Biotechnology Bulletin

Research Progress on Response Mechanism of Transcription Factors Involved in Plant Cold Stress

XIAO Yu-jie, LI Ze-ming, YI Peng-fei, HU Ri-sheng, ZHANG Xian-wen, ZHU Lie-shu

2018, 34(12): 1-9. doi:10.13560/j.cnki.biotech.bull.1985.2018-0240

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Cold stress has an important influence on the geographical distribution and growth of plants. Under cold stress,transcription factor activate the cold responsive gene expressions by binding to the cis-acting elements of gene promoter,therefore improving the cold resistance of plants by regulating signal transduction pathways in plants. This review focuses on recent research progresses on transcription factors AP2/ERF,NAC,WRKY,MYB,bZIP,and ZFPs involved in plant cold stress,and proposes a regulatory network that transcription factors participates in the cold stress by interacting with other factors and promoter elements while in gene expression.

Research Advances on Plant Antifreeze Proteins

WANG Yu-han, LI Zi-hao, LI Shi-biao, CHEN Chi-hang, WANG Yu-lin, ZHANG Yang

2018, 34(12): 10-20. doi:10.13560/j.cnki.biotech.bull.1985.2018-0511

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Low temperature stress threatens the growth of plants,resulting in the reduction in the yield of crops,subsequently people’s needs for production and life cannot be met;therefore,it is of great significance to enhance the ability of plants to resist cold. Antifreeze protein is a protein that improves the ability of organisms to adapt to low temperatures. At present,the discovered antifreeze proteins can increase the adaptability of organisms to low temperature and prevent organisms from damages by reducing the freezing point of a plant system,changing the morphology of ice crystals,and inhibiting the growth of ice crystals. Proteins with antifreeze properties have been discovered and isolated from fish,insects and plants,and the genes related to antifreeze proteins from plants or animals or fused animals and plants are transferred to receptor plants,their cold resistances increase,and consequently crop yields increase. This paper summarizes the progress of various antifreeze proteins in recent years in a table form,and reviews the research of antifreeze protein from several aspects of discovery,effect,evaluation method of activity,mechanism,research progress,and application of antifreeze protein. In addition,some literature shows that there are still some substances that regulate the accumulation of antifreeze proteins in some plants,such as jasmine acid and ethylene,which are also important for improving the ability of plants to live at low temperatures,and further research is needed. Second,other antifreeze proteins have also been uncovered to have other functions,but the homology of these antifreeze proteins is not high,and further studies are needed.

Research Progress on Circular RNA

LI Hong-yu, XU Wen-tao

2018, 34(12): 21-31. doi:10.13560/j.cnki.biotech.bull.1985.2018-0127

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Circular RNA（circRNA）appears in the splicing process of transcription,and single-stranded RNA molecules form a ring via covalent bonding. The development of high-throughput sequencing technology allows a comprehensive study of noncoding linear RNA（NCL）,this makes the NCL re-entering into the researcher's vision,especially the circRNA. circRNA has been proved to have features of rich,highly expressed,and evolutionary conservative. Some circRNAs have been demonstrated to have the functions that can affect the regulation of microRNA to genes,can be used as the miRNA sponges,can regulate the transcription of parent gene,and also have translation function and some other functions. It plays an important role in some diseases,and has a regulating effect on Alzheimer's disease,diabetes,ischemic heart disease and some other cancers. It can not only regulate the progression of diseases but also can be used as a biological marker for disease detection. Therefore in this paper,we give the overview about biogenesis,function,detection methods of circRNAs,novel circRNA identification and their associations with diseases,and we prospect its further study.

Research Advance on PPARα Gene in Livestock and Poultry

WANG Zhao-yang, LAN Li-ming, BAI Ding-ping, ZHANG Fu-jun, LI Ang

2018, 34(12): 32-40. doi:10.13560/j.cnki.biotech.bull.1985.2018-0504

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The peroxisome proliferator activator receptor（PPARs）is the ligand activator receptor in the nuclear hormone receptor family,and controls the metabolic process in many cells. As an important member of the peroxisome proliferator activated receptor family,PPARα is a key hub for regulating body lipid metabolism,and plays a critical role in regulating lipid metabolism in the liver of livestock and poultry. PPARα genes are composed of 4 structural domains and are mostly expressed in the liver and adipose tissue of body. PPARα as a nuclear receptor is activated by binding exogenous and endogenous specific ligands,then binds to the target gene to exert a regulatory role in the liver lipid metabolism of body. In this paper we respectively review the structure characteristics of PPARα and its expression pattern,the regulation mechanism of PPARα gene on lipid metabolism in liver,and the research progress of PPARα in livestock and poultry at present stage,aiming at raising awareness of PPARα regulation in lipid metabolism and providing theoretical supports for studying mechanism of lipid metabolism in the livers of livestock and poultry and the treatment of related diseases.

