Table of Content

26 July 2020, Volume 36 Issue 7

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Advances on the Molecular Action Mechanisms of Plant miRNA

ZHANG Cui-ju, MO Bei-xin, CHEN Xue-mei, CUI Jie

2020, 36(7):  1-14.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0262

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microRNAs（miRNAs）are a class of 20- to 24-nucleotide（nt）endogenous noncoding single-stranded RNAs widely exisitng in eukaryote. miRNAs in plants negatively regulate the expressions of target genes through mRNA cleavage or translation repression and play important roles in cell proliferation and differentiation,growth and development of an individual,and responses to environmental stresses. Since the first identification of plant miRNAs in 2002,research on miRNAs has become a hotspot in the field of plant molecular biology. Decades of research have elucidated the mechanisms of miRNA biogenesis and degradation and revealed the regulatory roles of miRNAs in numerous biological processes in plants. However,mechanisms underpinning the action of plant miRNAs are still not well understood,especially with respect to miRNA-mediated translation repression. This review summarizes both historical perspectives and the latest progress on the plant miRNAs-mediated mRNA cleavage and translation repression,discusses the influencing factors and relationship of two mechanisms,and suggests the research direction and thoughts in future,aiming at providing a theoretical basis for in-depth understanding of the mechanism of plant miRNA and promoting the basic research and practical application of miRNA.

Non-canonical Auxin Signaling Pathway in Plants

MA Jun, XU Tong-da

2020, 36(7):  15-22.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0523

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Phytohormone auxin is involved in various processes of plant growth and development,including embryogenesis,organogenesis,tropism and so on. Plants precisely regulate growth and development by coordinating auxin biosynthesis and metabolism,polar transport and signal transduction. The biological function of auxin relies on the diverse downstream responses converted by signal transduction cascades after being sensed. The canonical auxin signaling pathway starts from its perception by the SCF^TIR1/AFB receptor in nucleus,followed by the ubiquitination-based degradation of Aux/IAA transcription repressors and the release of ARF transcription factors,finally regulates gene transcription. Although this canonical signaling pathway illustrates the molecular mechanisms of auxin in regulating many plant developmental processes,there still have many auxin actions that cannot be explained by this pathway. This review focuses on the regulatory mechanisms of non-canonical auxin signaling pathways and its critical role in plant growth and development,as well as discusses and prospects the challenges and the future research directions in this field.

Research Progress on DNA Guanine Quadruplex

FENG Yi-long, ZHANG Wen-li

2020, 36(7):  23-31.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0205

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G-quadruplex（G4）refers to a four-stranded secondary structure formed by guanine-rich nucleic acid sequences through Hoogsteen hydrogen bonding in the DNA or RNA strand. Studies of G4 in humans and animals demonstrate that G4 is involved in a wide range of basic biological functions such as DNA replication,transcription,translation,and maintenance of telomeric structure. By contrast,researches on the biological functions of G4 in plants have lagged way behind. This paper reviews the research progress on the methodologies,biological functions and possible mechanisms of DNA G4s in humans and animals,summarizes the research status and possible biological functions of G4 in plants,and finally prospects the application of G4 in diagnosis and treatment of human diseases as well as molecular crop breeding.

Transcriptome Analysis of Saliz matsudana Under Cadmium Stress

CAO Ji-min, LI Shuang-cai, HE De

2020, 36(7):  32-39.  doi:10.13560/j.cnki.biotech.bull.1985.2019-1018

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With the expanded application of heavy metal cadmium,the soil cadmium pollution is increasingly serious. Saliz matsudana,which has potential of phytoremediation,was selected as research object,and the changes of gene expression and metabolic pathway were investigated after 1 d,7 d and 30 d under 2.5 mg/L and 50 mg/L concentrations of cadmium stress. The result of transcriptome sequencing showed that 102 595 Unigenes were obtained,and there were 26 623 and 32 154 differential expressed genes（DEGs）in the same concentration while at different stress time,and 8 550,3 444 and 11 428 DEGs in different concentrations while at the same stress time. Total 25 genes closely related to cadmium stress responses were screened from them. The changes of genes expression（such as metallothionein,ABC transporter,zinc and manganese transporter）depended on both concentration of cadmium stress and stress time. The expressions of several genes were obviously up-regulated after cadmium stress,for example,3,6-deoxyinosinone ketolase（ROT3）in brassinolide synthesis pathway,as well as flavonoid synthase（FLS）and flavanone-3-hydroxylase（F3H）in the synthesis pathway of flavonoids. In addition,GO analysis showed that GO entries were mainly enriched in metabolic processes,cellular processes,membranes,cell organelles,cells,cellular fractions,catalytic activation and binding proteins in response to cadmium stress,and the number of DGEs involved in these GO entries increased along with cadmium concentration and stress time. The analyzed response mechanisms of S. matsudana after cadmium stress in this study by transcriptome sequencing provide theoretical guidance for the remediation of cadmium pollution in soil by S. matsudana.

