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Table of Content

    26 February 2019, Volume 35 Issue 2
    Prokaryotic Expression,Purification and Antibody Preparation of Transcription Factor CNR in Tomato Fruit
    LI Ling, BAI Juan-juan, GUO Mei, WANG Si-yu, WANG Xiao-yue
    2019, 35(2):  1-8.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0664
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    Transcription factor CNR(Colourless non-ripening)plays a key role in the process of fruit development and maturity. At present,the study on tomato transcription factor CNR is mainly at the transcriptional level;however,the protein level is where CNR functions. In this study,the expression pattern of transcription factor CNR in tomato fruit was studied at protein level by prokaryotic expression,purification of CNR,and preparation of polyclonal antibody. The results confirmed that the pET-CNR recombinant vector had the correct reading frame and the sequence was in full agreement with the published CNR coding sequence in tomato. There was CNR protein expression at 27 kD,indicating that pET-CNRprokaryotic expression vector was successfully constructed. The optimal concentration of IPTG inducer was 1.0 mmol/L. The purified CNR protein was collected while eluting chromatographic column by the elution buffer of 500 mmol/L imidazole,then used as antigen to immunize mice,and the CNR protein polyclonal antibody with high potency and favorable specificity was obtained. Finally,the expression patterns of CNR protein in tomato fruit at different maturity stages were studied,and it was discovered that transcription factor CNR played a crucial role in the color-breaking stage of tomato fruit.
    Distribution of Fusarium Species in Vegetable Rhizosphere Soil of Hubei Province
    FANG Qing, TANG Liu, XIE Yuan-li, ZHANG Yi-jing, SHI Xue, HUANG Wen-kun, PENG Huan, PENG De-liang, LAN A-feng, KONG Ling-an
    2019, 35(2):  9-15.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0771
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    To clarify the species and distribution of Fusarium species in vegetable growing areas in Hubei province,55 rhizosphere soil samples were collected from 13 cities of Hubei province,and Fusarium species strains from the samples were isolated,purified and identified. Total 271 Fusarium strains were obtained. Among these Fusarium strains,according to molecular biological techniques such as species-specific primers and sequencing,118 strains were identified as Fusarium oxysporum accounting for 43.6% of the total,46 strains as F. equiseti for 17.0% of the total,45 strains as F. commune for 16.6% of the total,31 strains as F. solani for 11.4% of the total,11 strains as F. fujikuroi for 4.1% of the total,6 strains as F. delphinoides for 2.2% of the total,5 strains as F. concentricum for 1.8%,3 strains as F. tricinctum for 1.1%,and 6 strains were Fusarium spp. for 2.2% of the total. Based on the confrontation culture with pathogenic F. oxysporum f. sp. lycopersici strain,16 strains of antagonistic Fusarium species were identified,including 4 strains of F. oxysporum,4 strains of F. solani,3 strains of F. concentricum,and 3 strains of F. tricinctum,while each of F. equiseti and F. fujikuroi was only one,respectively. In this study,the species and diversity of Fusarium species in vegetable producing areas of Hubei province were confirmed,which provides the theoretical basis for the accurate pathogen identification and scientific control of Fusarium disease.
    Research on the Growth,Browning and Flavonoid Accumulation of Ginkgo biloba Callus
    WANG Jun-yi, DONG Jin-jin, LIU Wei, CAO Fu-liang, WANG Gui-bin, WANG Yi-qiang
    2019, 35(2):  16-22.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0672
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    The aim of this study is to establish a stable callus succession system of Ginkgo biloba,to screen the condition of culturing cells with high activity and flavonoids,and thus to provide references for industrial production. Using MS medium as the basic medium,we investigated the G. biloba callus growth,browning and flavonoid accumulation under the interactions among explant month,exogenous hormone and anti-browning agent by an orthogonal experiment of 40 d succession period,and compared with the contents of G. biloba leaves’ flavonoids in April,May,and June. The results showed that the condition for optimal growth of G. biloba callus was callus induced by April leaf + 3.0 mg/L NAA + 0.5 mg/L KT + 5 mg/L VC. The optimal anti-browning condition was callus induced by April leaf +1.0 mg/L NAA+1.0 mg/L KT+10 mg/L VC. The flavonoid content of G. biloba leaf tended to increase from April to June,and the flavonoid content of callus at different month was positively correlated with its explants,the condition of callus induced by June leaf + 2.0 mg/L NAA + 1.0 mg/L KT + 5 mg/L VC was the optimal for flavonoids accumulation. Combined with the dry weight of callus,the maximum flavonoid yield was from the combination of callus induced by May leaf + 2.0 mg/L NAA + 0.5 mg/L KT + 5 mg/L VC. The appropriate succession period for G. biloba callus was 24-32 d,with large growth and low browning rate.