Advances and Reflect on Dominant Factors Determining the Sex of Pelodiscus sinensis

GAO Li-li, DIAO Xiao-ming, LI Yun, ZHAI Xu-liang, ZHOU Chun-long

2018, 34(12): 41-49. doi:10.13560/j.cnki.biotech.bull.1985.2018-0519

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The diversity of inputs that guide sexual fate during development is both intriguing and daunting. Reptiles,as poikilothermal animals,differing from mammalian conservative genotypic sex determination （GSD）,are in the transition period of environmental sex determination （ESD）and GSD. Its sex determination is sensitive to environmental factors,such as temperature and contaminants. Pelodiscus sinensis,a precious aquatic economic animal,is of great significance in research on sex determination in reptile biology. With further research on the machinery of sex determination in P. sinensis over the world,several studies have provided evidence of P. sinensis can be classified as temperature-dependent sex determination（TSD）,largely due to the sex ratio can be truly impacted by temperature;however,the results still vary,20%-30% individuals fail to develop along the direction of temperature. Second,the sex determination mode of P. sinensis is generally categorized as GSD,as evidenced by the existence of heteromorphic ZZ/ZW micro-sex chromosomes. However,little is known about the exact genetic mechanisms. In addition,many previous reports have focused on conserved genes with known function in sexual differentiation across vertebrates,such as Dmrt1,Sox9,Mis,Amh,Cyp19a1 and Foxl2,very few other genes have been investigated in detailed,leading to a limited understanding of overall gene expression throughout TSD. Furthermore,exogenous hormones have been demonstrated to induce sex direct differentiation during a specific embryonic period known as the temperature sensitive period（TSP）. Taking all together,these results indicate that the boundary line between GSD and ESD is blurred,a critical question of exactly how temperature cues and sex hormones trigger sex development has remained open. This review provides coherent information on the recentlydiscovered mechanisms underlying reptiles sex determination,aiming at overcoming the single breeding technology and providing scientific basis for the selection of high-quality breeding in P. sinensis.

Review on the Application of Integrated Transcriptome and Proteome Analysis in Biology

JIANG Ke-ren, MA Zheng, ZHENG Hang, LIU Xiao-Jun

2018, 34(12): 50-55. doi:10.13560/j.cnki.biotech.bull.1985.2017-0929

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The main steps of gene expression in an organism include transcription and protein synthesis,mRNA is an intermediate product of gene expression,and protein is the executive of gene function. For understanding the specific regulatory process of gene expression,the mRNA and protein should be synchronously monitored,then transcriptome and proteome data are compared,and thus both may be linked while an integrated analysis is conducted at the transcriptional and protein levels. In theory,the correlation between transcriptome and proteome data obtained from the same growth conditions and the cells,tissues or organs of the same state should be high;however,a lot of literature reported the irrelevant or negative correlations of expression trend between transcriptome and proteome data. Here we reviewed the principles and features of transcriptome and proteome sequencing and the advantages of transcriptome and proteome integrated analysis,as well as its applications in animal,plant and microbial. Meanwhile,we analyzed the reasons that might cause inconsistence of gene expression trends between transcriptional and protein levels,providing a reference for future study on molecular biology and multi-omics analysis.

Application of Environmental DNA Technology in the Study of Fish Resources

HAO Ya-bin ZHANG Ai-ju LIU Jin-dian GU Zhi-min

2018, 34(12): 56-62. doi:10.13560/j.cnki.biotech.bull.1985.2018-0580

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Environmental DNA（eDNA）refers to the sum of DNA fragments of different species extracted directly from the living environment of organisms,and eDNA is becoming more and more popular in the study of fish resources. It is a novel investigation approach for biological resources,i.e.,whether or not existence of fish species in a water environment can be determined,based on the identification of specific gene fragments of fish and by aligning with the identification fragments obtained via molecular methods to detect eDNA. Compared with traditional methods,eDNA technology has the advantages of high sensitivity,low cost,and no damage. It can quickly detect invasive species,endangered species,and rare species. However,there are some deficiencies for it,for instance,the real-time data whether target species exist or not cannot be acquired,biological characteristics such as the growth stages of species as well as population structure cannot be obtained,“purebreds” cannot be distinguished from hybrids. The eDNA technology is of subversive significance to the study of fish resources and shows a good momentum of development. This article mainly reviews the application of eDNA technology in the research of fish species diversity,resource estimation and population distribution,aiming at providing some ideas for the study of fishery resources. It can be expected that the combination of eDNA technology and traditional survey methods will become a development trend for fish resources research.

Research Progress on Pharmacological Actions and Extraction Methods of Polysaccharide from Phellinus igniarius

LIU Shuai, MO Jun-kai, PAN Dan-yang, LIU Gao-qiang

2018, 34(12): 63-67. doi:10.13560/j.cnki.biotech.bull.1985.2018-0685

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Phellinus igniarius is a precious medicinal fungus in China. Researches have shown that its medicinal ingredients include polysaccharides,flavonoids,triterpenoids,terpenoids,polyphenols,pyrones,alkaloids,and so on. Among them,polysaccharide is an important active ingredient of P. igniarius,and has broadly pharmacological effects such as anti-tumor,anti-oxidation,immune regulation,anti-fatigue,liver protection,blood sugar lowering,antibacterial and anti-inflammatory,and has no toxic and side effects,thus it has important research value. In recent years,many scholars have carried out a large number of experimental studies on the extraction of polysaccharides from P. igniarius. The results show that the solvent extraction method,ultrasonic extraction method and salt extraction method are suitable for mass industrial production,but the solvent extraction method consumes a large amount of resources. In terms of microwave extraction and enzymatic extraction,it is more suitable for small-scale extraction for experimental research. Its advantages are high extraction rate,convenient operation and fewer impurities. Based on the related research contents and results at home and abroad,this paper summarizes the pharmacological effects and polysaccharide extraction technology of polysaccharide of P. igniarius,and prospects its development trends,aiming at providing a reference for its further study.

