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Table of Content

    01 September 2017, Volume 33 Issue 9
    CONTENTS
    Research Progress on YSL Transporters Gene Family
    LIU Yuan-feng,LI Su-zhen,GUO Jin-jie,CHEN Jing-tang
    2017, 33(9):  1-9.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0375
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    YSL(yellow stripe-like protein)are widely present in plants for the absorption and transporter of heavy metal. They are mainly involved in the absorption of Fe3+ and the transport of metal ions such as Fe2+,Zn2+,Cu2+,Ni2+ and Mn2+. At present,the research on the expression pattern,subcellular localization and the mutant of YSL in plants reveals its role in plant growth and development. In this paper,the research progress of YSL genes in plants in recent years are reviewed,which aims to lay the foundation for studying the mechanism of plant absorbing iron and the iron content in cereal grains by the biological enhancement.
    Research Advances on the Pathogenicity of Cucumber Mosaic Virus
    QIU Yan-hong,WANG Chao-nan,ZHU Shui-fang
    2017, 33(9):  10-16.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0278
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    Cucumber mosaic virus(CMV)is the type member of single stranded RNA viruses. As it infects wide host species and induces serious symptoms,it has been considered as one of the most impact plant virus and also the severe plant disease in the world. Many domestic and overseas scholars have investigated extensively about the CMV genes,gene-encoded products and plant-virus interactions since it was reported in 1916. With the development of high-throughput sequencing technology and proteomic technology,the molecular mechanism of viral virulence has been made great progresses. In this review,we introduce the role of viral encoding proteins,its associated satellite RNA and host factors that are involved in the development of symptoms,and also discusse the further study on viral virulence of CMV,which will provide a useful reference for CMVrelated studies.
    Research and Development Progress on Plant-made Pharmaceuticals
    LIU Rong-rong
    2017, 33(9):  17-22.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0177
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    Compared with the traditional production methods,the use of transgenic plants as bioreactor for the production of pharmaceutical proteins and vaccines is considered to be low-cost,safer and more convenient. At present,a number of plant-made pharmaceutical proteins have been approved for commercialization,while a group of plant-made vaccines have entered clinical trials. Research and development of plant-made vaccines and pharmaceutical proteins are reviewed,main problems and challenges are discussed,and the future technology development is prospected.

    Research Progress on Fish Genomics
    XU Gang-chun,DU Fu-kuan,BIAN Chao,SHI Qiong,XU Pao,
    2017, 33(9):  23-31.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0290
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    Fish genome sequencing is utilized for searching the genomics sequence information,subsequently clarifying the fish biological characteristics,and the relationship between the phenotypic variation and genome structures.. It has great applicable potential in fish research and economic fields,such as germplasm identification,genetic breeding,animal nutrition,environmental toxicology,disease control,and so on. Since the first report of zebrafish genome sequencing in 2001,nearly 40 fish genomes have been sequenced and published,which also illuminated their related biological characteristics and provided comprehensive interpretation of genetic evolution with high-yield and low-cost development of the genome sequencing technologies. The main aim of this review is to summarize research progress on the whole genome sequencing technology and the deep data analyses of fish genomes,and to further understand the recent development of fish genome researches and application prospects,so as to provide comprehensive references for subsequent fish genome researches.
    Research Progress on Red Fluorescent Protein
    WANG Fei,YANG Hai-tao,WANG Ze-fang
    2017, 33(9):  32-47.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0401
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    Since the first discovery of fluorescent proteins,its ever-increasing fluorescence spectra and the increasing number of photochemical properties have led to an increasing position in biological research. The red fluorescent protein emerged in 1999 dominated the biology study with its superiority. This paper focuses on the growing importance of fluorescent protein discovery from now on,as well as the significant advantages of red fluorescent protein. In addition,we discuss the characteristics of several common red fluorescent proteins and their advantages and disadvantages in biology research,providing an alternative for the future research of red fluorescent protein.
    Research Progress on the Function and Application of Membrane Protein Mms6 of Magnetosome
    MA Kun,ZHAO Hong-xin,LI Qian,WANG Jia-rong,SUN Hong-bin
    2017, 33(9):  48-55.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0170
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    The magnetosome is a special biomineralized product formed by the invagination of the magnetotactic bacteria(MTB)inner membrane. Magnetosome is composed of membrane-enclosed magnetite crystals ordered into chains along the cell. Moreover,these magnetic nanocrystals are highly homogeneous in size and morphology. The magnetosome membrane protein has been demonstrated to play an important role in the formation of magnetosome with recruitment and redox of iron and regulation of the nucleation and growth of magnetic nanocrystals. This article reviews that the main function of protein in magnetosome membrane in the process of the formation of magnetic nanoparticles from the absorption of iron ions into mineralization. Especially,the article introduces the structure characteristics and functions of the magnetosome membrane special protein 6(Mms6). Furthermore,the article summarizes the application of Mms6,as a biomimic addictive in the new-type magnetic nanomaterials,and discusses its possible mechanism of molecular regulation during the process of the formation of magnetosome,for providing a better understanding of biomineralization.
