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    26 November 2020, Volume 36 Issue 11
    Cloning of ZmENA1 and Its Responses to Metal Ions in Maize
    ZHANG Xin, ZHOU Xiao-jin, PANG Sen, LI Su-zhen, HUANG Jia-xing, CHEN Ru-mei
    2020, 36(11):  1-8.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0192
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    Nicotianamine(NA)is the precursor of mugineic acid(MA)which is a phytosiderophore of plants,and nicotianamine effluxer(ENA)is an important protein mediating NA transport in plants. Thus,exploring the functions of ZmENA1 may provide bases for understanding metal ions transport and storage mechanisms in maize. In this study,ZmENA1 was homologously cloned and the basic characteristics of the encoded protein were analyzed via bioinformatics. Also the expression patterns of ZmENA1 during kernel development process under different metal ions concentrations were examined using real-time quantitative PCR. The results showed that the ORF of ZmENA1 was 1 356 bp,and the encoded protein had a deduced isoelectric point of 8.06 and molecular weight of 48.998 kD. In addition,ZmENA1 contained 53.98% proportion of α-helix,four phosphorylation sites,10 transmembrane regions,and 1 N terminal signal peptide. The expression of ZmENA1 in the mixture of endosperm and pericarp was higher than that in the embryo,reached a peak at 16 DAP,and decreased with the developmental process. Besides,the expression of ZmENA1 presented fluctuations in response to deficiency of Zn2+,Mn2+,and Cu2+,as well as excess of Zn2+,Mn2+,and Cd2+,suggesting that ZmENA1 played a role in mediating transport and storage of these metal ions. This study may provide a basis for further revealing the function of ZmENA1.

    Functional Analysis of Ferric Reductase Gene ZmFRO2 in Maize
    QIAO Meng-xin, LI Su-zhen, CHEN Jing-tang
    2020, 36(11):  9-20.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0281
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    Fe is one of the most important micronutrients in plant growth and development,and iron deficiency in plant leads to leaves yellowing,which in turn affects yield. There are two mechanisms for plant absorption and transport of iron,that is,mechanism I and mechanism II,the corresponding plant called mechanism I plant and mechanism II plant. Dicots and non-graminaceous monocots employ the mechanism I,and graminaceous plants adopt mechanism II. As the key gene in mechanism I plant,gene FRO(ferric reduction oxidase)has been found and studied in a variety of plants,including graminaceous plant rice. In order to understand the function of FRO in maize,functional analysis of ZmFRO2,a ferric reductase gene in maize,was done,including bioinformatics analysis,expression pattern and subcellular localization. The results indicated that ZmFRO2 had the structural basis of iron reductase and had a closer kinship with OsFRO1,MtFRO1,AtFRO6 and AtFRO7. ZmFRO2 was located in the plasma membrane and cytoplasm. ZmFRO2 gene was expressed in large quantities in leaves and the expression of it was first induced and then inhibited by iron deficiency. In addition,transgenic maize plants with over-expression of ZmFRO2 were obtained using the method of agrobacterium-mediated infection. The iron reductase activity and Zn/Fe content determination of them showed that the over-expression of ZmFRO2 increased the iron reductase activity in maize roots and the iron content in the root,leaf and grain all increased at varied degrees. Through these analyses and determinations,the foundation for the further study of gene function verification is laid,and a new functional gene and a new way for improving iron content of maize will be provided.

    Screening and Identification of LCD-interacting Proteins in Rice
    HAO Xiao-hua, DAI Jia-li, JI Wen-jin, HUANG Dan, LI Dong-ping, TIAN Lian-fu
    2020, 36(11):  21-29.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0510
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    Low cadmium(LCD)gene is involved in grain Cd accumulation of rice. To screen LCD-interacting proteins is beneficial for revealing the molecular mechanism of LCD-mediated grain Cd accumulation and regulation. Using Oryza sativa ssp. japonica cv. Nipponbare as experimental material,the cDNA libraries of yeast two hybrid membrane system and nuclear system were constructed,and LCD was used as bait protein to screen the membrane system and nuclear system respectively. A total of 29 genes were obtained,and the gene ontology(GO)analyses were conducted to annotate their molecular function. Further,these genes were cloned and one-to-one interaction verification with LCD was performed. Finally,a total of 21 LCD-interacting proteins including 16 from membrane system and 5 from nuclear system were identified. Among them,6 proteins were related to the tolerance and transport of metal ions,5 were related to the stress responses,4 were related to plant development responses,and the rest 6 were related to cellular and physiological metabolisms.

