Biotechnology Bulletin

Recent Advances on Inhibition Mechanisms of Calcium on Plant Diseases

WANG Fang, LI Zhen-lun, CHEN Yan-li, YANG Shui-ying, XU Yi

2017, 33(2): 1-7. doi:10.13560/j.cnki.biotech.bull.1985.2017.02.001

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Calcium is not only an essential nutrient for plant growth,also plays an important role in the growth and development of plants. The prior studies have revealed that calcium may reduce the occurrence of plant diseases. Here we summarize the mechanisms of calcium eliminating plant diseases：Calcium involves in the formation of organizational structure and increases the stability of plant cells,induces plants to produce defense response,and regulates plant antioxidant system,therefore enhancing the disease-resistance of plant;calcium inhibits the growth of pathogen;calcium plays a synergetic role with biocontrol agents on the control of plant diseases and pathogens. In addition,the potential issues and application prospects of mineral elements on controlling plant disease are also discussed,aiming at providing the reference for further research.

Research Advances on Colonization of Plant Growth-promoting Rhizobacteria

SUN Zhen, ZHENG Liang, QIU Hao-bin

2017, 33(2): 8-15. doi:10.13560/j.cnki.biotech.bull.1985.2017.02.002

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The PGPB（plant growth-promoting bacterium）presents a promising prospect in agriculture due to its advantages such as inhibiting rhizosphere pathogens,promoting plant growth,and increasing crop production. This paper first summarizes the species of plant growth-promoting rhizobacteria and the promoting mechanisms of involved colonization,and demonstrates the colonization process of rhizobacteria as well as the factors influencing the colonization of bacteria in rhizosphere. Then the paper reviews the detection methods of microorganisms in the colonization of bacteria,including antibiotics marking,immunological methods,and exogenous gene marking methods. Further,the paper discusses high-throughput sequencing technologies widely used in the rhizosphere colonization. Finally,it is concluded that the future researches on interpreting strains in genetic level,gaining microbial community diversity in plant rhizosphere and the functional genes should be of significance to better predict the interaction of PGPB and its host plant,and to apply bio-fertilizers in field.

Research Progress on Optimizing the Expression of Exogenous Proteins in Escherichia coli

SU Peng, GONG Guo-li

2017, 33(2): 16-23. doi:10.13560/j.cnki.biotech.bull.1985.2017.02.003

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Escherichia coli expression system has become the first choice of expression of exogenous protein,the expression system for recombinant protein presents many advantages,and however,sometimes expressed exogenous protein is easily attacked by host protease or cannot form insoluble inclusion bodies because of incorrect folding,thus by which the expression is limited. This paper summarizes some optimization strategies used in the process of the expression of exogenous proteins in E. coli,mainly including the expression of rare codons,application of fusion tags,change of E. coli strains for expression,and reducing the formation of inclusion bodies,a single protein production system,auto-induction,fed-batch cultivation,high cell-density culture,etc.,which aims at exploring the optimal strategy for the expression of exogenous protein.

Research Progresses on the Role of Transport Proteins NupC and NupG During the Nucleoside Secretion Processing in Microbial Cells

WANG Meng-jiao, FANG Hai-tian, LIU Hui-yan, HE Xiao-guang, WU Qing, ZHAO Bei-bei

2017, 33(2): 24-29. doi:10.13560/j.cnki.biotech.bull.1985.2017.02.004

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The nucleosides are used extensively,and microbial fermentation is one of main methods of producing it. The secretion of nucleoside in microbial cells plays a critical role for high-yield nucleoside. There are many transport proteins as the role of transportation in the cell membrane,which are involved in the secretion of metabolites. Here,this article mainly elaborates the role of nucleoside transport proteins such as NupG and NupC during the nucleoside secretion process in microbial cells. Additionally,the effects of modification for transport protein on nucleoside biosynthesis were introduced,aiming at providing evidence for the breeding strategy of engineering high-yield strains.

Research Progress on Structure and Function of GLP-1R and Screening for Small Molecule Drugs

HU Zhong-ping, CHENG Nian, YANG Fan, SU Zheng-ding

2017, 33(2): 30-40. doi:10.13560/j.cnki.biotech.bull.1985.2017.02.005

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Glucagon-like peptide-1 receptor（GLP-1R）as an important target for type 2 Diabetes mellitus（T2DM）therapy,presents clinic significance. The breakthroughs on GLP-1R structures and functions have been made via structural biology and protein engineering. However,it is still unknown on the analysis of its full length structure,the molecular mechanism of polypeptide binding receptors,and the intrinsic mechanism of receptor activation. Owing to the rapid research progresses relating to GLP-1R,this article briefly describes the structure and function of the GLP-1 receptor and the leading compound of existing small molecule drugs,also discusses the developing direction and application prospects of action mechanism of the GLP-1 receptor molecule structure,aiming to provide structure base for the treatment of T2DM.

