Table of Content

26 May 2018, Volume 34 Issue 5

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The Advantages and Limitations of CRISPR/Cas9-based Gene Editing Technology

WU Yan, HAO Ya-qiao, WEI Xuan, SHEN Qi, LIU Ye-fei, WANG Sheng-hou, ZHAO Hong-xin

2018, 34(5):  1-8.  doi:10.13560/j.cnki.biotech.bull.1985.2017-1100

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CRISPR/Cas9 is becoming a hot spot in genome editing,and it provides a more efficient way for gene targeting than zinc finger endonuclease（ZFN）and transcription activator-like effector nuclease（TALEN）. CRISPR/Cas9 system exists widely in bacteria and archaea,carrying out the adaptive immune responses specifically recognizing the invaded genetic elements from phage infection,plasmid conjugation and transformation as well as and degrading exogenous DNA. CRISPR/Cas9 is a precise editing technology by which the target sites are recognized by a segment of 20 bp small RNA. CRISPR/Cas9 has the advantages of simple design and operation,high efficiency,and broad generality,thus it is a novel and revolutionary precise gene editing technology. This review induced and summarized the aspects of the discovery of CRISPR/Cas,mechanisms,gene editing,and application limitations,aiming at providing references for understanding its mechanisms and applying precise gene editing technology.

Research Progresses on the Development and Application of CRISPR Technology

LIANG Li-qin, YAN Jing, ZHANG Xin, HAO Ze-ting, DUAN Jiang-yan

2018, 34(5):  9-16.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0608

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CRISPR,an adaptive immune defense system based on RNA,whose space sequence has homologous with the sequences of phage or plasmid,can use RNA that has specificity in target point to guide the Cas proteins to target the genetic error genes in almost all organisms and cells. This gene editing technique,compared with zinc finger nuclease（ZFN）and transcription activator-like effector nuclease（TALEN）,is cheaper and more efficient. Therefore,since its first application in 2013,CRISPR has been accepted quickly and applied to the studies by the masses of Chinese and foreign researchers,becoming a new hot spot in today's life science research. Regarding the great significance of CRISPR in future scientific studies and practical applications,this article reviews the recent research progress on CRISPR and its applications in gene therapy,and discusses the limitations of this technology and the solutions. Finally,the foreground of CRISPR is prospected for providing useful reference for relevant researchers.

Probe into Application of CRISPR/Cas9 Technology in Epigenome

HE Shuai, WANG Shu-hui, SUN Xiu-zhu

2018, 34(5):  17-21.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0358

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CRISPR/Cas9（clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins）technology is the most widely used novel gene editing tool in the past five years. With the advantages of low cost,high efficiency,multi-site target and predictable off-target effects,it is becoming a powerful tool for designing genomes in different organisms. The continuous development of epigenetics challenges the central principles in molecular biology,including the modification of bases and histones in DNA and the crucial role of non-coding RNA in the regulation of genome and chromatin structure. The alteration of CRISPR/Cas9 system may modify the epigenome. This article summarizes the application of CRISPR/Cas9 system in modifying epigenome,discusses the problems in the directional modification of the genome,and provides references for the application of CRISPR/Cas9 system in the field of genome editing.

Advances in Bacterial Multiplex Genome Engineering and Its Applications in Synthetic Biology

GUO Ming-zhang, LIU Hai-yan, DU Ruo-xi, XU Wen-tao

2018, 34(5):  22-31.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0089

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Multiplex genome engineering refers to the technology that can simultaneously edit multiplex genome targets in one cell by a single experiment. High-throughput is a key direction of gene editing technologies,and the occurrence and development of multiplex genome engineering is an important intermediate stage of high-throughput genome engineering. So far there are mainly three strategies for multiplex genome engineering in bacteria,including iteration based editing,CRISPR/Cas9-based newly developed high-efficiency tools editing,and large synthetic DNA-based editing. In this article,the principles and developments of these three kinds of multiplex genome engineering technologies,as well as their applications in synthetic biology of microorganism are reviewed,and the shortcoming of these technologies are discussed. Besides,we propose the future development directions of multiplex genome engineering.