    Identification and Antifungal Activity Determination of an Endophytic Fungus from the Twigs of Vaccinium dunalianum
    YAN Dong, ZENG Wei-lin, LUO Xu-lu, CHEN Xiao-xue, LIU Hui-min, ZHAO Ping
    2020, 36(11):  30-38.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0200
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    An endophytic fungal strain Z-78 isolated from the twigs of Vaccinium dunalianum Wight was identified as Pestalotiopsis cocculi(Guba)G.C. Zhao & N. Li by morphological classification and molecular biological study. The plate confrontation method was used to determine its antifungal activities against eight plant pathogens including Botrytis cinerea,Fusarium solani,Fusarium graminearum,Fusarium oxysporum,Alternaria brassicicola,Gloeophyllum trabeum,Poria placenta and Coriolus versicolor,and the growth rate method was applied to conduct the acid-base stability test of the fermentation broth inhibiting the activity of A. brassicicola. The results showed that Z-78 strain presented strong inhibitory effects on all of the eight plant pathogens,while the activity against A. brassicicola remained stable under different pH conditions of fermentation broth.

    Cloning,Expression and Bioinformatics Analyses of HbCPA Gene from Hevea brasiliensis
    YANG Hong, HU Yan-ling, YUE Yi-fan, DENG Zhi, DAI Long-jun, LI De-jun
    2020, 36(11):  39-47.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0294
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    N-carbamoylputreseine amidohydrolase(CPA)is the final reaction enzyme for higher plants to synthesize putrescine through the arginine pathway. A CPA gene from Hevea brasiliensis was cloned with RACE and RT-PCR and designated as HbCPA. The full length of HbCPA was 1 342 bp with an open reading frame of 906 bp and encoded 301 amino acids. The molecular weight and isoelectric point of HbCPA were 33.5 kD and 6.01,respectively. Phylogenetic analyses showed that HbCPA and MeCPA in Manihot esculenta,RcCPA in Ricinus communis and JcCPA in Jatropha curcas clustered into one branch. qRT-PCR analyses demonstrated that HbCPA expressed in each tissue,with the highest expression in barks,followed by leaves,female flowers,male flowers and stem apexes. The expression of HbCPA varied in different developmental stages of leaves,with the highest in the light green period,and the same and lowest expression in the bronze and senescence stages. The expression of HbCPA was significantly up-regulated in barks of tapping panel dryness(TPD)tree compared with the healthy rubber tree. HbCPA expression was also regulated by ethephon,MeJA,low temperature,drought,high salt and hydrogen peroxide. All these results suggest that HbCPA may be involved in the regulation of rubber production and debinding in rubber tree and the response to abiotic stress.

    Study on Antifungal Activity of an Endophytic Bacterium of Polygonatum sibiricum Delar. ex Redoute
    GUO Xiao-ping, LIU Xing-fei, LI Xiao-nan, LÜ Xue-ru, XI Shao-mei, TIAN Yuan
    2020, 36(11):  48-54.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0184
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    This work aims to isolate and screen endophytic bacteria of Polygonatum sibiricum Delar. ex Redoute with antifungal activity,and to lay foundation for exploring bio-control microbial resources and development and utilization of P. sibiricum Delar. ex Redoute. The bacterial strains were isolated from the roots and stems of healthy Polygonatum using conventional separation method,and then screened using the plate confrontation method. The strain with the best activity was identified based on phenotypical and biochemical properties as well as its 16S rDNA gene sequence. The antifungal effect of volatile compounds produced by the strain was measured by plate back-off method,and the inhibitory activity of cell-free fermentation broth to pathogen fungi was measured by agar diffusion method. An endophytic bacterium HJ-12 with broad-spectrum antifungal activity was obtained,and was identified as Brevibacillus brevis. The inhibition rate of HJ-12 to 6 phytopathogens ranged from(18.57±0.25)% to (48.33±0.15)%. The inhibition rate of the volatile compounds produced by HJ-12 was from (21.05±0.17)% to (83.53±0.19)%,and the cell-free fermentation liquid also demonstrated obvious antifungal activity after 2 d fermentation. In summary,the B. brevis HJ-12 presents potential application value in biological control of plant fungal diseases.

    Isolation and Identification of an Algicidal Bacterium CBA02 and Its Algae-lysing Characteristics
    YANG Bing-jie, XIANG Wen-zhou, JIN Xue-Jie, CHEN Zi-shuo, WANG Ling, WU Hou-bo
    2020, 36(11):  55-62.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0394
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    Microalgae is susceptible to be contaminated by algae-lysing microorganisms in large-scale culture. This work aims to isolate and identify an algae-lysing bacterial strain CBA02,and to study its algae-lysing effect and mechanism. The 2216E plate method was used to isolate and purify this strain,and 16S rDNA sequences to identify the strain. The algae-lysing characteristics and approaches were determined using Dunaliella salina. The strain CBA02 was identified as Ponticoccus sp. CBA02 by 16S rDNA sequences. The algicidal efficiency of CBA02 to D. salina increased with the increasing of the fermentation time and the dose of bacterial solution,while decreased with the increasing of the initial concentration of algae. The concentration of chlorophyll a of D. salina significantly decreased in the group of bacterial solution,cell-free supernatant and high-temperature treated cell-free supernatant(P<0.05). The algicidal efficiency of the cell-free supernatant after pH treatment increased with the increasing of pH. The strain Ponticoccus sp. CBA02 presented a strong algicidal effect on D. salina,and its algicidal effect was influenced by fermentation time of bacterial solution,the dose of bacterial solution,initial concentration of algal cells and pH. CBA02 indirectly causes the lysis of D. salina by releasing water-soluble algicidal substances which have thermal stability and suitable for high pH.