Transcriptome-metabolomics Analysis and Its Application in Studying Drug Action Mechanism

JIN Yu, LI He-jian, FENG Cheng-qiang

2018, 34(12): 68-76. doi:10.13560/j.cnki.biotech.bull.1985.2018-0497

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With the development of high-throughput sequencing technology,high-throughput integrated analysis technology is an essential means to obtain the final biological information,and it is a revolutionary change to the research method for traditional drug action mechanism. Transcriptomics,metabolomics and the use of their combination are important components of systems biology. In recent years,they have been widely used in various fields related to drug mechanism research,such as development of new drug,improvement of drug efficacy,evaluating drug toxicity,and guiding therapy by combined drugs,etc.,it has become an indispensable screening stage in studying the drug action mechanisms. Here we review transcriptomics,metabolomics and 4 transcriptome-metabolomics conjoint analysis methods,analyze the advantages and disadvantages of the different methods as well as the existing problems,and briefly describe the application of the combined analysis method in the study of the drug action mechanism in recent years. Concurrently,we forecast its next development and challenges,with a view to exploring the strategies of transcriptomics and metabolomics and their combination in studying drug action mechanisms for future drugs. We expect that this review may provide the reference for understanding the molecular mechanism of drug action and subsequently for exploring new research methods and approaches based on the existing research foundation.

An Optimization on Identification of Irradiated Food by Fluorescence Quantitative PCR

LIU Mian-xue, FU Yi, ZHENG Yu, WANG Yan, PAN Lin, LI Hua, HUANG Min

2018, 34(12): 77-83. doi:10.13560/j.cnki.biotech.bull.1985.2018-0481

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Purpose of this study is to realize the identification ability of false positive samples and reduce template amount interference with real-time PCR results in optimizing irradiation food identification. Potato tuber was selected as material and treated with 60-Co in gradient dose（1-12 kGy）,irradiating,heating and freezing-thawing respectively. Analysis methods,including primer screening of transposon,detection limit analysis,comparison of DNA damage characteristics and dose curve regression,were used to explore the optimized effects of transposon primers in potato irradiation identification. As results,transposon primer combination of LTR2-5\18S-8 and LTR2-2\ACT-5 can be used for the identification of false positive samples,and the combination of LTR2-5\18S-5 can be used for irradiation identification. In the optimized method,the irradiation dose was linear with the exponential function of the initial cycle difference with the standard equation Y=-0.1581X+ 4.611 and determination coefficient R² 99.24%,which was superior to the original initial cycle difference-irradiation dose model. In conclusion,by the optimized method,the dose-effect relationship with real-time PCR in irradiation identification was improved with less possibility of false positive,and the result interference caused by template amount was eliminated.

Optimization of EDU Pulse Method for Tracing the Migration of Mouse Second Heart Field Cells

YUAN Bai-yin, LIU Zhong-ying

2018, 34(12): 84-89. doi:10.13560/j.cnki.biotech.bull.1985.2018-0518

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Outflow tract（OFT）malformation accounts for about 30% of detected human congenital heart diseases at birth. OFT and right ventricle are developed from second heart field（SHF）. Deployment and migration of SHF cells to heart tube is essential for sufficient lengthening of heart tube and subsequent heart morphogenesis. However,the development,migration and deployment process of SHF is still unclear. Establishing a method of marking and tracing the migration of second heart field cells in the mouse heart is of great significance in elucidating the cellular biological processes of SHF migration and deployment in mammalian hearts. Firstly,we adapted EDU（5-ethynyl-2'-deoxyuridine）pulse-chase assay with dose（10 mg/kg）reported in the literature,and the most embryo were not labeled by EDU. Following,we optimized the dosage and pulse time by analyzing the labeling effects of EDU in SHF cells and the migration of EDU-labeled SHF cells to OFT. The dosage of 40 mg/kg demonstrated effective labeling on SHF cells（31 embryos from 6 pregnant mice were efficiently labeled）,and no toxicity to embryos and pregnant mice. Moreover,2 h- and 4 h- EDU pulse period showed no difference in EDU labeling efficiency and the migratory distance to the distal OFT. Taking above results together,the 40 mg/kg EDU pulse dose and 2 h EDU pulse period presented effective EDU labeling of SHF cells in embryos,and many EDU labeling SHF cells can be detected in the distal OFT after 19 h tracing period.

Optimization of Culture Conditions for MDCK Cell Micro-carriers

MA Qi-cai, WU Wen-li, MA Fu-long, PAN He-ping

2018, 34(12): 90-95. doi:10.13560/j.cnki.biotech.bull.1985.2018-0491

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Studying the effect of cell inoculation quantity,stirring speed and microcarrier concentration on the MDCK cells in micro-carrier culture and optimizing the conditions of culturing MDCK cells micro-carrier during the age of the largest proliferation is of great significance for the separation of vaccine from virus,aiming at increasing the efficiency in separating vaccine from viruses. In this study,MDCK cells were cultured with a micro-vector and cultured at different stirring speed,concentration of the micro-vector,and amount of cell inoculation,and the optimal culture conditions were determined by sampling and counting every 24 h. The results showed that MDCK cells proliferation was faster and cell density was larger up to 16.5 x 10⁶cells/mL while cell inoculation in 20 balls,stirring speed in 45 r/min,and carrier concentration in 2 g/L,which is suitable for MDCK cells proliferation and growth.