CRISPR/Cas9 Genome Editing Technology and Its Application in Streptomyces

QIAO Long-liang, PANG Jian-hu, DANG Chen-yang, HUANG Hai-long, ZHU Peng

2018, 34(5):  32-40.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0877

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Streptomyces are known to produce a variety of antibiotics and have long been a research focus in life sciences and medicine,pharmacy,and industry. However,the genetic manipulation of Streptomyces is very difficult,thus the traditional means of genetic editing due to low efficiency,long experimental cycle and cumbersome steps and other reasons are gradually replaced by CRISPR/Cas9 system called as “magic scissors”. This technique allows for targeted editing of genome-specific sites,including indel,repair and replacement. By comparing the current gene editing methods in Streptomyces,it is concluded that CRISPR/Cas9 system has the advantages of simple operation,high efficiency,low cost,short experimental cycle and the ability to simultaneously knock out multiple genes in the application of Streptomyces. We also review the application of the system in Streptomyces and summarize the target space of CRISPR/Cas9 toolbox. Finally,we prospect the potential of CRISPR/Cas9 system in Streptomyces orientated-breeding and synthetizing new antibiotics,aiming at providing references for the work of related fields.

Research Progress of Gene Editing Technology on Cattle

TANG Bo, SUN Zhao-lin, DAI Yun-ping

2018, 34(5):  41-47.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0413

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Gene editing technology is an emerging biotechnology for site-directed modification of the genome,including zinc-finger nuclease（ZFN）technology,transcriptional activator-like effect nuclease（TALEN）technology and the recently developed CRISPR/Cas9 technology,which has exerted unprecedented influence in the breeding of the animal and plant,biopharmaceuticals and gene therapy,etc. In this review,we summarize the brief history of gene targeting and gene editing technology,and highlight the research progress of gene editing technology in the fields of the resistance of disease,the improvement of milk quality and lean meat percentage,and the cultivation of new varieties such as hornless cattle. Finally,we also propose the establishment of a gene editing plants and animals’ supervision system to speed up the industrialization of genetic engineering animals in China.

Applications and Challenges of Gene Editing in Non-human Primate Models

BI Yan-zhen, XIAO Hong-wei, ZHANG Li-ping, REN Hong-yan, HUA Zai-dong, HUA Wen-jun, WANG Zheng, NIU Min-jie, LIN Zheng-yun, REN Xi-dong, SUN Li-hua, ZHENG Xin-min

2018, 34(5):  48-56.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0891

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There is a pressing urgency in the medical field to establish an effective animal model to study human diseases. Non-human primates,which are evolutionary,physiologically and pathologically closer to humans than any other animal,are thus considered to be excellent animal models for studies of human diseases. With the development of gene manipulation technologies,researchers have successfully created a variety of non-human primate models via gene editing. Yet,there are still some challenges we have to face,such as off-target,mosaic mutants,and low efficiency of gene knock-in in CRISPR/Cas9. In this paper,we summarize the applications of gene editing in non-human primate models,as well as the existing problems and improvements,aimed to provide a reference for further research in non-human primate models mimicking human diseases.

Research Progress of CRISPR System and Its Application in Mice

DONG Wei-peng, WANG Jun-shi, ZHANG Shao-hua, YAN Jiong

2018, 34(5):  57-63.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0530

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As a gene editing tool,CRISPR system has been in rapid development since the first application in mammal in 2013, and iIt has been widely used in the field of molecular biology with its simple design,short time consuming,and high efficiency advantages. So far,its applications have far exceeded ZFNs and TALEN technology. With the continuous exploration of varied techniques from the CRISPR system,mice as a basic experimental animal play an important role in the application of CRISPR system. At present,the application tools developed from CRISPR system have been successfully applied to a variety of cells cultured in vitro. The technology for producing animal models by editing embryonic cells is also maturing,and it has its unique advantages in the treatment of diseases,especially in the treatment of hereditary diseases. And its scope of application is still expanding,and the mode of application is also in constant innovation. This paper mainly introduces the structure and mechanism of CRISPR system;also describes several major CRISPR application tools acting on DNA for gene knockin,knockout,gene silencing,transcriptional regulation,etc.,discuss several improvement measures focusing on its major deficiency,i.e.,the off-target effects,and its important role in the establishment of mouse disease models and treatment of diseases.

Gene Editing in Mouse Lin^-Sca1⁺Kit⁺ and Double Negative（DN）Primary Cell

WANG Yue-qiang, Avinash Bhandoola

2018, 34(5):  64-70.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0903

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The immune system of mice（Mus musculus）contains a large number of cell groups with different functions. In order to study the development mechanism of mouse immune system,it is necessary to establish efficient genetic manipulation techniques. Firstly CD4^-CD8^- double negative thymocytes（isolated from Cas9 knock-in mice）was chosen for this study,then sgRNAs（single guide RNA）were designed to target GFP and mouse CD4 cell surface antigen genes,and murine stem cell virus was used as a vector to deliver sgRNA into the cell,and by which gene editing was realized. The successful knockout of GFP and CD4 were detected by flow cytometry analysis. Based on these results,using Lin^-Sca1⁺Kit⁺ cell（isolated from Cas9 knock-in mice）as study materials,sgRNAs were designed and constructed with the same method for gene editing. After gene editing,the LSK cells did not differentiate into downstream T cells,but their myeloid differentiation potential was not affected. All these results suggest that we succeed in establishing mouse DN and LSK primary cell gene-editing platform.