Cloning,Sequence Analysis and Expression Characteristics of Gene Ty-6 Related to Tomato Resistance to Yellow Leaf Curl Disease

ZHU Xiao-lin, WEI Xiao-hong, WANG Bao-qiang, WANG Xian, ZHANG Chao-yang

2020, 36(7):  40-47.  doi:10.13560/j.cnki.biotech.bull.1985.2019-1042

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This work aims to further explore the yellow leaf curl disease of tomato at the molecular level and expand its disease resistance gene pool. The experiment was carried out by cloning the gene Ty-6 related to tomato yellow leaf curl disease,then using bioinformatics to conduct sequence analysis of its primary,secondary and tertiary structures,further constructing a phylogenetic tree,and finally detecting the expression characteristics of the gene Ty-6 in different tissues at different stages after infecting yellow leaf curl virus. The results showed that the gene Ty-6 encoded 305 amino acids,the encoded product belonged to the basic protein,and the secondary structure was dominated by random coils. The protein was predicted to be localized on the plasma membrane;and it contained 51 phosphorylation sites and 39 glycosylation sites. The promoter region was mainly TATA-box,and contained multiple elements of light response,jasmonic acid,salicylic acid and abscisic acid to induce gene expression. Additionally,phylogenetic tree results indicated that its affinity relation with potato was the closest. q-PCR results demonstrated that the expression level of this gene in the leaves after infection was significantly higher than that of the control group,and it expressed tissue specially. The results of studying gene Ty-6 not only expand Ty gene toolkit availability to breeders,and also provide a feasible approach of resisting disease.

Response to NaCl and ABA in Arabidopsis thaliana of the Double Silent Gene VHA-c2&c4

SU Jie, GUO Rong-qi, GAO Yang, YU Xiu-min, LI Guo-jing, WANG Rui-gang

2020, 36(7):  48-54.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0098

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For studying the functions of vacuole H⁺- ATPase subunit c gene（VHA-c2 and VHA-c4）in plant growth and response to abiotic stress,we constructed an RNAi expression vector of VHA-c2&c4 and then transfected it into wild-type Arabidopsis thaliana plants by the floral dip method. Transgenic homozygotes were screened and identified by kanamycin,the expression levels of VHA-c2&c4 were analyzed by semi-quantitative RT-PCR,and the treatments of NaCl and ABA on them were conducted. The results showed that 7 T₂ lines of double silent gene homozygous were obtained,and their mRNA expression levels of the two genes were lower than the wild type. Three lines with high silencing efficiency were selected for NaCl and ABA treatment. The relative elongation of primary roots and seed germination rate were both lower than that of the control group under NaCl treatment,indicating that the lines of VHA-c2&c4 gene silencing were sensitive to NaCl stress. The cotyledon expansion and the relative elongation of the primary root were both higher than the control under ABA treatment,indicating that the lines of the VHA-c2&c4 gene silencing were not sensitive to ABA inhibition. The results of NMT experiment showed that ABA promoted the H⁺ inflow ability of double-silenced gene homozygote lines. We hypothesized that the transcriptional levels of the genes have an impact on plant responses to salt stress and ABA-mediated signaling pathways.

Cloning of CiNAC038 Gene Promoter in Caragana intermedia and Profiling of Its Responses to Hormones

HONG Ge-riqiqige, WANG Yan-fei, GAO Xian-ling, PANG Cai-xia, SHANG Xiao-rui, LI Guo-jing, WANG Rui-gang

2020, 36(7):  55-61.  doi:10.13560/j.cnki.biotech.bull.1985.2019-1078

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The NAC transcription factor family is one of the large specific families in plants and play essential roles in plant development. To understand the molecular mechanism of CiNAC038 expression regulation would lay the foundation for the study of CiNAC038 function in Caragana intermedia. Genome walking method was used to clone the promoter sequence of CiNAC038 gene from genomic DNA of C. intermedia,and Cis-acting elements of the promoter were analyzed. The GUS expression vector was constructed and transformed into Arabidopsis thaliana,and the tissue-specific expression analysis of A. thaliana was performed. Transgenic plants were induced with ABA and the relationship between ABA and CiNAC038 was investigated. The result showed that the cloned fragment was 1 800 bp and contained multiple cis-elements. The vectors ProCiNAC038∶GUS was constructed successfully,and transformed into wild-type A. thaliana by floral dip method. The results of GUS histochemical staining showed that the roots of transgenic Arabidopsis seedlings were darkly stained while the hypocotyls were unstained. The mature veins,fruit pod ends,petals,anthers and other tissues of the transgenic Arabidopsis were darkly stained while the stems were unstained. The GUS reporter gene driven by the CiNAC038 promoter was mainly expressed in tissues and organs of plant leaves,roots and flowers. ABA-induced expression revealed that GUS stains became lighter with increasing concentration. CiNAC038 promoter might be an ABA-inhibitive promoter.