    Research Progress on Antibacterial Mechanism of Lactoferrin
    PEI Jie,CHU Min,BAO Peng-jia,YAN Ping,GUO Xian
    2017, 33(9):  56-63.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0354
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    Lactoferrin(LF)is a monomer glycoprotein in mammalian colostrum with multi biological activities,thus it plays a vital role in initial immunologic reconstitution for young mammals. Previous studies have discovered that enzymatic hydrolysates of lactoferrin have enhanced antibacterial activity compared to lactoferrin because more active antibacterial peptides were produced by the enzymolysis. At present,molecular mechanism of antibacterial function of lactoferrin is still unclear although many antibacterial activities of lactoferrin were reported. In this paper,combination of the bacterial cell membranes and the antibacterial activity of enzymatic hydrolysates were taken as the breakthrough point,and the process of LF acting in antibacterial function was explored. It was considered that the mechanism of the antibacterial function of lactoferrin was that the structure of lactoferrin changed following binding to bacterial surface,then the sensitive restriction enzyme cutting sites of lactoferrin were exposed,and many antibacterial peptides were released after enzymolysis;subsequently the peptides destroyed the membrane structures of bacterial cells,and thus the purpose of the bacteriostasis or sterilization achieved. In summary,the elucidation of antibacterial mechanism of lactoferrin provides the theoretical basis for developing active proteins with stronger antibacterial ability.

    Research Progress on Histone Demethylase JMJD3
    HU Shan,HAN Cong,HU Jian-hong,ZHU Hai-jing
    2017, 33(9):  64-72.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0148
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    Histone methylation state is dynamically regulated by different groups of histone methyltransferases and demethylases. H3K27me2/3 is controlled by the polycomb group proteins(such as methyltransferase EZH2),and its demethylation can activate gene expression. At present,two H3K27me3 demethylation enzymes,JMJD3 and UTX,have been identified. Moreover,a large number of studies have found that JMJD3 may promote cell differentiation and aging,and involve in the regulation of the occurrence and development of tumor. Here we summarize the roles and regulation mechanisms of JMJD3 in embryonic development as well as the occurrence and development of tumor,and the broad prospects in tumor diagnosis and treatment,for laying a theoretical basis for the future research work.

    Advancements in Quantitative Proteomics Technologies Based on Mass Spectrometry
    MU Yong-ying,GU Pei-ming,MA Bo,YAN Wen-xiu,WANG Dao-ping,PAN Ying-hong
    2017, 33(9):  73-84.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0343
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    Quantitative proteomic analysis is one of the key technologies in proteomics research. With the development of quantitative techniques,quantitative proteomics based on mass spectrometry has become an important branch of proteomics research. Quantitative proteomics technology is generally classified into two categories,untargeted quantitative proteomics and targeted quantitative proteomics. Targeted quantitative technology includes MRM and PRM models,and untargeted quantitative technology includes label-free and in vivo or vitro labeling. The most commonly used isotope labeling reagents are iTRAQ and TMT. According to the data collection model,the quantitative technology can be grouped into DDA and DIA. Through the collection and analysis of relevant literature,this article systematically introduces the main characteristics and current situation of quantitative proteomics based on mass spectrometry,aiming at providing help for the researchers on life science to better apply the quantitative proteomics technologies.
    Dynamic Changes of Microbial Community in the Process of Natural Fermentation of Puerariae lotaba Monitored by PCR-DGGE
    RAN Gan-qiao,ZHANG Hong-yan,REN Ping
    2017, 33(9):  85-93.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0251
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    In this study,the extraction yield of total isoflavones from Puerariae lotaba increased by 5.7% after pretreated with natural compost fermentation. After that,in order to determine the microorganisms playing a major role in this fermentation,the dynamic changes of the microbes in the process of natural fermentation of P. lotaba powder were analyzed using traditional flat culture method and PCR-DGGE technology respectively. The results showed that the number of bacteria showed an “increase-decrease-increase” trend in the whole fermentation process:increased significantly in the temperature-rising stage,then decreased slightly in the thermophilic stage,and finally rose again in the temperature-cooling stage,but the number of fungi presented a continuous increase trend. Besides,several microorganisms isolated from P. lotaba were initially identified to have positive effects on improving the extraction yield of total isoflavones,and these microbes included:Bacillus licheniformis,Rhizoctonia solani,Bacillus,Bacillus subtilis,non-cultivable bacteria,Penicillium japonicum,Aspergillus niger,Aspergillus,Asparagus,and non-cultivable Aspergillus.
    Study on Refolding and Purification of Plectasin MP1106
    WANG Peng-ju,TAN Huan-bo,SU Wen-cheng,ZHANG Wen-yu,ZOU Pei-jian
    2017, 33(9):  94-100.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0287
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    This work aims to establish an efficient and simple Escherichia coli expression system for the antimicrobial peptide,plectasin MP1106. The recombinant MP1106 fusion protein was obtained by genetic fusion with the artificially designed bio-surfactant,DAMP4. Then,the recombinant DAMP4-MP1106 was expressed in E. coli and the protein was isolated,moreover the formation of the disulfide bonds within the protein was identified. The results showed that the recombinant DAMP4-MP1106 was over-expressed in inclusion body. The protein was obtained by the Ni2+-NTA chromatography under the denature condition. The total yield of the fusion protein after the first chromatography was 118 mg/L in flask and the purity was 94.7%. The refolding buffer condition was screened by 96-well plate and the soluble recombinant DAMP4-MP1106 was obtained by a refolding procedure. The soluble recombinant DAMP4-MP1106 was cleaved by TEV protease and further purified by another Ni2+-NTA column. The purity of final MP1106 was 99% and the recovery of the protein was 38.4%. The comparison of the behaviour of the plectasin derivative,MP1106 with the standard plectasin on electrophoresis indicated that the disulfide bonds in MP1106 were folded correctly. In conclusion,the established E. coli expression system for the plectasin derivative MP1106 was efficient and successful.