Applications of Riboswitch-based Novel Regulation System of Gene Expression

XIONG Ying-zhe, CAO Yuan-qin, XIAO Ling-hui, LI Zhao-fa

2017, 33(2): 41-46. doi:10.13560/j.cnki.biotech.bull.1985.2017.02.006

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Riboswitches are cis-acting elements that can respond to the environmental changes of the cell by altering its conformation to achieve the regulation of gene expression. Due to its characteristics of simple regulation system without proteins involved,response fast,short sequences with simple structure,and easy to design and modify,applying it in biomedical area should be in superiority. The review addresses the advance of the structure and regulation mechanism of riboswitches,as well as the applications of this novel regulation system of gene expression in gene therapy,the development of novel target for antibiotic,the safety switch for virus vaccine,and the new riboselectors and biosensors,aiming at inspiring the application of riboswitches in China.

Technology of Artificial microRNA and Its Application in Breeding Disease-resistant Plants

LIN Yi-hua, ZHENG Tao, GAO San-ji, CHEN Zhen-dong

2017, 33(2): 47-52. doi:10.13560/j.cnki.biotech.bull.1985.2017.02.007

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Endogenous microRNAs（miRNAs）are recognized as important regulators of gene expression in eukaryotes. Artificial microRNA（amiRNA）is an effective strategy of silencing endogenous gene based on natural miRNAs. Using amiRNA to replace the miRNA/miRNA* sequence in plant’s endogenous miRNA precursor,the generated amiRNA match the target gene,specifically suppressing the expression of target gene. Here,we summarize the synthetic process and action mechanism of natural miRNA,the basic design principles and construction methods of amiRNA,and the application of artificial miRNA technique in breeding disease-resistant plant.

The Latest Research Progress on CRISPR/Cas System

LIU Ni, LU Qin, LIU Juan, CHEN Hang

2017, 33(2): 53-58. doi:10.13560/j.cnki.biotech.bull.1985.2017.02.008

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The CRISPR/Cas is a system of immune mechanism for bacteria and archaea against exogenous virus and plasmid DNA invasion. As a novel genome editing technology,it possesses the advantages of a simple design,strong specificity,and high efficiency,bringing revolutionary breakthrough for genome directional modification,regulation and application. We introduce the research background,the existence type of the CRISPR/Cas system,and basic action mechanism of Type II and Type III CRISPR system and new research breakthrough in detailed,for providing the reference for readers and researchers in related fields.

Improvement of Alkaline Lysis Method for DNA Extraction from Tobacco

XU Zong-chang, WANG Meng, KONG Ying-zhen

2017, 33(2): 59-65. doi:10.13560/j.cnki.biotech.bull.1985.2017.02.009

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To expeditiously extract DNA templates from a large number of transgenic tobacco samples,the alkaline lysis method of extracting DNA from tobacco was improved in this study. Solution of 0.01 mol/L NaOH was used as the extraction buffer and the supernatant could be directly used as a template for PCR reaction,after crushing transgenic tobacco leaf tissues without heating/boiling and neutralizing procedures. The results showed that DNA template produced by NaOH solution of 0.01-0.1 mol/L was amplified successfully. The DNA template produced by the improved method did not rely on particular reaction condition,a variety of types of PCR Mix buffer all amplified successfully. Compared with the traditional SDS extraction method or commercial kits method,PCR results showed no significant differences. In addition,the improved method presented promising results while it was applied to other crops such as wheat,sorghum,rice and maize. The PCR products amplified from the DNA template by the improved method was able to be directly used for sequencing analysis. In conclusion,the improved method is in need of fewer instruments and less sample consumption,rapid,simple,low cost,no cross-contamination,and very efficient to handle a large number of samples,thus,it may rapidly extract and identify the DNA from a large number of transgenic tobacco samples.