The Competitiveness of Transgene Flow Between Indica and Japonica Rice Pollen Donor

ZHAO Jian-fa, HE Guang-liang, HE Mei-dan, KE Zhi, ZHAI Nan-xin, FU Ya-xiaoning, PEI Xin-wu, YUAN Qian-hua

2018, 34(12): 96-101. doi:10.13560/j.cnki.biotech.bull.1985.2018-0446

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Gene flew is an important part of the research on the safety of transgenic rice（Oryza sativa L.）. It is of great significance to study the effect of pollen competition on gene flow in establishing and improving the gene flew model. In this experiment,the Japonica /Indica of transgenic rice and the common rice plants were used as pollen donors,and the male sterile line BoA was a pollen receptor. The results showed that the gene flow frequencies of dual donors of the same species was positively correlated with the quantity of pollen sources,and the relationship between the gene flow frequency and the pollen quantity of donor was consistent with S curve. Gene flow is a comprehensive result of pollen quantity competition and genetic competition.

Responses of PVC-pipe Seedlings and Their Root Tip Microstructures of Different Drought-Resistant Potato Varieties to Drought Stress

QIN Tian-yuan, SUN Chao, BI Zhen-zhen, WANG Han, LI Xin, ZENG Wen-jie, BAI Jiang-ping

2018, 34(12): 102-109. doi:10.13560/j.cnki.biotech.bull.1985.2018-0674

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Two varieties of potato（C16：CIP397077.16 and C119：CIP398098.119）with the same growth period but different drought tolerance were selected from the resources in the International Potato Center（CIP）. Potatoes were planted in the PVC pipes where the root growth would not be restricted. The morphological characteristics,stress-resistant biochemical indicators,root tip microstructure,and ultrastructure of two potato varieties were systematically analyzed under different drought stress conditions. The results showed that drought stress led to significant increases in root length,root vigor,catalase activity,content of soluble sugar and proline,significant decreases in plant height,leaf-to-stem angle and root relative water content,and some visible changes in the core structure and the integrity of cell wall in the root tip of two varieties. These results indicated PVC-pipe-planting was ideal for studying potato root response to drought stress. Most of above indicators in C119 presented significantly better performance than C16 no matter under normal watering condition or drought stress treatment,i.e.,C119 had better adaptability to drought stress. In addition,the diameter and number of axillary xylem vessels of both varieties decreased under drought stress;moreover,the number of axillary xylem vessels of C119 with better drought resistance was significantly less than that of C16,suggesting that the potato may also resist drought stress by changing the structure of the water transport organization.

Construction of E-box Motifs and the Analysis of Its Interaction with BplMYB46 Transcription Factor of Betula platyphylla

WANG Bo, WANG Lian-ping, YANG Chun-yu, GUO Hui-yan, WEI Ji-cheng

2018, 34(12): 110-115. doi:10.13560/j.cnki.biotech.bull.1985.2018-0496

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MYB transcription factor is one of members in the largest family of transcription factors in plants;it is mainly involved in the regulation of plant secondary metabolism,the response of hormones and environmental factors,and thus plays a crucial role in plant growth and development. A study reveals that the core sequence of E-box is CANNTGN（N：A/G/C/T）,which is a cis-element associated with the response to light and phenylpropanoid biosynthesis pathway. A library of cis-acting elements was constructed in our previous study,and results by a transcription factor-centered yeast one-hybrid technology showed that the BplMYB46 transcription factor bound to the E-box cis-acting element with a core sequence of CAAATG in birch;however,it is unclear whether BplMYB46 may bind to other core sequence of E-box. Therefore,in this study,the renaturation of the double-stranded DNA of every core sequence of E-box cis-acting element was performed and connected to the pHIS2 vector. Further,the yeast one-hybrid technique was used to select E-box cis-acting elements that specifically bound to the BplMYB46 transcription factor. The results showed that the yeast colony grew on TDO/3AT medium when the third nucleotide of the core sequence of E-box was A/T/C and the fourth was A/G/C,respectively,indicating that the specific sequence of E-box cis-acting element bound by BplMYB46 transcription factor was CA（A/T/C）（A/G/C）TG. This study provides a data basis for improving the genetic traits of birch by analyzing the regulation of BplMYB46 transcription factor to downstream genes and screening downstream genes via the binding of BplMYB46 transcription factor with E-box cis-acting element,

Comparative Analysis on Transcriptome Among Different Sugarcane Cultivars Under Low Temperature Stress