    Enhanced Methanol Utilization in Genetically Engineered Escherichia coli by Directed Evolution
    WANG Xiao-lu, WANG Yu, LIU Jiao,ZHENG Ping,LU Fu-ping
    2017, 33(9):  101-109.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0369
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    Methanol is an important organic chemical raw material as well as an attractive alternative of sugar for biochemical industry because of its cost advantage and higher degree of carbon reduction. However,commonly used industrial microorganisms such as Escherichia coli cannot utilize methanol as a carbon source,which has become a bottleneck for the application of methanol in bio-production processes. In this study,a methanol utilization pathway(ribulose monophosphate pathway)was constructed in E. coli using an engineering genetically engineered E. coli as a starting strain,the random mutation occurred by continuous passage and direct evolution. Then the mutants were screened using methanol as the carbon source,and 2 mutants that grew well on methanol were obtained. When methanol used as an accessory carbon source,biomass of the mutant 25113ΔfrmA/pZWM1-13 increased by 27.6% compared to the starting strain 25113ΔfrmA/pZWM1. 13C labeling of proteinogenic amino acids detection showed that 13C labeled amino acids increased obviously in the mutant 25113ΔfrmA/pZWM1-13. Among them,13C labeled methionine increased by 7.236%. Therefore,directed evolution improved methanol utilization efficiency of the genetically engineered E. coli.
    Map-based Cloning of a Curly Leaf Gene CLF1 in Rice
    XING Bao CHEN,Zhen-hua,ZHANG Zhi-guo
    2017, 33(9):  110-115.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0694
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    Leaf is one of the most important photosynthetic organs and plays an important part in the productivity. We screened the rice leaf morphological variation mutants and isolated a curly leaf mutant(clf1)showing abaxial leaf rolling. The more bulliform cells numbers on leaf adaxial sides in clf1 mutnat were the main reason leading to leaves curling. Using Map-Based cloning methods,the site controlling CLF1 gene was mapped in chromosome2,tightly linking with two polymorphic markers(InDel51 and InDel57). There were 44 genes in this region and we predicted LOC_OS02G45250 as the target gene by bioinformatics. We found that there were 20bp deletion in the sixth exon of LOC_OS02G45250 and the nucleotide deletion created a premature stop codon in the predicted coding region. The clf1 was another allele of the reported rice curly leaf gene Roc5(Rice outermost cell-specific gene5),whose encoded transcription factor including GL2 homologous structure domain. Different from the oul1 mutant(loss-of-function in Roc5),the clf1 was valuable for the production because of a better agricultural characters.
    Monosaccharide Components of the Male Sterile Mutant gsl5 in Rice
    LIU Qi-ming,CHEN Zhen-hua,HAN Xiao
    2017, 33(9):  116-119.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0696
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    Pollen wall plays vital role during microspore development and pollination in rice(Oryza Japonica). Abnormal pollen wall formation usually leads to male sterility. Callose wall,microspore primary wall development and sporonpollenin accumulation are key step to control pollen wall formation. Recent studies focus on callose wall formation during microspore development. Neither less,the knowledge about monosaccharide composition in the pollen wall is limited. In this study,we compared the monosaccharide composition in the pollen wall of the wide type rice with a male sterility mutant gsl5.
    Cloning and Expression Analysis of MoSOS1 Gene in Melilotus officinalis
    HUANG Kun-yong,LI Shan-shan,GUO Qiang,MAO Pei-chun,TIAN Xiao-xia,MENG Lin
    2017, 33(9):  120-130.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0364
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    Plant salt overly sensitive 1(SOS1)gene,encoding a Na+/H+ anti-port protein,plays an important role in biological processes of plants against salt stress. Using the total RNA extracted from the young leaves of Melilotus officinalis(L.)Lam. as the template,the full-length sequence of SOS1 gene was amplified by the reverse transcription polymerase chain reaction(RT-PCR)method and rapid amplification of cDNA ends(RACE),named MoSOS1. The bioinformatics analysis showed that MoSOS1 was in a length of 3931 bp with a complete open reading frame(ORF 2 874 bp),encoding a 957 amino acid residues of MoSOS1 protein with an estimated molecular weight of 112.8 kD and a calculated isoelectric point(pI)of 5.31. The prediction of transmembrane region of MoSOS1 by TMHAM software indicated that MoSOS1 had 8 transmembrane regions and N-terminal and C-terminal were located outside the cell. Amino acid alignment analysis demonstrated that the protein of MoSOS1 contained a conserved Na+/H+ exchanger superfamily,a cNMP(Cyclic nucleotide-monophosphate)and a CAP_ED(Catabolite gene activator protein-effector domain)superfamily. The bioinformatics analysis showed that MoSOS1 protein belonged to unstable acidic protein with mainly secondary structure of α spiral and random coil,and in which there was no signal peptide. Quantitative real-time RT-PCR analysis showed that expression levels of MoSOS1 presented an increase trend in both shoots and roots with the increase of external NaCl concentrations,and its levels in roots were higher than those in shoots,indicating that the expression of MoSOS1 was induced and regulated by salt.