Plant Bionic Remediation and Phytoremediation of Heavy Metal-contaminated Soil

HAO Da-Cheng, ZHOU Jian-qiang, WANG Chuang, HAN Jun

2017, 33(2): 66-71. doi:10.13560/j.cnki.biotech.bull.1985.2017.02.010

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Soil is essential for human survival and development. With the rapid advance of industrialization and urbanization,China is undergoing the increasingly serious soil pollutions. The aim of this study is to explore the feasibility of the novel plant bionic remediation and compare the effects of plant bionic remediation and phytoremediation on the heavy metal-contaminated soil. The effects of different filling materials of the bionic device,i.e.,sepiolite,kaolin,activated carbon,and diatomite,on the same metal ion（cadmium,chromium,zinc,or lead）,as well as the effects of the same filling material on the different metal ions,were investigated,and the effects of bionic remediation and phytoremediation were compared. Results showed that the bionic device differentially reduced four heavy metal ions. Cr of brown earth reduced most significantly in the sepiolite group,Zn and Cd dramatically reduced by all four types of filling materials,while diatomite-containing equipment was the most efficient in reducing Pb. This was correlated with the different features of heavy metals and the type of filling materials,closely correlated with the heavy metal abundance of soil and the saturated adsorption sites. Compared with single phytoremediation,the combined use of bionic remediation and phytoremediation substantially decreased Cd and Pb in red earth,and this approach caused no negative effects on the Cd and Pb accumulation in Brassica campestris,while Pb and Cd of filling materials were higher in sepiolite+diatomite+kaolin+activated carbon than in other types of filling materials. The bionic technology may be combined with phytoremediation,microbial remediation,and physicochemical remediation to perform the hybrid restoration of contaminated soil. The plant bionic remediation,as a novel restoration technology,may cost-effectively reduce the content of heavy metals in soil. Under the same conditions,the bionic remediation demonstrates higher efficiency and much better adsorption of heavy metals than phytoremediation.

Cloning and Identification of Seed-specific Promoter Ha ds10 G1 from Oil Sunflower（Helianthus annuus）

SUN Li, ZHOU Xi-ping, QI Zi-yun, WANG Meng-yao, CHEN Fu-long

2017, 33(2): 72-79. doi:10.13560/j.cnki.biotech.bull.1985.2017.02.011

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The objective of this work is to clone and identify the seed-specific promoter of Ha ds10 G1 in a LEA（late embryogenesis abundant）protein gene family from oil sunflower. The upstream regulatory region of Ha ds10 G1 gene was isolated from the genomic DNA of sunflower “Aidatou” by PCR method. The cloned region was fused to the GUS reporter gene,and plant expression vector pBI121-PHa ds10 was constructed,which was introduced into Nictiana tabacum NC89 by medicating Agrobacterium tumefaciens. The expression of GUS gene in the transgenic tobacco plants was detected by PCR,RT-PCR and GUS. As results,the gene promoter of Ha ds10 G1 was 1 417 bp,and it had homology with the reported sequence at 89.42%. Cis-acting elements search in the segment revealed that in addition to kernel regulatory sequences of promoter,there was the presence of a number of motifs related to tissue specificity,hormone,and adverse environment,such as RY-repeat elements,ABRE motif and TC-rich element,etc. PCR results showed that the transgenic plants were obtained successfully. GUS activity assays indicated that this promoter drove the expression of GUS gene only in tobacco seed embryo,and not in the other tissues such as roots,stems and leaves. Conclusively,the cloned 1 417 bp upstream regulatory region of Ha ds10 G1 gene from the LEA protein gene family of oil sunflower is seed-specific promoter. The results may provide the tissue-specific promoter for genetic improvement of oilseed crops such as oil sunflower.

Cloning and Expression Profiles of a Transcription Factor Gene ZjDREB4.1 in Zoysia japonica Under Adversity

LI Jing, WU Qi, ZHANG Lin-jie, LI Xu-ting, ZHOU Min-qi, WEI Shan-jun

2017, 33(2): 80-88. doi:10.13560/j.cnki.biotech.bull.1985.2017.02.012

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A novel DREB（dehydration responsive element binding）transcription factor gene,designated as ZjDREB4.1,was isolated from cultivar ‘Meyer’ of Zoysia japonica. The open reading frame（ORF）of ZjDREB4.1 was 651 bp in length,encoding 216 amino acid residues. The putative protein ZjDREB4.1 was 22.9 kD in molecular weight and with a theoretical isoelectric point（pI）of 5.74. Amino acids from the 50^th to the 110^th were predicted to build up a conserved AP2 domain. The phylogenetic tree analysis showed that ZjDREB4.1 was grouped with AtTINY of Arabidopsis thaliana and ZmDBF2 of maize,belonging to the A-4 group of DREB subfamily. ZjDREB4.1 was expressed constitutively in leaves. The expression was up-regulated under cold stress,and was first down-regulated then returned to normal level under drought and salt stresses.

