Table of Content

26 September 2018, Volume 34 Issue 9

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Research Progress on Functional Nucleic Acid Nanomachine Biosensors

ZHANG Qian, TIAN Jing-jing, LUO Yun-bo, XU Wen-tao

2018, 34(9):  2-14.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0397

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Functional nucleic acid nanomachine is a nucleic acid assembly that is naturally or artificially designed and self-assembled by nucleic acid base sequence-specific interactions. The operation of a functional nucleic acid nanomachine can be activated by transforming its structure or conformation in two ways. One way is for the functional nucleic acid to interact with specific signal molecules to change its structure,and the other way is to change the conformation of the functional nucleic acid under the stimulation of the external environment（such as changing the pH,adding ions,irradiation with different light,etc.）. The current researches mainly include functional nucleic acid nanomachines mediated by G-quadruplex functional nucleic acid,aptamer functional nucleic acid,deoxyribozyme,and Holliday junction functional nucleic acid,as well as based on non-metallic,metallic materials,strand-displacement,click-response,triplex nucleic acid,pH-response,and light-induced. This paper reviews the specific operating mechanisms of these classic functional nucleic acid nanomachines,and discusses the practical significance and existing problems in the drug targeted delivery,bioimaging application research. Finally,the paper prospects the application of functional nucleic acid nanomachines and the challenges that may be encountered.

Advances in Biosensors Based on Triplex Nucleic Acids

TIAN Jing-jing, LUO Yun-bo, XU Wen-tao

2018, 34(9):  15-28.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0381

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A super-molecular nucleic acid assembly,termed Triplex Nucleic Acids（TNAs）,is formed between the third oligonucleotide strand and classical Waston-Crick base-pair-formed double strands through the non-classical Hoogsteen hydrogen bond at the major groove site. Among recently developed biosensors,TNAs-based sensing platforms have been attracting more attentions on account of the advantages of rapidity,simplicity,sensitivity,and reversibility. In this review,we generalized and exemplified different TNAs-based biosensors from their classifications,characteristics to applications. We also highlighted the development prospects of the TNAs biosensor.

Research Progress on Functional Nucleic Acid Biosensor Mediated by Isothermal Technology

ZHANG Yuan, XIAO Bing, TIAN Jing-jing, XU Wen-tao

2018, 34(9):  29-38.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0574

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With the development of society,food diversity,and food circulation,consumers’ attention to food safety is increasing,which makes the demands of detection technologies for food safety risk factors rise. Functional nucleic acids play an important role in the field of detection by forming specific spatial structures and playing a variety of functions besides storing genetic information. Functional nucleic acid biosensor is based on functional nucleic acid to recognize target,to amplify signal or to put out signal with high sensitivity,high specificity,time-saving,and low cost. In order to avoid the dependence on temperature-changing equipment and to conduct on-site detection,functional nucleic acid biosensor mediated by the isothermal technology develops rapidly. Compared with temperature-changing technology,isothermal technology does not need temperature-changing equipment and some of them can be carried out at room temperature,which reduces the cost of detection and shortens the detection time to some extent. According to the function of isothermal technology in functional nucleic acid biosensor,it can be divided into isothermal-mediated signal recognition technology,signal amplification technology and signal output technology. Isothermal technologies in functional nucleic acid biosensors are introduced from above three aspects in this review and prospects are also summarized from these three aspects.

Research Progress on Duplex-specific Nuclease Mediated Biosensors

XIAO Xing-ning, ZHU Long-jiao, LI Xiang-yang, LUO Yun-bo, XU Wen-tao

2018, 34(9):  39-47.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0786

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Duplex-specific nuclease（DSN）is a kind of protein enzyme that can effectively identify and cleave double-stranded DNA or DNA strand of hybridized DNA/RNA complex,but not affect the single strand DNA and single/double strand RNA. Thus,the DSN has been widely used in the areas of biology and medicine. Based on the property of DSN specifically cleaving DNA and RNA and equipped with signal output and different amplification,a series of biological sensing technology,such as colorimetric method,fluorescent method,electrochemical method,etc.,have been established. By these methods a series of biomarkers and food safety risk factors,like miRNAs,mRNA and heavy metal ions,may be detected. In addition,DSN-mediated biosensors with emerging nanomaterial also display a great advantage in analyzing the RNA and other material sensing. Firstly,we present an overview of the basic knowledge of DSNs,including the structure,properties and cleaving ways. Secondly,we give a classified overview of DSN-mediated biosensor based on the various readout signals,like optical sensors,electrochemical sensors and magnetic sensors,etc. The detailed contents covered the principles of varied sensors,and the detection ranges and sensitivity of each method;meanwhile,the types of all the targets that can be detected by DSN-mediated biosensors are summarized,expecting that this is conducive to further deeply apply DSN. Finally,the disadvantages of the DSN-mediated biosensors are summarized and the future development trend is prospected,aimed at guiding people to avoid relevant problems in the future when they use the DSN-mediated biosensors,and to develop faster,more convenient and more sensitive biosensors.