Effects of Different Regulatory Elements and Their Combinations on Transient Expressions of Exogenous Proteinsin Nicotiana benthamiana

LIU Wen-hao, WANG Rui-feng, LIU Wan-lin, XU Jie

2020, 36(7):  62-71.  doi:10.13560/j.cnki.biotech.bull.1985.2019-1226

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The plant transient expression is an efficient way to have foreign gene expressed,and this system can be used for analyzing the protein interactions,identifying the localizations of proteins,investigating the functions of regulatory elements,producing proteins of interest,etc. To further improve the expressions of foreign genes in transient expression system,this study focused on the optimization of various functional elements and transformation conditions of the transient expression vectors. Firstly,the commonly used binary vector pCambia1300 was used as backbone for the insertions of functional elements that can increase the specific transcription and the expressions of stable proteins,and thenrecombinant vectors pREU-EF,pREUR-EF,and pREUR-p24-EF were constructed. Next,the qualitative and quantitative methods were applied to evaluate the effects of different element combinations and different agrobacterial suspension concentrations on the expression of reporter gene Enhanced Green Fluorescent Protein（eGFP）. The results of laser scanning confocal microscope showed that the eGFP in the recombinant vectors expressed rapidly in the cell membrane,cytoplasm,and nucleus of tobacco leaves. The quantitative PCR results indicated that the expression ofthe eGFPin thepREU-EF,pREUR-EF and pREUR-p24-EF increased by 4 times,20 times,and 28 times,respectively,while compared with the pCambia1301-eGFPvector. Moreover,the results from protein level analysis suggested that compared with pREU-EF,pREUR-EF and pREUR-p24-EF could obtain more proteins 48 hours post agroinfiltration. Furthermore,the expression of the foreign protein reached the highest when the concentration of agrobacterial suspension OD₆₀₀ was about 0.4. In addition,the transcription regulation analysis in dual-luciferase reporting system implied that the process was more rapidly and efficiently while using the modified recombinant transient expression vectors. Therefore,this study demonstrates that the combinations of regulatory elements in transient expression vectors can effectively increase the expressions of foreign genes at the transcription and translation levels.

Preparation and Potential Application of High-titer Polyclonal Antibody Against Capsid Protein of Porcine Circovirus Type Ⅱ

ZHANG Wen, LIU Zhao-zhen, LI Jun-shuo, SHANG Ying-li

2020, 36(7):  72-79.  doi:10.13560/j.cnki.biotech.bull.1985.2019-1209

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Capsid protein（Cap）is the major antigen protein of porcine circovirus（PCV）and plays critical roles in viral infection and elicitation of host immune responses. To prepare a high-titer polyclonal antibody against PCV2b Cap,a PCV2b Cap truncated protein without nuclear localization signal peptide was expressed by Escherichia coli prokaryotic expression system,and soluble recombinant protein was acquired. White rabbits were then immunized with the purified soluble recombinant protein to generate antiserum and anti-Cap IgG was further purified by caprylic acid-ammonium sulfate precipitation. ELISA analysis showed that the titer of the purified anti-Cap antibody was 1∶10⁷,indicating that the antibody was really in high-titer. The antibody was then applied to infect PCV2b-infected PK-15 cells or mouse tissues as well as clinical samples,and then they were detected by immunoblotting,indirect immunofluorescence,or immunohistochemistry. The results showed that the antibody specifically bound with target antigens. In summary,the prepared high-tier anti-Cap polyclonal antibody in this study may provide a candidate tool for the diagnosis and research of PCV2b-assciated diseases.

Molecular Characteristics of Bosgrunniens FAF1 Gene and Its Expression in Ovaries,Fallopian Tubes and Uterus at Different Stages

WANG Jing-yu, WANG Meng, PAN Yang-yang, WANG Jing-lei, ZHANG Rui, MA Rui, HU Xue-quan, QIU Xiao-fei, CUI Yan, YU Si-jiu, XU Geng-quan