Polymorphism of TMEM-18 Gene and Its Correlation with Production Traits in Yak

ZHANG Huan, ZHANG Quan-wei, WANG Qi, MA You-ji, ZHANG Yong, ZHAO Xing-xu

2017, 33(2): 89-96. doi:10.13560/j.cnki.biotech.bull.1985.2017.02.013

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The TMEM-18（transmembrane protein 18）gene is the one with oligomer in its terminal,and closely related to the growth and development of animal. This study aims to study the correlation between polymorphism of TMEM-18gene and production traits in yak. Firstly,constructing DNA mixing pool with blood samples from 192 female Tianzhu yaks,and the intron1 and exon5 of TMEM-18 gene were amplified. Then,BLAST and Chromas were used to analyze the mutation sites in TMEM-18 gene,high-resolution melting curve（HRM）to determine the genotype,software SHEsis to calculate and the frequencies of genotypes and alleles and to analyze the linkage disequilibrium and haplotypes in the SNPs of TMEM-18 gene,and software SPSS21.0 to understand the correlation between gene polymorphic loci and product traits. The experimental results showed that the intron1 of yak TMEM-18 gene contained 2 polymorphic loci of 861（A/G）and 1 267（C/T）,and the exon5 contained 1 polymorphic locus of 4 447（C/T）. Correlation analysis indicated that the different genotypes at locus 861（A/G）resulted in the significant differences on body height,body weight,carcass weight,and slaughter rate（P<0.05）;while the varied genotypes at locus 1 267（C/T）caused significant differences on body height,body weight and slaughter rate（P<0.05）;and the various genotypes at locus 4 447（C/T）let to significant differences on body length,tube girth,chest girth,body weight,carcass weight,meat weight,net meat rate,and slaughter rate（P<0.05）. There were 3 haplotype combinations in the yak populations,namely ACC,GTC and GTT. Among them,the number of ACC was significantly more than that of others,i.e.,it was dominant haplotype. The results suggested that the TMEM-18 gene could be used as molecular-marker candidate for developing the production traits of yak,providing a scientific basis for the utilization and development of yak genetic resources and breeding of new varieties.

Cloning and Expression Profile of Interleukin-1β Gene in Schizothorax prenanti

DUAN Hui-qin, WANG Li

2017, 33(2): 97-101. doi:10.13560/j.cnki.biotech.bull.1985.2017.02.014

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This work aims to characterize interleukin-1β（IL-1β）gene of Schizothorax prenanti. First,RT-PCR was used to amplify IL-1β gene with cDNA of S. prenanti spleen as template. Then,the IL-1β gene sequence was analyzed with various bioinformatics tools,and the expressions in 6 tissues of S. prenanti were detected used RT-PCR. As results,the length of IL-1β gene was 1 252 bp,and it encoded 276 amino acids. Its GenBank accession number was KU886235,the molecular weight of IL-1β was 31.25 kD,and pI was 5.45. It was induced as a hydrophilic protein,and its secondary structure mainly consisted of random coil and extended strand. There were 4 N-glycosylation sites and 16 phosphorylation sites in this protein. Phylogenetic relationships revealed that S. prenanti presented a closest genetic relationship with Cyprinus carpio and Carassius auratus. The analysis of tissue expression indicated that IL-1β gene was mainly expressed in the spleen and kidney. It provides theoretical evidence for further studying the biological function of IL-1β gene.

Construction of the Recombinant Expression Plasmid and Expression Pattern of Chitinase Gene MDCII from Musca domestica

YANG Yu-jin, GUO Guo, WU Qin-yi, LI Yan, FU Ping, ZHANG Yong

2017, 33(2): 102-108. doi:10.13560/j.cnki.biotech.bull.1985.2017.02.015

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The aims of this study are to isolate the chitinase gene MDCII from Musca domestica EST sequencing database,to clone the gene and analyze its molecular characteristic,and to explore the temporal-spatial expression patterns of the gene in different tissues and different developmental stages as well as after induced by different microorganism. Firstly,EST sequencing technology was employed to screen the chitinase gene MDCII from the constructed cDNA plasmid library of M. domestica larvae,the bioinformatics method to analyze the gene sequence and physicochemical properties of the encoded protein,and the neighbor joining to build phylogenetic tree. Then,the target gene amplified by PCR then was constructed into the recombinant plasmid pEASY-E1-MDCII,the recombinant plasmid was transformed in clonal cell Trans1-T1,and the real-time PCR technology was to detect the expression difference of the MDCII gene in different developmental stages and different tissues. Finally,injection method was used to induce different microorganisms into the 3 instar larvae of M. domestica,and real-time PCR to detect the changes of expression level in different time points after inducing. The results indicated that the ORF length of the MDCII gene was 1 374 bp,encoding 457 amino acids,and the molecular weight 51.6 kD. The alignment analysis of the phylogenetic tree revealed that the genetic distance of MDCII was close to the adult growth factor of Drosophila. The recombinant plasmid pEASY-E1-MDCII with correct gene sequence was successfully constructed. The MDCII gene expressed in different developmental stages of M. domesticaat different expression levels,and the highest in salivary glands and fat body of 3 instar larva. The MDCII gene presented up-regulated expression at 3 h after induction of Candida albicans,Staphylococcus aureus,and Escherichia coli. In conclusion,the MDCII gene is the adult growth factor in chitinase,participating in growth and development of M. domestica,and also playing a certain role in immune defense process.