Research Advances on Apurinic/Apyrimidinic Endonuclease 1 Mediated Functional Nucleic Acids Biosensors

HE Wan-chong, HUANG Kun-lun, XU Wen-tao

2018, 34(9):  48-54.  doi:10.13560/j.cnki.biotech.bull.1985.2017-1015

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Apurinic/apyrimidinic Endonuclease 1（APE1）is an enzyme widely existing in living organisms and can effectively and specifically identify and cleave DNA in AP site in the process of base excision repair（BER）. Meanwhile,the activity of APE1 in some tumor cells is significantly higher than that in normal cells,thus it is also a kind of tumor biomarker. At present,by designing AP sites in DNA strand artificially and utilizing the activity of APE1 to produce ideal functional nucleic acids,combined with different methods of signal output and amplification,researchers have established some APE1 mediated electrochemical and fluorescent functional nucleic acids biosensing techniques,by which the detection for the activities of some enzymes such as DNA glycosylase can be conducted. In addition,some functional nucleic acids biosensing techniques for the detection of the activity of APE1 were also established. In this paper we review the research status of APE1 mediated functional nucleic acids biosensing techniques and other biosensing techniques for the detection of APE1,discuss the significances and problems of APE1-relevant biosensing techniques,and prospect development trends of APE1 mediated detecting techniques for the detection of more targets,aiming at promoting APE1 to become a common enzyme tool in functional nucleic acids biosensing techniques.

Research Progress on Exonuclease III-assisted Functional Nucleic Acid Biosensors

SONG Huan, LUO Yun-bo, XU Wen-tao

2018, 34(9):  55-69.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0278

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Exonuclease III（Exo III）is a kind of nuclease that can specifically recognize dsDNA and catalyze the release of mononucleotides by stepwise hydrolysis from 3'-hydroxyl termini of dsDNA strands;while hydrolysis on ssDNA is limited. Based on its exonuclease activity,Exo III has been successfully employed for amplified detection of DNAs,small molecules and metal ions while combined with a series of signal outputs. Here we mainly give a detailed classifying description of the Exo III-assisted biosensors based on various target substances,and also expound the working principles and sensitivity of each sensor. In addition,we discuss the recent progresses on the integration of Exo III-assisted target-recycling strategy with other signal enhancement strategies or emerging nanomaterials in detail as well. Finally,we summarize the advantages and disadvantages of the Exo III-assisted biosensors and prospect the future development,which will be conducive to further application and continuous development of Exo III-assisted biosensors in environmental management,biological analysis and clinical diagnosis.

Research Progress on Magnetosome-mediated Biosensors

LI Shu-ting, ZHOU Zi-qi, TIAN Jie-sheng, XU Wen-tao

2018, 34(9):  70-78.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0223

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Bacterial magnetosomes（BMs）are special organelles synthesized by magnetotactic bacteria（MTB）and used for geomagnetic navigation in their aquatic habitat. It consists of an external lipid bilayer membrane and internal magnetic nanocrystals. Because of its narrow particle size distribution,uniform morphology,single magnetic domain,paramagnetism,large specific surface area,and high biocompatibility,it is widely used in the medical field as an anticancer drug delivery vehicle,nuclear magnetic resonance imaging contrast agent and imaging probe,etc. At present,there are relatively few researches on the preparation of biosensors based on BMs and applied in the biological detection technology field,and they are mainly biased toward the covalent or non-covalent modification of antibodies on the surface of BMs membranes,and the use of antigen-antibody-specific immunoreactions to achieve the detection of target substances. This article reviews the structure and composition,basic characteristics,extraction and purification,morphological structure characterization and application of BMs in the enrichment of target substances,and highlights several types of BMs-based biosensors such as electrochemical biosensors,fluorescent biosensors,magnetic biosensors,etc. Finally,the practical significance and existing problems of magnetosome-mediated biosensors in application research are discussed. The prospects for the development of magnetosome-mediated biosensors and the opportunities and challenges that may be faced are anticipated in order to provide reference for promoting the application of magnetosome-mediated biosensors in practice.