Functional Study of miR-22 in Mouse Embryonic Stem Cell with CRISPR/Cas9 System

ZHANG Xue, CHEN Liang-liang, DAI Hong-xia, ZHANG Wen-sheng, REN Wen-yan

2018, 34(5):  71-79.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0964

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This study aims to construct neomycin-resistant sgRNA expression plasmids and to generate miR-22 knockout mouse embryonic stem cells（mESCs）with CRISPR/Cas9 for examining miR-22’s regulatory roles. First,point mutation and overlap PCR were performed to construct neomycin-resistant sgRNA expression plasmids. Then,sgRNA expression plasmids of miR-22 knockout were electroporated into mESCs with stable Cas9 expression. After neomycin selection and PCR genotyping,the miR-22 homozygous knockout mESCs were screened. The homozygous deletion was confirmed by RT-qPCR experiments and the efficiency of homozygous knockout was about 6.67%. In addition,the deletion of miR-22 presented no obvious effect on the morphology of mESC as well as the expressions of pluripotent markers including Oct4,Sox2 and Nanog. Together,miR-22 may be not necessarily required for the maintenance of embryonic stem cell,while the roles of miR-22 in controlling mESC differentiation and cell fate decision need further identification.

Construction of FOXA2 Expression Tracer System Based on CRISPR/Cas9 System

CHENG Hao-jie, ZHANG Zhen-wang, TAN Gui-xiang, LIU Yu-xiang, PIAO Hui-tong, TAN Yong-jun

2018, 34(5):  80-86.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0111

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This study enabled the tracing of FOXA2 gene on and off by using CRISPR/Cas9 system to insert a green fluorescent protein（GFP）nucleic acid sequence following the FOXA2 protein coding region. The study was carried out from the following five aspects：targeting sequence selection,cloning of Cas9-targeting plasmids and donor fragment,cells transfection and flow cytometry fluorescence sorting,limited dilution of positive cells,as well as enrichment culture and identification,transcription and translation level verification of monoclonal cells. The results showed that the expression of GFP was positive in the edited breast cancer cell line MCF-7. The expression level of GFP in breast cancer cells was positively correlated with the expression level of FOXA2 protein,and the expression of GFP and FOXA2 decreased synchronously after the induction by EGF,an inducing factor in the process of tumor cells epithelial-mesenchymal transition（EMT）. The results indicate that CRISPR/Cas9 targeting FOXA2 and using intracellular homology directed repair（HDR）to insert GFP sequence are success. Using the expression level of reporter gene protein for tracing the target gene on and off as well as its expression is significantly accurate and convenient.

Sequence Analysis on the CRISPR Sites of Lactobacillus bulgaricus

CHENG Na, CAI Zhen-hua, LÜ Jia-ping, JIA Zhen-hu, PANG Xiao-yang

2018, 34(5):  87-93.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0952

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CRISPR（Clustered regularly interspaced short palindromic repeats）sequence in the genome of Lactobacillus is its important part to recognize and resist phage,and CRISPR and cas protein constitute the acquired immune system. Currently there are few studies on the CRISPR sequence of Lactobacillus. In this study,clone sequencing was performed in the CRISPR of 8 strains of L. bulgaricus from different sources,and then the evolutionary genetics analysis to the repetitive sequence was also conducted,and the secondary structure was predicted;moreover the homology analysis in the spacer sequence was also carried out. Results showed that the lengths of CRISPR in 8 different strains of L. bulgaricus varied,among which the longest one was 1820 bp with 30 spacer sequences,and the shortest one was 408 bp with 5 spacer sequences. Two different structures were found in the prediction on the secondary structure of CRISPR repetitive sequence：one was the typical RNA mainly based on “ring” shape,another was the one mainly based on “stylo” shape. The former one had typical crRNA after cleaving,while the function of the latter needs to be further studied. This paper laid the foundation for exploring the molecular mechanism of L. bulgaricus against phage and has great significance in screening anti-phage fermentative strains in the dairy industry via analyzing the CRISPR sequence of L. bulgaricus.