Cloning of β-amyrin Synthase Gene from Panax japonicus var. major and Its Bioinformatics Analysis

WU Ya-yun, ZHAO Xiao-long, CHEN Ping, ZHANG Shao-peng

2017, 33(2): 109-117. doi:10.13560/j.cnki.biotech.bull.1985.2017.02.016

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β-amyrin synthase（βAS）,the rate-limiting enzyme saponin of directional shunt oleanane and dammarane types,plays an important role in biosynthesis of triterpenoid saponin in medical plants. The primers were designed based on the transcript sequence of βAS from the transcriptome dataset of P. japonicus var. major. And the full-length cDNA sequence of βAS was cloned from the rhizome of P. japonicus var. major by utilizing RT-PCR method,named as pjβAS. The length of this gene was 2 655 base pairs containing a complete open reading frame of 2 286 base pairs and encoding 761 amino acids. The accession number in GenBank is KX254566. The bioinformatics analysis showed that the putative molecular weight and theory isoelectric point of protein encoded by pjβAS were 87.90 kD and 5.84,respectively. The protein contained no transmembrane domain,was a non-secretory protein with 38 phosphorylation sites,it played a physiological role in chloroplast and endoplasmic reticulum. The secondary structure of the protein indicated that α-helix accounted for 39.82,extended strand for 16.56%, β-turn for 10.38%,and random coil for 33.24%. Meanwhile,three feature conserved motifs of oxido squalene cyclase（OSC）were found in pjβAS,i.e.,DCTAE,QW（QXXXXXW）,and MWCRCY. The results of the sequence homology comparison concluded that the protein encoded by pjβAS was in 100% similarity with Panax japonicus C. A. Mey（AKN23431.1）,and pjβAS gene was classified in the same branch with Panax japonicus C. A. Mey（AKN23431.1）,Panax quinquefolius（AGG09939.1）,and Bupleurum chinense DC（ABY90140.2）in phylogenetic analysis. Real-time fluorescence quantitative PCR was used to analyze the relative expression quantity of pjβAS gene in different tissues of P. japonicus var. major,the results showed that there were different expression trends in flowers,leaves,stems and rhizomes,with the highest in leaves but the lowest in rhizomes.

Biology and Expression Characteristics of Trehalose-6-Phosphate Synthase Gene from Banana

XING Wen-ting, LI Ke-ming, JIA Cai-hong, SHU Hai-yan, WANG An-bang, SUN Pei-guang, XU Gui-ying

2017, 33(2): 118-124. doi:10.13560/j.cnki.biotech.bull.1985.2017.02.017

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Our aim is to explore the role of trehalose-6-phosphate synthase gene in the stress tolerance of banana plant by studying its biological function. A gene from banana rhizome’s transcriptome,designated as MaTPS5,was reaped using the method of random cloning and sequencing. Bioinformatics analysis showed the full length of MaTPS5 cDNA sequence was 3 081 bp,containing a complete open reading frame of 2 559 bp,encoding 852 amino acids. Protein MaTPS5 was an unstable and hydrophobic protein with iso-electric point of 6.52,in which there were two structure domains of GT1-TPS and TPP. It was located in cytoplasm and had two trans-membrane regions but no signal peptide. Comparing MaTPS5 protein sequence to the known plant TPS amino acid sequence,the homology level was 77.31%. Phylogenetic analysis showed MaTPS5 protein sequence was clustered on a common branch with the TPS protein sequence of wild banana,indicating that they were the close relatives and had a common ancestor. Tissue-specific analysis indicated MaTPS5 expression level was high in corm,leaves,and flowers,but the lowest in the roots. qRT-PCR analysis showed MaTPS5 expression increased when banana plantlets were treated with ACC and salt,and peaked at 24 h. After treating banana plantlets with Foc TR4 pathogen,the MaTPS5 expression level rose and was constant in three hours. The results showed MaTPS5 may depend upon the ethylene signaling transduction pathways to induce itself expression,and synthesize trehalose,and therefore participate in stress tolerance of banana plants.

Expression and Analysis of an Amylase with Agarase Activity from a Marine Bacterium

LIN Bo-kun, SONG Yan, LU Guo-yong, ZHAO Min, ZHONG Ming-qi, HU Zhong

2017, 33(2): 125-130. doi:10.13560/j.cnki.biotech.bull.1985.2017.02.018

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Our aim is to confirm that there is amylase with agarase activity in the novel marine bacterial strain ZC1. The amylase gene amy440 of strain ZC1 was cloned and expressed in recombinant prokaryotic vector and the amylase and agarase activity of the recombinant protein was determined using the method of activity staining electrophoresis. Results indicated that the recombinant protein of gene amy440 showed amylase and agarase activity in the activity staining electrophoresis. Conclusively,the encoded protein of gene amy440 of strain ZC1 is an amylase with agarase activity.