2020, 36(7):  80-89.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0958

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FAF1（fas-associated factor-1）binds to Fas protein in a variety of cells,mediating the initiation of apoptosis,and its gene mutation maylead to the death of embryos in the mitotic phase. The purpose of this study is to explore the molecular characteristics of Bosgrunniens FAF1 gene and its biological role in reproductive cycle. The FAF1 gene was cloned from ovaries,fallopian tubes and uterus of female B.grunniensat 3 different stages（luteal,follicular and the third month of pregnancy, the third month of pregnancy is hereinafter referred to as pregnancy）. The expression levels of genes and proteins were detected and located by real-time fluorescence quantitative PCR（qRT-PCR）,immunohistochemistry and Western blot. As results,the coding region（CDS）of B. grunniens FAF1 gene was successfully cloned in this study,its length was 1 953 bp（GenBank：MK416195）,encoding 650 amino acids. Genetic traits analysis showed that the gene was highly conservative,its encoded protein was non-transmembrane and soluble protein containing 5 protein binding sites,mainly distributed in the nucleus（52.2%）,mitochondria（26.1%）,the cytoplasm（13.0%）,golgi body（4.3%）,and cytoskeleton（4.3%）. The results of qRT-PCR demonstrated that the expression of FAF1 gene in the ovary was the highestat the follicular stage,followed by the pregnancy stage and the lowest attheluteal stage.The expressionin the oviductwas the highestat theluteal stage. The expression in the ovarywas the highest atthe follicular stage,the second atthe pregnancy stage,and the lowestat the luteal stage. The protein level showed that the relative expression of FAF1 protein in the fallopian tube and uterus during pregnancy was significantly higher than that atthe follicular and lutealstages. The expression of FAF1 protein in the ovary was the highest in the lutealstage,followed by the follicular stage,and the lowest in the pregnancystage. Immunohistochemistry（IHC）results revealed that there was no significant difference in the expression sites of FAF1 protein in the same tissue at different stages. The main expression sites in ovary were ovarian reproductive epithelium,granulosa cells,follicular membrane cells and corpus luteum cells（follicular phase）. The main expression sites in the fallopian tube were mucosal epithelial cells. The main expression sites in the uterus were endometrial cells and uterine glands. In conclusion,the expressions of FAF1 gene and protein in different reproductive cycles of the same reproductive organ were significantly different,which indicated that it was of great significance to the physiological regulation of B.grunniens reproduction.

High-throughput Screening High-yield Bacitracin Strain from Bacillus licheniformis DW2

MI Liang-bo, ZENG Wei-zhu, HUANG Ke-xue, WANG De-ming, DU Guo-cheng, ZHOU Jing-wen, CHEN Jian

2020, 36(7):  90-96.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0080

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Bacitracin,as secondary metabolite with complex biotransformation pathway,is a widely used broad-spectrum peptide antibiotic. An industrial strain Bacillus licheniformis DW2 was used for this study,and strains of high-yield bacitracin were obtained using the Atmospheric and Room Temperature Plasma（ARTP）mutagenesis and an established high-throughput screening strategy based on flow cytometry（FCM）. Finally,the 5 best performing strains with regard to bacitracin yield were determined by screening,and they were 8#6D,7#5E,6#7E,5#8D,and 2#5F. The yield increased by 20.9%,20.9%,20.8%,19.7%,and 22.2%,respectively,compared with that of starting DW2 strain. The highest titer of the bacitracin reached 912 U/mL. With the help of high-flow sorting by flow cytometry,high-throughput screening characterized of simple and safe can quickly increase bacitracin production.

Isolation,Identification and Antimicrobial Activity Determination of Actinobacteria from the Lakes in Tibet

PAN Wen-juan, LIN Jia-fu, WANG Xiao-tao, GUO Yi-dong, CHU Yi-wen, LIU Chao-lan

2020, 36(7):  97-103.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0970

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To study the diversity and antimicrobial activity of actinobacteria isolated from the sediment samples of lakes in Tibet aims to discover the medical actinobacteria producing novel active materials. Seven sediment samples were collected from lakes in Tibet,and from which actinobacteria were isolated using dilution plating method with 4 different media and then identified by 16S rRNA sequence analysis. Antimicrobial in vitro activities of the isolates were tested by paper disk diffusion method. In vivo activities of the isolates were evaluated for their ability to rescue Caenorhabditis elegans infected with tested pathogens. Results showed that the 73 actinobacteria were distributed in 8 genera affiliated with 5 families of 4 orders,Streptomyces and Micromonospora of them were the dominant genera. The 55 of 73 strains demonstrated positive results in antimicrobial assay,and the positive rate was 75.3%. Eight isolates presented fine antibacterial activity in vivo based on C. elegans infected with pathogens. Strain PF188 showed favorable antimicrobial activity both in vivo and in vitro,and deserved further research. The 16S rRNA sequence similarity between strain PF188 and the nearest Nonomuraea guangzhouensis NEAU-ZJ3^T was 98.13%,indicating that strain PF188 is a potential new species of Nonomuraea. The sediment samples from lakes in Tibet are rich in cultivable actinobacteria,which could be a potential source for the discovery of new antibiotics.