Enhanced Production of Cellobiohydrolase in the Recombinant Saccharomyces cerevisiae by MRP8 Overexpression

WAN Qing-qing, LI Jie, ZENG Yu, ZHANG Ming-ming, ZHAO Xin-qing, BAI Feng-wu

2018, 34(5):  94-100.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0380

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Cellulase production and secretion using budding yeast Saccharomyces cerevisiae as a host is one of the strategies for the production of cellulosic ethanol by consolidated bioprocessing（CBP）. However,the ability of cellulase secretion by S. cerevisiae still needs to be improved. Therefore,the aim of this study is to improve cellulase production by the recombinant yeast. It was reported that elevation of genes involved in oxidative stress tolerance improved cellulase production,we thus investigate the effect of overexpression of MRP8,which encodes the mitochondrial ribosomal protein in the recombinant yeast producing CBH1. MRP8 was overexpressed through CRISPR/Cas9 genome editing in the recombinant S. cerevisiae strain producing CBH1. The underlying mechanisms for improved CBH1 production were explored. Results showed that omparing with the control strain,the CBH1 activity was improved by 80% in the MRP8 overexpression strain,and no significant effect on growth ability was observed by MRP8 overexpression. Real-time quantitative PCR analysis showed that the transcription level of CBH1 was increased in the recombinant strain overexpressing MRP8,whereas,the transcription levels of key genes related to protein folding and secretion exhibited no change. No difference on cell growth in the presence of Congo red,Tunicamycin（TM）or Dithiothreitol（DTT）was observed. In addition,no significant difference in ATP content and reactive oxygen species（ROS）accumulation was detected. These results indicate that overexpression of MRP8 enhanced production of CBH1.

Analysis of Transcriptional Activity and Interaction Proteins of WRKY31 Transcription Factor in Arabidopsis thaliana

WANG Yi-qiao, ZHAO Xin-jie, NIU Fang-fang, GUO Xiao-hua, YANG Bo, JIANG Yuan-qing

2018, 34(5):  101-109.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0918

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In order to deepen our understanding on functionally-unknown members in AtWRKY gene family,and to explore the roles and regulatory mechanisms of WRKY genes in Arabidopsis thaliana in response to abiotic stresses,we chose to study AtWRKY31 gene. First,we performed the subcellular localization assay of AtWRKY31 and analyzed its transcriptional activity. Then,we examined the expression changes of AtWRKY31 under different stress treatments for 1 h,3 h and 12 h through qRT-PCR. At last,we used yeast two-hybrid（Y2H）and bimolecular fluorescence complementation（BiFC）assays to screen and validate the interacting partners of WRKY31. The results showed that AtWRKY31 protein was located in the nucleus and acted as a transcriptional repressor. qRT-PCR showed that the expression of WRKY31 was significantly inhibited under heat,low potassium and low nitrogen treatments. Through yeast two-hybrid and BiFC assays,WRKY31 protein was identified to interact with itself,WRKY6 and WRKY42,respectively. Conclusively,WRKY31 in Arabidopsis thaliana,as a transcriptional repressor,may be involved in the regulation of some abiotic stress responses by the interactions with transcriptional factor WRKY6 and WRKY42.

Analysis of Mineral Elements in Maize Kernel Treated with Leaf Fertilizers

LI Wen-zong, LI Wei-hua, XU Miao-yun, WANG Lei

2018, 34(5):  110-116.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0957

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Iron（Fe）,zinc（Zn）and selenium（Se）are essential trace mineral elements in organism and play an important role in the growth and development of plants,animals and human body. In order to increase the contents of Fe,Zn and Se in maize seeds,we sprayed iron,zinc,selenium fertilizer and their combinations on leaves,and explores their effects on iron,zinc,selenium and other mineral elements in maize kernels. Different concentration of Fe,Zn and Se fertilizers and their combinations were sprayed to leaves of maize cultivar JK2010. The results showed that spraying Fe,Zn and Se fertilizers significantly increased Fe,Zn and Se contents in JK2010 seeds. Fe and Zn promoted each other to increase their contents in seeds. Selenium promoted the Zn accumulation,whereas Fe and Zn slightly inhibited Se accumulation. Application of Fe,Zn and Se fertilizers and their combinations promoted Ca accumulation in maize seeds,and presented different effects on Cu accumulation. Overall,Fe,Zn and Se fertilizer didn’t affect the accumulations of Mn,Mg and P elements. The mixed Fe/Zn/Se fertilizer extremely significantly increased Fe,Zn and Se contents in maize seeds,which provides a novel strategy for increasing Fe,Zn,and Se contents in crop grains. The usage of Fe/Zn/Se fertilizers also presents varied influence on the contents of several other mineral elements in maize seeds.