Isolation and Identification of Endophytic Fungi from the Rhizomes of Xinjiang-specific Plant Brassica rapa

LIANG Han-qiao, Dilidaer Kudereti, BAI Fei-rong, MIAO Lei, LI Nan-nan, LIU Su-hui, CHEN Jian-guo, LIU Yang, SUN Yu-ping

2017, 33(2): 131-136. doi:10.13560/j.cnki.biotech.bull.1985.2017.02.019

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The endophytic fungi in plants are closely related to their own growth and biological defense,therefore,the root tissue of healthy Brassica rapa was selected for isolating and purifying the endophytic fungi,the community diversity of the endophytic fungi were preliminarily studied with morphology and molecular identification method. Twenty-nine strains of endophytic fungi were isolated,and identified as 11 species in 8 genera. The preponderant fungi were Acremonium sp.（34.48%）and Penicillium sp.（27.59%）. This is the first research of isolating endophytic fungi in Xinjiang-specific medical plant B. rapa L.,and its taxonomic status is determined.

Species Identification and Growth Characteristics of Endophytic Anti-infective Bacteria from Herba siegesbeckiae

CHEN Shao-hong, ZHAO Peng-chao, CHEN Ming-zhao, LI Luo-yi, LI Yue, JIA Jing-jing

2017, 33(2): 137-142. doi:10.13560/j.cnki.biotech.bull.1985.2017.02.020

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Herba siegesbeckiae has many healing values,and our aim is to isolate and screen highly active antagonistic endophytes from its fruits. The biological characteristics of anti-infective bacterium C-5-1 were determined by antagonistic experiment,morphological observation,physiological and biochemical tests,sequence alignment of 16S rRNA gene,analysis of phylogenetic tree,measuring OD₆₀₀,drug sensitive tests,and ultra-filtration centrifugation. The results showed that the C-5-1 was identified as Brevibacillus laterosporus and was sensitive to various pathogenic bacteria,especially Pseudomonas aeruginosa. The strain C-5-1 had limited tolerance to low pH and highly saline environments,i.e.,inhibited completely or even died in pH≤5 or more than 15% NaCl,but it grew well in pH≥7 and 0-5% NaCl,suggesting that C-5-1 had good alkali resistance,but not resistant to acid and salt. Screening experiment revealed that the antibiotic ampicillin was highly sensitive to C-5-1,but resistant to 5 pathogenic bacteria. Fermentation supernatant of C-5-1 contained two antibacterial substances and relative molecular mass of both was less than 2 000. Conclusively,H. siegesbeckiae having excellent microbial resources will be a key resource for the development of anti-bacteria drugs.

Biological Characteristics of Bacillus subtilis Bacteriophage and Mutagenesis Breeding of Resistant Strains

LIU Xiu-xia, XU Hai-yan, XIN Guo-qin, MU Xi-jun, SUN Xue-sen, GU Wei

2017, 33(2): 143-148. doi:10.13560/j.cnki.biotech.bull.1985.2017.02.021

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Bacteriophage infection usually occurs in industrial fermentation,thus this work aims to isolate and screen bacteriophage with Bacillus subtilis as host,to analyze its biological characteristics,and to screen anti-phage strains via spontaneous mutation. With B. subtilis KC-260 as the host bacterium,a bacteriophage,designated as P260,was isolated and purified from the fermentation broth. Its titer was measured after enrichment culture,and then its biological characteristics such as thermal stability,optimal multiplicity of infection,host range,inhibitor,disinfectant-tolerance,etc. were investigated. In order to prevent the contamination from phage P260,anti-phage 260 strains were screened by spontaneous mutations. As results,titer of P260 reached 3.45×10¹⁰ PFU/mL,it had a certain host specificity,and not sensitive to Bacillus coagulans and Bacillus pumilus. P260 easily lost its activity at high temperature,i.e.,not heat-resistant,and its survival rate under 70℃ condition for 15 min decreased significantly. ClO₂ in 25 mg/L had a strong exterminate effect on P260. The 0.5% ammonium oxalate and ammonium citrate presented inhibition to P260. Optimal multiplicity of infection of phage P260 to the host was 0.01,one step growth curve showed that the incubation period was 15 min and the lytic stage started after 45 min. An anti-phage strain ZF-260 was obtained by spontaneous mutation. ZF-260 was not affected when adding phage P260,which indicated that ZF-260 was an excellent strain of anti-phage P260. In conclusion,the biological characteristics of phage P260 was studied,and ZF-260 with stable genetic and high viable cell count was obtained.