    Effect of Sowing Date on Heterosis of Wheat Ecotypes Hybridization
    KONG De-zhen, NIE Ying-bin, XU Hong-jun, MU Pei-yuan, CUI Feng-juan, TIAN Xiao-ming, SANG Wei
    2019, 35(2):  23-28.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0683
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    In order to clarify the optimal sowing dates of different ecotype hybrids,3 sowing dates(October 2,2014,November 3,2014 and March 21,2015)were set up with 6 winter wheat and spring wheat parent materials and 8 hybrid combinations. The two-factor split design was used to study the heterosis changes of agronomic traits and yield by using strong heterosis combination. The results showed as such:All the ecotype hybrid combinations and their parents were able to overwinter normally,and the winter parents sown in spring was not heading. The differences of yield and agronomic traits under different sowing dates all reached the significant level. With the delay of sowing date,the yields of both hybrids and parents decreased significantly,the yields of hybrids decreased more than that of parents,the yield of spring parents decreased less than that of winter parents,and the yields of reciprocal combinations between winter and spring ones were not significant. Wheat ecotypes hybrids may demonstrate their high-yield potential during normal autumn sowing seasons. Four strong heterosis combinations were selected:Xinchun38×Xindong41,Xindong41×Xinchun38,Bachun 02-509×Xindong33,and Xindong33×Bachun02-509. Correlation analysis revealed the yield of strong heterosis combination community was in T1>T2>T3. Screening strong heterosis combination between winter wheat and spring wheat ecotypes can be conducted by adjusting the sowing dates to solve the problem of adaptability in winter and spring.
    Effects of Micro-fertilizer Combined with Small-Molecule Organic Matter on Growth and Quality of Flue-cured Tobacco
    XIONG Ya-nan, HAN Dan, WANG Fa-zhan, MA Xiao-han, JIN Yi-nan, CHEN Biao, CAO Li-jun, WANG Xiao-li, REN Zhi-guang, XU Zi-cheng
    2019, 35(2):  29-38.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0729
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    In order to improve the utilization rate of micro-fertilizer by flue-cured tobacco and improve the quality of tobacco leaves,using cultivar Yunyan 87 as the material in the field spray test,different treatments(CK:spraying water;T1:spraying micro-fertilizer;T2:spraying micro-fertilizer + sorbitol;T3:spraying micro-fertilizer + sodium gluconate;T4:spraying micro-fertilizer + glycine)were carried out to study the effects of micro-fertilizer combined with different small-molecule organic matter on the growth and quality of flue-cured tobacco. The results showed that the plant height,stem circumference,maximum leaf area,and dry matter accumulation of tobacco plants under different growth stages increased at different degrees in combination of micro-fertilizer and small-molecule organic matter while compared with the control and sole spraying micro-fertilizer. Fertilization with small-molecule organic matter effectively increased the chlorophyll content of tobacco leaves,and the photosynthesis indexes Pn,Gs,and Tr of leaves were significantly higher than that of the control,while Ci was significantly lower(P < 0.05). Physical characteristics such as leaf width,single leaf weight,tensile force and filling value increased,while the tobacco stem stalk rate reduced. The chemical constituents such as reducing sugar,total sugar,potassium,sugar-to-alcohol ratio and potassium-chloride ratio of tobacco leaves rose at varied degree,while the contents of nicotine,total nitrogen and chlorine decreased,and the contents of Ca,Mg,B,Mo,Fe,Mn and Zn all increased at varying degrees. Maillard reaction products,carotenoid degradation products,solanone and content of neutral aroma components such as phytoene were high,and the quality of sensory evaluation was also greatly improved. The above results revealed that micro-fertilizer combined with small molecular organic matter may effectively improve the photosynthetic characteristics of leaves,promote the growth and development of tobacco plants,and improve the quality flue-cured tobacco leaves. In this experiment,the effect of glycine treatment combined with micro-fertilizer on the growth and quality of flue-cured tobacco was the best.
