Table of Content

26 January 2019, Volume 35 Issue 1

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Preliminary Analysis of Functional Diversity of RALF Peptide Family in Arabidopsis thaliana

QIANG Xiao-nan, LI Xin, CHEN Jia, LIAO Hong-dong, YU Feng

2019, 35(1):  2-10.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0921

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The Rapid Alkalinization Factor（RALF）,which exists as a gene family,is a kind of conservative peptide molecule in green plant. There are at least 35 RALF genes in model plant Arabidopsis thaliana. Previous studies show that certain members of RALF family,such as RALF1/23 and RALF4/19 function as the ligand of CrRLK1L members FERONIA and BUPS1/2 respectively,thus regulate cell elongation,plant immune response and double fertilization. However,whether other RALF members have biological activity and whether distinct RALF have varied functions are still unclear. Here,we have expressed 19 heterogeneous representative RALFs and analyzed their biological activities and functional diversity. The experimental results showed that all 19 RALF proteins inhibited root growth at various extents. Some representative RALF members were further selected for flg22-induced ROS release and MAPK phosphorylation level assays. From the results,we confirmed that 11 RALF proteins participated in the response of MAPK signal,and also confirmed that 16 RALF proteins inhibited the ROS release caused by flg22. In addition,there were significant differences in the regulation of hypocotyl elongation,for instance,RALF10 promoted hypocotyl elongation. The above results suggest that different RALF have both functional redundancy and differences. This study complements and improves the understanding of the complexity and diversity of RALF functions.

Functional Verification and Bioinformatics Analysis of Cotton GhDMT3 Gene

YANG Xiao-min, WANG Jun-juan, WANG De-Long, LU Xu-ke, CHEN Xiu-gui, GUO Li-xue, WANG Shuai, CHEN Chao, WANG Xiao-ge, HAN Ming-ge, YE Wu-wei

2019, 35(1):  11-16.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0724

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The function of the gene in cotton and higher plants was explored by building VIGS silencing vector of GhDMT3 and infecting cotton. Bioinformatics was used to analyze the structure characteristics of DNA sequence of cotton DNA methyltransferase gene GhDMT3（CotAD_46796）as well as the physicochemical properties,hydrophobicity,and secondary structure of the protein encoded by this gene. The gene was cloned and the pYL156：GhDMT3 silencing vector was constructed. The relative molecular weight of the protein was 71 107.02 kD,the pI was 4.85,and the instability coefficient was 36.33. Cotton seedlings of VIGS silenced GhDMT3 treated with different stress（cold and dry）showed significant phenotypic differences. The expression pattern analysis of qRT-PCR showed that the expression levels in the different parts of pYL156-infected cotton did not vary under different stress,while the transcriptional Level of GhDMT3 in pYL156：GhDMT3-infected cotton decreased significantly compared with the control. Compared with cotton seedlings without GhDMT3 silenced,cotton seedlings with GhDMT3 silenced were significantly resistant to stress.

Effects of Exogenous Silicon on Growth,Leaf Photosynthesis and Physiological Indexes of Tobacco Seedlings Under Drought Stress

ZHANG Huan-wei, CHEN Biao, WEN Xin-yi, ZHANG Jie, WANG Xiao-dong, LI Ji-wei, XU Zi-cheng, HUANG Wu-xing

2019, 35(1):  17-26.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0691

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The effects of different concentrations of exogenous silicon（0,0.5,and 1.0 mmol/L）on the growth,Leaf photosynthetic characteristics and physiological indexes of tobacco seedlings under drought stress（10% PEG and 20% PEG）were studied using nutrient solution culture method. The results showed that drought stress severely inhibited the growth and photosynthesis of tobacco seedlings,and decreased membrane stability and induced oxidative stress response. Application of different concentrations of exogenous silicon effectively improved the growth of tobacco seedlings under drought stress,showing that the growth indices such as plant height,leaf area,root volume,dry weight of root and shoot increased;the contents of chlorophyll a,chlorophyll b,chlorophyll a+b,and carotenoids increased;the net photosynthetic rate（P_n）and transpiration rate（T_r）and stomatal conductance（G_s）significantly increased,while intercellular CO₂concentration（C_i）decreased;MDA content of membrane peroxidation product significantly reduced;and water content of leaves,membrane stability coefficient,and osmotic adjustment substance（proline and soluble sugar）content significantly increased. The activities of antioxidative enzymes such as SOD,POD and CAT increased. The 1.0 mmol/L Si treatment significantly affected the growth and physiological characteristics of tobacco seedlings under drought stress better than 0.5 mmol/L Si treatment. All above results revealed that the application of exogenous silicon may improve the photosynthesis,antioxidation and osmotic adjustment ability of tobacco seedlings under drought stress,relieve drought damage on tobacco seedlings,and promote its growth.

