explanation of interactions in the strain metabolic level and gene level. In addition,with the development of genome sequencing and omics engineering technology,an increasing number of researches aimed to improve the metabolic synthesis ability and the tolerance of biobutanol were reported. These researches included transforming metabolic network structure,blocking non-butanol metabolism pathway,clarifying the mechanism of regulations to stress,and removing related metabolic regulation,which were for endogenous transformation of butanol-producing Clostridium. In this paper,based on the development of biobutanol fermentation biomass resources,macro-control strategy of metabolism and strain breeding,the bottleneck in the process of biobutanol production and metabolism was discussed,and the biobutanol development was also prospected. The aim is to provide theoretical basis for the construction of engineering bacteria and metabolic regulation based on process engineering technology.

Research Advances on Integrating Metagenomics and Synthetic Biology in Discovering Novel Biocatalysts

WANG Ye, JIA Zhen-hua, SONG Shui-shan

2018, 34(8):  35-42.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0027

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Biocatalysts refer to cells or enzymes with catalytic activity,and they are widely applied in industry,agriculture,medicine,and energy fields due to their high efficiency and environmentally friendly. Traditional biocatalyst mining technology relies on bio-separation and in vitro culture technology,which to a certain degree hinders the development of novel biocatalysts. Discovering novel biocatalysts by metagenomics is as such,directly extracting the DNA of all microorganisms from the target sample,constructing genomic library,and then screening novel biocatalysts based on functional activity and sequence analysis,which avoids the necessity of isolation and laboratory cultivation of microorganisms. Synthetic biology refers to redesign biological pathways,organisms or biological systems via the design,assembly and construction of components by employing engineering principles. This paper reviews the advances on minng new biocatalysts by metagenomics methods and discusses the efficiency of synthetic biology approaches to improve biocatalyst identification in metagenomic library screening,aiming to improve the efficiency of mining new biocatalysts.

Research Advances in the Major Facilitator Superfamily

LI Chun, SUN Chun-yu, CHEN Jing, LIN Yan-ping, WANG Yi, ZHANG Mei-ping

2018, 34(8):  43-49.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0069

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As one of the largest superfamilies of membrane transport proteins,Major Facilitator Superfamily（MFS）included more than 1 million sequenced members. MFS,with a majority of members contained 400-600 amino acid residues,has been expanded to 95 families according to Transporter Classification Database（TCDB）. MFS promoted the transport of solutes such as sugars,drug molecules,peptides,metabolites of tricarboxylic acid cycle,organic anions,and inorganic anions across the cell membrane in response to electrochemical gradient. Recently,there have been more and more studies focusing on the crystal structure and transport mechanism of MFS. Usually,the MFS were reported to have 12 transmembrane helical segments and special fold method called MFS fold. Like a “rocker switch”,MFS proteins exhibited intracellular and extracellular open or closed conformations to transport the solute. In addition,there were many studies focusing on the physiological functions of MFS in animals and plants. At present,the MFS transporter of human was increasingly concerned as their distinctive physiological functions. For example,the deletion of some members of MFS might lead to brain shrinkage and developmental delay. Besides,some MFS proteins were also reported to regulate acid-base balance of cells,promote the absorption of anti-cancer and anti-inflammatory drugs. Overall,the study of human MFS transporters provided the basis for the occurrence and prevention of many diseases such as diabetes,fatigue syndrome,cardiovascular disease,and cancer. In this study,to provide theory basis for further study of MFS,the development of MFS,its crystal structure,transport mechanism and physiological function were reviewed,and on such a basis,the development trend of MFS was also prospected.