TANG Shi-yun, YANG Li-tao, LI Yang-rui

2018, 34(12): 116-124. doi:10.13560/j.cnki.biotech.bull.1985.2018-0522

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In order to comparatively analyze the variations of molecular mechanism among different cultivars in cold resistance,RNA-seqs of GT08-1180 and ROC22 were conducted under cold stress and normal temperature. The results showed that a total of 57.41 G clean reads were produced and 183515 unigenes were obtained after de novo assembly. All unigenes were annotated in NR,NT,Swiss-Prot,PFAM,KOG/COG,KEGG,and GO,and 110 021 unigenes were functionally annotated. There were 16 145 and 20 317 differentially expressed genes（DEGs）in GT08-1180 and ROC22 respectively in the cold stress. In addition,there were more up-regulated DEGs than down-regulated DEGs in both GT08-1180 and ROC22. Total 13 113 DEGs were found to be the same in both GT08-1180 and ROC22,while 7 204 specific DEGs in ROC22,and 3 032 specific DEGs in GT08-1180. The result of GO enrichment analysis of the common DEGs in 2 varieties showed that common DEGs were enriched in sub-groups of cold stress response,membrane system,and photosynthesis. GT08-1180 enriched more in DEGs related to DNA integration,RNA polymerase,ADP binding,and metabolic enzymes. ROC22 presented more enrichment in DEGs related to synthesis and transfer of organic substances,and transporter proteases.

The Expression Analysis of Sucrose Transporter Genes in Disease Free Sugarcane

ZHAO Ting-ting, WANG Jun-gang, YANG Ben-peng, SHEN Lin-bo, FENG Xiao-yan, WANG Wen-zhi, FENG Cui-lian, XIONG Guo-ru, ZHANG Shu-zhen

2018, 34(12): 125-131. doi:10.13560/j.cnki.biotech.bull.1985.2018-0626

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Sucrose transporters are closely correlative with sugar storage and crop yield formation.It is helpful to clarify the molecular mechanism on increasing yield and sucrose content by analyzing the expression pattern of sucrose transporter genes in disease free sugarcane seedlings. The expression differences of three sucrose transporter genes,SUT1,SUT4 and SUT6 were analyzed between sugarcane diseases free seedlings and ordinary seedlings during the whole growth period by Real time PCR. The results showed that SUT1,SUT4 and SUT6 were up regulated in immature leaves of diseases free sugarcane plants at seedling growth stage,however SUT1 and SUT4 were down regulated and SUT6 were up regulated in mature leaves of diseases free sugarcane plants at seedling growth stage. SUT1,SUT4 and SUT6 were up regulated in leaves of diseases free sugarcane plants at tillering growth stage. SUT4 and SUT6 were up regulated in immature leaves and internodes of diseases free sugarcane plants at elongation growth stage. The expression of SUT1 was not different between diseases free and ordinary sugarcane plants at elongation growth stage. Moreover,SUT1,SUT4 and SUT6 were up regulated in leaves and immature internodes of diseases free sugarcane plants at mature stage. In conclusion,SUT1,SUT4 and SUT6 were up regulated in actively metabolism and rapid growth tissues of diseases free sugarcane plants. It postulated that the up-regulated sucrose transporter genes by disease-free treatment promoted sucrose partition and utilization to ultimately improve the cane yield and sucrose content.

Construction of Eukaryotic Expression Vector of SLA-2-HB01 from Hebao Pigs and Its Expression in PK15 Cells

ZHAI Xiao-xin, GAO Hua, JIANG Ping, XU Chong-bo, ZHANG Zong-hui, LI Wen-zhe, GAO Feng-shan

2018, 34(12): 132-139. doi:10.13560/j.cnki.biotech.bull.1985.2018-0695

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This work is to construct a eukaryotic expression vector of SLA-2-HB01 in Hebao pigs and observe its expression in PK15 cells. Firstly,a pair of primers was designed based on the sequence of coding region of SLA-2-HB01 gene,then the gene fragment of coding region of SLA-2-HB01 gene was amplified using SLA-2-HB01-pMD 18-T as template. The positive clones were sequenced after TA cloning and screened by colony PCR. The clones with correct sequences were cleaved and the target genes were recovered to ligate with the eukaryotic expression vector pEGFP N3. The recombinant plasmid SLA-2-HB01/pEGFP N3 was transformed into Escherichia coli firstly and to clone at a high copy way,then a large amount of extracted positive plasmids were transfected into PK15 cells by liposome method. The expression of the SLA-2-HB01 protein was detected by observing the fluorescence intensity of PK15 cells with fluorescence microscopy and by Western blotting. As results,the SLA-2-HB01 with 1 104 bp was successfully amplified,and the recombinant plasmid SLA-2-HB01/pEGFP N3 was constructed successfully. The inserted fragment was proved to be 1 092 bp by enzymatic cleavage. The SLA-2-HB01/pEGFP N3 recombinant plasmid transfected into PK15 cells displayed green fluorescence. Western blotting showed that the molecular weight of the SLA-2-HB01-EGFP fusion protein was 70 kD,which was consistent with the theoretical value. In conclusion,the recombinant plasmid SLA-2-HB01/pEGFP N3 was successfully constructed and expressed in PK15 cells as fusion protein SLA-2-HB01-EGFP,which lays a base for further studying the presentation of peptide epitopes for SLA-2-HB01.