Activity Detection of Chloroplast Promoter Based on Tetracycline Regulatory System in Escherichia coli

YU Xiao-jun, CAO Shao-yu, DONG Yu-mei, BI Bao-liang, ZHANG Ying-hua, XU Jun-qiang

2017, 33(2): 149-154. doi:10.13560/j.cnki.biotech.bull.1985.2017.02.022

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In order to study tetracycline regulatory system regulating the activity of promoter in chloroplast genetic engineering,having the tetracycline（Tet）gene and the core regulatory area of TetR（Tet repressor）specific recognition sequences as the basis,the promoter prrnO1O2 with tetracycline core regulatory area was synthetized,then ligated to expression vector Bio3-GFP,and the activity of the promoter was validated in Escherichia coli. The expression of the GFP gene driven by the promoter led colonies to be bright green. The expression vector Bio3-TetR-prrnO1O2-GFP for GFP gene induced by Tet was constructed,and screening experiment confirmed that the highest concentration of tetracycline for the proper growth of E. coli was 5 μg/mL. Finally,GFP expression vector based on tetracycline regulatory system was constructed,the function of prrnO1O2 promoter was suppressed while without adding tetracycline. GFP gene expressed to be green fluorescent protein after adding tetracycline. These results indicated that tetracycline regulatory system controlled the activity of chloroplasts promoter prrnO1O2 in E. coli,thus avoiding the regulation interference of nuclear genome to plastid genome,and which provides effective ways and methods to plastid genetic engineering breeding further.

Construction and Biological Characterization of Gene dlp Deletion Mutant of Deinococcus radiodurans R1

LIU Xiao-li, JIANG Shi-jie, XUE Dong, LIU Ying-ying, WU Xiao-li, FENG Shuai, HAN Jia-hui, WANG Yu-zhou, PING Shu-zhen, WANG Jin

2017, 33(2): 155-163. doi:10.13560/j.cnki.biotech.bull.1985.2017.02.023

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Deinococcus radiodurans（DR）has drawn growing attentions for its superior resistance to abiotic stress. To investigate the effect of the hydrophilic protein Dlp in cell tolerance to abiotic stress,the dlp deletion mutant strain（Δdlp）was obtained by fusion PCR and homologous recombination in vivo. The assays of wild strain DR and mutant Δdlp were analyzed under abiotic stress. The results showed that the deletion of dlp gene in D. radiodurans led cells to be more sensitive to high-salt and oxidative stresses. The in vitro protective effect of Dlp protein on the activities of MDH and LDH was measured under oxidative stress condition;and the data demonstrated that the addition of Dlp protein alleviated the loss of activities of MDH and LDH under oxidative stress. Our findings indicated that hydrophilic protein Dlp enhanced the resistance of D. radiodurans to abiotic stresses,and functioned as chaperone-like protein to protect enzyme activity under abiotic stresses.

Effect of Ferritin DrfE on Antioxidant Enzyme Activity in Deinococcus radiodurans

WU Xiao-li, LIU Ying-ying, JIANG Shi-jie, CHEN Yun, LIU Xiao-li, WANG Yu-zhou, PING Shu-zhen, WANG Jin

2017, 33(2): 164-171. doi:10.13560/j.cnki.biotech.bull.1985.2017.02.024

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To investigate the function of the ferritin DrfE encoded by gene drfE in Deinococcus radiodurans,we constructed the complemented strain pdrfE∷Δ,and compared the stress resistances of the wild-type WT,mutant ΔdrfE and complemented strain pdrfE∷Δ under different doses of hydrogen peroxide（H₂O₂）and NaCl. Also,the Fe²⁺concentration and antioxidant enzyme activities of three strains were measured under oxidative stress. The results showed that the deletion of gene drfE led the strain to be more sensitive to oxidative and high-salt stresses and the complementation of gene drfE restored the resistance of the strain to oxidative and salt stress. Furthermore,the deletion of drfEresulted in the Fe²⁺ concentration in D. radiodurans cell increased from 232 μmol/L to 293 μmol/L in vivo. After treated with 80 mmol/L H₂O₂for 30 min,the CAT and SOD activities of the mutant ΔdrfE decreased by 32.74% and 41.3%,respectively,than that of wild-type. The above findings implied that the ferritin DrfE might be involved in iron metabolic pathway of D. radiodurans and played a key role in antioxidant system.

Patterns of Biofilm Formation in Pseudomonas stutzei Under Abiotic Stresses

YAN Ning, YANG Zhi-min, SHANG Li-guo, DAI Shu-ling, ZHAN Yu-hua, LU Wei, LIN Min, YAN Yong-liang

2017, 33(2): 172-178. doi:10.13560/j.cnki.biotech.bull.1985.2017.02.025

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In order to systematically investigate the effects of abiotic stress factors on the growth and biofilm formation of Pseudomonas stutzeri A1501,we measured the variation patterns of growth and biofilm formation of A1501 under different NaCl concentrations,pH and temperature. The results revealed that above 3 environmental factors did influence the growth and the biofilm formation of A1501 strain with certain extent of regularity. The optimal growth condition of A1501 with those factors was 2 mmol/L NaCl,pH6.8,and 30℃,respectively. However,the biofilm formation reached the maximum in the stress condition of 0.6 mol/L NaCl,pH6.0,and 40℃ with 7.8-,7.0- or 2.0-fold increment compared to the optimal growth condition,respectively. Among the three factors,NaCl showed the most significant effect on the biofilm formation of A1501. In a conclusion,under the tested conditions,the biofilm formation of A1501 strain was negatively correlated with the growth of strain,i.e.,high salt,weak acid and high temperature were adverse to the growth of A1501,however,it stimulated the biofilm formation.