Research Progress of Cold Tolerance Mechanism and Functional Genes in Fish

LIU Li-li, ZHU Hua, YAN Yan-chun, WANG Xiao-wen, ZHANG Rong, ZHU Jian-ya

2018, 34(8):  50-57.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0098

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The ability of fish to adapt to ambient temperature varies broadly,as a result of long-drawn adaptation to habitat environment and evolution. The suffertibility of different fish species to cold stress is the manifestation of the specific expression of genetic information,as well as the reflection of differences in physiological and biochemical performance. Currently,fish physiology responses under low temperature have been in-depth researched while the underlying molecular mechanisms attract increasing interest. The low cost and extensive application of high-throughput sequencing technologies together with bioinformatics analysis allow researchers using omics method to study the metabolic and molecular signaling pathways of the fish under cold stress. The biological molecular mechanisms and related functional genes of fish to cold stress are thus enabled to be further explored. Recent studies prove that many polar fishes have evolved with functional genes gain,loss and large-scale amplification during the long-term adaption to low temperature. The transcription and regulation patterns in fish are conservative to a certain extent while possess obvious specificity in species and tissues under cold stress. Some proteins including antifreeze（glycoprotein）protein,chaperone,metabolic enzyme and transmembrane channel protein are involved in cold-responsive process in fish. However,the corresponding genes characterization and function research remain jagged. This review summarized the molecular mechanisms of fish adaption and tolerance to low temperature from the perspectives of evolution,the genetic expression and epigenetics. Then,cold tolerance related genes were classified and introduced according to their molecular function. Last but not least,we predicted potential research focus in mechanisms,gene mining and application of fish under cold stress,hoping to provide background and reference for researchers and those interested in the field.

Research Progress on Peptidoglycan Recognition Proteins in Fish

XIA Hong-li, CHENG Jun, YU Da-peng, CHEN Wen-jie, LU Yi-shan

2018, 34(8):  58-66.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0218

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Peptidoglycan recognition proteins（PGRPs）are a family of highly-conserved pattern recognition receptors（PRRs）,they may recognize peptidoglycans,an essential component of bacterial cell wall,consequently activate and regulate innate immunes of a body. At present there are 23 reported fish PGRPs,and they were classified into three groups based on the length of the amino acid sequence,named short-PGRPs,intermediate-PGRPs and long-PGRPs. Its C-terminal PGRP domain is highly conserved although fish PGRPs have some differences in protein size,domain organization and subcellular localization. Fish PGRPs widely express in different tissues,its expression levels display notable changes in various immune tissues under the stimulation of different bacteria,thus we speculate that fish PGRPs are involved in the immune response. In addition,fish PGRPs also play a vital role in the physiological process and innate immune regulation of embryonic development. Currently,researches on the functions of fish PGRPs mainly focus on pathogen recognition,amidase activity,bactericidal ability and antimicrobial activity,as well as immune regulatory mechanisms;however,researches on the mechanisms of fish PGRPs remain swallow,and needs further deep researches. This paper reviews the structure,express and function of fish PGRPs,aiming at providing for further studying the function and application for PGRPs in fish.

Separation and Extraction Process of 3β,7α,15α-trihydroxy-5-androsten-17-one in Biological Preparation

NI Yu, YIN Si-qi, LI Hui, SHI Jin-song, XU Zheng-hong

2018, 34(8):  67-74.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0106

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3β,7α,15α-trihydroxy-5-androsten-17-one（7α,15α-diOH-DHEA）is a key intermediate of drospirenone,the active ingredient of the fourth generation of oral contraceptive Yasmin for women,thus it has the significant value in the market. The substrate dehydroepiandrosterone（DHEA）can be converted to 7α,15α-diOH-DHEA through biotransformation,which has advantages of high conversion rate and environmentally friendly. In this study,the extraction and separation of the product in the fermentation broth were optimized,and the optimal process of extracting and separating 7α,15α-diOH-DHEA were determined. Results showed that fermentation broth was pretreated with 4%（V/V）sodium hydroxide-ethanol,and its supernatant and sediment were extracted with 1.5 times and 2 times of ethyl acetate and ethanol respectively. When dichloromethane：ethanol = 15∶1 was select as eluent,the purity of product reached 98.1% after separation,and the yield of purification reached 91.4%. The structure of the product was characterized by MS,IR and¹H-NMR,and it was consistent with the structure of standard 7α,15α-diOH-DHEA.

Antibodies Preparation and the Establishment of Chemiluminescence Immunoassay Detection Method for Cystain C Polypeptide

WANG Yun-long, XU Rui-fang, LI Yu-lin, WANG Ji-chuang, CHENG Chun-jie, GAO Yu-hong

2018, 34(8):  75-79.  doi:10.13560/j.cnki.biotech.bull.1985.1017-1084

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The objective of this work is to prepare cystain C antibody and establish the chemiluminescence detection method for the early diagnosis of renal damnification. Cystatin C monoclonal antibody was prepared through chemical synthesis of cystain C polypeptide,then competitive inhibition assay was used for preliminary screening of paired antibody,and chemiluminescence immunoassay detection method of cystain C was established. Totally,39 strains of cystatin C monoclonal antibody cells were obtained,and paired antibody was screened. By analyzing the linear range,minimum detection limit,precision,accuracy and other performance evaluation analysis,chemical luminescence detection method for detecting cystatin C was established,with linear range of 50--20 000 ng/mL and the minimum detection limit of 3 ng/mL. Conclusively,cystatin C paired antibody was prepared and chemiluminescence detection method was established successfully,which provides a new detection technique and method for early renal damnification.