    Construction of Aeromonas hydrophila acrA Deficient Strain and Determination of Its Physiological Function
    LI Xiao-yan, LI Ze-qi, WANG Yu-qian, YU Jing, LIN Zhen-ping, LIN Xiang-min
    2020, 36(11):  63-69.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0380
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    AcrA is a membrane fusion protein in Gram-negative bacteria,and scientists mainly study the regulation of AcrA protein in the process of drug efflux and other membrane transport in bacteria. To elucidate the effects of acrA deficient strain ΔacrA in the bacteria on physiological functions such as hemolytic,toxicological,stress and antibiotic sensitivity,acrA(AHA_2911)deleted strain was constructed in A. hydrophila using traceless based homologous recombination technology. The physiological phenotypes,such as hemolytic,extracellular protease activity,antibiotics susceptibility,high temperature stress,growth trends,and biofilm formation ability for both wild strain and ΔacrA were measured. The results showed that the hemolytic and extracellular protease activities in the ΔacrA were not affected,while the acrA deletion increased the antibiotics sensitivity of the ΔacrA to kanamycin,erythromycin,balofloxacin and acriflavinium chloride,and significantly improved the biofilm formation. But,the growth trend of ∆acrA and its biofilm formation ability at 42oC were significantly lower than wild strain. The above studies proved that acrA has a certain function in drug efflux pumps and suggested that it may play an important role in the high temperature tolerance and biofilm formation.

    Soluble Expression and Enzyme Activity Analysis of African Swine Fever Virus K196R and A240L Proteins
    LI Peng-hao, LIANG Yan-yu, WANG Yan-wei, GUAN Yang, PANG Wen-qiang, TIAN Ke-gong
    2020, 36(11):  70-75.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0216
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    The objective of this work is to provide effective antigens for the clinical diagnosis and immunoprophylaxis of African swine fever virus(ASFV). K196R and A240L coding sequences derived from ASFV SY18 were executed codon optimization and co-expression with soluble-tag in their N terminals. The target proteins were purified by affinity chromatography after TEV digestion,and their reactivity and enzymatic activity were evaluated through Western blot and enzyme activity detection kit,respectively. As results,the soluble expressions of K196R and A240L proteins were obtained by severally fusing with SUMO and MBP tag,and the size was about 22 kD and 27 kD after tag removed. Western blot results showed both K196R and A240L proteins well specifically reacted with the ASF positive serum. Enzyme activity detection results demonstrated the K196R and A240L proteins all presented definite enzyme catalytic activity in vitro. In conclusion,the soluble K196R and A240L proteins primarily expressed in Escherichia coli all show better reactivity and certain enzymatic activity in vitro,which will be the basis for the ASFV clinical diagnosis and protein relevant functions investigation.

    Composition Analysis of Prokaryotic Flora in the Marine Biofilm Correlated with Ascidian Settlement
    LIU Qian-qian, SHI Hong-wei, GUO Chang-lu, ZHANG Zhi-zhou
    2020, 36(11):  76-84.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0187
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    Using iron oxide red paint and acrylate as coating base,4 different nanomaterials(0.1%,W/V)were added to make 2 types of antifouling coatings. The two types of coating performed similarly in the context of antifouling scores,but significantly more Ciona intestinalis attached on the iron oxide red paint coatings than acrylate ones. Biofilm samples were collected from marine hanging plates,the metagenome DNA samples from the 28th day biofilm were subjected to high-throughput sequencing of full-length 16S rRNA,and the composition differences of prokaryotic flora between 2 coatings were analyzed. The results indicated that:1)the alpha diversity of prokaryotic flora in the acrylate coating was higher than that in the iron oxide red paint coating;2)interestingly,nanomaterials didn’t result in the significant composition changes in the prokaryotic flora on the both coatings;and 3)there were significant differences in the abundance of several bacterial strains between two types of coatings,especially for two dominant bacteria Neptuniibacter sp. CAR-SF and Cellvibrionales unclassified at 4% on the iron oxide red paint coating but almost zero on the acrylate coating. The observed OTU numbers of the two bacteria on 2 coatings were of over 100-fold difference,and the qPCR results confirmed that the contents of Neptuniibacter genus on 2 coatings differed remarkably. Whether the decrease in the amount of ascidian attached to the acrylate-based coating is related to the above bacterial content difference is worthy of further study.