Applications of Optical and Photothermal Nanomaterials in Biosensing,Drug-targeted Delivery and Bioimaging

ZHENG Li-rong, LUO Yun-bo, XU Wen-tao

2018, 34(9):  79-89.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0388

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Nanomaterials are one of the important development directions in nano-science and technology. The structure of nanomaterials has endowed it unique optical properties,the nanometer size facilitates its targeting to tumor tissue via EPR effect or surface modification,and some nanomaterials can absorb the energy of an external light source and convert it into heat energy. Therefore,nanomaterials are widely applied in optical sensors,bioimaging,drug-targeted delivery,and tumor photothermal therapy. This review mainly describes the excellent properties of optical nanomaterials and photothermal nanomaterials,and illuminates their applications in the above fields. Finally,the future development trend of nanomaterials is prospected.

Research Progress on the Sensors of High Analytical Performance Based on the Graphene and Its Related Material

LEI Xin-you, ZUO Guo-fang, WANG Peng, ZHANG Jian-bin

2018, 34(9):  90-96.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0452

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Based on the excellent electrical conductivity,large specific surface area and fine biocompatibility of graphene and graphene-related materials,the constructed sensors from graphene exhibit better properties than that by other materials. The application of graphene in sensing field is generally conducted by functionalization. The combination of graphene with polymer or nanoparticles may significantly enhance the response of the sensor and improve the sensitivity of detection. This paper reviews the recent advances in the application of graphene and its related materials in the sensing fields such as clinical analysis,environmental monitoring and food safety control. When the sensors are based on the graphene oxide and its derivatives（containing oxygen groups）with fine water dispersibility and biocompatibility,larger specific surface,flexible surface modification,and simple preparation,and their analytical performances are comprehensively evaluated by the analysis data of sensitivity,detection limit,etc. At the same time,the research trends of novel sensing material graphene and its derivatives is prospected：Precisely controlling the size and shape of graphene monodisperse,studying the sensing mechanism of the reaction between graphene and analyte molecular electrode under catalytic action,and reducing the aggregation of graphene sheet and precisely controlling the microstructure of graphene-based sensing system. It is expected as such：Developing portable chip-based sensors with more powerful characteristics to achieve the multi-analysis of complex environmental samples in a shorter time,to further increase the detection sensitivity and selectivity,to enhance the stability and reusability of the sensors,and to overcome toxicity and biological intolerance.

Research Advances on Aptamer-based Quartz Crystal Microbalance Sensors

WU Li-ting, SU Xue, LIN Jun-sheng

2018, 34(9):  97-103.  doi:10.13560/j.cnki.biotech.bull.1985.2017-1099

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Aptamers are short-chain oligonucleotides sequence selected from synthetic random single-stranded oligonucleotide libraries via a technology of Systematic Evolution of Ligands by Exponential Enrichment（SELEX）. They have the advantages of low molecular weight,simple structure,easily modified,and wide-ranging target,as well as high specificity and affinity with target molecules. Correspondingly,these characteristics of aptamers may be applied for preparing a variety of sensors. Accordingly to the different principles of various aptamers-based sensors to detect their targets,here we focus on electrochemical,optical,and piezoelectric crystal sensors that are commonly used for detecting the affinity between aptamers and target. These three methods all have the advantages of short detection time and low detection limit. Piezoelectric crystal sensor is also called quartz crystal microbalance（QCM）. In addition to characterization of candidate aptamers during SELEX process,aptamers-based quartz crystal sensors can be built up with QCM to detect their targets with high sensitivity and high specificity. Thus,the basic principle of aptamer-based quartz crystal microbalance sensor is briefly introduced. Further,the recently published reports of QCM on the characterization and detection of interactions between aptamers and their targets,including the small molecules,ions,proteins,cells,bacteria,and viruses,are reviewed. Moreover,the advantages and disadvantages of QCM technology are summarized and analyzed. This review is aimed at providing a reference for the screening of aptamers and their further application in basic research,clinical diagnosis and disease treatment.

Research Progress on Biosensor Detection Technology for Mg²⁺ Functional Nucleic Acids

WANG Lian-zhen, ZHANG Qian, LI Kai, HE Wan-chong, LUO Yun-bo, HUANG Kun-lun, XU Wen-tao