Establishment and Optimization of Cell Suspension Culture System for Vitis vinifera

WANG Ling, LI Yan, DAI Wei-na, YAN Jing, ZHANG Chao-hong

2018, 34(8):  80-86.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0075

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In order to establish a rapid and stable cell suspension system of grapevine callus,the stems,leaves and petioles of cv. Thompson seedless and cv. Pinot Noir were used as explants. Through the selection of basic medium,the ratio and concentration of plant growth regulators and the presence or absence of PVP,the induction method of loose callus was optimized,and a stable cell suspension culture system was established. The results showed that the MS as the basic medium combined with 2.0 mg/L NAA and 0.3 mg/L 6-BA was suitable for the induction of loose callus of grapevine while the stems of cv. Thompson seedless and cv. Pinot Noir were as explants. Under the optimal culture conditions of B5-based medium supplemented with 1.0 mg/L 2,4-D,0.5 mg/L 6-BA and 0.2% PVP,a rapid and stable suspension culture system for cv. Thompson seedless was established. The growth curve of grapevine suspension culture cells was S-shaped,the logarithmic phase was on the 6-18 d after inoculation,and the cell growth reached the maximum at the 21 d. The results of both cell viability assay and TTC staining showed that the cell viability was the strongest on the 9 d after inoculation. Conclusively,a rapid and stable suspension culture system of cv. Thompson seedless is established,and it is more efficient to select 7-9 d cells with the stronger cell viability and faster growth to carry out the genetic transformation.

Cloning and Identification of a Promoter with Root Tissue-specific Expression in Common Wild Rice

HUANG Ke, HUANG Ge-ge, XUE Man-de, LONG Yan, YUAN Qian-hua, PEI Xin-wu

2018, 34(8):  87-92.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0382

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Based on previous transcriptome data,a root-specific expression gene of common wild rice（Oryza rufipogon Griff.）was screened,and the promoter sequence was cloned and designated as OrRSGp. It was 896 bp in length and contained main functional elements such as CAAT-box and TATA-box,MeJA-responsiveness and some elements involved in defense and stress responsiveness. It was fused with the β-glucuronidas（gus）reporter gene and transformed into Arabidopsis thaliana. GUS histochemical staining and quantitative analysis showed that OrRSGp regulated gene gus specific expression in roots.

Transcriptome Sequencing of Stamen in Muskmelon Male Sterile Lines at Different Developmental Stages

DAI Dong-yang, YUAN Li-wei, SHENG Yun-yan, ZHENG Qun

2018, 34(8):  93-100.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0306

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In order to explore the mechanism of male sterility in muskmelon,the correlation between pollen abortion and differentially-expressed genes at different stages of male sterility and stamen development was studied at the molecular level. The transcriptome sequencing of bud（while its diameter was 2 mm and 5 mm）stamen from male sterile（MS）line of muskmelon was conducted,differentially-expressed genes were screened,and differentially-expressed genes were analyzed by fluorescent quantitative method. Totally 224 differentially-expressed genes were identified. GO function analysis of them showed that all the genes belonged to 3 branches of biosynthesis,molecular function and cellular components. Among them,9 differentially-expressed genes were related to various enzymes,2 related to carboxylesterase homologous,4 related to carbohydrate enzymes synthesis,and 1 gene related to signal transduction and 1 gene related to zeaxanthin biosynthesis,respectively. qRT-PCR verification revealed that the expression levels of carbohydrate-related enzymes and zeaxanthin biosynthesis-related genes in 2 mm buds and stamens were lower than those in 5 mm buds and stamens. The expressions of cell signal transduction-related genes in MS line 2 mm stamens were higher than that of 5 mm stamens. The above results indicate that there is a certain correlation between the occurrence of male sterility and the increase of energy metabolism-related gene expression and the reduction of the expression of genes related to cell signaling.