    CRISPR/Cas9 System-mediated CSN2 Gene Knock-out for Preparation of Goat Fetal Fibroblasts Mutation Cells
    JIA Qi-peng,SHEN Pei-lei,ZHANG huan,ZHANG Yong
    2017, 33(9):  131-138.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0218
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    To study the effects of beta casein(CSN2)on lactation performance of goat,the CSN2 gene of goat fetal fibroblast(GFFs)were knocked out by CRISPR/Cas9 system for providing the nuclear donors for the construction of transgenic animal. Five sgRNA targeting sites(T1,T2,T3,E1,and E2)were designed for CSN2 targeting at second and eighth exons and ligated to vector PX330-eGFP,thus PX330-eGFP-sgRNA expression vector was constructed. The knockout efficiency was evaluated by T7E I enzyme digestion experiment. Moreover,the sgRNA with the high knock-out efficiency of eighth exons was selected for constructing the PX330-Neo-sgRNA expression vector. Then the PX330-eGFP-sgRNA T and PX330-eGFP-sgRNA E combined with PX330-Neo-sgRNA E were transfected into cells,and mutant cells with CSN2 knock-out were obtained by flow sorting and G418 screening. The expression of CSN2 in the transfected cell was measured by Western blot. Sequencing results showed that the sgRNA was correctly ligated to the PX330 expression vector,T7E I digestion experiments showed that the knock-out efficiency of sgRNA T1 and sgRNA E2 was 34.9% and 25.9%,respectively. The proportion of CSN2 knock-out cell in eGFP+Neo transfection group was significantly higher than that in eGFP+eGFP transfection group(P<0.05). In summary,we draw the conclusion that CRISPR/Cas9 system would be efficiently applied to the gene editing of GFFS and the targeting mutation cell will be of the potential resourse of nuclear donor in generating CSN2 gene knock-out goat.
    Function of Osa in the Differentiation of Drosophila Ovary Germline Stem Cell
    LI Min,QU Jie,LI Meng-jie,HU Xiao-long
    2017, 33(9):  139-144.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0289
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    The Drosophila ovary germline stem cell(GSC)is recognized as an ideal model system for studying stem cell fate regulation in vivo. The epigenetic mechanism is involved in the Drosophila ovary GSC fate regulation. Identification and characterization of apparent epigenetic regulators involved in this process will contribute to reveal underlying regulatory mechanisms. The aim of this study is to investigate the possible functions and the molecular mechanisms of Osa,the chromatin remodeling complex BAP-specific subunit,in Drosophila GSC differentiation. GAL4/UAS binary system combined with RNAi technology was used to specifically down-regulate osa expression in escort cells(ECs),and immune fluorescent dyeing was for detecting the relevant indexes. The results showed that knock-down of osa led to a significant increase of undifferentiated germ cells(UGCs)in the germarium of ovarian tissue,the positive cells of pMad and Dad-lacZ,two BMP signaling activity reporters,increased significantly. Moreover,EC morphology was abnormal,and they failed to wrap germline cells. In conclusion,Osa might be involved in the regulation of GSC differentiation by BMP signaling pathway-depending and specific morphological process of ECs.

    Mining the Cryptic Bioactive Secondary Metabolites from Streptomyces vietnamensis Using a‘Tree-Removal’Strategy
    LIN Hai-zhou, CHEN Zhou-qin WANG Yan GUO Jun ZHU Hong-hui DENG Ming-rong
    2017, 33(9):  145-152.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0276
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    This study aims to discover novel bioactive substances by activating the cryptic biosynthetic gene clusters of secondary metabolites from Streptomyces vietnamensis with appropriate strategies. The study was carried out in a S. vietnamensis mutant DMR1. In this mutant,the key biosynthetic genes,gra-orf1-3,of the dominant product were knocked out,resulting in deficiency in granaticin production. The strain was subject to nutrient condition changing,inducer adding and heat shocking. The antimicrobial activities of the fermentation crude extracts were tested using agarose diffusion method and TLC-bioautography. Results showed that the fermentation crude extracts of Gause No.1 and ISP3 presented significant inhibitory effects against Bacillus subtilis ATCC6633,Staphylococcus aureus ATCC6538 as well as multidrug resistant Staphylococcus aureus strains ATCC43300 and 206. With adding 3% DMSO to the media,not only did the activities of the fermentation crude extracts of Gause No.1 and ISP3 against the afore said strains increase,but also the spectrum broadened into against Escherichia coli ATCC8739,Candida albicans ATCC10231 and multidrug resistant E. coli and Salmonella sp. strains. The newly discovered bioactive substances from S. vietnamensis showed extensive activities against both gram-positive and gram-negative bacteria as well as yeast. More importantly,some of the crude extracts were significantly active against all the tested drug-resistant strains. These results indicate a possibility of a novel type of antibiotic. The results presented in this paper illustrate that the so-called ‘tree-removal’ strategy can improve the mining efficiency of the cryptic bioactive secondary metabolites.

    Effects of Novel Antifungalmycin N2 Crude Extract from Streptomyces sp. N2 on Germination of Rice Seed
    WU Zhi-ming,ZHONG Min,LU Cheng-jian,ZHU Man-ning,DU Qi-feng,LI Kun-tai
    2017, 33(9):  153-159.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0337
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    To investigate the effects of a novel antifungalmycin N2(AN2)crude extract from Streptomyces sp. N2 on germination of rice seed,the multi-index of germination,amylase activity,soluble sugar contents,soluble protein contents and the contents of malondialdehyde(MDA)in rice seeds were measured using ultraviolet-visible spectrophotometry. The results showed that the influence of AN2 on each index was correlated with the treated AN2 concentration. It was found that AN2 at low concentration promoted rice seed germination,while it at high concentrations had an inhibitory effect. When treated with 0.23-1.15 μg/mL of AN2,the characteristics of seed germination was effectively improved,and the growth of seedlings was significantly promoted,especially under the treatment with the concentration of 0.58 μg/mL. In addition,the content of soluble sugar,soluble proteins and amylase activity of optimal concentration treated groups was increased significantly than control group by 28.27%,7.73% and 6.49%,respectively. In the 0.23-1.15 μg/mL of AN2 treatment group,the MDA content decreased significantly. Therefore,proper concentration treatments promote germination capacity,improve enhance material metabolism and enhance ability of osmotic regulation.