Screening and Identification of an Effective Agarase-producing Marine Bacterium

ZHOU Qin-mao, XIE Yan-chun, CHEN Yan-mei, ZHANG Yang, KE De-sen, CHEN Qiong-hua

2018, 34(12): 140-146. doi:10.13560/j.cnki.biotech.bull.1985.2018-0512

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This work is to isolate,screen and identify the effective agarase-producing strain from Gracilaria lemaneiformis collected from Nan'ao Island,Guangdong province,for building a foundation for the utilization of agarase. Four different screening culture-mediums were used to screen the agarase-producing bacterium,the morphology,physiological and biochemical properties were employed to identify the isolated strain,and phylogenetic tree was constructed. DNS was adapted to determine the enzyme activity of the agarase,to study the type of the agarase from the strain,and to draw its growth curve and fermentation curve. As results,an effective agarase-producing strain ZQM2017 was isolated from the samples of G. lemaneiformis,which was a gram-negative bacterium,its 16S rRNA sequence was 99% homologous to 52 strains of Vibrio species in the database of NCBI. Combining with the morphological and physiological-biochemical characteristics,it was identified the strain ZQM2017 as Vibrio alginolyticus. The type of agarase by V. alginolyticus ZQM2017 was both α-agarase and β-agarase. Under the condition of 180 r/min and 28℃,the logarithmic phase of the strain was between 3 h to 9 h and the enzyme obviously produced at 5 h,and the enzyme activity of agarase from V. alginolyticus ZQM2017 reached 109.87 U/mL after fermenting for 46 h. In conclusion,the marine bacterium ZQM2017 isolated and screened independently from Nan'ao Island was identified as V. alginolyticus ZQM2017,and can secret α-agarase as well as β-agarase with the initial enzyme activity of 109.87 U/mL.

Screening and Identification of Actinomycetes with Antifungal Activity from the Guts of Periplaneta americana

ZENG Huan-xiong, FANG Xia, LIU Ling-yan, JIN Xiao-bao

2018, 34(12): 147-151. doi:10.13560/j.cnki.biotech.bull.1985.2018-0606

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This study aims to screen and identify actinomycetes strains with antifungal activity from guts of Periplaneta americana. Antifungal activities of 159 actinomycetes strains of Periplaneta americana were screened using the Oxford cup and confrontation co-culture method in vitro while using 4 species of human pathogenic fungi of Candida albicans,Trichophyton rubrum,Aspergillus niger,and Aspergillus fumigatus as the research object. Subsequently 16S rDNA PCR,Blast homology alignment and phylogenetic tree were used to identify target strains. Results showed that 45 actinomycetes had antifungal activity,many of them had a wide antifungal spectrum,and 10 actinomycetes strains were antagonistic to all 4 tested fungi. There were 32 strains Streptomyces that were dominant actinomycetes among them,accounting for 71.11%;6 strains were Gordon’s genus Microbacterium,2 strains were Mycobacterium,and there was 1 Achromobacter,Micromonospora,Cellulomonas,and Agromyces respectively. It is suggested that there are rich actinomycetes with antifungal activity on the guts of Periplaneta americana.

Mutant Construction and Functional Validation of NHX1 in Saccharomyces cerevisiae BJ3505

WANG Li-guang, CHEN Jun, YE Chun-lei, LUO Jun-jie

2018, 34(12): 152-158. doi:10.13560/j.cnki.biotech.bull.1985.2018-0561

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A Saccharomyces cerevisiae mutant with deficient NHX1 gene was obtained for verifying its function in S. cerevisiae BJ3505's resistance to stress and vacuole fusion. The BJ3505Δnhx1,the mutant of BJ3505 with inactivated NHX1,was cloned via homologous recombination. Then the complementary strain BJ3505Δnhx1-C was constructed to validate the function of NHX1 in stress tolerance and vacuole fusion. As results,mutant BJ3505Δnhx1 had a much lower ability in stress tolerance and an increase in cells harboring multiple vacuoles was observed,compared with the BJ3505Δnhx1-C and the wild one. Conclusively,the NHX1gene-deleted mutant strain was constructed successfully by homologous recombination. As a crucial ion antiporter,NHX1 plays an important role in the stress resistance and vacuole fusion of S. cerevisiae BJ3505.

Identification of the Interacting Surface Between ZMYND8 and RBBP4,a Core Components of the Nucleosome Remodeling and Deacetylase（NuRD）Complex

XIE Chang-lin, WU Ji-hui, TANG Ya-jun

2018, 34(12): 159-165. doi:10.13560/j.cnki.biotech.bull.1985.2018-0567

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Nucleosome remodeling and histone deacetylase（NuRD）complex contains multiple subunits and plays a critical role in transcription regulation and DNA damage repair. Human ZMYND8（Zinc finger MYND-type containing 8）participates in transcription regulation and DNA damage repair by forming complex with NuRD. Rbbp4, a core submit of NuRD, was expressed in baculovirus expression system in vitro and purified by Ni-NTA column and molecular sieve chromatography column to electrophoretic purity. Isothermal titration calorimetry（ITC）experiment was used to identify the interacting surface between ZMYND8 and RBBP4,and results from which demonstrated that RBBP4 bound to the basic amino acids（residues 43-54）at the N-terminus of ZMYND8. The site-directed mutation experiment at the amino acid residues of RBBP4 further confirmed the interacting surface of both. Combined with the previous findings,it is revealed that ZMYND8 binds to the surface of NuRD complex using the N-terminal basic region and C-terminal MYND domain by surrounding way.