Effects of Silver Nitrate on the Synthesis of Phenolic Compounds and the Expression Levels of Related Genes in Callus Browning Process of Paeonia ostii ‘Fengdan’

MAO Pei-qi, LI Hou-hua, LI Ai, CAO Zhi-xiu, HAN Mei-ling, ZHANG Yan-long

2018, 34(8):  101-107.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0307

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Tissue culture plantlets of Paeonia ostii “Fengdan”were used as material and inoculated in the basal medium containing 0 - 5.0 mg/L silver nitrate. By measuring the total phenols content,types and content of free phenol and the expression of related genes,the inhibition effects of different concentrations of silver nitrate on callus browning were determined. The results showed that adding silver nitrate in medium inhibited the expressions of related genes,significantly reduced the content of polyphenols in the callus and subsequently reduced browning,of which the comprehensive results by 2.0 mg/L was the best. The main phenols contributing callus browning were chlorogenic acid,rutin,coumaric acid,p-coumaric acid,epicatechin,and dihydro quercetin. In silver nitrate-treated callus,there were significant correlations between the expressions of transcription factors MYB2,WD40-1,and WD40-2,the structural gene phenylalanine ammonia lyase（PAL）,flavanone reductase（DFR）,chalcone synthase（CHS）,chalcone isomerase（CHI）,3-dihydroflavonol hydroxylase（F3H）,flavonol 3'-hydroxylase（F3'H）and browning grade as well as total phenols content（P < 0.05）.

Difference Analysis on the Responses of Three Peltigera Species to Cu²⁺ Stress

Jimilamu·JIAMALI, Guhainisha·MAIMAITI, Rouxianguli·NAIZAIER, Pazilaiti·BAIHETI

2018, 34(8):  108-114.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0271

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This work is to analyze the response differences of 3 Peltigera specie Peltigera rufescens,Peltigera elisabethae,and Peltigera canina to Cu²⁺ stress based on the induced glutathione（GSH）variation and the copper adsorption,in order to understand the role of glutathione in the resistance of low organism lichen to heavy metals. Spectrophotometry and flame atomic absorption spectrometry were used to determine the content of glutathione and the content of intracellular and extracellular copper. The results showed that GSH in 3 lichen species increased gradually at low Cu²⁺ concentrations（1- 4 mmol/L）and reached the highest in the 4 mmol/L. With the increase of the stress（5 - 8 mmol/L）,the GSH contents in both P. rufescens and P. canina decreased significantly,but still higher than that in the control. The GSH content in P. elisabethae varied little under 3 - 5 mmol/L Cu²⁺ stress condition,and decreased significantly（P<0.05）at Cu²⁺ >5 mmol/L. Under the highest tolerant concentration 4 mmol/L Cu²⁺,the P. canina increased gradually with the prolongation of treated time,but the GSH contents of the P. rufescens and P. elisabethae fluctuated（increased in 6 h,decreased in 12 h,and increased gradually in 18 - 24 h）. The contents of adsorbed extracellular copper in 3 lichen species were positively correlated with Cu²⁺ concentration and treatment time and were significantly higher than that of the adsorbed intracellular copper（P < 0.01）. The changes of adsorbed intracellular copper corresponded to the changes of GSH contents in the lichens as an up-down-up pattern. The results of this study indicate that the lichen species may induce the synthesis of GSH to alleviate the toxicity of Cu²⁺ on lichen thallus,and the tolerance of 3 Peltigera to copper is closely related to their copper adsorption and GSH synthesis ability.

Screening and Characteristics of a Broad Spectrum Fungus Degrading Polycyclic-aromatic Hydrocarbons：Aspergillus flavus AD-X-1