2018, 34(9):  104-115.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0205

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Mg²⁺ is one of the important divalent metal ions in human body and plays an important role in the reactions of catalyzing nucleic acids in cells. The loss or excess of Mg²⁺ in human body can harm people’s health. At the same time,Mg²⁺ also has varied functions in the natural environment. Therefore,the detection of Mg²⁺ has received people’s attention,of which Mg²⁺ instrument detection technology has matured,but there are still some drawbacks. In recent years,functional nucleic acids have been uncovered to have the advantages of easy sequence modification,high specificity,high stability,low cost,and rapid detection on site with biosensors,and are attracting more and more attentions. Currently,a variety of specific Mg²⁺ functional nucleic acids biosensors have been established and the detections of various biomarkers are achieved. Meanwhile,the combination with novel nanomaterials further increases the detection limit and the broad spectrum of detection. This review first introduced the action and regularity mode,in vitro screening methods and structural properties of Mg²⁺ functional nucleic acids,and summarized the principles and applications of Mg²⁺ specific functional nucleic acid sensor. Secondly,based on the way of signal amplification and the combination with different nanomaterials,Mg²⁺ functional nucleic acid sensors are classified into temperature sensors,thermostatic sensors,gold nanosensors,and carbon nanosensors. The main content covers the sensing principles,application areas,sensitivities and detection limits of different sensors. Finally,the applications of Mg²⁺ functional nucleic acids in food and biomedicine are summarized and the existing problems and future applications are prospected,aiming at providing a theoretical basis for the future development of more portable,sensitive and accurate biosensors.

The Development of Sensors for the Detection of Hg²⁺

FENG Yu-xiang, LI Xiang-yang, SHAO Xiang-li, XU Wen-tao

2018, 34(9):  116-128.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0429

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Of all the heavy metals that contaminate the soil and water,Hg²⁺ is the most common one. Atmospheric deposition and ocean circulation of Hg²⁺ result in its entering into fresh water and ocean. With biological accumulation through food web,Hg²⁺ consequentially risks human health. Therefore,the rapid and sensitive detection of Hg²⁺ is central to the environmental monitoring and human health. This review demonstrates the origin,biotransformation of Hg²⁺ and its effects on human health,and summarizes the mainstream methods of detecting Hg²⁺ based on its interaction properties with other substances,including fluorescence molecules,nanoparticles,DNAzyme,antibodies,proteins,and transgenic microorganisms, in order to provide a basis for rapid detection of Hg²⁺.

Research Progress on Metal-organic Framework-mediated Functional Nucleic Acid Detection Technology

LI Shu-ting, HE Wan-chong, XU Wen-tao

2018, 34(9):  129-138.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0055

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Metal-Organic Frameworks（MOFs）are also known as Coordination Polymers（CPs）. They are crystalline molecular functional material that are composed of metal ions or metal clusters and organic ligands and formed under relatively mild conditions. It has the characteristics of high porosity,excellent structural adjustability,huge internal surface area,structural diversity,high chemical stability and strong thermal stability. It has been used in various fields such as chemical engineering and medicine. At present,the research on the combination of MOFs and Functional Nucleic Acids（FNA）in the field of detection technology mainly focuses on two kinds of fluorescence biosensors and electrochemical biosensors,and other types of biosensors have rarely been reported. In addition,the preparation of MOFs has also become increasingly miniaturized to improve its performance. In this paper,the recent progress on MOFs-mediated functional nucleic acid biosensors is reviewed. The practical significance and problems of MOFs-mediated functional nucleic acid detection technology in applied research are discussed. The prospects for the development of the technology,the opportunities and challenges it may face are prospected,and it is aimed that it will further promote the practical application of the MOFs-mediated functional nucleic acid detection technology.

Research Progress on Mesoporous Silica Mediated Functional Nucleic Acids-based Detection Technologies

LI Shu-ting, HE Wan-chong, HUANG Kun-lun, XU Wen-tao

2018, 34(9):  139-148.  doi:10.13560/j.cnki.biotech.bull.1985.2017-1075

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Mesoporous silica nanoparticles（MSNs）are porous inorganic nanoparticles,they are characterized by adjustable particle and pore size,large surface area and pore volume,surface modification and fine biological compatibility and others,and are widely used as a delivery carrier for anticancer drug in medical field. At present,MSNs and functional nucleic acids（FNA）are combined to prepare biosensors for building detection technologies,the study of which is mainly focused on the FNA immobilization on the surface of MSNs. Through the change of FNA structure,the controlled release of guest molecules in mesopores can be realized and further converted into fluorescent signals,electrical signals,and the like for detection. Here we reviewed the basic properties,preparation and applications of MSNs,highlighted several types of functional nucleic acid biosensors based on MSNs,and discussed the practical significances and issues while applying mesoporous silica mediated functional nucleic acids-based detection technologies. Finally,we prospected this technology and the potential opportunities and challenges,aiming at further promoting the practical application of mesoporous silica mediated functional nucleic acids-based detection technologies.