    Effects of pH on the Lentinan Content and the Transcriptional Levels of Genes Related to the Synthesis of Key Enzymes in Lentinula edodes
    WANG Jie, ZHANG Yang, QIN Peng, HE Mao-lan, LI Jin, GU Yun-fu, ZENG Xian-fu, XIANG Quan-ju
    2019, 35(2):  39-45.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0842
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    Lentinula edodes is a fungus as both medicine and food. Its main metabolite,lentinan,has great medicinal value. It is of great significance to study the metabolic pathways of lentinan biosynthesis for improving the content of lentinan. With Xin808 as the material,phenol-sulfuric acid method was used to measure the lentinan content under different pH fermentation conditions,and fluorescence quantitative PCR and three software(GeNorm,NormFinder and BestKeeper)were adapted to screen the stably expressed reference genes. Based on those,the transcriptional expression levels of 3 key enzyme genes in polysaccharide metabolic pathways were analyzed. The results showed that the biomass and intracellular polysaccharide of mycelium increased with the increase of pH,while the extracellular polysaccharide decreased. 18S and rpl4 were determined as the most stable and unstable reference genes,respectively. In the range of pH 5.0-7.0,the relative expression level of UGP was obviously higher than that of PGM and PGI,low-level and stable expression profile of PGM was detected,and the relative expression of PGI was low,but fluctuated with the variation of pH value. In sum,the variation of pH value resulted in the various effects on the biomass and polysaccharide content of L. edodes,and there was no correlation between the transcriptional expression level of 3 key enzyme genes and polysaccharide content in this study.
    Effects of Exogenous Paclobutrazol and GA3 Treatment on the Accumulation of Endogenous IAA and Transcriptional Level of Related Genes in the Development of Tuberous Toot of Pseudostellaria heterophylla
    ZHENG Wei, ZHOU Tao, JIANG Wei-ke, LI Jun, XIAO Cheng-hong, YANG Chang-gui, ZHANG Chen, GONG An-hui, WEI De-qun, BI Yan
    2019, 35(2):  46-52.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0754
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    ELISA and Real-iime quantitative PCR experiments were conducted for investigating the dynamics of endogenous IAA and transcriptional level of related genes in response to exogenous paclobutrazol and GA3 treatment on P. heterophylla. The results showed that after PBZ treatment,the content of endogenous IAA by PBZ treatment at 10 d,20 d and 30 d was higher than the control groups after the first treatment,while lower than the control groups after 30 d. The content of endogenous IAA in the tuberous root by GA3 treatment showed fluctuation,i.e.,it was always higher than the control groups in other periods except for the 20d after treatment. In addition,the expression levels of genes related to endogenous IAA biosynthesis were analyzed in tuberous roots of P. heterophylla treated at 50 d after exogenous PBZ and GA3 treatment. The results showed that the Unigene37777 was up-regulated by PBZ treatment,while the expression of Unigene43146 and Unigene43412 were inhibited and the expression was down-regulated,but the three genes were induced by exogenous GA3;however,Unigene49937 presented opposite expression pattern in the treatment of exogenous PBZ and GA3,it was induced by PBZ,but inhibited by GA3. Overall,PBZ and GA3 induced the accumulation of endogenous IAA during the development of P. heterophylla,and had varied responses to the expressions of synthesis related-genes.