    Isolation of Brevibacillus laterosporus A60 and Its Greenhouse Control Efficiency Against Phytophthora capsica
    HAO Nan,TONG Zan-Hua,QIU De-Wen
    2017, 33(9):  160-165.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0210
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    A60 strain with high inhibition rate against Phytophthora capsici was screened from 700 strains isolated from 43 soil samples in Hainan province. The A60 strain was identified as Brevibacillus laterosporus based on physiological and biochemical characteristics and a 16S rDNA gene sequence analysis. The control effects of A60 strain against Phytophthora on Capsicum annuum and Capsicum fructescens were determined in greenhouse. The results showed that the biocontrol efficacy of A60 strain on C. annuum L. were 100%,95.71% and 92.22% at 3rd,5th and 7th day,respectively. The control efficacy on C. fructescens were 100%,75.26% and 73.20% at 3rd,5th and 7th day,respectively. Our results highlight that the application of B. laterosporus A60 significantly reduce morbidity and disease index,i.e.,control efficacy is solid.
    Effects of Different Herbicides on Weed Control and the Growth,Yield and Quality of Highland Barley
    YAN Dong,LUO Zhen-hua,HU You-liang,WANG Jun-cheng,SI Er-jing,REN Pan-rong,YAO Li-rong,LI Bao-chun,MA Xiao-le,MENG Ya-xiong,WANG Hua-jun,
    2017, 33(9):  166-171.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0341
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    This work is to select the suitable type and dosage of herbicide for highland barley in alpine region,and to determine the effects of different herbicides on the growth,yield and quality of highland barley. Using the single-factor experiment and random-plot selection method,we studied the control effects of herbicide Aixiu,Yanmaiwei,and Dabiaoma on main weed wild oats in highland barley field as well as the effects on the growth,yield and quality of barley in field conditions in Gannan alpine region. The treatments with 3 herbicides significantly reduced the number of weed wild oats,while some barley seedlings were a slightly yellow,but they recovered to normal after half a month. The best control effect of Aixiu was found in the 3-leaf stage of weed wild oats,as the average control effect per plant was 82% and the control effect in fresh weight reached 87%. The control effect with soil treatment of Yanmaiwei was poor and the Dabiaoma was the worst. The plant height,ear length,grain number per ear,and 1 000 grain weight of highland barley after different treatments were significantly or extremely higher than contrast. The yield increase of highland barley by Aixiu and Yanmaiwei treatments was obvious. There were no significant variations in protein contents,water contents and starch contents of highland barley among different treatments. In summary,Aixiu treatment presented better control effect on weed wild oats,increased yield significantly,and caused no impacts on the quality of highland barley,thus it could be considered as a suitable herbicide in highland barley field of alpine area.

    Genome-wide Identification and Expression Characteristics of Invertase Gene Family in Jatropha curcas
    WANG Hai-bo,XIN Hu,LIU Chao,TANG Li-zhou
    2017, 33(9):  172-178.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0303
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    Plant invertase is one of critical enzymes in sucrose metabolism and stress resistance formation. The 16 invertase genes were identified based on complete genome data of Jatropha curcas,then the evolutionary relationship,gene structure,physical and chemical characteristics,conserved motif,and expression feature were analyzed. The results showed that invertase gene family in J. curcas was clustered to three subfamilies according to phylogenetic analysis. The α subgroup of neutral/alkaline invertases and acidic invertase gene owned 6-7 exons,and β subgroup owned 4-5 exons in gene structure. Signal peptides in N-terminal and conserved motifs of -NDPNG/A- and -MWECV/P- belonging to GH32 family were identified in acidic invertase protein sequences. qRT-PCR analysis revealed that JcC-INVD expressed differently in different tissues,with abundantly in cotyledon,but scarcely in root,and remarkably cold-induced expression in leaf.
    Prokaryotic Expression and Polyclonal Antiserum Preparation of Gn Protein in Tomato Zonate Spot Virus(TZSV)
    ZHENG Kuan-yu,SHANG Wei-na,ZHANG Jie,ZHENG Xue,DONG Jia-hong
    2017, 33(9):  179-183.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0242
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    Bioinformatics prediction showed that Gn protein in tomato zonate spot virus(TZSV)is a transmembrane protein consisting of 3 transmembrane domains. Antigen epitope prediction showed that there was a high antigen index region,thus non-transmembrane region of Gn was selected to conduct the prokaryotic expression and to prepare polyclonal antiserum. The Gn gene fragment of TZSV was amplified from TZSV-infected tomato sample by RT-PCR,and then cloned into prokaryotic expression vector pET-30a. The recombinant plasmids pET-30a-Gn were transformed into Escherichia coli BL21(DE3),and then induced to express with IPTG. The result of SDS-PAGE analysis showed the specific fusion protein with molecular weight of 40 kD was highly expressed. Using the purified fusion protein,rabbit was immunized to obtain polyclonal antiserum against TZSV Gn protein. The titer of antiserum was 1/16384 determined by ID-ELISA. The specific band was detected by Western blot while applying the prepared antiserum to the TZSV-infected samples. These results reveal that the specificity of the antiserum is fine,and it is applicable for immune-reaction test related to TZSV Gn.