Regulation of Efflux Pump MdrL by LadR in Listeria monocytogenes

XU Ya-meng, JIANG Xiao-bing, YU Tao

2018, 34(12): 166-171. doi:10.13560/j.cnki.biotech.bull.1985.2018-0430

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The aim of this study is to investigate the role of LadR in the regulation of efflux pump MdrL in Listeria monocytogenes. The mutant strain ΔladR was constructed from the wild type strain EGD-e by homologous recombination. Thetranscriptional levels of mdrL in the wild type strain EGD-e and the mutant strain ΔladR were tested by quantitative reverse transcriptase PCR. The expression strain BL21（DE3）-ladR was constructed,and then the recombination protein His₆-LadR was purified. Electrophoretic mobility shift assay（EMSA）was employed to assess the binding activity of LadR to the mdrL promoter. As results,the mutant strain ΔladR was constructed successfully. Compared to the wild type strain EGD-e,the transcriptional level of mdrL in ΔladR showed about 51-fold up-regulation. The expression strain BL21（DE3）-ladR was also constructed successfully,and the concentration of the purified recombinant protein His₆-LadR was 1 mg/mL. Results of EMSA showed that His₆-LadR protein was able to bind to the promoter of gene mdrL specifically. Conclusively,the regulator LadR negatively regulates the efflux pump MdrL by specifically binding to the promoter of mdrL.

Absence of dbpA Results in the Delayed Initiation of DNA Replication in Escherichia coli

ZHANG Shu-jun, Wunier, DI Jian-jun, ZHANG Guo-wen, XU Ya-nan

2018, 34(12): 172-178. doi:10.13560/j.cnki.biotech.bull.1985.2018-0538

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This work is to investigate the effect of ribosome maturation factor dbpA on the initiation of DNA replication in Escherichia coli. The phenotype changes such as DNA replication pattern,doubling time and cell size of ΔdbpA deletion mutants were observed,and the temperature sensitivity test and protein quantitative experiment were used to study the action mechanisms of dbpA. The results showed that the mild phenotypic change such as the delayed initiation of DNA replication,prolonged doubling time,declined cell size occurred in ΔdbpA cells compared with wild type BW25113. Moreover,the change in LB medium was more obvious than that in ABTGcasa medium. The temperature sensitivity test demonstrated that DbpA did not interact with DnaA,DnaB or DnaC protein. The protein quantitative experiment revealed that the amount of total protein and DnaA protein in ΔdbpA cell decreased,and the decrease degree was greater in LB medium than in ABTGcasa medium. Thus,the absence of dbpA leads to the delayed initiation of E. coli DNA replication,the reason of which may be that dbpA affects the protein synthesis through influencing the assembly or translation activity of ribosome,and subsequently reduces the total protein and DnaA protein in cells. This study provides a reference to clarify the function of dbpA.

Wdr1 Knockout Inhibits Migration and Proliferation of Mouse Primary Vascular Smooth Muscle Cells

YUAN Bai-yin, HU Ji-sheng, LIU Zhong-ying, HUANG Xia

2018, 34(12): 179-185. doi:10.13560/j.cnki.biotech.bull.1985.2018-0578

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Cytoskeletal actin plays an important role in cell proliferation,migration,and other biological processes. However,the role of WDR1,a major cofactor for actin depolymerizing,has not been reported in smooth muscle cells（SMCs）. Inducible Wdr1 conditional knockout mouse model was constructed,and then sacrificed to isolate mouse primary vascular smooth muscle cells（VSMCs）and induced to detect the morphology,proliferation,and migration. The PCR results suggested that Wdr1^f/f;ERT2Cre mouse model was successfully constructed. The results of immunofluorescence revealed that the improved separation method can effectively separate primary smooth muscle cells. A significant effect on cell morphology after Wdr1 knockout was observed. Concurrently,the scratch and CCK8 test results also showed that Wdr1 conditional knockout obviously inhibited the migration and proliferation of SMCs. The changes of smooth muscle cell morphology and cell biological processes after Wdr1 knockout indicate that it is closely related to the occurrence and development of vascular diseases. It is of great significance to uncover the pathophysiological process of blood vessels.

Research and Prospect of the CRISPR Technology Based on Global Patent Information

WANG You-hua, ZOU Wan-nong, ZHANG Yi, FAN Jun-li, SUN Guo-qing

2018, 34(12): 186-194. doi:10.13560/j.cnki.biotech.bull.1985.2018-0204

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Since the advent of the technology system for gene editing by clustered regularly interspaced short palindromic repeats（CRISPR）in 2012,it has been developed rapidly in the fields of medicine,agriculture and environmental protection in just five years due to its characteristics,such as editing multiple sites simultaneously,high efficiency of editing and easy operation of design process,etc. A statistical analysis of the world-wide patents in the field of the CRISPR from 2006-2017 is accomplished based on patent search and service system of SIPO,and the future CRISPR gene editing technology is prospected,aiming at understanding the current situation,development trends,hotspot,distribution and competition pattern in this field.