    Cloning,Prokaryotic Expression and Activity Research of Panax ginseng Thionins
    ZHANG Peng-fei, BIAN Shuai, ZHAO Yue, ZHAO Yu, WANG Jia-wen
    2019, 35(2):  53-57.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0702
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    Antibacterial peptides play an important role in bacteriostasis and resistance to stress in the growth process of ginseng. Studying the antibacterial activity of ginseng thionins may lay a foundation for large-scale production and biological pesticide research in the future. First,the gene sequence of ginseng thionins was analyzed and prokaryotic expression plasmid was constructed to induce the expression of the protein.. Then the affinity and purification of inclusion bodies was performed,the anti-fungal activity of the purified protein was detected by paper tablet diffusion method and membrane permeability assay. The ginseng thionins was 228 bp in length and encoded 75 amino acids,and its mature peptide region(29-75 aa)was selected for subsequent expression and purification. The prokaryotic expression plasmid pGEX-4T1-thionins was successfully constructed,the efficient expression of the target protein was obtained in Escherichia coli BL21,and the target protein was purified by inclusion body and then renatured and concentrated. The growth inhibition rate of ginseng thionins on Alternaria panax was 36.0% and 51.1% at 3 μmol/L and 9μmol/L,respectively. The half-inhibitory concentration of the fungal spores was 0.87 μmol/L. In conclusion,the ginseng thionins has a significant effect of inhibiting the fungus A. panax.
    Screening of Straw-degrading Bacteria and Study on Their Cellulose-degrading Performances
    YU Hui-juan, GUO Xia-li
    2019, 35(2):  58-63.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0731
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    High-efficiency straw-degrading bacteria were screened by straw powder plate,Congo red staining method and degradation rate of wheat straw. The extracellular cellulase activities of the strains were determined to study the degradation ability of the straw cellulose. The results showed that among the 4 strains of fungi screened,the strain z-5 had the highest degradation rate of 47%,and that of strain 4-1,3-1 and 2-2 were 45%,32% and 31%,respectively. The filter paper-decomposing test results were similar to the straw degradation test results. Four strains all had exo-1,4-β-D-glucanase,endo-1,4-β-D-glucanase,and β-glucosidase,but their degradation trends were different. The three enzyme activities of strain z-5 and strain 4-1 showed better synergy,and that of strain 2-2 and strain 3-1 were less synergistic. In sum,the target strain can be obtained more accurately and quickly through the straw powder plate test,straw degradation rate determination and filter paper hole method,and the cellulase plays a leading role in straw degradation.
    Isolation,Identification,and Biological Characteristics of a Cadmium-Resistant Endophytic Bacteria Bacillus aryabhattai
    LIU Jun-sheng, XIE Xiu-chao, LUO Yang-lan, DENG Bai-wan, BAI Qiu-yue, YAN Meng-chen, BAI Xing
    2019, 35(2):  64-72.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0803
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    A cadmium-resistant endophytic bacterium GR42,which may grow in the culture medium with 900 mg/L of Cd2+ concentration,was identified and studied for its biological characteristics in order to provide theoretical basis for its application. 16S rDNA sequence analysis,morphological,as well as physiological and biochemical experiments were used to identify the species,to explore the influences of culture conditions on the growth of the strain,and to test its cadmium-removed rate,its tolerance to lead and zinc,growth promoting characteristics,and the promotion effect on the cadmium-resistance of wheat seeding. As results,the strain GR42 was identified as Bacillus aryabhattai,and the optimal temperature,pH,loading volume,and salt concentration was 28℃,7.0,50 mL/250 mL,and 5-10 g/L,respectively. Cadmium-removed rate was 41.1% at 800 mg/L Cd2+concentration;toleration of Pb2+ and Zn2+ was 2000 mg/L and 1200 mg/L respectively;siderophore activity was 76.74%,and ACC deaminase activity was 0.627 U/mg;the capacity of the strain dissolving phosphorus and potassium was 13.801 mg/g and 8.868 mg/g,respectively;it also showed significant promoting effects on the cadmium-resistance of wheat seedlings. In conclusion,the cadmium-resistant endophytic bacterium GR42 is B. aryabhattai,its lead-zinc-cadmium resistance and cadmium-removed rate are high,and it has ability of promoting growth,therefore it owns application potential in promoting plant growth and remediating cadmium-contamination soil.