TIAN Jing, XU Xiao-lin, KANG Yan-shun, TANG Wei-hua, LIU Si-qi

2018, 34(8):  115-122.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0125

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This study is aimed to screen a broad spectrum strain degrading polycyclic-aromatic hydrocarbons（PAHs）and investigate its degradation characteristics for providing strain supply and technical support in the management of combined pollution. An efficient PAHs-degradation fungus was screened with PAHs as the sole carbon and energy source and identified and designated as Aspergillus flavus AD-X-1. The experiment convinced that the degradation was predominant in the removal of PAHs by AD-X-1,while mycelium adsorption also played a part role. The degradation condition was optimized using anthracene as the substrate,and the removal rate of anthracene reached 88% in 72 h by AD-X-1 under the condition of 50 mg/L anthracene,temperature 35℃,rotator speed 170 r/min and pH 7. Further AD-X-1 was found to have promising salt-tolerance,and the removal rate of anthracene still remained at 50% when the salinity increased to 9%. Moreover,AD-X-1tolerated higher concentration of heavy metal ions（Cr³⁺,Cu²⁺,and Pb²⁺）. In addition to anthracene and phenanthrene,the removal rate of tetracyclic pyrene,benzo[a]anthracene,and dibenzo[a,h]anthracene by AD-X-1 were 71%,68%,and 63% respectively. Conclusively,AD-X-1 has broad spectrum ability of removing PAHs,and concurrently it has the excellent resistances to salt and heavy metals.

Screening of Microsatellite Markers Associated with Furfural Tolerance of Saccharomyces cerevisiae

LI Sha-sha, ZHANG Liang, SHI Gui-yang

2018, 34(8):  123-129.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0107

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Two phenotypically different haploid strains were screened as parents and were crossed to obtain two hundred segregants. The polymorphic microsatellite markers were used to amplify offspring yeasts（39 strains）,and t-test was used to analyze the significance of difference among the genotypes of different sites in two gene pools,which is aimed to have microsatellite markers associated with furfural tolerance. The results revealed that two microsatellite markers associated with furfural tolerance were identified. Among them the marker 2P3 had significant differences（P = 0.039 < 0.05）and marker 2P5 had extremely significant differences（P = 0.000 ﹤ 0.01）in the 2 gene pools. Furthermore,72.2% of high tolerant individuals contained the marker 2P5（333 bp）derived from P1,the high tolerant parent;100% of low tolerant individuals contained the marker 2P5（318 bp）derived from P2,the low tolerant parent. These results suggested that the significant difference of 2P5 genotype in population segregation may be linked with the gene controlling this trait and can be used to select the favorable allele in molecular design and breeding so as to improve the accuracy and predictability of breeding.

Gene Cloning,Prokaryotic Expression and Enzymatic Analysis of the Phosphite Dehydrogenase from Soil Pseudomonas Species

YUAN Hang, LUO Zhu, YANG Yu-mei, LIU Yan-juan, GAO Yan-xiu, LIU Xian, GONG Ming, ZOU Zhu-rong

2018, 34(8):  130-137.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0250

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Phosphite dehydrogenase（PTDH）catalyzes the oxidation of phosphite to produce phosphate and NADH,using NAD⁺ as the cofactor. This enzyme is of large application potentials in the areas of coenzyme regeneration and phosphite-based phosphorus utilization,and etc. Herein,the full-length of a putative PTDH gene（PsPtx）was obtained by two rounds of PCR from the soil metagenome as DNA template,which was then subcloned into plasmid pET32a（+）by enzyme digestion to generate the prokaryotic expression vector pET（PsPtx）. Sequence analysis showed that the PsPtx gene had an entire coding region of 1 011 bp,encoding a protein of 336 aa and 36.5 kD. The results of the conserved domain prediction demonstrated that the deduced protein PsPtx was a member of the PTDH family,and had the conserved NAD⁺-binding motif as well as the crucial catalytic residues. Furthermore,phylogenetic analysis indicated that the PsPtx gene was originated from an undefined soil Pseudomonas species. In addition,PsPtx efficiently expressed in Escherichia coli strain BL21（DE3）by IPTG induction. The recombinant protein PsPtx was purified by His-tag affinity chromatography,and then detected to have a specific activity of 3.75 U/mg towards its substrate,sodium phosphite. Certainly,this cloned PsPtx gene with verified activity is of fundamental importance for its future application studies.

Antitumor Effect of Water Extracts from Fruiting Bodies of Different “Sanghuang”Fungi

WU Xiao-lin, WANG Chao-yi, BAO Hai-ying

2018, 34(8):  138-143.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0145

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The antitumor activities of water extracts from 6 “Sanghuang” fungi were studied with H₂₂ mice in vivo. The results showed that the high and low dose groups of the water extracts from Inonotus hispidus,Fomitiporia ellipsoidea,and Phellinus yamanoi and high dose group of the water extracts from Phellinus igniarius,Phellinus baumii,and Phellinus vaninii presented significant antitumor effect with over 40% of antitumor rate,except the low dose group of it from P. baumii. Among them,the antitumor effect of the water extract from F. ellipsoidea（1000 mg/kg）was the best. Compared with positive and control group,the thymus index of the tested mice in the high dose group of water extract from I. hispidus showed a very significant difference（P < 0.01）,but ones in the other groups had not. The water extracts from 6 “Sanghuang” fungi all can promote the expression of Bax in mouse tumor cells,and inhibit the expression of Bcl-2.