Effects of NaCl Stress on the Germination of Reaumuria soongorica and Evaluation of Salt Tolerance at Germination Stage

LIU Xiao-wei, YANG Xiu-yan, WU Hai-wen, ZHI Xiao-rong, ZHU Jian-feng, ZHANG Hua-xin

2019, 35(1):  27-34.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0686

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This work is aimed to explore the effects of salt stress on seed germination,growth and physiological and biochemical indexes of Reaumuria soongorica,and to provide data support for the analysis of salt tolerance mechanism and further relevant research. The seeds of halophytes R. soongorica were used as experimental materials and NaCl solution was for simulating salt stress,the germination of R. soongorica seeds,changing trend of root length,seedling height and physiological and biochemical indexes after germination were investigated under the treatment of different concentrations of NaCl（0,50,100,150,200,300 and 400 mmol/L）.The results showed that：（1）Compared with the control,the germination rate of R. soongorica treated with 50 mmol/L increased,the root length and plant height increased significantly. With the increase of concentration,the germination time of R. soongorica delayed,the germination rate decreased,the salt damage rate increased,and the root length and plant height decreased significantly. When the salt concentration increased to 400 mmol/L,the R. soongorica did not germinate,but still remained the germination vigor. The relative germination rate of seeds after 35 d of 400 mmol/L stress was 105.43% by re-geminating treatment. （2）With the increase of NaCl concentration,the contents of malondialdehyde（MDA）and proline（Pro）in R. soongorica increased significantly,and the activities of peroxidase（POD）and superoxide dismutase（SOD）increased in varying degrees. （3）Using the relative germination rate and the plant length decreased to 50% of those in the control as a standard,the germination salt tolerance threshold was 273 mmol/L and the optimal salt concentration was 45.78 mmol/L,and the growth salt tolerance threshold was 388.19 mmol/L and optimal one was 50.59 mmol/L respectively. R. soongorica remained germination vigor for a long time under the stress environment,and concentrated a large number of germination quickly when the germination conditions were suitable. The growth threshold concentration of R. soongorica was higher than the germination threshold concentration,indicating that the germination period was the key period for it to complete its life cycle under salt stress. With the increase of salt concentration,the growth of R. soongorica was inhibited to varying degrees,the plants scavenged MDA by increasing POD and SOD activities and thus increased Pro content in a certain degree of stress to maintain the normal physiological metabolism.

Cloning and Functional Analysis of a Potato Ubiquitin-conjugating Enzyme Gene StUBC17

LEI Zhao-xia, LIU Jing, BAI Yi-ping, TANG Wei, WANG Hong-yang

2019, 35(1):  35-41.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0763

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Ubiquitin-conjugating enzyme E2 is closely correlated with plant growth and disease resistance. To understand the contribution of ubiquitin-conjugating enzyme gene in plant resistance to late blight（Phytophthora infestans），an E2 ubiquitin-conjugating enzyme gene（designated as StUBC17）was cloned from potato cultivar “C88”，and its induced expressions by P. infestans and functions were analyzed. The transcriptions of NbUBC（homolog of StUBC17）was silenced in Nicotiana benthamiana using virus-induced gene silencing（VIGS）method，and the resistance to disease was validated by inoculating P. infestans. Furthermore，the disease-resistant gene and corresponding non-toxic genes of R3a+AVR3a、R3b+AVR3，Rx+CP，and INF1 were transiently expressed in NbUBC-silenced plants. The 447 bp coding region of StUBC17 encoded 148 amino acid residues with 16.52 kD of an estimated molecular mass and 7.72 of a calculated pI. Quantitative reverse transcriptase PCR analysis showed that the expression of StUBC17 was up-regulated after P. infestans infected it. By inoculating leaves in vitro，the identification of disease resistance showed that NbUBC-silenced plants presented an increased susceptibility to P. infestans；however，silencing of NbUBC demonstrated no effect on the hypersensitive responses. Our results suggest that the StUBC17 is necessary for potato plant to defense P. infestans，but its silencing do not affect the R3a-，R3b-，Rx- and INF- triggered hypersensitive responses

Isolation and Identification of a Uranium-resistant Strain and Effect of Its Characteristics on Growth Promoting