Development Progress of Digital PCR in the Precise Detection of Functional Nucleic Acid

LIU Xiao, ZHU Peng-yu, WANG Yao, ZHU Shui-fang, FU Wei

2018, 34(9):  149-162.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0568

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Digital PCR,as a quantitative analysis technology,has developed rapidly in recent years. The analysis of results from it doesn’t depend on the cycle of threshold（Ct）of the amplification curve and is also not affected by the amplification efficiency,it has a very promising accuracy and reproducibility,and the absolute quantitative analysis can be achieved. Digital PCR has shown great advantages and application prospects in the detection and identification of functional nucleic acids. Based on the introduction of the basic principles and quantitative methods of digital PCR technology,this paper reviews the main application fields and forecasts the research prospect of digital PCR in the detection of functional nucleic acids.

A DNA Biosensing System Assisted by Comb-type Cationic Copolymer for the Detection of Single-base Mismatch

ZHANG Ming-ke, HAN Jia-lun, LIU Chen, WU Jin-cai, DU Jie

2018, 34(9):  163-169.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0468

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In order to quickly and accurately detect the single-base mismatch in the system,using the difference of absorptivity between ssDNA and dsDNA and the excellent fluorescence quenching ability of the oxidized graphene,assorted with comb-type cationic copolymer PLL-g-Dex that produces high nucleic acid companion activity while coordinated with the oxidized graphene,an enzyme-free,accurate and efficient analysis system was established. First,the feasibility of experiment design was confirmed by fluorescence detection. Then in the detection of optimal concentration of every component in the process,it was found that the optimal experimental position occurred when the concentration of T-DNA was 20 nmol/L,GO was 9μg/mL,cDNA was 90 nmol/L,and PLL-g-Dex was 96 nmol/L. Finally,the selectivity of the detection system to single-base mismatch was collaborated,and the results revealed that the fluorescence intensities from different base mismatch systems differed. Moreover,compared to other bases,PLL-g-Dex presented stronger selectivity to C-C base mismatch.

Advances in Gene Engineering Technologies for Aspergillus oryzae

ZHANG Zhi-min, ZHUANG Miao, JIN Feng-jie

2018, 34(9):  170-176.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0252

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The filamentous fungus Aspergillus oryzae is an important strain in fermentation industry. It has a strong capacity of secreting protein and recognized food safety,therefore is widely used as a cell factory for the expression of foreign proteins. However,the production and secretion of foreign proteins by A.oryzae are limited by a number of bottlenecks,including transcription,translation,protein folding,translocation,degradation,transport,and secretion,etc. The existence of these problems makes it difficult to achieve the desired target in the production of foreign proteins by A.oryzae. In recent years,with the decipherment of the whole genome sequence,the basic research and the genetic engineering technologies related to the production of A. oryzae have been well developed,for example,improvement of the homologous recombination efficiency,application of selectable marker genes,large chromosome deletion technology,hyphal fusion technique,DNA microarray,etc. The development and establishment of these technologies provided a great deal of technical support for the industrial production and application of A. oryzae. Regarding the bottlenecks in the heterologous protein expression of A. oryzae,this article mainly elaborated several aspects such as the optimization of genetic transformation system and the increase of transformation efficiency,the construction of protease gene-deleted strains,the fusion expression strategy,and the optimization of its exogenous expression system to improve the production of foreign proteins. These advances in basic research and genetic engineering technology have greatly improved the production and application of A.oryzae,and also provide more effective ways and research space for the breeding of A.oryzae production strains in the future.

Optimization of Extracting Method for Microbial Genome DNA in Coal Geological Environment

YANG Xiu-qing, CHEN Yan-mei, WU Rui-wei, WANG Bao-yu, HAN Zuo-ying

2018, 34(9):  177-183.  doi:10.13560/j.cnki.biotech.bull.1985.2017-1145

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High-quality DNA is the basis for molecular biology research. By improving the traditional method of extracting DNA（phenol-chloroform method）and optimizing the cell disruption conditions while having Rhodococcus sp.R04 and pulverized coal as experimental materials,a modified method of efficiently extracting genomic DNA from microbes in coal geological environment has been established. The microbial genomic DNAs extracted by this modified method and the commercial kit were evaluated by agarose gel electrophoresis and PCR amplifications of bacterial and archaea specific fragments. The results showed that the microbial genome DNAs from coal geological environment were obtained by both the modified method and the kit method,and used as temple for various specific PCR amplifications. Compared with the extracted DNA with the commercial kit,the DNA by the modified method had obvious main band,accounted about 50% of total DNA content,and the molecular weight of the DNA was nearly 23 kb. Also,the extraction amount of DNA was very large,and was about 5-10 times that by the commercial kit. Moreover,DNA by the modified method can be used in many purposes such as DNA library construction and metagenomic sequencing. Consequently,the modified method for extracting microbial genomic DNA from coal geological environment is not only cheap and effective,also the extracted DNA is in high-quality,thus it is very suitable for laboratory and scientific researches.