Identification of the Mycoparasites from Aecidium pourthiaea and Screening

SUI Guo-qiang, ZHANG Deng-yun, KONG Lei, CHEN Yu-hui, LI Jing

2020, 36(7):  104-111.  doi:10.13560/j.cnki.biotech.bull.1985.2019-1154

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Three Pestalotiopsis strains PG52,PG53 and PG90,which are mycoparasites of Aecidium pourthiaea,will be identified to species,and the optimal toxigenic medium of the 3 mycoparasites are screened in this study,for laying a foundation for the isolation and identification of toxins. The phylogenetic trees of 3 strains were constructed by PAUP software,and combined with morphological characteristics,they were identified to species. The crude extracts prepared from different medium were used to treat the aeciospores of A. pourthiaea and Cronartium ribicola,and the optimal toxigenic medium were screened by Trypan blue staining and bioassay. The results of molecular identification showed that PG52 and PG90 belonged to the same branch,and they were in the same big branch with Pestalotiopsis kenyana and Pestalotiopsis oryza. PG53 and Pestalotiopsis telopeae belonged to the same branch. On the morphological characteristics,the morphological description of PG53 was basically consistent with that of P. telopeae. The morphological characteristics of PG52 and PG90 were consistent with the morphological description of P. kenyana. The effects of treating spores by the crude extracts from PG52 in the improved Fries medium were the best,and also that by the crude extracts from PG53 and PG90 in the modified M-1-D medium were the best. In conclusion,PG52 and PG90 belong to P. kenyana. PG53 belong to P. telopeae. The optimal toxigenic medium for PG52 strain is the improved Fries medium,and the optimal toxigenic medium for both PG53 and PG90 strains was the modified M-1-D medium.

Multiple-site Mutations in Escherichia coli Capable of High-density Growing Induced from the Biosynthesis of Polyhydroxybutyrate

CHEN Qiao, WU Hai-ying, WANG Zong-shou, XIE Yu-kang, LI Yi-qing, SUN Jun-song

2020, 36(7):  112-118.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0004

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Biosynthesis of polyhydroxybutyrate（PHB）in Escherichia coli can be achieved by the overexpression of the operon phaCAB. Improving strain performance is regarded as the one of the key factors to lower the cost of PHB bio-production. An E. coli mutant S17-3,when having synthetized plasmid pBhya-CAB of expressing PHB,is able to grow at high density,and the maximal OD₆₀₀ reached about 40 when growing in shake flasks. Comparative analysis of whole-genome scan sequencing and transcriptome sequencing revealed that mutations in reading frame sequence or at transcription level occurred in the carbon metabolic flux-related proteins,such as pgi of encoding phosphoglucose isomerase and zwf of encoding glucose-6-phosphate 1-dehydrogenase. Null deletion of zwf in S17-3 diminished the growth density of the strain,while pgi knockout in BW25113 promoted the transformant cells’ growth density. Therefore,it is revealed that the high-density growth of S17-3 is closely related to the optimization of itself carbon metabolic flux.

Celastrol and Apoptin Mutant Exert Synergistic Anti-tumor Effects by Enhancing Nur77-induced Apoptosis Pathway

YIN Xiao-meng, CAO Xue-wei, WANG Fu-jun, ZHAO Jian, ZHANG Hui-zhan

2020, 36(7):  119-129.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0944

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Both celastrol and apoptin can exert anti-tumor effect through the Nur77 pathway. We named a truncated variant of apoptin as tApoptin. tApoptin co-administered with esculentoside A（EsA）is known simply as tApoptinE. Combination of cleastrol with tApoptinE is applied to enhance the inhibiting tumor cell growth activity of these two drugs,and the molecular mechanism for these two drugs synergistically exerting anti-tumor ability is studied. The cytotoxicity of the combination of celastrol with tApoptinE on tumor cells was detected by MTT assay,and the molecular mechanism of cell apoptosis was observed by flow cytometry and Western blot analysis. The results showed that celastrol whose concentration is above 300 nmol/L could effectively increase the anti-tumor activity of tApoptinE in cell line SMMC-7721 in comparison with the free tApoptinE. Under the combined conditions above,the combined index values of all combined groups were < 1,which showed that celastrol and tApoptinE synergistically inhibit the growth of tumor cells in vitro in this case. In addition,600 nmol/L celastrol in combination with tApoptinE had the best synergistic effect. Flow cytometry analysis revealed that the synergistic effect was due to cell apoptosis enhancement. Western blot analysis showed that after combined administration,celastrol and tApoptinE synergistically targeted their common Nur77 pathway which will have Nur77 phosphorylated. And the phosphorylated Nur77 can significantly regulate the expression of Caspase and Bcl-2 family proteins,hence enhancing the induction of apoptosis. Therefore,the combination of celastrol and tApoptinE can further induce tumor cell apoptosis by enhancing the apoptosis pathway induced by Nur77,so as to exert synergistic anti-tumor effect.

Outdoor Cultivation Oil Extraction of the Salt-tolerant Microalga,Eustigmatos sp.