WANG Zhuo, LUO Xue-gang, DING Han-lin, YANG Hao

2019, 35(1):  42-50.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0577

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A uranium-resistant and plant growth-promoting bacterial strain was isolated from the root of a native Rumex acetosa in a uranium mining by enrichment culture and LB plate. Its growth characteristics and the performance of plant growth-promoting under different experimental conditions were analyzed by characteristic reaction and average absorbance method. The results showed that the strain grew well at the uranium concentration of 350 mg/kg,it produced indole-3-acetic acid（IAA）and IAA reached 40.21 mg/L in 24 h at normal condition. The optimal condition for IAA production was the temperature 30℃,pH 7,rotating speed 150 r/min,yeast extract as nitrogen source,and glycerol as carbon source. The strain also had deaminase capacity of ACC,and ACC enzymatic activity was 0.32 U/μg for 24 h at normal condition. The optimal condition for ACC enzyme activity was temperature 35℃,pH 7,and rotating speed 180 r/min. The strain was identified as Achromobacter xylosoxidans while combined morphological characteristic with physiological and biochemical preliminary characteristics and 16s rDNA sequence. Dry biomass of Medicago sativa increased by 17.9%-110.4% and U was enriched by 12.2%-180.6% under different U concentration.

Construction of Promoter Probe Vectors,and Screening and Identification of Oidium heveae Promoters

XU Liang-xiang, LIU Yao, LIAO Xiao-miao, WANG Yi, M.J.N. RAJAOFERA, HE Qi-guang, LIU Wen-bo, LIN Chun-hua, MIAO Wei-guo

2019, 35(1):  51-57.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0584

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This study aims to construct a probe vector suitable for rapidly detecting the promoter of Oidium heveae HO-73. The kanamycin-resistance gene was used as a reporter gene to construct the promoter probe vector pUC19-K in the pUC19 backbone vector,and it was ligated with the CaMV35S promoter to verify the function of the promoter probe vector. The promoter probe vector pUC19-K was employed to screen and identify the predicted promoter fragments LY1,LY2,LY3,and LY4. The CaMV35S promoter was ligated into the promoter probe vector to obtain the promoter probe vector pUC19-K that can detect the promoter activity. Using bioinformatics software to predict the whole genome data of Oidium heveae HO-73,4 theoretically active promoter sequences,LY1,LY2,LY3,and LY4,were obtained,and their activities were compared using the constructed promoter activity probe vector. The Kana tolerance test showed that strains containing LY2 and LY3 were more tolerant with increasing concentrations of kanamycin,resulting in two more active promoters,LY2 and LY3.The above results indicate that the constructed promoter activity probe vector can be effectively and sensitively used for screening the strong promoter of HO-73 and detecting promoter activity.

Precursor Cloning and Tissue Expression Analysis of Yak miR-378

JI Hui, WANG Hui, CHAI Zhi-xin, WANG Ji-kun, LUO Xiao-lin, JI Qiu-mei, XIN Jin-wei, ZHONG Jin-cheng

2019, 35(1):  58-66.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0671

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This work aims to clone the precursor sequence of yak miR-378,to elucidate its tissue expression pattern,and to explore the regulatory function of miR-378 in the growth and development of yak combined with bioinformatics prediction and analysis of bta-miR-378 target gene. PCR was employed to successfully clone the miR-378 precursor sequence of leiwuqi yak,and real-time quantitative PCR（RT-qPCR）was used to detect the expression patterns of miR-378-3p in various tissues. Further,bioinformatics software and databases such as TargetScan,DAVID,NCBI,miRbase,etc were together applied to perform conservative analysis,target gene prediction and biological function analysis of miR-378. The results showed that miR-378 was highly conserved among different species and miR-378-3p was widely expressed in all tissues,the expression level was the highest in gluteus maximus,which was significantly higher than that in other tissues（P < 0.01）. Expression in hip fat was higher than in ovary,brain,breast and liver. The obtained 272 target genes were mainly involved in many biological processes such as cell differentiation,cell development,and macromolecular metabolism,involved in progesterone-mediated oocyte maturation and gonadotropin releasing hormone（GnRH）signaling pathways. It is speculated that miR-378 may play a key role in follicular development and oocyte maturation,which may affect the reproductive performance of female calves.

Isolation,Identification,Inhibition Spectrum and Safety Test of Antagonistic Bacteria to Major Pathogenic Bacteria in Larimichtys crocea