    Screening of Strains for Enhancing Oil Recovery in Northern Shaanxi and Evaluation of Their Degradabilities
    DENG Zhen-shan, SHANG Rong-fang, CHEN Kai-kai, WANG Xiao-jiang
    2019, 35(2):  73-79.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0654
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    In order to screen the strains that can enhance oil recovery by using metabolites,using enrichment and dilution plate method,strains of degrading crude oil as sole carbon source were screened from nearby long-time petroleum-polluted waters and oily sludge in the northern Shaanxi,and their characteristics,including their growth condition,oil displacement capability,surface active substance of metabolites,surface tension and emulsification,and the degradation of crude oil were analyzed,respectively. Ten strains of bacteria were screened from wastewater containing oil and oily sludge. Further,6 dominant bacteria were selected as test ones for follow-up study,6 strains of bacteria resulted in the physicochemical properties of crude oil changed. Crude oil viscosity reduced by 49.02% after strain B and crude oil reacted,the composite bacteria of strain A-08,strain 5-13,strain 1-2,strain A,and strain A-08 the crude oil emulsified into circular small particles,and the degradation rate reached 43.25%-60.00%.
    Antifungal Activities and Stability of Fermentation Broth from Streptomyces corchorusii Strain AUH-1
    YANG Yong, ZHANG Shuai-wen, ZHANG Yong, LIU Qun, LI Kun-tai
    2019, 35(2):  80-84.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0638
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    This work aims to study antifungal activity of fermentation broth from Streptomyces corchorusii AUH-1,and to verify the effects of biochemical and physical factors such as temperature,pH and ultraviolet on the stability of antifungal active substances. With 8 plant pathogenic fungi as the tested phytopathogen,inhibition rate of mycelial growth was used to determine antifungal activities of fermentation broth. With Rhizoctonia solani as indicator phytopathogen,the antifungal activities of fermentation broth under different conditions were measured. The antifungal activity results showed that the fermentation broth of strain AUH-1 had inhibitory effects on 8 pathogenic fungi at varied degrees. The inhibition rate of the fermentation broth to Fusarium oxysporum f.sp. niveum,Phytophthora parasitica var. nicotianae,Rhizoctonia solani,and Phytophthora capsici was over 81%,78%,68%,and 67%,respectively. The stability test showed that the antifungal active substance produced by this bacterium had favorable temperature stability and UV stability. The above results suggest that S. corchorusii AUH-1 have broad-spectrum in suppressing the growth of various fungal plant pathogens,and have promising prospects for the development of microbial pesticides.
    Optimization of Fermentation Medium for Streptomyces roseoflavus NKZ-259
    MA Jin-jin, GE Bei-bei, SHI Li-ming, LIU Bing-hua, WEI Qiu-he, ZHANG Ke-cheng
    2019, 35(2):  85-92.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0761
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    Streptomyces roseoflavus NKZ-259 is a biocontrol strain and plant growth regulator indole-3-acetic acid(IAA)is produced in the process of secondary metabolism. The fermentation medium was optimized in this study in order to increase the content of IAA produced by a strain. The 6 optimal nutrients of fermentation medium were determined by single factor experiment,and they were glucose,soluble starch,peptone,potassium nitrate,dipotassium hydrogen phosphate,and L-tryptophan,respectively. Plackett-Burman design method was applied to screen major factors affecting the fermentation production of IAA,which was L-tryptophan,glucose,and dipotassium hydrogen phosphate. Central combination design(CCD)and response surface methodology were used to analyze the interactions of all factors. Finally,the optimized fermentation medium was composed as follow:L-tryptophan 2.24 g/L,glucose 20.7 g/L,dipotassium hydrogen phosphate 0.5 g/L,soluble starch 10 g/L,peptone 3 g/L,potassium nitrate 4.5 g/L. The concentration of IAA reached 45.377 4 μg/mL after the medium optimized,which was increased 3 times compared to the original fermentation medium.