Variation Analysis of DNA Methylation in Different Development Stages of Cassava

XUE Jing-jing, CHEN Song-bi

2018, 34(5):  117-123.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0893

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DNA methylation plays a key role in the regulation of gene expression and the silencing of transposons in plants. In this study,two cassavas’ germplasm SC5 and Cas36-12 with large differences in starch content were used as study material. The methylation-sensitive amplified polymorphism（MSAP）technique was used to analyze the DNA methylation changes of two cassavas’ germplasm in the different development stages by LabChip GX Touch microfluidic capillary electrophoresis. The results showed that 345 and 339 methylation bands were amplified from SC5 and Cas36-12 by 14 pairs of primers,respectively. The methylation rate of SC5 was higher than that of Cas36-12 in the three developmental stages,and all were over 50%. Comparing the DNA methylation changes in the 3 key developmental stages of SC5 and Cas36-12 root tuber,it was found that the overall changing trend of DNA methylation of two materials was consistent. From the formation period to the expansion,the DNA methylation mainly occurred,while the DNA de-methylation mainly occurred from expansion to maturation.

Quantitative and Distribution Characteristics of LTR Retrotransposons in Tetraploid Gossypium barbadense

LIU Zhen ZHANG, Shu-lin, SHI Ying-hui, PENG Ren-hai

2018, 34(5):  124-130.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0940

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Cotton is leading natural fiber-producing crop in the world. The fiber quality of Gossypium barbadense is the best among the four cultivated species of cotton. Understanding the number and distribution of LTR retrotransposons will promote the genome study of G. barbadense. In this study,we searched,classified and analyzed the LTR retrotransposon sequences from the genome of G. barbadense by many different methods. The results showed that LTR retrotransposon families shared by both of subgenome A and D accounted for 95% of all families,and the copy number and specific families of LTR retrotransposons in subgenome A were more than that in the subgenome D. The distribution characteristics of LTR retrotransposons superfamily Copia and Gypsy were significantly different;however,the same superfamily showed similar characteristics on the same subgenome chromosomes. In addition,the GO annotations of genes around LTR retrotransposons were mainly enriched in binding,catalytic,metabolic process,etc. In summary,the distribution characteristics of LTR retrotransposons on G. barbadense chromosomes and the functional enrichment of their surrounding genes are revealed in this paper.

Genome-wide Identification and Expression Analysis of TPS Gene Family in Pear

ZHAO Rong-rong, LIU Bin, GUO Chao, LIU Heng, ZHANG Yuan-hu

2018, 34(5):  131-141.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0804

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Terpene synthases（TPS）are the key enzymes in the synthesis of terpenoids. The TPS gene family was identified and subfamilies were clustered based on the genome-wide database of Pyrus bretschneideri,then the structure of TPS genes,the characteristics of encoded proteins and expression patterns were analyzed by means of bioinformatics and molecular biology. The results showed that 33 TPS gene family members were identified and they were clustered into five subfamilies. Among them,TPS-a subfamily had the largest number of members and owned 4-7 exons;most of TPS proteins were located in cytoplasm,and a few of them were distributed in mitochondria,chloroplasts and endoplasmic reticulum. All pear TPS proteins were hydrophilic proteins,the secondary structure of most TPS proteins was α-helix and random coil,and the folded and extended strands scattered in it. TPS-a subfamily had the largest number of conserved motifs and less in TPS-e/f. Proteins in each subfamily were highly similar in tertiary structure. The qRT-PCR analysis of 10 TPS genes showed that 9 PbrTPS genes except PbrTPS24 expressed in roots,stems,young leaves and sprouts,each gene expressed highly in different part,indicating that the expression of PbrTPS gene was tissue-specific.

Establishment of Cell Suspension Culture System Based on the Leaf Callus in Noni

ZHANG Zheng-xue, LAN Zeng-quan, WU Tian

2018, 34(5):  142-147.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0871

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This work aims to get the secondary metabolites from the cell suspension culture system of Morinda citrifolia L.（noni）leaves. In the experiment,the cell suspension system of noni leaf was established based on the callus induced from the sterilized leaves and determination of the subculture cycle by changing the collocation of hormone in liquid medium and inoculation amount of callus,and measuring some relevant parameter with the process of the culture. Liquid medium was MS+2.0 mg/L NAA+0.2 mg/L KT.,and the initial inoculation amount of callus was 60 g/L. During the 14th -22nd d,cells were in the linear growth phase,then grew slowly,and reached a peak of 11.8×10⁵/mL at the 26th day,and then declined. The best cell viability appeared at 21st day when the OD₄₈₅=1.8. The primary cell was irregular rod-shaped and showed regular clusters of small cells and spheres when in the sixth generation. The best subculture cycle of noni cell suspension culture system was 22-26 d,and the cell survival rate in the liquid suspension culture of noni leaves was 77.9%. In conclusion,a stable cell suspension culture system of noni leaves is established in the experiment.