FU Chao-ying, WANG Jian-ping, SUN Chen, CHEN Lin, QIAN Dong

2019, 35(1):  67-75.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0565

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Large yellow croaker Larimichtys crocea is an important marine cultivated fish in China. With the rapid expanding of large yellow croaker culture,the disease is increasing in recent years. Bacteria are the important agent causing diseases outbreak and the death of large yellow croaker. Vibrio harveyi,V. alginolyticus,Nocardia seriolea,Pseudomonas plecoglossicida and other bacterial agent were reported to cause the severe economic loss in the culture. Antagonistic bacteria were considered as a potential alternative measure for bacterial diseases instead of antibiotics,with its green and ecological friendship merits. Using V. harveyi and P. plecoglossicida as the indicator bacteria and via disk method（KB）and colony inoculation,10 antagonistic bacteria were screened from the large yellow croaker and soil,algae,and sea bass around fish culture ponds,and then identified as Bacillus sp.,Alcaligenes sp.,Paenibacillus sp.,Lysinibacillus sp.,Vibrio sp.,Daenococcus sp. by 16S rDNA sequence analysis. A. faecalis NBPa-7 and B. amyloliquefaciens NBlm-36,the antagonistic strains with strong inhibition to V. harveyi and P. plecoglossicida,the main bacterial pathogens of large yellow croaker,were used in this study. The results showed that NBPa-7 gave fine antagonism against V. alginolyticus and P. plecoglossicida,with inhibition zone of 23.20 mm,12.00 mm,respectively. The NBlm-36 showed antagonistic effects on V. vulnificus,A. hydrophila,Escherichia coli,and Staphylococcus sp.,with the inhibition zone of 6.00 mm-14.00 mm. Extracellular product presented favorable tolerance to heat,acid and alkali. The ICR mice were used for injection intraperitoneally with 1×10⁸ CFU/ind of 2 antagonistic strains,and no toxicity and death occurred in 48 h after injection. There was no morbidity within 2 weeks,no lesion in the viscera of the large yellow croaker,and there was no obvious increase in white blood cells when the large yellow croakers were injected intraperitoneally with 3×10⁸CFU/ind of two antagonistic strains,indicating that antagonistic strains are non-pathogenic to mice and large yellow croaker.

Screening and Identification of Soil Mycelial Pellets and Application of Immobilized Mycelium

LIN Sheng-hong, PAN Xiao-mei, SHI Xiao-ling, IHSAN Ali, LIU Zhen-fei, YU Shuang, ZHANG Jin-feng, NIU Zhen-feng, TIAN Yong-qiang

2019, 35(1):  76-81.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0542

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A fungus isolated and purified from soil was identified as Aspergillus fumigatus L-3. Using strain L-3 as immobilized carrier,on which Bacillus Licheniformis was immobilized to form an immobilized system. The degradation of Congo red dye wastewater by the composite mycelial cake,mycelia,fermentation mixture,and crude laccase enzyme solution,as well as the toxicity test of the dye wastewater were conducted. The results showed that the degradation effect on the dye wastewater by mycelial pellets was the most significant,the degradation rate reached 99.96%;and the degradation rate by mycelia cake was 91% with only 20 s,while the degradation effect by the fermentation mixture and the crude laccase solution was not so significant. The detoxification rate from dye wastewater by this system was obvious,especially the detoxification rate by mycelial pellet reached 78%. It can be seen that the immobilized system has not only high degradation ability but also a high detoxification rate.

Breeding of Strain Producing L-isoleucine and Medium Optimization for It

WANG Zhuang-zhuang, WEI Jia, YU Hai-bo, XU Jian-zhong, ZHANG Wei-guo

2019, 35(1):  82-89.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0634

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This work aims to breed a strain yielding high L-isoleucine by mutagenesis,and explore the optimal fermentation conditions for the mutant strain. The original strain Brevibacterium flavum I-12 was mutagenized by conventional chemical mutagenesis and atmospheric and room temperature plasma（ARTP）biological breeding system. A mutant strain having high resistance to 2-thiazolyl alanine（2-TA）and sulfaguanidine（SG）and capable of rapid growth on a succinic acid plate was selected. Subsequently,the response surface design based on the single factor experiment was used to optimize the optimal parameter level of the shake flask fermentation medium components for the target mutant strain. Results showed that a strain producing L-isoleucine was obtained after multiple rounds of random mutagenesis and screening,which was resistant to 40 g/L of 2-TA and 5 g/L of SG and rapidly grew on the medium with succinate as the sole carbon source,and designated as B. flavum TA-6. The L-isoleucine production by this mutant strain reached 26.2±0.5 g/L,which was 44.75% higher than that by the original strain（18.1±0.5 g/L）;while the accumulations of side product L-valine and L-leucine significantly reduced. The L-isoleucine production increased to 27.8±0.5 g/L after optimizing the fermentation conditions by response surface method,which was 6.1% higher than that before optimization. In conclusion,a strain yielding high L-isoleucine but producing low other acids,TA-6,is obtained by conventional chemical mutagenesis and ARTP biological breeding system,thus it has potential production and application value in L-isoleucine fermentation industry.