Development of a Colloidal Gold Immunochromatographic Test Strip for the Detection of Moraxella catarrhalis

HUANG Ying-qi, ZHANG Qiu, TAO Ye, HU Zheng, ZHANG Gai-ping, ZHANG Hua-shan

2018, 34(9):  184-189.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0184

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The objective of this work is to develop a colloidal gold immunochromatographic test strip for the expeditious detection of Moraxella catarrhalis in the respiratory tracts of infected patients,and then to establish the corresponding approach. By bioinformatics analysis,the linear antigenic epitope on UspA1 of Moraxella catarrhalis was discovered and named UspA1Line1. The UspA1Line1 protein then ligated with KLH to generate the UspA1Line1-KLH protein. The already-constructed engineering strain for expressing UspA1 was used to express and purify the recombinant UspA1-His protein,which then was used to immune New Zealand rabbits for preparing the polyclonal antibody. Then the serum from the rabbits was purified by two steps of Protein A and polypeptide affinity chromatography,and the polyclonal antibody with high-purity and strong-specificity was acquired. The 40 nm colloidal gold particles were prepared by trisodium citrate reduction method,which then bound with the polyclonal antibody to form a double antibody sandwich,thus the goal of rapidly detecting M. catarrhalis was achieved. As a result,by the established approach M. catarrhalis was specifically detected in 10 minutes,and no cross-reactivity with common pathogens in upper respiratory tract occurred. The strips may be stored at 24℃ and their repeatability and stability remain well. In sum,a colloidal gold immunochromatographic test strip to rapidly detect M. catarrhalis is successfully developed.

Duplex Real-time PCR Methods for Quantitative Detection of Bovine Derived Materials in Animal Products

GAO Zhi-qiang, WANG Lin, PU Jing, YIN Yi, ZHANG Wei, ZHAO Xiang-peng, YAO Zhen-yu

2018, 34(9):  190-194.  doi:10.13560/j.cnki.biotech.bull.1985.2017-1129

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The objective of this work is to develop a duplex real-time quantitative PCR method for detecting the amount of bovine derived material in animal product. Using bovine satellite IV DNA as detection target gene and myostatin gene as an internal control to synthesize primer probe,the standard freeze-dried meat mixture powder with known beef proportion was detected by duplex real-time PCR based on TaqMan,and the linear model was established. Then the model was verified by detecting mocked and delivered samples. The method detected 0.1% bovine material in freeze-dried meat powder,and no cross reaction with the tissues of other animal species occurred. The relative standard deviation of repeatability test was < 5%,and the amplification efficiency of the method was 93.6%. Conclusively,the established method is suitable for quantitative detection of bovine derived material in meat products,and can be used to determine the content of bovine derived material in animal products on the market.

Establishment of Molecular Identity for Wolfberry Cultivars Based on SSR Markers

YIN Yue, ZHAO Jian-hua, AN Wei, Li Yan-long, HE Jun, CAO You-long

2018, 34(9):  195-201.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0155

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Using 16 cultivars as experimental materials,wolfberry molecular ID was constructed by SSR marker,aiming at providing references for identification and protection for wolfberry cultivars. Twenty-four primer pairs with high polymorphism,repeatability and distribution on the 12 chromosomes of wolfberry were screened from 600 pairs of SSR primers,and amplified 16 wolfberry cultivars. A total of 155 alleles were detected,the number of alleles per pair of primers ranged in 3-10,averagely 6.5 alleles per locus. Two hundred and eight genotypes were obtained ranging from 3 to 14 with average of 8.7 genotypes per locus. Polymorphic information content（PIC）ranged from 0.461 to 0.848 with an average of 0.682 per locus. Shannon’s information index ranged from 0.939 to 2.055 with an average of 1.487. Based on the principle of identifying the most cultivars using the minimum pairs of primers,the number of screened alleles,the number of genotypes,and PIC values were great than the average,and the value of Shannon’s information index was great than 1.7. Finally,the combination of primers LBSSR0052 and LBSSR0423 distinguished all tested cultivars. Based on these two pairs of primers,the alleles of all the cultivars were sequenced in descending order,and then the values of each cultivar in these two SSR loci were sequentially combined,thus the molecular ID of 16 wolfberry cultivars were established. The results indicate that SSR marker can be used as an effective technique for the variety identification and molecular ID construction of wolfberry.