    Biosynthesis of 3-(4-Hydroxyphenyl)Propionic Acid in Saccharomyces cerevisiae
    JIANG Jing-jie,ZHUANG Yi-bin,YIN Hua,LIU Tao,LIU Shao-wei
    2017, 33(9):  184-190.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0208
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    3-(4-Hydroxyphenyl)propionic acid(HPPA)is an aromatic compound with importantly economic value,because it is an intermediate mainly used in medicine,food and chemical industry. In this work,we aim to achieve the De novo synthesis of HPPA in Saccharomyces cerevisiae,and it was conducted via overexpressing a tyrosine ammonia-lyase from Flavobacterium johnsoniaeu(FjTAL)and a p-coumaroyl-CoA ligase from Arabidopsis thaliana(At4CL),as well as using super long-chain enoyl-CoA reductase(ScTSC13)and acyl-CoA thioesterases from S. cerevisiae BY4742. The yield of HPPA reached 70 mg/L. Bioconversion rate of tyrosine into p-coumaric acid was 36% under the condition of 4 mmol/L tyrosine enhancement in shake flask after 72 h fermentation at 30℃ and galactose inducing FjTAL overexpression. While in the recombinant S. cerevisiae overexpressing At4CL combined with enzymes ScTSC13 and acyl-CoA thioesterases,94% of 4 mmol/L p-coumaric acid was converted to HPPA in shake flask after 72 h fermentation at 30℃,and 91 % of 4 mM caffeic acid was converted to DHPPA. This work laid a foundation for the biosynthesis of HPPA and its derivatives.
    Improvement of Inhibitor Tolerance of a Xylose-Fermenting Industrial Saccharomyces cerevisiae Strain Through UV Mutation and Acclimation
    ZHANG Yi-zhi,GOU Min,TANG Yue-qin
    2017, 33(9):  191-199.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0285
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    The inhibitor tolerance of a xylose-fermenting industrial Saccharomyces cerevisiae strain was improved through direct acclimation or ultraviolet mutation combined with acclimation. During the repeated batch incubations,the concentration of the mixed inhibitors,containing nine kinds of typical inhibitors,was gradually increased to make cells adapt to the high inhibitor concentration. UV mutation combined with acclimation improved the tolerance of cells to inhibitors with high concentrations more effectively than direct acclimation. By the isolation and screening of mutation strains,three mutation strains with better inhibitor tolerance than the original strain were obtained. They showed 11.3%-23.2% higher xylose consumption ratios than the original strain when fermenting xylose under the inhibitory condition of 100% MI(formic acid 1 g/L,acetic acid 3.5 g/L,levulic acid 1.5 g/L,furfural 1.5 g/L,5-hydroxymethylfurfural(5-HMF)1.5 g/L,syringaldehyde 0.1 g/L,vanillin 0.1 g/L,coniferaldehyde 0.025 g/L,cinnamic acid 0.025 g/L). UV mutation combined with acclimation improved the tolerance of S. cerevisiae strain to mixed inhibitors.
    Construction and Fermentation Testing of an O-acetyl-homoserine Producing Escherichia coli Strain
    YANG Jing,LI Qing-gang,XU Guo-dong,ZHENG Xiao-mei,ZHENG Ping,MOU Hai-jin
    2017, 33(9):  200-209.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0310
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    O-acetyl-homoserine(OAH)is an important industrial ingredient for homoserine and methionine production. We used the modified threonine-producing strain Escherichia coli ThrL as the host strain,to overexpress homoserine dehydrogenase gene thrA and homoserine acetyltransferase gene metX that are key genes for OAH production. The promoter combination was designed to optimize the transcription of these two key genes. Then,the concentrations of threonine and yeast extract in the fermentation medium were also optimized. After the promoter combination optimization,it showed that the OAH yield of the recombinant strain E.coli OAH4,in which the thrA and metX genes were separately controlled by J23110 promoter,was the highest and reached up to 1.54 g/L after 50 h incubation. When the concentrations of threonine and yeast extract were 2 g/L and 6 g/L,respectively,the OAH yield of E. coli OAH4 was the highest and further increased to 1.98 g/L after 50 h incubation. In this study,the synthesis of OAH was achieved in E. coli for the first time. The expression of thrA and metX,and fermentation conditions of OAH were optimized,which paved the way for the further OAH production research.
    Preliminary Probe on Antagonistic Mechanisms of the Pichia pastoris G5 Against Botrytis cinerea
    LUO Lin,ZHOU Ling-xuan,LIU Ya
    2017, 33(9):  210-215.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0238
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    Considering the Pichia pastoris G5,a biocontrol strain isolated from red grape in Xinjiang and using Botrytis cinerea as target strain,we studied the effects of G5 on the spore of B. cinerea and the activities of five enzymes in the red grape fruit,and primarily explored the antagonistic mechanisms of G5 against B. cinerea by in vitro and in vivo experiment. Results showed that the G5 obviously inhibited the germination of B. cinerea spores and the growth of mycelium,and the rate of inhibiting mycelium maximally reached 80.20% and the rate of inhibiting spore germination was 86.89%. Nutritional competition existed between the G5 and B. cinerea. In the course of antagonism,the heavy parasitism phenomenon occurred. Experiment sindicated that G5 itself did not secrete antimicrobial substances. In addition,the strain induced and significantly increased the enzymes’ activities in fruit,such as peroxidase,polyphenol oxidase,phenylalanine ammonia lyase,chitinase,and β-1,3-glucanase. The inhibition mechanisms of G5 to B. cinerea include nutrition competition and inducible resistance;whether or not there is heavy parasitism needs to be further validated.