    Synergistic Effect of Musca domestica Cecropin and Gentamicin Against Bacillus proteus vulgaris
    CHEN Qing-ru, GUI Shui-qing, LU Xue-mei
    2020, 36(11):  85-93.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0245
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    The combined antibacterial action and the synergistic antibacterial mechanism of Musca domestica cecropin(MDC)and gentamicin on Bacillus proteus vulgaris were investigated. Micro broth dilution,chessboard,and crystal violet staining were used to study the synergistic antibacterial and antibiofilm effects of the two drugs. Fluorescence staining,flow cytometry and transmission electron microscopy(TEM)were employed to observe the antibacterial mechanism of the combination of the two drugs. The fractional inhibitory concentration of MDC and gentamicin was 0.313,indicating that the 2 drugs presented promising synergistic antibacterial effect. According to the results of crystal violet staining,the two drugs demonstrated a synergistic effect on the initial biofilm,mature biofilm and extracellular protein of the Bacillus proteus vulgaris. The results of fluorescence and flow cytometry showed that the two drugs efficiently killed bacteria and increased the number of bacterial deaths. TEM observation revealed that most bacterial cell walls were damaged,their cell membranes were incomplete,and intracellular necrosis caused cell swelling and rupture. The MDC and gentamicin act synergistically against Bacillus proteus vulgari,and the mechanism of the two drugs is complementary.

    Effect of Site-directed Mutagenesis of Amino Acids in Lid Region on the Enzymatic Properties of T1 Lipase
    ZHU Cai-lin, LÜ Xiang, XIA Xiao-le
    2020, 36(11):  94-102.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0220
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    T1 lipase from strain Geobacillus zalihae is often used as a biocatalyst in various fields. In order to comprehensively improve the enzymatic properties of T1 lipase,three mutants L188M,A190L and A190Y with better effect were obtained by rational design of lid domain in the early stage of study. On this basis,two combined mutants L188M/A190L and L188M/A190Y were obtained by combining single point mutants. Then the enzymatic properties of the lipase mutants were studied,and the effects of the single and combined mutants were compared. The results showed that the optimal pH of wild ones wt-T1 and mutants was 9.0,and the optimal temperature of the combined mutants increased by 5℃. For other enzymatic properties,both single and combined mutation led to the enzymatic properties of T1 lipase improved at varied degrees,and the effect of the combined mutants was more significant. The combined mutants significantly increased the temperature and pH stability of lipase,and the tolerance to organic solvent dimethyl sulfoxide and n-hexadecane of L188M/A190L was 1.88 times and 2.73 times that of the wt-T1,and that of L188M/A190Y was 1.96 times and 2.58 times that of the wt-T1. The selectivity to C16 and C18 substrates of L188M/A190L was also significantly enhanced,which was 1.65 and 2.7 times that of the wt-T1,and that to L188M/A190Y was 1.5 times and 2.25 times that to the wt-T1. The results broaden the application prospect of T1 lipase to a certain extent,and provide certain theoretical basis for the modification of other lipases.

    Advances on Alkaline Tolerance of Rice
    LENG Chun-xu, ZHENG Fu-yu, ZHAO Bei-ping, LIU Hai-ying, WANG Yu-jie
    2020, 36(11):  103-111.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0263
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    In China,with the degree of land salinization aggravating,the crop production is impeded. Rice,as a moderately salt-sensitive crop,as its roots absorb salt and secrete organic acids,may dilute the salinity-alkalinity of the soil surface in its growing water environment,thus making it the first choice for improving saline-alkali land. Salt and alkali stress cause both osmotic stress and ion toxicity to rice plants. Furthermore,alkali stress results in the rice suffering from double damages of high pH value and high salinity,the damage is more severe and the regulation mechanism is more complex than salt stress. At present,most of the researches had focused on salt stress,but few studies were on the response and regulation mechanism of alkali stress. This paper systematically reviewed the effects of alkali stress on the growth,development,physiology,and biochemistry of rice,as well as the alkali-resistance mechanism and related genes,and finally proposed a strategy of enhancing the alkali tolerance of rice. It is aimed to breed new alkali-resistant rice varieties through the molecular biological technique for improving saline-alkali soil,increasing rice plating area and rice yield,and thus protecting national food security.