Research Trend and Technical Composition of Cannabis sativa Based on Patent Analysis

CHANG Li, DENG Xin, TANG Hui-juan, LI Jian-jun, HUANG Si-qi, CHEN An-guo, ZHANG Cui-ping, ZHAO Li-ning, LI De-fang

2018, 34(12): 195-201. doi:10.13560/j.cnki.biotech.bull.1985.2018-0663

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The objective of this work is to analyze the research trend and technical composition of Cannabis sativa-related patents from 1985 to 2017 in PatSnap database. We designed a suitable query formulation,moreover,translated and screened the retrieved patents manually or by more detailed logic strategy during searching. Results showed that the total number of applied C. sativa-related patents was 28 542,of which 24 521 were invention patents,7 565 were valid patents and 13 233 were invalid patents. The top 3 countries in the world by patent applications were China,the United States,and Germany;while the top 3 provinces in China were Jiangsu,Zhejiang and Shandong. The vast majority of inventions were in the hands of corporations,while universities owned few patent applications. The scope of patent protection related to C. sativa was mainly concentrated in such fields as medical,food,textile,building materials,etc. This analysis is conducive for China to promptly discovering technology blanks in C. sativa-related field and conducting research more scientifically.

Technology Situation and Hotspot Analysis in Using Polygonatum sibiricum Based on Innography

DENG Xin, MEI Shi-Yong, CHEN Xiao-Jun, XIAO Qing-Ming, LI Yu

2018, 34(12): 202-206. doi:10.13560/j.cnki.biotech.bull.1985.2018-0658

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As a traditional Chinese medicine,Polygonatum sibiricum has important medicinal value,edible value and economic development value. Based on the Innography patent analysis platform,this paper not only discussed the development trend of global and Chinese P. sibiricum from the perspectives of patent applications,patents layout,technology source,competitive situation and IPC technology category,but also further analyzed source and patentees of core patents,and technical hotspots based on exploring the core patents in this field. Studies have shown that worldwide P. sibiricum research is in a period of rapid development,and China owns a large number of patents in the field and controls the most core patents. P. sibiricum related patents are mainly applied by enterprises and related technologies are mainly concentrated in 4 enterprises in Qingdao,Zhejiang and Shandong. The application area is mainly distributed in IPC-A department and the main directions are herbal formula containing P. sibiricum,extraction of active substances and development of alcoholic beverages. The result may provide an important reference for comprehensively understanding the development of P. sibiricum research and formulating scientific industrial development policies.

Development Trends and Technical Hotspots in Feed Processing Technology of Bast Fiber Crops Based on the Analysis of Patents

YANG Jing, DAI Qiu-zhong, HOU Zhen-ping, WANG Yan-zhou, WANG Man-sheng

2018, 34(12): 207-214. doi:10.13560/j.cnki.biotech.bull.1985.2018-0628

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With the rapid development of animal husbandry in China,the situation of "human and livestock fighting for food" occurs. In the meanwhile,using bast fiber crops as feeds may expand the feed source of livestock and poultry,thus it is expected to effectively alleviate such situation. In order to better understand and grasp the development status of feed processing technology of bast fiber crops,the relevant patents of using bast fiber crops as feeds was searched from the Innography patent database while techno-sphere of feed processing technology for the bast fiber crops chosen as research object. Further,the patent portfolio,major patent technologies,the core patents,and the competences of patentees in global were completely analyzed. In the meanwhile,the 5 main research fields of raw materials,flax seed,hemp,flax harvester and animal feed were analyzed by means of the text clustering function of patent analysis software. The research results are aimed to provide references for the transformation of bast industry and using bast fiber crops as feed in China.

Research on the Patent Retrieval Strategy for Agricultural Biotechnology

XU Qian, LI Xiao-man, HAO Xin-ning, SUN Wei

2018, 34(12): 215-220. doi:10.13560/j.cnki.biotech.bull.1985.2017-1087

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Agricultural biotechnology is not only the basis of the biotechnology research and development, but also the most direct, extensive and important field in biotechnology application. Agricultural biotechnology makes great importance on agricultural production. Patent documents have great value in technological forecasting and technological innovation for the technical, economic and legal information contained. In order to make a comprehensive analyze on the agricultural biotechnology,this paper discussed the existing problems and difficulties in patents retrieval, identified International Patent Classification（IPC）codes related to agricultural biotechnology, constructed the patent retrieval strategy based on the literature features of agricultural biotechnology patents and characteristics of IPC system, and finished the data collection, which can serve as the data base of the global agricultural biotechnology.

Analysis of Development Trend of Agricultural Biotechnology Based on Patent Bibliometrics

LI Xiao-man, SUN Wei, XU Qian, HAO Xin-ning

2018, 34(12): 221-231. doi:10.13560/j.cnki.biotech.bull.1985.2018-0751

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As one of the fastest growing high-tech fields in current world,agricultural biotechnology has gradually rewritten the history of species evolution in centuries. The research of its development trend is of great significance to the government and relevant researchers. On the basis of Incopat patent database,this paper analyzes application years,national patent layout,main R&D organizations,main techniques and high value patents with the aid of DDA,EXCEL and other tools,and summarizes the current situation and the development trend of agricultural biotechnology. The results show that：（1）The development trend of agricultural biotechnology in China is not in the same rhythm as that of international agricultural biotechnology. Particularly,the research ardour of international agricultural biotechnology is declining,while that in China is gradually heating up. （2）Though international corporations are still in a dominant position,Chinese institutions are breaking this situation. （3）Chinese agricultural biotechnology is developing rapidly;however,the quality needs to be improved.（4）In the field of GM technology,applying patent in gene sequences and improving genotypes methods is more active than that in other sub-technologies. Producing new varieties and improving safety by utilizing transgenic plants is blank in the patent protection.

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2018, 34(12): 300.

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