    Prokaryotic Expression,Purification and Polyclonal Antibody Preparation of Neurospora crassa ERG-11 Protein Antigen
    LIU Yan-xia,LI Shao-jie,FAN Zhen-chuan
    2017, 33(9):  216-222.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0258
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    This work aims to construct the recombinant plasmid pET-28a(+)-ERG-11 and express the 6 × His-ERG-11 fusion protein for preparing ERG-11 polyclonal antibody. The target fragment amplified by PCR was inserted into pET-28a(+)prokaryotic expression vector,and then transformed into the competent cells of Escherichia coli BL21(DE3)for expressing the fusion protein. Further,the fusion protein was purified by affinity purification and gel filtration chromatography,and then immunized to New Zealand white rabbit for preparing polyclonal antibody. Taking serum,the titer and specificity of the polyclonal antibody were detected through indirect ELISA and Western blot,respectively. As results,the pET-28a(+)-ERG-11 expression vector was successfully constructed. SDS-PAGE showed that the 6 × His-ERG-11 fusion protein was induced successfully in the inclusion form. After two-step purification,the antigen with high purity was obtained. ELISA showed that the polyclonal antibody titer reached 1∶512 000,and Western blot confirmed its high specificity. Conclusively,the prokaryotic expression of ERG-11 gene was successfully conducted,and a polyclonal antibody against Neurospora crassa ERG-11 was prepared,providing a foundation for further study on the genetic structure and function of ERG-11 in N. crassa.
    Antimicrobial Activity and Anti-MRSA Mechanism of the Fermentation Broth of Bacillus amyloliquefaciens AF1
    YANG Yan-hong,YU Ying HU,Yong-qiang,YU Xi,LI Qing-qing
    2017, 33(9):  223-230.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0312
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    This study aims to measure the antimicrobial spectrum of fermentation broth of Bacillus amyloliquefaciens AF1 and to explore the activity stability and mechanism of anti-MRSA,for providing an experimental basis in seeking new anti-MRSA compounds. The drug-resistant S. aureus SA46 was used as indicating pathogen,the rest antimicrobial activities of AF1 fermentation broth were detected after it was dealed with different pH,temperatures and proteases. The antmicrobial mechanism was studied preliminarily by observing the morphologic change of SA46 tread with AF1 fermentation broth under both optical microscope and transmission electron microscope,and by detecting the protein and DNA leakage of treated SA46 with AF1 fermentation broth with SDS-PAGE and agarose gel electrophoresis. The results showed that AF1 fermentation broth had a broad antimicrobial spectrum. It presented a strong inhibitory ability to all tested clinical drug-resistant Staphylococcus aureus,common clinical Escherichia coli and S. aureus;also it inhibited the most of the tested phytopathogen. The antimicrobial activity of AF1 fermentation broth remained more than 95% in pH 2.0 - 9.0 for 120 min from 4 to 80℃,and it was stable when adding protease K,pepsin and trypsin into the broth respectively. The cell walls of SA46 treated by AF1 fermentation broth(MIC)were destroyed at varied level while observed under both optical microscope and transmission electron microscope. Furthermore,the leakage of protein and DNA of treated SA46 with AF1 fermentation broth were checked by SDS-PAGE and agarose gel electrophoresis. The electrophoretic bands of experimental groups were clear,while there was no bands in control groups on both DNA and protein electrophorograms. Thus,a preliminary deduction was that the cell walls of MRSA were damaged by AF1 fermentation broth. In conclusion,B. amyloliquefaciens AF1 might be developed as a potential new source for the novel anti-MRSA medicines.
    Effect of Cryoprotectant on the Survival Rate of Freeze-dried Pseudomonas sp. Powder
    LI Li,ZHAO Peng-nian,ZHU Xi-kun
    2017, 33(9):  231-234.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0215
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    The cryoprotectant presents remarkable effect on increasing the survival rate and stability of freeze-dried Pseudomonas sp. powder. Here single-factor and orthogonal experimental designs were conducted to screen the optimal combination of cryoprotectant. The results showed that the survival rate reached 90% when the concentration of skim milk powder,sorbitol,glycerol,and L-sodium ascorbate were 10%,3%,1%,2%,and was 50.2% while stored for 6 months at 25℃.
    Characterization of Clavulanic Acid High-yield Streptomyces clavuligerus Industrial Strain by Transcriptome Analysis
    QIN Rong-huo,FU Jia-fang,ZONG Gong-li,WANG Zhi-yu,CAO Guang-xiang,
    2017, 33(9):  235-243.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0275
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    This work aims to explore the molecular mechanism of clavulanic acid(CA)high-yield Streptomyces clavuligerus industrial strain. Transcriptome high-throughput sequencing was performed to compare the differential gene expressions between standard strain ATCC27064 and high-yield industrial strain F613-1,and comprehensive analysis was carried out combined with the phenotypic differences between them. The phenotypic results showed that F613-1 grew differently in growth rate,aerial mycelium morphology and spore formation in solid medium,compared with the standard strain. In the liquid culture,the growth rate of the two strains demonstrated no difference,but the sugar consumption of F613-1 significantly decreased,and the CA yield was 11.7 times higher than that of ATCC27064.Transcriptome analysis indicated that the expressions of both CA cluster genes and pathway-specific regulatory genes significantly increased;while the expressions of holomycin(CA by-product)cluster genes significantly decreased. On the other hand,the transcriptional levels of genes associated with arginine and glyceradldehyde-3P(raw materials for CA synthesis)metabolic pathways changed significantly,i.e.,up-regulated for arginine and down-regulated for glyceradldehyde-3P. Besides,the expressions of over80 genes involved in ABC transport system obviously changed,and 42 remarkably up-regulated and 38 down-regulated. The main reason of F613-1 highly produces clavulanic acid possibly lies in the increased transcription of CA cluster genes and relative regulatory genes,the silence of cluster genes involved in CA by-products,and the accumulation of arginine and alyceradldehyde-3P.