Isolation,Identification of Carbon-fixing Bacteria and Determination of Their Carbon-fixing Abilities

GUO Jun, FAN Fang-fang, WANG Li-ge, WU Ai-Lian, ZHENG Jun

2019, 35(1):  90-97.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0544

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Currently,as a biological carbon sequestration strategy,microbial recovery and fixing of CO₂ gas,has become the focus of interest in dealing with the major environmental problem of greenhouse effect. The purpose of this study is to separate and screen efficient carbon-fixing microbial strains. By using the carbon-free source inorganic medium,24 kinds of strains with CO₂ as carbon source were separated from facilities soil,activated sludge and biogas slurry. Then,the 8 strains of bacteria that grew faster were selected to conduct their morphological observations,physiological and biochemical responses as well as to perform cbbL gene and 16S rDNA sequencing analysis. Further,the sequencing results were compared with the data in BlAST database and the molecular characterization of the 8 carbon-fixing strains was carried out along with the detection of the strain content and the RubisCO enzyme activity. Finally,the C2-8R with the highest RubisCO activity among the 8 strains was selected for soil application test,and the carbon sequestration capacity of the C2-8R was determined through the detection of the RubisCO enzyme activity in soil. The above results revealed that carbon sequestration strains can be screened through carbon-free medium. The screened carbon-fixing bacteria are subordinate to the strains of Pseudomonas sp. and Methanotrophs sp.,and can be used to indicate the microbial carbon-fixing ability through RubisCO enzyme activity.

Fermentation Condition and Phosphate-dissolving Capacity of Phosphate-solubilizing Bacterium

HAN Lei, YANG Le, TANG Jin-ming, ZHANG Ya-qian

2019, 35(1):  98-104.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0733

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The aim of the present work is to isolate a highly effective phosphate-solubilizing strain PS-3 for improving phosphorus supply of saline-alkali soil in Xinjiang. The single factor experiment and the orthogonal experiment were used to analyze the fermentation conditions and the phosphate-dissolving capability of strain PS-3. The results showed that the appropriate fermentation conditions of the strain PS-3 were temperature 35℃,pH 7 and salt concentration 1%. The order of the strain utilizing carbon and nitrogen was glucose > maltose>fructose>sucrose>lactose,and ammonium nitrate>sodium nitrate>ammonium chloride>ammonium sulfate>urea,respectively. The optimal conditions for the solubilization of strain PS-3 were 2% glucose,0.01% ammonium nitrate,temperature 35℃,initial pH 7.5,salt content 2 g/L,and inoculation amount 4%. The phosphate-solubilizing capability reached the level of 1 002.95 mg/L under this condition. The strain PS-3 is tolerant to salt stress and has desired phosphate-solubilizing capacity,and thus has a better potential for microbial inoculants with phosphorus-solubilizing ability.

Construction of Co-expression System of S-imine Reductase and Glucose Dehydrogenase and Synthesis of Chiral Amine

LI Ji-xuan, YU Lei, LI Jing-mei, ZHENG Gui-lan, WANG Hong-zhong

2019, 35(1):  105-111.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0806

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This work aims to construct a co-expression system of S-imine reductase（S-IRED）and glucose dehydrogenase（GDH）in Escherichia coli Bl21,and to efficiently synthesize chiral secondary amine by regenerating coenzyme NADPH. A co-expression system with double promoters in a single plasmid was designed and constructed by seamless cloning. Whole cells of the recombinant strain were adapted as a catalyst to synthesize chiral secondary amine S-2-methylpyrrolidine（S-2MP）,and the effects of temperature,pH and organic solvent on the reaction were studied. As results,the recombinant co-expressed plasmid was successfully constructed and intracellular co-expression of S-IRED and GDH in E. coli was achieved. Using imine 2-methyl pyrroline（2MPN）as the model substrate and catalyzed by whole cells of the strain,S-2MP was synthesized;both the production rate and optical purity of chiral amine were higher than 95%. The optimal temperature and pH were 37℃ and 8 respectively. Methanol of <10% had a positive role in promoting the reaction. In sum,the construction of co-expression system in E. coli Bl21 allows in-situ regeneration of coenzyme NADPH,which reduces the synthesis cost of chiral amine catalyzed by imine reductase and provides a basis for further scale preparation of chiral amine.

Research Advances in Proteomics of Ammopiptanthus in Responses to Abiotic Stresses

LAN Yu-ting, WANG Shuang-Lei, LI Zheng-zhen, FENG Jin-chao, WANG Xiao-dong, SHI Sha

2019, 35(1):  112-119.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0780

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Ammopiptanthus is one kind of rare relic plants，showing many unique features of stress resistance and thriving in many extreme environments，such as draught stress，cold stress，high salinity stress and so on. Many studies have been proven that Ammopiptanthus is an ideal model material for studying plant resistance mechanisms as well as screening natural stress-resistant gene. Abiotic stress is one of two major factors which limits the growth and the geographical distribution of Ammopiptanthus. Studying proteomics of Ammopiptanthus in response to abiotic stress may lay a foundation for discovering stress-resistant protein，mapping stress-resistant interactive protein network mapping，and exploring stress resistance mechanism. This review summarizes the recent advances in proteomics investigation for Ammopiptanthus in response to abiotic stresses，such as low temperature，drought，and salinity，focusing on different physiological statuses of Ammopiptanthusduring abiotic stresses，to reveal protein dynamics，specific protein interactive network，and relevant stress response mechanisms. The application prospect of proteomics is also viewed here to provide a reference for in-depth and comprehensive study on the resistance molecular mechanism of Ammopiptanthus.