Improving the Thermal Stability of Lactate Oxidase by ETSS

HUA Chen, LI Xin-xin, TU Tao, YANG Hong, LUO Hui-ying, CHEN Jia-ming, YAO Bin, BAI Ying-guo, PENG Shu-chuan

2018, 34(8):  144-150.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0177

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Lactate oxidase can catalyze the oxidation of lactic acid to pyruvate,thus it has important industrial application value. However,lactate oxidases from different sources share the common problem of poor thermal stability,which impacts its application in industry. The researches on lactate oxidase are less reported,and there was no report on the improvement of its thermal stability,therefore improving the thermal stability of lactate oxidase is of great significance. In this study,we cloned and expressed highly specific lactate oxidase EgLOD,and used bioinformatics and surface charge analysis software ETSS to analyze the protein structure of lactate oxidase. We designed 9 mutants for the experiment of improving their thermal stability based on the distribution and optimization of protein surface char,2 mutants D250N and D281N presented efficient enhanced thermal stability after screening. After incubated under 60℃ for 30 min,they remained more than 50% of the enzyme activity,while the wild type EgLOD remained only 20%.

Analysis of Free Amino Acids During Fermentation of Iturin A by High Performance Liquid Chromatography

ZHAO Jie, YUE Hua, GOU Xue-lei, ZHOU Jin-yan, TAN Hong

2018, 34(8):  151-158.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0012

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A fast and efficient HPLC with phenylisothiocyanate（PITC）as the pre-column derivatization to detect the concentrations of free amino acids was established and utilized to analyze the dynamic change patterns of free amino acids in the fermentation process of iturin A. The method using PITC as pre-column derivatizing reagent for HPLC analysis,liquid chromatography conditions were optimized. Amino acids were separated on a Vensuil-AA amino acid analysis column（4.6 mm × 250 mm,5 μm）,with optimal conditions：0.1 mol/L sodium acetate buffer（pH 6.4±0.1）-acetonitrile（66∶5）as mobile phase A and acetonitrile -water（4∶1）as mobile phase B by gradient elution,the flow rate was 1.0 mL/min,the UV detection wavelength was set at 254 nm,and chromatographic column temperature was 40℃. The results showed that gradient elution program,pH of the mobile phases and chromatographic column temperature had important effects on the analysis time of amino acids,peak separation and peak shapes. Sixteen amino acids were separated drastically in 35 min under the optimal chromatographic conditions. In addition,each amino acid was separated with fine linearity（R² > 0.9986）in a range of the test concentration,and the average recoveries were 83.84% to 108.02% with RSD less than 2.77%. The method was accurate and reliable with favorable precision and stability. Based on this method,the dynamic changes of free amino acids during the fermentation of iturin A was studied,by which three different changing trends of various amino acids were revealed.

Transcriptomics Analysis of High-xylose-tolerance Klebsiella pneumonia Strain and Optimization of Fermentation Conditions for 2,3-butanediol Production

GUO Xue-wu, ZHANG Yu, GUAN Xiang-yu, NI Xiao-feng, WANG Qing, CHEN Ye-fu, XIAO Dong-guang