LI Tao, ZHAO Wei, YANG Bing-Jie, CHEN Zi-Shuo, WU Hua-lian, WU Hou-bo, XIANG Wen-zhou

2020, 36(7):  130-138.  doi:10.13560/j.cnki.biotech.bull.1985.2019-1050

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Due to the fast growth and the ability of accumulating high content of microalgal oil and polyunsaturated fatty acids,Eustigmatos sp. has received widespread attention from microalgal scientists. Assessing its outdoor cultivation and exploring oil extraction from Eustigmatos are considered as the key to its industrialization. Two kinds of light path self-made flat-plate photobioreactors were used to evaluate the productivity of Eustigmatos sp. SCSIO-45821,which was isolated from the Ningxia Autonomous Region by measuring its growth,lipid accumulation,and fatty acid composition. Moreover,the method of oil extraction by ethanol was investigated. Results showed that Eustigmatos sp. SCSIO-45821 grew normally in the flat-plate photobioreactors. The light path of 6 cm flat-plate photobioreactors was suitable for the production of Eustigmatos biomass being rich in eicosapentaenoic acid（EPA）. The highest average productivity of biomass and EPA was 93.5 mg/L·d and 0.6 mg/L·d,respectively. However,the light path of 4 cm flat-plate photobioreactor was more conducive to oil accumulation,and the lipid productivity was 22.0 mg/L·d. Ethanol was used for the extraction of oil from the biomass of Eustigmatos. The optimal condition for oil extraction included：100% ethanol,room temperature and more than 36 h for extraction,and under which the highest extraction rate reached 94.3%. The extracted oil contained > 70% neutral lipid in total lipid and 4.0% EPA in total fatty acid. However,the Eustigmatos oil showed black color and needed to be decolorized and refined. In sum,the outdoor flat-plate photobioreactor is an ideal device for cultivating Eustigmatos sp. However,it is necessary to select a suitable light path according to the target product. Ethanol can be used as an ideal solvent for extracting oil from Eustigmatos oil.

Research Trend and Hotspot Analysis of Avian Influenza Virus Prevention and Control in China-Based on OIE Reference Laboratories Impact

WUJI Siguleng, LI Xiao-man, ZHANG Xue-fu

2020, 36(7):  139-147.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0514

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In order to explore the current situation and trend of avian influenza prevention and control research in China,and understand the hotspots and problems in related research in China. Based on the web of science database and incoPat patent database,this paper analyzes the research trend of avian influenza virus prevention and control in China from the perspective of bibliometrics,including the impact situation,theme situation and citation situation. Select one OIE Reference Laboratories in China,and three OIE Reference Laboratories as benchmarking teams,and reveal the research trends and hotspots of avian influenza prevention and control in China through the benchmarking analysis. In the dimension of impact situation,China is following the pace of international research,with advantages in the growth rate of papers,excellent performance in the frequency of cited patents,but insufficient performance in patent intensity. In the dimension of theme situation,the R&D hotspots in China involve “diagnosis” and “prevention”. In the dimension of citation situation,the overall distribution of China is relatively concentrated in institutions and years,unlike other countries,its citation situation is more diffuse.

Research Progress of Demethylase ROS1 in Plants

WANG Jie, XIE Li-nan

2020, 36(7):  148-157.  doi:10.13560/j.cnki.biotech.bull.1985.2019-1017

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DNA methylation status is the result of de novo synthesis of methylation,maintenance methylation and dynamic demethylation of DNA,and is catalyzed by various regulatory pathways targeting various enzymes. 5-methylcytosine DNA glycosylase/lyase ROS1（Repressor of Silencing1）is a DNA demethylase that can initiate DNA active targeting by initiating a base excision repair pathway. In this review,we describe demethylases and regulatory factors for active DNA demethylation in plants,ROS1-mediated DNA demethylation,the roles of DNA active demethylase ROS1 in different plant development processes,including negative regulation of imprinted gene expression and seed dormancy,regulation of rice grain quality,and affecting plant stomatal development.

Review for Research on Polysaccharides Produced by Endophytic Fungi Derived from Plants

PAN Feng, HOU Kai, LIU Yun, WU Wei

2020, 36(7):  158-169.  doi:10.13560/j.cnki.biotech.bull.1985.2019-1104

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Endophytic fungi from plant,as an important microbial resource,not only can live in the internal tissues of plants without causing any symptoms,but also produce a large number of bioactive natural products. The polysaccharide（PS）produced by endophytic fungi derived from plant is one important kind of macromolecular natural products,and the research value of PS from endophytic fungi is confirmed by increasing reports. The source,culture and fermentation of endophytic fungi,the physical and chemical properties and structural characteristics of homopolysaccharides from endophytic fungi are closely related to their physiological activities. The strain selection,strain culture condition,polysaccharide separation and purification,physicochemical characteristics,biological activity and effects on plants of endophytic fungal PSs are summarized in this report,aiming to develop the endophytic fungal polysaccharides,and to provide some references for further research on the physiological and biochemical effects of endophytic fungal PSs,

Physiological and Molecular Mechanisms of Plant Co-evolution Responses to Phosphorous Deficiency and Aluminum Toxicity