    Research Progress on Chloroplast Genome of Major Gramineous Crops
    LI Yu-hua, REN Yong-kang, ZHAO Xing-hua, LIU Jiang, HAN bin, WANG Chang-biao, TANG Zhao-hui
    2020, 36(11):  112-121.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0285
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    Chloroplasts are unique organelles in plant cells and the main sites of photosynthesis. The chloroplasts are semi-autonomous,possessing independent extranuclear genomes that can encode some of the genes associated with their own functions. The chloroplast genomes(cpDNA)contains a large number of functional genes,and their application values in the study of species identification and phyletic evolution have been widely accepted and gradually recognized by researchers. Gramineae is one of the most concentrated families in chloroplast genome researches. In this paper,we elaborated the studies on the characteristics of the chloroplast genome,the distribution of gene types,gene sequence analysis,RNA editing,and the application of chloroplast genomes in the phylogenetic development of major Gramineae crops,so as to lay a foundation for better analyzing the chloroplast genomes of Gramineae in terms of species evolution,genetics,and phylogenetic relationships.

    Research Progress of Plant DNA Methylation Under Abiotic Stress
    LIU Zhi-min, YANG Zhi-yi, JI Feng-dan, MEI Zhi-chao, YU Jia-hui, XIE Li-nan
    2020, 36(11):  122-132.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0330
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    Abiotic stress,including high temperatures,droughts,colds,floods,and salinization,is usually not conducive to plant growth and development. In the long-term natural selection and evolution of plants,plants have formed a complete system to cope with the constantly changing environment. In eukaryotic cells,the expression of protein-coding genes is largely influenced by the state of chromatin. Epigenetic modifications,mainly including DNA methylation,histone modifications,histone variants,and some non-coding RNA(ncRNA)changes,will affect the structure and accessibility of chromatin,thereby altering the activity of gene expression. Recent studies have shown that epigenetic modification is widely involved in plant abiotic stress response and is important for plants to adapt to changing environments. As one of the most important epigenetic modifications,the role of DNA methylation in regulating abiotic stress responses has attracted widespread attention. This paper reviews the recent advances in plant DNA methylation and abiotic stress under DNA stress,with a view to providing a reference for using epigenetic variation to improve plant resistance to stress.

    Research Status on the Stress Resistance of Moringa oleifera and Its Application
    PU Tian-lei, HAN Xue-qin, LIAO Cheng-fei, DEN Hong-shan, LUO Hui-ying, JIN Jie
    2020, 36(11):  133-140.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0392
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    Moringa oleifera Lam is tropical and subtropical fast-growing tree species. It has excellent resistance to high temperature,barrens,and drought. It is rich in nutrient and functional ingredients,and thus it can be used in various fields such as food,health product,and fodder. The changes of global environmental and human activities have aggravated abiotic stress. The resistances of M. oleifera to abiotic stress,including drought,low temperature,and salt,have been attracted growing attentions. At the same time,the extract from M. oleifera can be used as a safe and effective biostimulant for alleviating the damage caused by abiotic stress,promoting plant growth,increasing stress resistance,and improving the yield and quality of plants. In either the study of abiotic stress resistance of M. oleifera or the study of M. oleifera growth regulators,the resistances of M. oleifera plant and species as well as the improvements to the plant damages by the stress were evaluated by the changes of relevant parameters,such as morphology,yield and quality,osmoregulatory systems,protective enzyme systems,photosynthetic systems,molecular levels,etc. This review summarizes the research status in last few years on the stress resistance of M. oleifera and the application progress of its extract in the stress resistance of plants,discusses the existing issues,and proposes the research prospects,aiming to provide references for studying the stress-resistant breeding,the mechanism of stress-resistance,as well as the application research of inducing plant stress resistance.

    Research Progress in Biosynthesis of Secondary Metabolites of Microorganisms
    ZHAO Jiang-hua, FANG Huan, ZHANG Da-wei
    2020, 36(11):  141-147.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0475
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    Microorganisms can produce a variety of natural secondary metabolites with biological activity,which have broad application prospects in medical,agricultural and food fields. The development of molecular biology, bioinformatics and sequencing technology has laid a theoretical foundation for the mining of secondary metabolites and provided operational tools. In recent years,there are more and more studies on the secondary metabolites of microorganisms. It is difficult to get satisfactory results by traditional methods. It will be a new direction to operate microorganisms at the molecular level,such as the regulation of secondary metabolite biosynthesis gene cluster.The strategies of finding new secondary metabolites and increasing the production of secondary metabolites were reviewed,by using the methods of heterologous expression of secondary metabolite biosynthesis gene cluster,ribosome engineering,epigenetic regulation,metabolic regulation and regulation of transcription and translation level,the aim is to excavate new secondary metabolites,improve the production of secondary metabolites,and provide a breakthrough for the exploration of new drugs.