Construction of Mutant Strain bamA,bamB and bamD of Aeromonas hydrophila and Their Effects on the Outer Membrane Protein Transportation

HUANG Fang, LIN Xiang-min

2018, 34(5):  148-153.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0771

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The BAM（β-barrel assembly machinery）complex of gram-negative bacteria is composed of five subunits of BamA-E,and plays a key role in the transporting and correctly folding of β-barrel outer membrane proteins. The current knowledge of this complex is based on the several researches on few bacterial species such as Escherichia coli and Neisseria meninyitidis,whereas its function in other bacteria remains largely not-studied. In this study,we firstly constructed the bamA-,bamB-,and bamD-deletion mutants in Aeromonas hydrophilausing homologous recombination method. The altered outer membrane proteins among mutants and wild type control strain were detected by SDS-PAGE,and then identified by mass spectrometry. There were 7 differentiated proteins,and 5 of them were outer membrane proteins and rest 2 were proteases in the inner membrane. In addition,the expressions of several selected outer membrane proteins were validated by Western blotting. Results showed that the mutation of BAM complex of A. hydrophila not only affected their expressions,but also their transportation of outer membrane proteins. The transportation and expression of outer membrane protein varied in the different subunits of the system. These results indicate that the different subunit of BAM transport system in A. hydrophila may have specific characteristics on outer membrane protein transporting and folding.

Effects of Nitrogen Concentration on the Growth,Lipid Accumulation and Fatty Acids Distribution of Oleaginous Chlorococcum sp.

LI Tao, XU Jin, WU Hua-lian, WANG Ming, XIANG Wen-zhou

2018, 34(5):  154-162.  doi:10.13560/j.cnki.biotech.bull.1985.2017-1134

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Oleaginous microalga with rich polyunsaturated fatty acids（PUFAs）is considered an optimal raw material for the production of high-valued microalgae oil. The key screening indexes of high-valued microalgae include oil yield,neutral lipid fractionation,and PUFAs distribution. Low-nitrogen stress is one of the most effective approaches in inducing oil accumulation in microalgae. The changes in growth,lipid composition,and PUFAs distribution of Chlorococcum sp. were determined under 17.6 mmol/L NaNO₃,5.9 mmol/L NaNO_3,and 3.5 mmol/L NaNO₃. The results showed the total lipid yield reached 2.55 g/L and 2.51 g/L at 3.5 mmol/L and 5.9 mmol/L NaNO₃ group,respectively,much higher than that 1.43 g/L at 17.6 mmol/L NaNO₃ group. Lowering nitrogen concentration increased the proportion of neutral lipids,i.e.,it was the highest at 3.5 mmol/L NaNO_3,accounted for 88.6% in total lipid,higher than 86.3% at 5.9 mmol/L NaNO_3,and 80.5% at 17.6 mmol/L NaNO₃. Decreasing nitrogen concentration also changed the fatty acids distribution in different lipid fractionations and promoted the fatty acids more distributed in neutral lipids. For instance,at 3.5 mmol/L NaNO_3,the proportion of α-linolenic acid in neutral lipids increased to 86.5% at the end of the cultivation from 33.9% at the beginning of cultivation. Conclusively,appropriate nitrogen concentration has a great role in the improvement of total oil yield,neutral lipid fractionation,and PUFAs distribution;moreover,the accumulated C18：3（ω-3）in Chlorococcum sp. tends to be distributed in neutral lipids under low nitrogen,and thus Chlorococcum sp. is a potential oleaginous microalgae for the production of high-valued microalgae oil.