WU Pei, LI Hao, ZAO Hao-long, WANG Yu-yun, YANG Jian-li, TANG Li, FAN Wei

2020, 36(7):  170-181.  doi:10.13560/j.cnki.biotech.bull.1985.2019-1211

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Acid soils comprise approximately 50% of potential arable lands worldwide. Phosphorus（P）deficiency and aluminum（Al）toxicity are two major nutritional stress factors limiting plant growth on acid soils. Studies have shown that organic acids（OA）,hormones and iron（Fe）homeostasis play a core role in co-evolution and signal crosstalk of plant responses to both stresses. This article systematically reviewed the physiological and molecular mechanisms of OA secretion,pleiotropic regulation of STOP1/ALMT1 and STAR1/ALS3,hormone signal transduction and cell wall-related kinases in regulating plant root development and root architecture to improve P availability and Al resistance on acid soils and also prospected the visions in this field.

Research Progress in Histone Modification of Plant Involved in the Regulation of Gene Expression Response to Abiotic Stress

ZHAO Lin, WANG Pu, WU Qi, SONG Rui-rui, LAN Tao, YUN Zhen-yu

2020, 36(7):  182-189.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0880

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It is unavoidable to suffer from adverse environment in the process of plant growth. Hence they have evolved a flexible mechanism for reprogramming gene expression to cope with abiotic stresses such as drought,high salinity,cold,heat or submergence. Recently,along with the intensive studies of epigenetics,histone modification has demonstrated to play a crucial role under abiotic stress in plant. The properties of histone modification after translation can be altered by environmental stress,which initiates the expressions of abiotic stress-responsive genes,or act as a downstream factor to participant in the transcriptional regulation. In this review,we summarize the existing information about histone modification involved in the transcriptional regulation of abiotic stress-responsive genes,which is expected to provide a reference for the research on abiotic stress tolerance of plants.

Research Progress on Biosynthesis of Polyhydroxyalkanoates from Waste Oils

PAN Lan-jia, LI Jie, LIN Qing-huai, WANG Yin

2020, 36(7):  190-199.  doi:10.13560/j.cnki.biotech.bull.1985.2019-1119

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Polyhydroxyalkanoate（PHA）,a kind of high molecular biopolymer,is synthesized by microorganisms and accumulated in microbial cells with the limitation of essential nutrients and excessive carbon source in the medium. PHA products are promising biodegradable materials with excellent physicochemical properties and are expected to be as potential alternatives of traditional plastics for avoiding the pollution of “white plastic waste”. However,the high cost of production limits its industrialization and application in large-scale. Exploring some cheap feedstock as carbon resource of microorganisms to produce the PHA is one of the feasible approaches to reduce the cost. Waste oil as carbon-hydrogen component presents the promising potentials of utilization by microorganisms. Recently,it has become a research hotspot to synthesize biodegradable plastics by microorganisms using waste oil as raw material. This strategy can not only reduce the costs of PHA production,but also achieve the high-value utilization of waste oil. The replacement of traditional fossil fuel plastics by biodegradable plastics conforms to the sustainable development strategy in China. This paper systematically summarizes various types of PHA and its applications,the latest progress in the synthesis of PHA from waste oil by microbes,and the extraction methods of PHA from microbial cells. Finally,some insights and outlooks are provided on the development of PHA production from waste oils.

Research Progress on Bioremediation of Saline-alkali Grassland：A Review

GUO Wei, XUE Shuai, ZHANG Zhe-chao, DIAO Feng-wei, HU Jie, ZHANG Min, LIU Mei-chun, DING Sheng-li, JIA Bing-bing, SHI Zhong-qi

2020, 36(7):  200-208.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0413

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With the intensification of climate change and the rapid development of social economy,the salinization of grassland ecosystem in China is becoming more and more serious. The salinization of grassland will not only seriously threaten the local ecological security but also restrict the social and economic development of pastoral area. It is imperative to repair the saline-alkali grassland. Bioremediation is a low-cost,effective technology with little environmental impact,and recently it has attracted much attention in salinized soil remediation. This paper comprehensively introduces the research contents and application progress in three fields of bioremediation technology,including phytoremediation,microbial remediation,and combined plant-microbe remediation. We focuses on the analysis and discussion of the role and potential application prospects of plant probiotics and AM fungi in improving the saline-alkali stress tolerance of forage grass and promoting the phytoremediation efficiency of saline-alkali grassland soil,aiming to provide theoretical basis and research ideas for the restoration and treatment of salinized grassland,which is of great practical significance to the restoration of salinized grassland ecosystem.