    Research Advances on Transcription Factor Yrr1p of Pleiotropic Drug Resistance in Saccharomyces cerevisiae
    CAO Wen-yan, WANG Xin-ning, SHEN Yu, LI Zai-lu, BAO Xiao-ming
    2020, 36(11):  148-154.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0401
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    Yrr1p(for yeast reveromycin A resistance)is a zinc finger transcription factor involved in the pleiotropic drug resistance(PDR),and plays important role in the drug resistance and inhibitor resistance of Saccharomyces cerevisiae. This paper reviewed the discovery process of Yrr1p,the relationship between Yrr1p structure and function,and the mechanism of resistances mediated by Yrr1p in S. cerevisiae to different inhibitors of 4-nitroquinoline-N-oxide,salicylic acid,mancozeb,and vanillin. Tolerance mechanism to vanillin stress mediated by Yrr1p displayed remarkable differences from other drugs. Further the paper outlined the regulatory relationship between Yrr1p and its homologous transcription factors Yrm1p,Pdr8p,and its upstream regulatory factors Pdr1p,Pdr3p. The research of Yrr1p provides scientific guidance for studying microbial tolerance and constructing highly resistant microbial cell factories.

    Advances in the Applications of Metabolomics Technologies in Aquatic Products Quality and Safety Research
    LI Rui, SUN Zu-li, YANG Xian-qing, LI Lai-hao, WEI Ya, CEN Jian-wei, WANG Jing, ZHAO Yong-qiang
    2020, 36(11):  155-163.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0448
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    As an emerging technology,metabolomics technology can perform qualitative and quantitative analysis of small molecule metabolites in samples,and thus is widely used in modern life science research. Concurrently,it provides us new methods for the study of molecular mechanism of changes in quality and safety of aquatic products. This article introduces the metabolomics technology and its application in the quality and quality safety control of aquatic products,and summarizes the recent research status of metabolomics technology in the fields of aquatic product breeding and nutrition,raw material identification,storage and transportation,and quality and safety of processed products. This article also investigate the interrelationship between omics technologies,and further prospects the future application of multi-omics technology in studying the factors influencing aquatic product safety as well as the mechanism of quality change,aiming at providing a reference for use of metabolomics technology and multi-omics technology in the quality and quality safety research of aquatic products .

    Advances in Nitrifying Enzymes
    ZHAO Lu-yao, CHEN Zhen-ya, HUO Yi-Xin
    2020, 36(11):  164-172.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0065
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    The nitration of organic compounds especially aromatic nitro-compounds play an important role in chemical production. Aromatic nitro-compounds have become high-value chemical intermediates and industrial products due to their high energy and stable structure. At present,chemical synthesis is the most commonly used nitrification method due to its simple operation,low cost and controllable production conditions. However,this method is of low conversion efficiency and of a lot of waste acid and waste water are produced,and thus causes environmental pollution. These drawbacks restrict the production and application of nitro-compounds. Therefore,it is necessary to seek more efficient and environmental synthesis methods. The application of enzyme catalysis is becoming more and more extensive,and there are abundant microorganisms in nature that can transform nitrogen-containing groups through ammonia or nitrification,providing a repository for enzymatic synthesis of high-energy materials. In this article,we first summarized the types and main functions of nitro-compounds as well as available methods and their deficiencies for synthesizing nitro-compounds. Then we reviewed the types and characteristics of the main nitrifying enzymes in the P450 superfamily,peroxidases and N-oxygenases,analyzed the protein structure and catalytic mechanism of nitrifying enzymes in depth,and listed the directed evolution of nitrifying enzymes. Finally we prospected the research direction of preparing nitro-compounds by using nitrifying enzyme.

    Strategies and Advances in the Molecular Modification of Substrate Binding Pocket of Lipase
    CAI Yu-zhen, BAI Qiao-yan, SU Min, TANG Liang-hua
    2020, 36(11):  173-180.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0123
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    Lipase is one of the important enzymes and widely used in industrial processes due to its high enantioselectivity,stereoselectivity and broad substrate specificity. In addition to optimize the reaction conditions of lipases,people can also molecularly modify the lipase and essentially improve the catalytic efficiency in order to improve the enzymatic and physicochemical properties of lipase as well as to meet the high industrial demand. Generally,semi-rational design and rational design are often applied in molecular modification of binding pocket or “lid” domain of lipases for changing their substrate specificities,stereoselectivities,enantioselectivities,regioselectivities,etc. In this article,the molecular modification of lipase substrate binding pocket in recent decades is reviewed,aiming at providing some clues for relevant researches in the future.

    Rapid Screening Strategy for Target Identification of Bioactive Natural Products
    CHEN Peng
    2020, 36(11):  180-187.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0400
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    Bioactive natural products from traditional Chinese medicine and other natural resources are the important origin of medicines. Identification of the protein target in bioactive natural products is the key step to clarify the pharmacology and toxicology of them and prepared medicines. The current traditional target screening method is based on the molecular marker tracing,which has a long cycle and also leads to changes in the structure and pharmacological effect of natural products,and thus it has drawbacks. The novel coronavirus diseases(Coronavirus Disease 2019,COVID-19)enlightens us:It is of great value to reveal the target of drug action for the promotion and application of natural products. In recent years,the rapid screening technology for the pharmacological targets of unlabeled natural products based on the protein physical and chemical properties and omics is maturing. In this article,we sort and summarize the major unlabeled and rapid screening strategies for the protein targets of natural products via the practical cases,and aim to provide a prospective reference for the practioners in this field.