Antigenic Epitope Analysis and Preparation of Antibody of Listeria monocytogenes CdaA

SUN Jing-juan, QIU Jing-xuan, ZENG Hai-juan, DING Cheng-chao, WANG Guang-bin, LI Jie, WANG Shu-juan, LIU Qing

2018, 34(5):  163-171.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0823

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Bioinformatics analysis showed that Listeria monocytogenes adenylate cyclase,CdaA,had solid intraspecific conservation,interspecific specificity,and antigen epitope structure. In this study,CdaA was used as a detection target to detect L. monocytogenes,and the immunogenicity of the protein was verified and monoclonal antibody was prepared. Given that the transmembrane domains might block CdaA expression,pET30a-Δ300cdaA was constructed and induced to express Δ100CdaA. The titer of polyclonal antibody prepared by the purified Δ100CdaA reached 1：128000. Western blotting analysis demonstrated that polyclonal antibodies recognized the CdaA extracted from L. monocytogenes. In addition,a monoclonal antibody,3F8,was screened,and its titer was 1∶512 000. Western blotting analysis showed that the 3F8 bound with the extracted proteins of 9 strains of L. Monocytogenes and 2 strains of non-pathogenic Listeria species,while not bound with the extracted proteins from 7 strains of other species,such as Escherichia coli,Staphylococcus aureus,and Salmonella,indicating it had promising specificity. In summary,we used bioinformatics methods to screen the detection target and to analyze the epitopes,and we prepared polyclonal antibody and monoclonal antibody successfully.

Isolation and Degradation Characteristics of a Efficient Tetracycline-degrading Strain

WU Xue-ling, WU Xiao-yan, LI Jiao-kun, SHEN Li, YU Run-lan, ZENG Wei-min

2018, 34(5):  172-178.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0723

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The wide use of tetracycline antibiotics in aquaculture causes drug residues,which brings serious impacts to environment and human health. In this study,a strain XY-1,which efficiently degraded tetracycline,was screened from the sediment of pig farm using tetracycline antibiotics as feed additive. After the Gram stain,morphological observation,physiological and biochemical and 16S rDNA sequence analysis,the strain was identified as Raoultella sp. and named as XY-1.The ability of strain degrading tetracycline was determined by high performance liquid chromatography（HPLC）. The results showed that the optimal degradation conditions were as followings：the temperature was 25℃ and the initial pH was 7.0. Under the optimal conditions,XY-1 grew at most and the highest tetracycline degradation rate reached 70.68% at the 8th day. Besides,the preliminary probe on the tetracycline-resistant genes was also conducted,and it was the first time to have two tetracycline-resistant genes tet（D）and tet（E）detected in the strain.

Sulfur Oxidation Model Construction of Extremely Thermoacidophilic Archaea Acidianus manzaensis

ZHU Wei, MA Ya-long

2018, 34(5):  179-186.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0620

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To understand the metabolic pathway of sulfur oxidation in extremely thermoacidophilic archaea,20 possible genes related to sulfur oxidation were preliminarily screened by NCBI database alignment based on the complete genome sequence of Acidianus manzaensis YN-25. Quantitative real-time PCR（RT-qPCR）was used to analyze the differential expressions of the screened genes under two different energy substrates,elemental sulfur（S⁰）and iron（Fe²⁺）. The results indicated that 15 genes were associated with sulfur oxidation from the compartive analysis of the experiment results ,including 5 genes encoding the enzymes of oxidizing S⁰ and sulfur-containing intermediates,4 genes encoding terminal oxidase,1 gene encoding sulfate transporter permease,1 gene encoding electron transfer protein,and 4 dsrE genes encoding sulfur reduction protein family closely related to sulfur oxidation. Based on the above result of analysis,a sulfur oxidation model of A. manzaensis was presented as below. Extracellular S⁰ was transported into the cell through membrane,and reduced by sulfur oxygenase-reductase（SOR）to be intermediates such as S₂O₃^2-,SO₃^2- and H₂S. Following,these sulfur-containing intermediates were further oxidized by relevant enzymes inside cells,and the electrons from the oxidation were transferred to oxidized quinone（Q²⁺）on the cell membrane,from which the reduced quinone（QH₂）was formed. QH₂ were eventually oxidized by terminal oxidase to be NADH and ATP providing energy for the growth of cells.

Biological Characteristic and Biodegradation Capacity of Ochrobactrum tritici WNP-3 for Carbendazim

CHEN Rui, SUN Xiao-yu, DENG Yuan, LU Peng-peng, ZHAO Ling-xia, QU Jia, SHEN Wei-rong

2018, 34(5):  187-194.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0675

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Pesticide pollution becomes major cause to environment safety and human health. Introducing microorganisms of degrading specific pesticide into polluted soil may efficiently reduce the concentration of this pesticide;moreover,this is a green technology and causes no secondary pollution. In order to reduce the residual carbendazim in the soil of a greenhouse,a strain WNP-3 decomposing and utilizing carbendazim was isolated via sole carbon source plate screening method from the greenhouse soil where carbendazim has been used for a long time. This strain normally grew in LB medium with carbendazim concentration of 200 mg/L. According to the results of morphology,physiological and biochemical property,Biolog identification,and 16S rDNA sequence analysis,WNP-3 was identified as Ochrobactrum tritici. The WNP-3 had broad growth range,i.e.,growing well at pH 4 - 9 in culture media containing 1%-3% NaCl,and it was resistant to several pesticides. Confirmed by UV spectrophotometry and HPLC,the carbendazim at concentration of 100 mg/L degraded 61% in mineral medium in 180 r/min flask for 120 h at 28℃.