Breeding of Transgenic Insect-resistant Sugarcane and Strategies for Preventing the Its Resistance to Insects from Loss

FENG Cui-lian, ZHANG Shu-zhen

2020, 36(7):  209-219.  doi:10.13560/j.cnki.biotech.bull.1985.2019-1265

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Due to the complexity of the sugarcane genome and the lack of insect-resistant germplasm resources,the conventional insect-resistant breeding of sugarcane is far behind other crops. Currently,genetic engineering provides a novel approach for insect-resistant sugarcane breeding. The recent development status of transgenic insect-resistant sugarcane was reviewed via literature collection and sorting combined with the research achievements in our team . First,the new development of transgenic sugarcane transformation system and studying genetic stability of transgenes were introduced,and then the breaks in studying transgenic insect-resistant sugarcane were focused. In particular,the strategies for preventing the insect resistance of transgenic sugarcane from decreasing or even losing by modifying Bt genes and utilizing gene stacking were introduced in detail,which will provide reference for insect-resistant transgenic sugarcane breeding in the future.

An Improved Method for Large Size DNA Recovery Facilitates Fosmid Library Preparation

ZHANG Cui-cui, ZHAO Sheng, WANG Yue, ZHANG Peng, CHANG Yu-xiao

2020, 36(7):  220-227.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0935

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The Fosmid library with inserted DNA of around 40 kb has been widely used in genomics research. However,the traditional method of agarose gel separation and electroelution into dialysis bag has long been used for large DNA fragment isolation,which makes it difficult to obtain sufficient amount of DNA fragments for ligation,thus dramatically reduces the success rate in Fosmid library preparation. In this study,a simple,convenient and efficient method for 40 kb DNA fragments recovery was established exploiting a modified program on SageELF,a fully automated nucleic acid / protein recovery system. The method was used to prepare Fosmid libraries with improved quality successfully. Through Sanger sequencing and restriction fragment analysis of 25 random selected Fosmid clones,we found the size of the inserted DNA was 37.9 ± 5.2 kb. These results showed that,our improved method could recover 40 kb DNA accurately,simply and efficiently;In addition,the inserts of the Fosmid library constructed with the recovered 40 kb DNA were more concentrated,which will facilitate the following genomics analysis.

Study on Evanescent Wave Fluorescence Aptasensor for Direct and Rapid Detection of Escherichia coli O157∶H7

FANG Shun-yan, SONG Dan, LIU Yan-ping, XU Wen-juan, LIU Jia-yao, HAN Xiang-zhi, LONG Feng

2020, 36(7):  228-234.  doi:10.13560/j.cnki.biotech.bull.1985.2019-1132

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By combining the advantages of evanescent wave fluorescence optical fiber biosensor and specific nucleic acid aptamer,a fast and direct method to detect Escherichia coli O157∶H7（E. coli O157∶H7）based on evanescent wave fluorescence principle and its size effect with pathogen was proposed. The basic principle is that when a certain concentration of fluorescence labeled E. coli O157∶H7 aptamer is added to the sample detection cell,the evanescent wave triggers the fluorescence molecules to emit fluorescence,and the evanescent wave all fiber biosensor is applied for the quantitative detection of fluorescence signal. After the fluorescence labeled aptamer is mixed with E. coli O157∶H7,the mixture is added to the sample detection cell. Because the penetration depth of evanescent wave is only 100 nm,the fluorescence molecules labeled aptamer specifically bind with cannot be triggered,which results in the decrease of detected fluorescence signal. The quantitative detection of E. coli O157∶H7 can be achieved by using the proportional relationship between the fluorescence intensity and its concentrations. The results showed that the detection limit of E. coli O157∶H7 was 610 CFU/ml,and the linear detection range was from 1.1×10³ to 1.4 × 10⁷ CFU/mL. The recovery of E. coli O157∶H7 spiked in real samples was 40%-180% and the relative standard deviation was within 10%. The matrix of water sample had no significant effect on the detection of E. coli O157∶H7. Based on the principle of evanescent wave fluorescence and its size effect on pathogens,this universal biosensing method can be applied for the direct and rapid detection of other pathogenic bacteria only using different fluorescence labeled bio-recognition molecules.

Screening and Evaluation Strategies of Cell-specific Nucleic Acid Aptamers

YUHAN Jie-yu, ZHU Li-ye, CHEN Xu, HE Xiao-yun, XU Wen-tao

2020, 36(7):  235-244.  doi:10.13560/j.cnki.biotech.bull.1985.2019-1115

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As recognition molecules of cell surface information,cell-specific nucleic acid aptamers have received extensive attentions. The screening method is different from ordinary small molecules and macromolecules. The maintenance of cell viability,the removal of dead cell interference and the feasibility of the application of the aptamer are all important in the screening process. Screening of cell aptamers has always been a focus of research. This paper reveals the screening methods based on cell surface markers,whole-cell,tissue and in vivo. Meanwhile,the screened aptamers will be evaluated from the aspects of affinity,specificity,cell viability,and the feasibility in clinical tissue and in vivo,aiming at providing reference for the selection and evaluation of cell-specific nucleic acid aptamers.