Cloning of Ferritin Gene Fth1 and Its Expression in the Bone Marrow Mesenchymal Stem Cells of Rat

LIU Wei, ZHU Xiu-liang, LI Qing-hai

2017, 33(2): 179-184. doi:10.13560/j.cnki.biotech.bull.1985.2017.02.026

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Aimsare to construct a lentivirus encoding rat ferritin heavy chain 1（Fth1）and to detect its expression in rat bone mesenchymal stem cells（RMSCs）. The rat Fth1 gene was amplified by PCR and cloned into the lentivirus expression vector pLenti-GFP-C. The lentivirus carrying pLenti-GFP-C-Fth1 or empty vector was then packaged in HEK 293T cells,by which the RMSCs were infected. GFP expression was observed under a fluorescence microscope. Fth1 expression was detected by Western blot. WST-1 reagent was used to analyze the proliferation of infected cells（RMSC-Fth1）and empty vector（RMSC-GFP）. Resultsof DNA sequencing showed that the pLenti-GFP-C-Fth1 lentivirus expression vector was successfully constructed. After RMSCs were infected by the packaged lentivirus,GFP was observed under a fluorescence microscope,indicating the infection was achieved. A specific band of Fth1 in the lysate of Fth1-lentivirus infected cells（RMSC-Fth1）was detected by Western blot. Cell proliferation assay showed that the growth of RMSC was not affected by overexpressing Fth1 compared with RMSC-GFP. As conclusion,the recombinant lentivirus vector carrying rat Fth1gene was successfully constructed,infected RMSC as expected,and expressed FTH1 protein even without significant impacts on the cell proliferation.

Effect of Regular Repetitive Sequence on the Thermal Hysteresis Activity of Antifreeze Protein ApAFP914 in Anatolica polita

WANG Jun, DU Rong-feng, LIU Zhong-yuan, MAO Xin-fang

2017, 33(2): 185-191. doi:10.13560/j.cnki.biotech.bull.1985.2017.02.027

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This work aims to explore the relationship between protein structure and thermal hysteresis activity of antifreeze protein ApAFP914 in Anatolica polita. A regular repetitive sequence was added in the ApAFP914 by gene synthesis. The synthesized gene was then expressed in prokaryotic cells and the thermal hysteresis activity of the protein was determined. Results showed that the gene ApAFP-C914 with a regular repetitive sequence added was 312 bp. The fusion protein TrxA-ApAFP-C914 was verified by SDS-PAGE and Western bolt,and its molecular weight was 34 kD. The measurement by differential scanning calorimetry showed that the thermal hysteresis activity of ApAFP increased significantly（P<0.05）by adding a regular repetitive sequence at the concentration of 50 μg/mL. Conclusively,adding repetitive sequence may significantly increase the thermal hysteresis activity of antifreeze protein ApAFP914 in A. polita.

Screening and Optimization of Co-crystallization Condition of α-Conotoxin GIC Complex with Ac-AchBP

LIN Bo, LIU Kun, ZHU Ming-yue, LI Wei, DONG Xu, LI Meng-sen

2017, 33(2): 192-198. doi:10.13560/j.cnki.biotech.bull.1985.2017.02.028

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The aim is to screen and optimize the co-crystallization condition of α-conotoxin complex with acetylcholine binding protein（AChBPs）,and to obtain co-crystals with high resolution. AChBP from Aplysia californica（Ac-AChBP）was expressed in insect expression system and purified by gel filtration chromatography. Then,the purified Ac-AChBP was co-crystalized with α-conotoxin GIC,further,the HAMPTON RESEARCH crystal kit and the crystal robot were applied screening and optimization of the growth condition of crystallization. Results showed that crystal grew well under the growth condition of 0.6 mol/L ammonium citrate dibasic and pH4.8 of 0.1 mol/L sodium acetate trihydrate,and the resolution of the crystal reached 2.1 Å. In conclusion,the co-crystal of α-conotoxin GIC complex with Ac-AChBP with a high resolution may be acquired via screening and optimization of crystallization condition（The molar concentration of ammonium citrate dibasic and the pH of 0.1 mol/L Sodium acetate trihydrate）.

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2017, 33(2): 300.

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