2018, 34(8):  159-169.  doi:10.13560/j.cnki.biotech.bull.1985.2017-1111

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This work is to breed a strain of Klebsiella pneumonia with high xylose tolerance,to analyze its transcriptomics,and to optimize the fermentation conditions of producing 2,3-butanediol（2,3-BD）. Firstly,the adaptive evolution method was used to screen a K. pneumonia strain KP2 with high xylose tolerance and high yield of 2,3-BD. Then,the transcriptome analysis and the optimization of fermentation conditions were conducted. Results showed that the xylose conversion rate and 2,3-BD production by KP2 increased by 174.5% and 227%,respectively. The transcriptome analysis of parental strain KPG and KP2 demonstrated that 242 genes were up-regulated and 263 genes were down-regulated in KP2. The pathway analysis further revealed that the changes of transcripts mainly occurred in signal transduction,carbohydrate metabolism,energy metabolism and other material metabolism. Under the initial concentration of xylose 120 g/L in 1 L reactor,the optimal fermentation conditions were as follows：35℃,230 r/min,0.7 L/min（aeration）,and pH 5.9. Under this optimal conditions,the maximal production of 2,3-BD reached 43.9 g/L,which was improved by 33.3% compared to that before optimization. In sum,we successfully obtained the strain of producing 2,3-BD by efficiently using xylose,the mechanism of phenotypic change was expounded at the molecular level,and the optimal fermentation conditions were determined. The result may provide reference for the production of 2,3-BD,and has a certain guiding significance for the biological production of 2,3-BD.

Construction and Phenotypic Analysis of Gene p53 Knockout Rat Model

HUO Gui-tao, YANG Yan-wei, WU Xi, LIU Su-su, LI Qian-qian, ZHOU Shu-ya, LIU Quan-ming, WANG San-long, SHEN Yue-lei, LÜ Jian-jun, FAN Chang-fa

2018, 34(8):  170-174.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0086

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The objectives of this work are to construct rat model with gene p53 knockout using TALEN,to establish colony,and to analyze the phenotypes of the rats. TALEN protein specifically recognizing exon 2 of gene p53 was designed and the corresponding vectors were constructed. In vitro transcribed TALEN mRNA was injected into fertilized eggs of SD rats. New born rats with right splicing at the target site were determined by sequencing. Together 11new-born rats were obtained after injection and 10 of them were having right splicing at target site by sequencing. Four rats were selected as founder to establish the colony. The homozygotes with gene p53 knockout presented tumor susceptibility and the main type of spontaneous tumor was malignant fibrous histiocytoma. In addition,the homozygous rats with gene p53 knockout also had eye developmental defects,such as retina degeneration and disorganized fiber of lens. In conclusion,rat model with p53knockout is efficiently constructed by TALEN,the model exhibits tumor susceptibility and eye developmental defects,indicating that the model can be used for studying the mechanisms of tumorigenesis and the function of gene p53 in eye development.

Eukaryotic Expression of DLL4 Protein and Preparation of Polyclonal Antibody

JIN Qiao, GAN Zhi-kai, ZHOU Peng, LIU Xia

2018, 34(8):  175-180.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0376

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This work is to clone,express and purify human DLL4 protein and to prepare corresponding polyclonal antibody. Bridging PCR was employed to construct a DLL4 gene with tPA signal peptide,and the gene then was cloned into pGZX expression vector. After identification by enzyme digestion and sequencing,it was transiently transfected into HEK293F cells. At 48 h after transfection,the supernatant was purified by nickel column affinity purification. Dot blotting,Western blotting,and Fortebio macromolecules were used to measure the biological activity of DLL4. Then,rabbits were immunized to prepare DLL4 polyclonal antibody. Antibody titer was detected by ELISA and specificity of antibody was tested by Western blotting. A 1.6 kb tPA-DLL4-6His target fragment was amplified by PCR and the sequencing result showed that it was consistent with DLL4 sequence（NM_019074.3）in the NCBI database. After one-step affinity purification,DLL4 protein with a purity of 95% was obtained. Dot blotting and Western blotting detected that the fermentation supernatant contained DLL4 protein,and the affinity between DLL4 protein and DLL4 antibody was 1.848E-10 and R² was 0.999. The titer of DDL4 antibody by ELISA was 1∶12 800,and the DLL4 antibody had solid specificity. In conclusion,the immunogenic DLL4 protein and its polyclonal antibody are successfully prepared.