The Augmentation Strategies and Mechanisms in the Phytoremediation of Heavy Metal-contaminated Soil

YUAN Jin-wei, CHEN Ji, CHEN Fang, LIU Wan-hong

2019, 35(1):  120-130.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0618

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Heavy metal pollution has posed a harmful influence on ecological environment,agricultural production,human health and many other aspects. Phytoremediation,a cost-effective and green ecological biotechnology,has been a research focus in the remediation of heavy metal-contaminated soil. Actually,most phytoremediation cases were limited to the lab research due to the factors such as heavy metal toxicity of plants and time consuming during the remediation process. Reasonable augmentation measures may enhance the phytoremediation efficiency,which may be a key to solve the problem mentioned above. Rhizobium,arbuscular mycorrhiza fungi,phosphate-solubilizing microbe,and endophytic fungi may construct the microbe-plants symbiotic system with plants as host,and the application of such microbe-plants symbiotic system in phytoremediation field is discussed. Meanwhile,the function of chelating agents for enhancing phytoremediation efficiency is reviewed,for example,EDTA and EDDS etc.,with the ability to change the heavy metal solubility in the soil,prompt the heavy metals to transfer into plants from the soil. Genes encoding proteins involved in heavy metals transfer and metabolism,including metal transporter protein,metallothionein,and phytochelatins,are introduced about their applications in phytoremediation. The main mechanisms of the above-mentioned augmentation strategies are summarized as microbes promoting plant growth,alleviating the toxicity of heavy metal plants,and increasing the bioavailability of heavy metals in the soil,thereby promoting the over-accumulation of heavy metals in plants and the increase of plant biomass. Finally,the key research points and existing problems in phytoremediation augmentation technology in future are summarized and forecasted. This article systematically reviews the main augmentation strategies and mechanisms used in phytoremediation technology,aiming at providing an important reference for the use of phytoremediation technology to solve soil contamination by heavy metal.

Research Progress on Functional Nucleic Acids for Detecting Pb²⁺

LI Chen-wei, DU Zai-hui, LIN Shao-hua, LUO Yun-bo, XU Wen-tao

2019, 35(1):  131-139.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0676

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Low concentration of Pb²⁺ can cause multiple damages to the nervous system and blood system of the human body,especially the damage to the nervous system of children is irreversible. In natural environment,Pb²⁺ can also pass into the human body through the biologic chain and affect the health of the human body. Therefore,the detection of Pb²⁺ has attracted wide attention. At present,the traditional methods of detecting Pb²⁺ mainly include Large instrument method,chemical reaction colorimetric method,and electrochemical method;however,there are still disadvantages such as operation complex,cost high,and detection time long. In recent years,using functional nucleic acid to detect Pb²⁺ shows the advantages of simple,rapid,sensitive,and high specialty;therefore,increasing attentions has been paid to Pb²⁺ biosensors based on functional nucleic acid. Firstly,the screening methods and structural properties of Pb²⁺-dependent functional nucleic acids in vitro are introduced. Secondly,the sensors are classified according to the way of signal output in Pb²⁺ functional nucleic acid sensors,including colorimetric biosensor,fluorescent biosensor,electrochemical biosensor,nanomaterial biosensor,and other type of biosensors. Concurrently,the principle,sensitivity,specificity and application fields of various sensors are compared,and then their advantages and disadvantages are evaluated. Finally,the shortcomings are analyzed and the future development direction of Pb²⁺ functional nucleic acid biosensor is prospected,aiming at providing a theoretical basis for developing more portable,more sensitive,and more accurate Pb²⁺ functional nucleic acid biosensors.