Fermentative Production of Rhamnolipid Using Waste Oil from Range Hood as Substrate

ZHANG Jing-bo, WU Jian-rong, JIANG Yun, Yang Di, ZHAN Xiao-bei

2018, 34(5):  195-200.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0736

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Rhamnolipid（RL）is a kind of anionic biosurfactant with great potential and could be applied in the fields of petroleum,food,agriculture,and daily chemicals industry. This study explores the feasibility of producing rhamnolipid by fermentation with waste oil from range hood. Pseudomonas aeruginosa WB505 was used as the starting strain,and by which the yield of rhamnolipid in 7 L fermenter reached 12.3±0.52 g/L. Matrix-assisted laser desorption time-of-flight mass spectrometry was applied to analyze the composition of produced RL. Results showed that the main contents were Rha-C₁₀-C₁₀ and Rha₂-C₁₀-C₁₀,and the total abundance of mono-rhamnolipid and di-rhamnolipid were 49.7% and 50.3%,respectively. Critical micelle concentration（CMC）of the produced RL was 45.0 mg/L and it reduced the surface tension of water from 60.5±0.81 mN/m to 25.3±0.68 mN/m. E24 for six kinds of common hydrocarbons were higher than 60%,and E24 for benzene reached 80.3±0.85%. Therefore,using waste oil from range hood as substrate for RL production can reduce the capital cost of substrate,and provides a recycling treatment for waste oil from range hood.

Expression of HFSC Marker in the Arbas Cashmere Goat Hair Follicle and Hair Follicle Stem Cells

NIMANTANA, ZHANG Yan-jun, LIU Dong-jun, LI Jin-quan

2018, 34(5):  201-205.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0885

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This study is aimed to detect the expression of widely-used hair follicle stem cell（HFSC）markers in the hair follicle of Arbas Cashmere goat,and to provide experimental evidence for further research on histology and cytology of hair follicle in Inner Mongolia Cashmere goat. Using Arbas Cashmere goat as study object,frozen section and immunohistochemistry of Krt15,Krt19,CD34 and Sox9 were performed. Results showed that the above-mentioned four markers all expressed in the outer root sheath;Krt15 and Krt19 positively expressed in the bulge region and non-contiguously in the suprabulbar region;CD34 had a strong expression in the bulge and suprabulbar region;Sox9 expression focused in the bulge region. Conclusively,Krt15,Krt19,CD34 and Sox9 all expressed in the bulge region of the hair follicle,thus they can be used as special markers of Arbas Cashmere goat hair follicle stem cells from bulge region according to their expressed location.

A Case Study of“Glyphosate Safety Controversy”

YUAN Zi-qing, LI Jing-yi, ZHU Long-jiao, LUO Yun-bo, HUANG Kun-lun, XU Wen-tao

2018, 34(5):  206-218.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0375

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The safety of glyphosate has been a subject of concern and debate since the release of IARC’s assessment report on the possible carcinogenic effects of glyphosate in 2015. In all kinds of cases,the “dispute over the safety of glyphosate” has become a representative event of agricultural inputs affecting food safety and ecological health because of its significance,complexity,and tortuosity. The reason why this case is so important is due to the widespread use of glyphosate and its dominance in the herbicide market. The complexity of the case is reflected in the breadth of its scope and impact with its inextricable links between economics,politics,education,and other areas. While tortuosity refers to following the carcinogenicity assessment of IARC,no risk assessment results released by the international agencies have made the fate of glyphosate runs dramatically. This article firstly reviews the development of the whole case,and states the participants of the event,then systematically introduces glyphosate from the development course,the physical,chemical properties and detection methods. Based on this,case analysis was conducted to reveal the relationship between glyphosate and GM crops. Next,combine this case with teaching methods and provide a way for the case to be integrated into the classroom. In the end,from multiple perspectives,the author develop a case study,explore the hidden social truth behind the case and give some enlightenment gained from it.