    Research Progress of CRISPR System in Translational Medicine
    YE Zhou-jie, WANG Xin-rui
    2020, 36(11):  188-197.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0250
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    CRISPR system is the most widely used technology for gene editing in the current scientific researches. In the post genomic era,CRISPR system is of great significance for gene function research,disease development mechanism and gene targeted drug research. At present,CRISPR/Cas9 editing technology has been used to successfully construct cell and biological models for clinical medical research. However,CRISPR/Cas9,as a powerful gene editing technology,is not only used for gene deletion or gene knock-in,but also applied in the gene high-throughput screening,research on disease caused by gene silencing,cross epigenetic modification activating gene expression,reversible interference of target gene expression and molecular diagnosis of pathogenic microorganism infectious diseases via the research and application of CRISPR/Cas9 in translational medicine in recent years,such as the screening system of CRISPR gene-knockout library,CRISPR/dCas9 gene activation system,CRISPR interference technology and CRISPR molecular diagnosis technology,etc.,which promotes the application and development of CRISPR in translational medicine research.

    Advances in Biotechnological Application of Elastin-like Polypeptides as Functional Nanomaterials
    ZHOU Yang, WANG Tao-tao, YAN Dan-dan, WANG Ying-ying, SHI Qin-xuan, SUN Li-hui, LIN Feng
    2020, 36(11):  198-208.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0042
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    Elastin-like polypeptides(ELP)are an elastic and temperature-sensitive synthetic protein polymer composed of a Val-Pro-Gly-Xaa-Gly repeat unit derived from human tropoelastin. ELPs undergo a sharp reversible phase transition at specific temperatures known as the inverse transition temperature(Tt). Below its Tt,ELP assumes a random coil shape,is highly solvated and hence highly soluble in aqueous solution. However,when temperature is raised above its Tt,the ELP becomes insoluble,forms large microns and begins to aggregate. ELP-based inverse transition cycle(ITC)is a non-chromatographic technique for the separation and purification of recombinant protein. Using the reversible phase change characteristics of ELP fusion proteins,ELP-labeled fusion proteins are specifically isolated from soluble protein supernatant after multiple ITC cycles. This technique requires only conventional centrifuges to obtain the target protein of the same purity as that by affinity chromatography,and it is low cost,easy to operate and suitable for lage-scale production. ELP and ELP-derived molecules present properties of fine elasticity,self-assembly,long-term stability and biological activity. The precise control in designing ELP is conducive to manipulating their stimulation responses to the external environment and other physical functional characteristics. The adjustable physical and chemical properties of ELP and its promising biocompatibility make ELP widely used in biotechnology. In this paper,the phase transition characteristics and mechanism of ELP,the design of ELP fusion protein and the effect of ELP to fusion proteins(peptides)are reviewed. Meanwhile,the application of ELP tag in protein purification,biomedicine and bioengineering,hydrogels and emerging fields is reviewed. Finally,the research directions and key issues and technologies of ELP also are discussed.

    Application and Prospect of Gene Editing Technology in Crop Breeding
    LI Shu-lei, ZHENG Hong-yan, WANG Lei
    2020, 36(11):  209-221.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0328
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    Genome editing is a group of technologies that allows the introduction of deletions,insertions or base substitutions by causing damage in targeted gene locus. This technology has now been utilized widely for editing the genome of various organisms,including bacteria,fungus,animals and plants. Compared to the Zinc-finger nucleases(ZFNs)and Transcription activator-like effectors nucleases(TALENs)editors,clustered regularly interspersed short palindromic repeats(CRISPR)/ CRISPR-associated protein(Cas)(CRISPR/Cas)system has developed as an efficient and versatile method for genome engineering. In this review,we describe the basic mechanism of ZFNs,TALENs and CRISPR/Cas system,and summarize the functions,limitation and application of CRISPR/Cas system in crop breeding. Finally,we discuss the potential challenges and opportunities of genome-edited crops in the future.