    Preparation and Characterization of Novel Heparinase from Sphingo spiritivorum
    YIN Si-yuan,BAI Jia-ke,LI Li,MA Xiao-lai
    2017, 33(9):  244-251.  doi:10.13560/j.cnki.biotech.bull.1985.2016-1162
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    This work aims to screen new type of strain producing novel heparinase,and to prepare novel heparinase and test its ability of degrading heparin. The bacteria in soil were cultured using the directional enrichment method,and strains producing heparinase were isolated. Microbial fermentation and purification technology were adopted to prepare novel heparinase,and to study the physical and chemical properties and the cleavage site of novel heparinase. Then,the heparinase were applied to degrade heparin,and the obtained low molecular weight heparins(LMWHs)were characterized. A new heparinase-producing strain belonging to the genus of Sphingo spiritivorum was isolated. The crude enzyme after fermentation was purified with multi-step column chromatography and novel heparinase SShepI and SShepII were obtained,with which two kinds of novel low molecular weight heparins were generated by the degradation of heparin. Conclusively,novel heparinase SShepI and SShepII isolated and purified from a strain of S. spiritivorum are adopted to degrade heparin for preparing low molecular weight heparins,which are expected to be developed into LMWH drugs with better anticoagulant effect or new clinical function.
    Preparation and Application of Soluble Human Squamous Cell Carcinoma Antigen Expressed by Escherichia coli
    CHEN Chun-ye,LIU Jian,ZHU Rui,LI Shu-xuan,YE Jiang-hui,WANG Wei,PAN De-quan,XU Fei-hai,CHENG Tong,XIA Ning-shao
    2017, 33(9):  252-258.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0228
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    The aims of this study are to establish a method for efficient soluble expression of human squamous cell carcinoma antigen(SCCAg)based on Escherichia coli expression system and obtain the recombinant SCCAg antigen in fine activity,then apply it in the detection method establishment of antigen. The study on the method of soluble expression and purification of recombinant SCCAg antigen was conducted based on pGEX-6P-1 vector and E. coli ER2566 strain. The activity of the purified antigen was evaluated by Abbott Kit and the specific monoclonal antibody was screened by indirect ELISA. It was proved that PGEX-6P-1 vector and E. coli strain ER2566 could be used to establish efficient soluble expression and purification method for recombinant SCCAg antigen. Moreover,the recombinant SCCAg antigen was proved to be in high purity and activity. Thus,the SCCAg detection method of chemical luminous tube was established with the specific monoclonal antibodies. In conclusion,an effective method for the expression and purification of SCCAg,which is based on the E. coli expression system,is established.
    Low Expression of PD-L1 Gene in Human Breast Carcinoma Cell Mediated by Lentiviral Vector
    OUYANG Jing,SUN Yuan-yuan,TANG Ze-min,TANG Zheng-shan,LI Fang-jun,YANG Yin-ke
    2017, 33(9):  259-266.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0181
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    This study aims at constructing three-plasmid system of the lentiviral vector carrying the programmed death ligant 1(PD-L1)gene and investigating the expression of PD-L1 in human breast carcinoma cell MDA-MB-231. A pMAGic 7.1 recombinant lentiviral expression vector,plasmid △8.91 and pVSVG were co-transfected into 293T cells for packaging virus,using polyethylenimine(PEI)as transfected solution. The lentiviral products were added to infect and extract stably-expressed MDA-MB-231 cells,and finally the PD-L1 expression of mRNA and protein levels were detected. The results showed as followings:(1)The PD-L1 expression in MDA-Mb-231 was the highest,compared with other two breast cancer cells. (2)At 48 h after co-transfecting three plasmids,the fluorescence intensity reached over 90%. Moreover,the fluorescence intensity reached almost 20% at 48 h after using virus infecting MDA-MB-231 cells. (3)There were low PD-L1 expressions of mRNA and protein levels in the stably-expressed MDA-MB-231 cell lines,and the pMAGic-sh1 had the best effect on it among the three plasmids. (4)After adding viruses,the cell proliferation declined in interfering groups,and the cell proliferation rate in the pMAGic-sh1 interfering groups was 64.77% at 7 days of adding virus compared with the negative group. (5)Mixed lymphatic experiments showed that the tumor inhibition rate of sh-1 group was 35.8%,which was 3.06 times of that in normal group,and the T lymphocyte proliferation rate significantly enhanced. In conclusion,the three-plasmid system of lentiviral vector containing PD-L1 gene using PEI reagent is successfully established. The study demonstrates that the PD-L1 expression of mRNA and protein is lower through the virus infecting system,and interfering groups have lower cell proliferation rate and they enhance the T lymphocyte proliferation rates and its ability to kill tumor cells.
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