Expression and Stress Tolerance Analysis of HbMC2 Gene from Hevea brasliensis in Yeast

LIU Hui, DENG Zhi, YANG Hong, DAI Long-jun, LI De-jun

2018, 34(9):  202-208.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0298

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Metacaspase-mediated programmed cell death plays an essential role in plant growth and development as well as resistances to biotic and abiotic stresses. Our previous study revealed that the rubber tree（Hevea brasliensis）metacaspase gene HbMC2 was regulated by abiotic stresses,such as drought,high salinity,etc. To investigate the function of HbMC2,the coding sequence of HbMC2 was amplified from rubber tree by RT-PCR and inserted into the pYES2 vector,and its yeast expression vector pYES2-HbMC2 was constructed. The plasmid of pYES2-HbMC2 was transformed into the yeast strain INVSc1 and the recombinant yeast INVSc1（pYES2-HbMC2）was obtained. PCR and RT-PCR analysis showed that HbMC2 gene was successfully introduced and expressed in the recombinant yeast when induced by galactose. Compared with the control yeast INVSc1（pYES2）,the recombinant yeast INVSc1（pYES2-HbMC2）demonstrated growth inhibition and its cell death rate increased under oxidative,drought,and salt stresses. These results indicate that expression of HbMC2 gene in yeast reduces tolerance to abiotic stress,i.e.,HbMC2 is a negative regulator of abiotic stress tolerance.

Cloning and Expression Analysis of CkP5CS Gene in Caragana korshinskii

ZHANG Teng-guo, SHI Zhong-fei, KOU Ming-gang, ZHENG Sheng, WANG Juan

2018, 34(9):  209-218.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0349

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The study aims to clone the full-length cDNA sequence of gene P5CS from Caragana korshinskii,analyze its tissue expression specificity and expression under the stresses of low temperature,high salt and PEG,and clarify its biological functions. The full-length cDNA of P5CS from C. korshinskii was isolated by RACE technique and analyzed by bioinformatics. Phylogenetic tree was constructed for studying homology with similar sequences. RT-PCR method was used to analyze the tissue expression specificity and the expression of CkP5CS under low temperature,high salt and osmotic stress. As results,the full-length cDNA sequence of P5CSgene of C. korshinskii was cloned and named as CkP5CS with a length of 2 604 bp,containing a 5'-UTR of 102 bp,a 3'-UTR of 363 bp,and a 2 139 bp opening reading frame,and it encoded 712 amino acids with the molecular weight of 46.8kD and theoretical isoelectric point of 8.6.The main secondary of the protein was a helix,extended strand and random coils. Comparison of amino acid sequences and phylogenetic analysis the CkP5CS showed high identity with those from Sophora davidii SdP5CS（85.1%）,Glycine max GmP5CS（84.2%）and Vigna unguiculata VuP5CS（82.6%）. Real-time PCR analysis revealed that CkP5CS expressed in roots,stems,leaves,indicating that there was almost no tissue specificity. The transcript level of CkP5CS was induced in response to low temperature,high salt and osmotic stress. As conclusion,CkP5CS gene isolated from C. korshinskii plays an important role in the adaptive process under low temperature,high salt and osmotic stress.

Correlation Analysis Between IRX3 Gene Expression and Intramuscular Fat Deposition in Tibetan Chicken

ZHANG Ya-nan, LIN Ya-qiu, XU Qing, XU Ya-ou, HE Qing-hua

2018, 34(9):  219-223.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0048

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The objective of this study is to establish IRX3 gene expression profiles in different tissues and muscle tissues of different developmental stages,and to analyze the correlations between the expression profile of IRX3 gene and the deposition of intramuscular fat in Tibetan chicken. The expression levels of IRX3 gene in different tissues and muscle tissues at different developmental stages were detected by real-time fluorescence quantitative PCR,intramuscular fat contents of the tissues were also measured,and its correlations with the expression level of IRX3 gene were analyzed. The results showed that the expression of IRX3 gene in lung tissue was significantly higher than that in other tissues（P < 0.01）. The relative mRNA levels of IRX3 in the leg and the chest muscle from 210 days-old cock were significantly higher than that of other ages（P < 0.01）,while the relative expression levels of IRX3 mRNA in the chest muscle from one day old hen were significantly higher than that from other ages（P < 0.01）. Correlation analysis showed that expression levels of IRX3 were positively correlated with intramuscular fat content in cock leg muscle（P < 0.01）,while the expression levels were negatively correlated with intramuscular fat content in female chest muscle（P < 0.01）. As conclusion,there is a correlation between IRX3 gene expression and intramuscular fat content,thus it is inferred that IRX3 might regulate intramuscular fat deposition.