Research Progress on 10-23 DNAzyme Mediated Biosensors

LI Kai, LUO Yun-bo, XU Wen-tao

2019, 35(1):  140-150.  doi:10.13560/j.cnki.biotech.bull.1985.2017-1101

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In the past two decades,many DNA-based biosensors emerged and developed rapidly. While the main role of DNA is genetic information container,its folded space structure is functional as well. The theories and applications of functional nucleic acids have been involved in many critical fields such as biosensor,bioimaging and medication. As one of them,10-23 DNAzyme of a functional nucleic acid was obtained using in vitro selection technology. It can be made to specifically recognize and cleave any targeted RNA substrate at cleavage site of phosphodiester bond between pyrimidine and purine at the presence of Mg²⁺. Because of its special identification and cleavage ability,10-23 DNAzyme mediated disease treatment has been applied widely;also 10-23 DNAzyme mediated biosensors have been established. In this paper,we present the structure,property,action mechanism,and modification of 10-23 DNAzyme;meanwhile,we summarize the establishment and application of 10-23 DNAzyme-mediated biosensors,aiming at providing theoretical basis to guide people to develop new 10-23 DNAzyme-mediated biosensors.

Research Progress on S1 Nuclease-mediated Functional Nucleic Acid Biosensors

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

2019, 35(1):  151-160.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0387

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The S1 nuclease is a highly single-stranded and specific endonuclease that degrades single-stranded DNA or RNA under optimal enzymatic conditions to produce single nucleotides or oligonucleotides with 5'-phosphate. It is relatively insensitive to double-stranded DNA,double-stranded RNA,and DNA-RNA hybrids. At present,based on the activity of S1 nuclease for carrying different signal outputs and amplification methods,a series of biosensors have been constructed to detect metal ions,single-stranded nucleotides,amino acids and other substances. Moreover,the S1 nuclease can be applied to the purification of nucleic acid reaction system,the construction of a multi-dimensional gene Library. In this paper,S1 nuclease is introduced from the aspects of structure to functions,and the representative biosensors mediated by S1 nuclease are summarized from the view of establishment and application. Then,based on the different target substances detected,various S1 nuclease-mediated sensors are classified and introduced. Finally,the current research status of S1 nucleases is analyzed,and the future development direction of S1 nuclease-mediated biosensors is prospected.

Research Progress on Functional Nucleic acid Biosensors Mediated by Terminal Deoxynucleotidyl Transferase

ZHANG Yuan, LIANG Jia-hui, LUO Yun-bo, TIAN Jing-jing, TIAN Hong-tao, XU Wen-tao

2019, 35(1):  161-169.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0502

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Terminal deoxynucleotidyl transferase（TdT enzyme）is a DNA polymerase that catalyzes the binding of deoxynucleotides to the 3’ hydroxyl end of a DNA molecule,and for which a specific template is not demanded. Currently,based on the characteristics of terminal deoxynucleotidyl transferases that can extend the ends of the template nucleic acid strands and carried on with different signal outputs and amplification methods,a series of biosensing technologies were constructed,such as electrochemical biosensors,fluorescent biosensor,and surface ion resonance biosensors. This article describes the basic design principles and applications of various sensors. A series of biosensors can be designed for the detection of metal ions,pathogens,and proteins according to the properties of TdT enzymes,with the advantages of simplicity,rapidity,low cost,high sensitivity,and fine specificity. At the end of this article,the current research status on TdT-mediated biosensors is summarized and its future development direction is prospected.

Research Progress on Fluorescent Copper Nanoparticles Mediated Biosensors

LIU Xing-yu, LI Chun-hui, TIAN Jing-jing, SHAO Xiang-Li, LUO Yun-bo, XU Wen-tao

2019, 35(1):  170-186.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0524

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Fluorescent copper nanoparticles（CuNCs）are nanometer-sized copper crystals generated by using deoxyribonucleic acid（DNA）as a template and divalent copper ions（Cu²⁺）and ascorbic acid as reactants,and they are fluorescent and can be used to be output signal in biosensors. The generation of CuNCs is fast,simple,and safe. Therefore,many researches on the principles and applications of CuNCs have emerged in recent years. Here CuNCs mediated biosensors are classified in terms of mediators supporting the sensor operation and signal output mode. A detailed description of the principles of various sensors and comparison of the advantages and disadvantages of the biosensors are included,for the purpose of understanding the feasibility and versatility of（CuNCs）mediated biosensors after reading the development and directions of these biosensors. Finally,this review also discusses the shortcomings in applying the CuNCs biosensors and looks into its future development trend. This is aimed to inspire future research and development in this area and thus to develop copper nanoparticles mediated biosensors into more practical and convenient biosensing tool.