    Biosafety Assessment Technology Research for Genetically Modified Rice Based on Metabolomics
    LAN Qing-kuo, ZHAO Xin, SHEN Xiao-ling, WEI Jing-na, LIU Shuang, CHEN Rui, TAN Jian-xin, WANG Yong
    2020, 36(11):  222-229.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0198
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    Currently metabolomics is becoming the hotspot in safety assessment research of genetically modified organism(GMO)because of its unbiased and untargeted analysis. Here metabolome extraction method,multivariate processing methods,and data analysis platform were explored while transgenic insect-resistant rice and its non-transgenic receptor were taken as the research object and technical process of metabolomics evaluation of genetically modified crops was used based on the ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry(UPLC-QTOF/MS). The results showed that metabolites in the leaves of rice seedlings were effectively extracted with extraction buffer containing 80% methanol. By UPLC separation,QTOF/MS analysis,and orthogonal partial least squares-discriminate analysis(OPLS-DA),the differential metabolites between genetically modified rice and its non-genetically modified rice were effectively discovered. The systematic analysis method of transgenic rice metabolome established in this study demonstrates the advantages of high resolution and easy to operate. This study enriches the current contents and methods of safe assessment for GMO as well as provides technical support for the safety management of GMO.

    Screening and Application of the Nuclear Dissociation Solutions of Soybeans Suitable for Flow Cytometry Analysis
    CHEN Lin, PAN Zhen-zhi, DAI Yi, SONG Li
    2020, 36(11):  230-237.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0426
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    Using soybean Williams 82 and other varieties as research materials,the nuclear separation effects of 6 different kinds of nuclear dissociation solutions were compared,aiming to prepare the high-purity nucleus suspensions from different soybean tissues suitable for flow cytometry analysis,as well as to detect the application of them in the regulation of soybean cell cycle. The fresh young leaves and roots of soybean were mechanically cut in different nuclear dissociation solutions to release the nuclei from the tissues. After passing through 400 mesh sieves,enriched by centrifugation,and DAPI staining,the characteristics of biological particles and DNA content in the nuclear dissociation solution were detected and analyzed by flow cytometry. The results showed that Galbraith’s and LB01 dissociation solutions were suitable for the dissociation of nuclei from the soybean leaves and roots,and their coefficient of variation values were quite low at 2.78% and 2.96%,respectively. Among them,a distinct G2/M cell cycle peak was found in the LB01 nuclear dissociation buffer,which was more suitable for cell cycle and genetic ploidy research. Moreover,the nuclear dissociation solution can be successfully applied to analyze the regulation of drought stress simulated by PEG on the cell cycle of different soybean tips.

    Development of a Real-time Fluorescent Double Reverse-Transcription Recombinase Polymerase Amplification Method and Its Application in Detecting SARS-CoV-2 in Food
    LV Ji-zhou, WU Shao-qiang, ZHANG Zhou, DENG Jun-hua, YUAN Xiang-fen, WANG Cai-xia, FENG Chun-yan, LIN Xiang-mei
    2020, 36(11):  238-244.  doi:10.13560/j.cnki.biotech.bull.1985.2020-0432
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    The goal of this research is to develop a fast and real-time fluorescent reverse-transcription recombinase polymerase amplification method(RT-RPA)method for detecting and monitoring SARS-CoV-2. Two pairs of RPA primers/probe were initially designed based on the conserved sequences of specific S gene and Ngene of SARS-CoV-2. Meanwhile,SARS-CoV-2 virus-like particles based on the recombinant plasmids containing targeted N gene fragment was developed to offer positive material for molecular biological detection. Finally a set of RPA primers and probes targeting S gene and N gene was selected for developing fluorescent RT-RPA test strips. The analytical sensitivity of this method was about 10 copies/reaction of S gene and 102 copies/reaction of N gene. The RT-RPA could not amplify templates of H1N1 influenza virus,PEDV,TGEV,FMDV,PPRV and RV,demonstrating its high specificity. The established method was revealed to exclusively detect SARS-CoV-2 in foods,animal tissues and clinical samples,suggesting that the RT-RPA method is fast(20 minutes),double-targeting,sensitive,easy-operating and not depending on the expensive equipment,which makes it an effective approach for the rapid detection of SARS-CoV-2 in foods,animal products and clinical samples.

    Research Progress of DNA Aptasensors for Foodborne Pathogen Detection
    WANG Qi, YAN Chun-lei, GAO Hong-wei, WU Wei, YANG Qing-li
    2020, 36(11):  245-258.  doi:10.13560/j.cnki.biotech.bull.1985.2019-1273
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    Aptamers are single-stranded DNA or RNA molecules which possess specific identification and binding ability to their targets. Aptamers present breathtaking potential in the field of foodborne pathogen detection and other bio-molecular recognition,owing to their excellent specificity,stabilization,selectivity,simplicity of modification,and high affinity. This review outlines the technologies of aptamer screening and aptamer application,including colorimetric assays,visible spectrum assay,fluorescence assays,electrochemical assays,surface-enhanced Raman assays,and gold-nanoparticle lateral flow assays in the determination of foodborne pathogens. Until now,the remarkable progress(more rapid,sensitive,and accurate)in the monitoring of foodborne pathogen on aptasensor has been achieved;however,there are still many challenges and flaws in their utilization to be overcome.

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    2020, 36(11):  259-262. 
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    Cover
    2020, 36(11):  263-263. 
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