Antigenic Epitope Analysis and Preparation of Polyclonal Antibody of Lepidopteran Pest-resistant Gene cry1C

JIN Yong-mei, CHEN Mo-jun, LIU Xiao-xiao, LIN Xiu-feng

2018, 34(9):  224-229.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0200

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In this study,cry1C antigenic epitope region of cry1C was predicted by bioinformatics analysis and designated as Δcry1C. The nucleotide sequence of the Δcry1C was synthesized after codon optimization,and then recombinant expression vector pET28b（+）-Δcry1C was constructed. To prepare cry1C polyclonal antibody,His₆-Δcry1C recombinant protein was purified after induction in Escherichia coli competent cell BL21（DE3）,and then was used to immunize New Zealand white rabbits as antigen. ELISA assay showed that the titer of cry1C polyclonal antibody reached 1：512 000.Western Blot analysis showed that cry1C polyclonal antibody was able to specifically detect the target protein of cry1C in transgenic pest-resistant rice. In summary,cry1C antigenic epitope region was screened by bioinformatics analysis and its polyclonal antibody was prepared successfully. This study provides a basis for further studying Lepidopteran pest-resistant transgenic plants.

Isolation of Rare Actinobacteria in 5 Ecodistricts of Tarim Basin and Distribution of the Genes Synthesizing Antibiotics

ZHU Rong-gui, GUAN Tong-wei, JIANG Xiu-juan

2018, 34(9):  230-236.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0011

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This work is for investigating the distribution of rare actinobacteria in different ecodistricts and assessing the potential of antibiotic production of these actinobacteria. Total 5 soil samples were collected from the Tarim Basin,and rare actinobacteria were isolated from these samples by 7 media. And then the potential of antibiotic productions was assessed by detecting type I PKS,type II PKS,NRPS,APH and HMG-CoA genes,related to antibiotics synthesis. The results showed that：（1）by molecular identification,we obtained 18 kinds of rare actinobacteria strains belonging to 10 different genera;（2）the riparian forest ecodistrict in the Tarim Basin had the most species of rare actinobacteria,and there were 8 genera;while the least one was the east of the Talimu river ecodistrict in Taklimakan desert,and there were 4 genera;（3）9 of these 18 strains had type I PKS gene,4 strains had type II PKS gene and APH gene,and 3 strains had NRPS gene;there was one Streptosporangium strain that contained type I PKS gene,type II PKS gene,NRPS gene and APH gene. Conclusively,5 ecological zones are found to have relatively more rare actinomycetes which also contain abundant genes in associated with the synthesis of antibiotics.

Effect of Glycosylation of Brucella P39 Antigen in Pichia pastoris on Macrophage Phagocytosis

LIU Si-yang, WANG Yan, FU Yu-qin, HU Xiang-kun, WANG Ming-yu, LU Tian-cheng, WANG Xiu-ran

2018, 34(9):  237-243.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0170

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P39 protein is one of the most predominant antigenic proteins identified from Brucella by proteomics. First,the dominant antigen protein gene P39 was expressed in Pichia pastoris,and the expressed protein was purified and identified by Western blot. Then,the glycosylation of the protein was identified by NMR（nuclear magnetic resonance）conjugated PNGaseF glycosylase,and the interaction speed between the protein and macrophage cell was studied. The results showed that P39 protein was secreted in P. pastoris GS115 and the expression level was up to 5.05 g/L. NMR results demonstrated that the recombinant P39 protein was modified by glycosylation. The macrophage capture effect of the modified protein was significantly stronger than prokaryotic protein,indicating that the glycosylation modification may affect the efficiency of the interaction between the target protein and the cell,which provides a reference for further exploring the effect of glycosylation on P39 protein and candidate antigen of Brucella glycosylated vaccine.

Morphological Identification and Antibacterial Activity on Metabolites of Fungi Isolated from Ascidean

LUO Guo-cong, HUANG Yun-yi, CHAI Hui-zi, LEI Xiao-ling, NIE Fang-hong

2018, 34(9):  244-248.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0173

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To seek new species of marine fungi with high antibacterial activity,ascidean was used as study material,and 14 fungi were isolated by plate spreading method,and their morphological identification and preliminary screening of their antibacterial activity was conducted. The results indicated that 14 fungal strains were isolated from ascidian,and preliminarily identified as Penicillium sp.,Aspergillus sp.,Cladosporium sp.,and Tritirachium sp. based on the morphological observation. Total 11 fungal strains of 14 presented antibacterial activity,and 4 strains of Asc-2-4（Penicillium sp.）,Asc-2-12（Penicillium sp.）,Asc-1-1（Penicillium sp.）,and Asc-2-1（Aspergillus sp.）had broad-spectrum and strong antibacterial activity. The antibacterial activity of Asc-2-12（Penicillium sp.）was the most obvious,with the diameter of maximum inhibition circle reaching 21.91 mm. Symbiotic fungal strains from ascidian are potential antibacterial resources and Asc-2-4,Asc-2-12,Asc-1-1,and Asc-2-1 have potential prospect to be further investigated.