Research Progress on Biosensing and Drug Delivery Mediated by the Staudinger Ligation

ZHANG Yang-zi, ZHU Long-jiao, SHAO Xiang-Li, XU Wen-tao

2019, 35(1):  187-198.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0096

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The Staudinger ligation is a type of metal-free catalyzed click reaction between organic azide and phosphine under the mild conditions like at room temperature and in the water solution. Due to its bio-orthogonality and no potential cytotoxicity,Staudinger ligation is now widely used in the surface functionalization of materials,synthesis and labelling of various biological macromolecules. At the same time,there is great development space for applying Staudinger ligation in the synthesis and delivery of drugs and biosensors. In this review,the Staudinger ligation is described in detail involving the reaction mechanism,the factors influencing the reaction kinetics,the yield of the ligation and the reaction process,and the biomarker technology mediated by the ligation. On these bases,the biosensing applications mediated by the Staudinger ligation are discussed,including fluorescent biosensors targeting nucleic acids and small molecules,the applications of cell imaging technology in nucleic acid and glycan,and the applications in drug synthesis and delivery. At last,the future development trend of the Staudinger ligation is forecasted and the application prospects in the biosensing are presented.

Progress in Microbial Fuel Cell Coupled Constructed Wetlands

WANG Guo-zhen, WEN Hong-yu, CAI Jia-ying, YUAN Zhen-ya, WANG Xiu-ying, SU Si-ting

2019, 35(1):  199-206.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0575

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Microbial fuel cell coupled constructed wetlands（CW-MFC）is a new type of wastewater treatment system that combines constructed wetland（CW）and microbial fuel cell（MFC）. The electricigen generate electrons in the wetland of underlying where an anaerobic environment（anode）is,and the electrons cross the external circuits to surface wetland（cathode）to complete the redox reaction. However,the research on the CW-MFC in recent years is rare and simple. In this paper,the effects of electrode materials,water conservancy conditions,wetland plants and microbes on wastewater treatment capacity and electricity-generating capacity of CW-MFC are reviewed. For electrode materials,the power generation and decontamination ability of CW-MFC could be effectively improved while using materials with high conductivity,adsorption,and large effective area as electrodes. Regarding water conservancy conditions,the up flow or up flow-down flow type of water inlet should be used in CW-MFC under the condition of HRT of 2-3 d. Considering wetland plants,the CW-MFC planted with wetland plants is superior to those without wetland plants in terms of decontamination and electricity production. On microbes,there are significant differences in the microbial community structure between the cathode and the anode;however,the composition of the electricigen is very similar. The types of common electricigen in CW-MFC include Geobacter,Desulfobulbus,Pseudomona and Desulfovibrio,etc.,respectively. Finally,the challenges and research directions of CW-MFC are analyzed briefly. This review aims to provide reliable experimental reference and theoretical basis for CW-MFC’s future application.

Research Progress on Exosomes Isolation Methods Based on Microfluidics Technology

LIU Na, DU Pan-pan, YANG Yang, LI Xiao-mao

2019, 35(1):  207-213.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0571

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Exosomes are nanoscale extracellular vesicles of diameter 30-150 nm secreted by cells. Exosomes contain many specific biomarkers such as proteins,mRNA,miRNA,and participate in many important biological processes such as intercellular communications,antigen presentation,and the transport of proteins,RNA,and other molecules. Exosomes are also associated with the occurrence of cancer and other diseases. Therefore,exosomes have significant application value in the investigation of disease mechanism,disease detection and diagnostics. However,these exosomes-based applications rely on the isolation and purification of exosomes because they are generally dispersed in body fluids. In recent several years,based on their physical or chemical properties many methods and techniques for the isolation and purification of exosomes have been developed,such as ultracentrifugation,polymer precipitation,immunoaffinity capture,and microfluidics-based methods. This article reviewed recent advances in methods and techniques for exosomes isolation and purification,including the brief introduction of conventional exosomes isolation methods,the detailed introduction of microfluidic-based exosomes isolation techniques,and the contrast of these methods in the mechanism,performance,and application prospects. By inducing and comparing recent advances in exosome isolation and purification,this review aims to provide a reference for the researchers in exosomes-related research work,and indirectly promote the exosomes research in disease detection and other biomedical applications.

Screening of Estrogenic Binding Activity of Phthalic Acid Esters

ZHANG Xiao-cong, ZHANG Dong-sheng, LIU Lan-hua, ZHOU Xiao-hong, LIU Chun, LU Yun, SHI Han-chang

2019, 35(1):  214-218.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0480

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Taking the evanescent-wave optical fiber fluorescent biosensor as a platform and the phthalic acid esters（PAEs）contaminants as targets,the biosensing analytical technology based on the estrogen receptor interaction theory was optimized,and qualitative screening of estrogen binding activity of PAEs was achieved. The order of the estrogenic binding activity of seven typical PAEs were BBP > DBP > DIPP. The other 4substances had almost no estrogenic binding activity,which was consistent with the widely reported conclusions in Literatures,and confirming the accuracy of the established method. Under the optimal test conditions,the optical fiber sensing surface can be regenerated more than 300 times,providing a Low-cost,automated method for the screening of estrogenic contaminants.