Analysis of miRNA Transcriptome in Early Developmental Stage and Identification of Growth-related miRNA of Siniperca chuatsi

ZHAO Yan, CAO Xiao-ying, ZHOU Hao-tian, SONG Ling-yuan, TU Han-qing, HUANG Si-ying, ZHAO Jin-liang

2018, 34(8):  181-189.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0261

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MicroRNA（miRNA）is a kind of non-coding small RNA molecules with the length of about 20-22 nt,and they are involved in regulating the expression of target genes at the post-transcriptional level. High-throughput sequencing technology was employed to analyze the miRNA transcriptome of the whole larvae of Siniperca chuatsi at day 3,day 17 and day 28 after hatching. A total of 1084 miRNAs were identified,including 432 known,624 conserved,and 28 new miRNAs. miRNAs of the 17-day larvae were identified the most with 926. The 628 miRNAs were ubiquitously expressed at all the days sampled,while 71,104 and 70 miRNAs were specially expressed at day 3,day 17 and day 28,respectively. Several miRNAs that were involved in development showed sustained up or down regulation patterns. Further results suggested that miR-199-5p/miR-203 were correlated with the growth and development of mandarin fish,and the corresponding target genes were WnT and MyoD,respectively.

Isolation and Identification of Human Urine-derived Stem Cells

WANG Fu-ping, ZHANG Feng, ZHAO Tong-biao

2018, 34(8):  190-198.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0312

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In order to establish an efficient and convenient system for the cultivation and identification of human urine-derived stem cells（hUSCs）,urine samples from young volunteers were collected under aseptic conditions to isolate and culture hUSCs. The clone assay,morphological observation,karyotype analysis,growth curve determination,surface marker detection,osteogenic and adipogenic differentiation were utilized to identify the hUSCs. The clones of hUSCs were in three types of fiber-like,pebble-like and silk-like. For the second passage hUSCs highly-expressed antigens CD44,CD73 and CD90,the positive rates were higher than 99.6%;while for the lowly-expressed antigens CD34 and CD45,the positive rates were less than 0.3%. For the sixth passage hUSCs highly-expressed antigens CD44 and CD73,and the positive rates higher than 99.5%;while for the lowly-expressed antigens CD34 and CD45,the positive rates were less than 0.5%. The sixth passage hUSCs still had strong proliferative ability and stable karyotype,and was induced to differentiate into osteocyte and adipocyte. By this research,the efficient and convenient hUSCs culture and identification systems are established.

Effects of the Characteristic Changes of Mice Hematopoietic Stem/Progenitor Cells on the Aged Immune Imbalance in the Aging Process

ZHANG Li-jie, LI Xiao-yu, CEN You-fei, ZHOU Zu-ping, PU Shi-ming

2018, 34(8):  199-203.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0149

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This work is to explore the age-related changes of hematopoietic stem/progenitor cells（HSPCs）in the aging process and to reveal the occurrence and development mechanisms of immune “aging” in the aging process. Flow cytometry was used to measure the cell abundance and proliferation activity of hematopoietic stem cells（HSCs）and its downstream progenitor cells. The results showed that the abundances of HSCs and multipotent progenitor cell（MPP）and granuiocyte/macrophage progenitor（GMPs）cells in bone marrow cells increased significantly with the rise of age,and the number of cells while entering the phase of division（G1,G2/S/M）also increased. In addition,the myeloid-derived suppressor cells（MDSCs）in the downstream cells of GMPs accumulated. In conclusion,the results revealed that HSCs were activated in the aging process,promoting cell proliferation and the differentiation and accumulation of the myeloid suppressor cells,and inhibiting the immune function of the body. This study may provide reference for understanding the decline of immunity in the aging process.

Safety Accident Disposal and Warning of Foodborne Pathogenic Bacteria：Taking “the Outbreak of Enterohemorrhagic Escherichia coli Infection in Germany in 2011” as a Case

TANG Chu-mei ZHU Long-jiao GUO Shun-tang MIAO Jing XU Wen-tao

2018, 34(8):  204-214.  doi:10.13560/j.cnki.biotech.bull.1985.2017-1106

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With the improvement of living standard,issues on food safety are drawing a growing concern worldwide. In regards to all food safety incidents in the world,those caused by foodborne pathogens are becoming highlighted due to their universality,frequency and severe pathogenicity. This article is organized in the following order：First it gives an overview of the characteristics of foodborne pathogens and their connections with food safety by several historic food safety incidents;then it takes the outbreak of enterohemorrhagic Escherichia coli（EHEC）infection in Germany,2011 as a case,introducing pathogenic characteristics,detection techniques,tracing methods of foodborne pathogens in detail,and summarizing control measures and guides to government and consumer response. At last,based on the case,the article discusses the possibilities of its role both played in teaching and popularization in science. In sum,this article gives multi-angle analysis on the case,aiming at promoting effect on the disposal and warning of foodborne pathogenic food safety accident.