    Effects of Site-directed Mutation of Gene lysC and Co-expression of lysC-asdA Cluster on L-threonine Accumulation in Corynebacterium glutamate
    HUANG Qin-qin, WANG Hui-mei, LIANG Ling, HUANG Qin-geng, WU Song-gang, HUANG Jian-zhong
    2019, 35(2):  93-100.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0624
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    Aspartate kinase(AK)and aspartate semi-aldehyde dehydrogenase(ASD)encoded by gene lysC and gene asdA are two key rate-limiting enzymes playing vital roles in L-threonine synthesis pathway,and AK was co-inhibited by lysine and threonine. Selecting Corynebacterium glutamicum T11 as the original strain,the lysC-asdA gene co-expression cassette were constructed,the key rate-limiting enzyme gene lysC was mutated by site-directed mutation at Ala279Th,and the anti-feedback inhibition mutant gene,named lysCr-asdA,was obtained,and was inserted into the shuttle expression vector pZ8-1 containing the strong promoter tac,thus the tandem expression plasmid pZ8-1-lysCr-asdA was constructed successfully and transformed into the starting strain,then,the engineering strain T11/pZ8-1-lysCr-asdA was obtained. The production of L-threonine by shaking flask was 7.18 g/L,which was 27.8% higher than that by the original strain. The results of further batch fermentation in 30 L fermenter showed that the yield of L-threonine reached 65.5 g/L for 60 h and the conversion rate of sugar and acid reached 39.5 g/L,increasing by 29.5% and 33.9% higher than by the original strain. It provides a strong basis for further constructing Corynebacterium glutamicum engineering strain highly yielding threonine.
    Homology Modeling and Molecular Docking of Cycloartenol Synthase in Siraitia grosvenorii and Speculated Mechanism of Catalytic Cyclization
    QIAO Jing, CUI Sheng-rong, SHI Hong-wu, LUO Zu-liang, MA Xiao-jun
    2019, 35(2):  101-108.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0693
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    Cycloartenol,a phytosterol compound,also one of the key precursor substances for biosynthesis of numerous sterol compounds,demonstrates a variety of pharmacological activities such as anti-inflammatory,antioxidant,anti-tumor and regulating cholesterol levels. The genes of cycloartenol synthase(CAS)from Siraitia grosvenorii cloned in our team’s previous work have been functionally characterized(GenBank accession number:HQ128566). However,its active sites and catalytic mechanism of cyclization remain unclear,which hinders its modification and application. Homology modeling was used to predict the 3D structure of CAS,and the interaction between CAS and substrates was analyzed by combining with molecular docking. Results showed that Asp491,Cys492,Cys570,Tyr540,and His265 were the key catalytic sites in CAS. The catalytic mechanism for the cyclization reaction were involved several reaction steps. First,4 cyclization reactions were triggered by Asp491 protonating 2,3-oxidosqualene and C20 cation intermediate was formed. Then,skeletal rearrangement of C cation in this intermediate resulted in the formation of C11 intermediate. Finally,deprotonating by His26 and Tyr540 led to the product cycloartenol via forming single bond between C27 and C11. In addition,there were a large number of hydrophobic amino acids in the active cavity,and the product was stabilized by hydrophobic reaction and the intermediate bound to the active cavity through the hydrophobic action. The discovery of enzyme active sites laid a foundation for the modification of enzyme activity by site-directed mutation technique and regulation of metabolic pathway in the future.
    Eukaryotic Expression of Antibacterial Peptide Domesticin from Musca domestica in Pichia Pastoris and Antibacterial Activity Identification
    LIU Jin-lan, YANG Xue, LI Shuang-shuang, ZHANG Yu-ming, LIU Feng-song, TANG Ting, LI Hong-quan
    2019, 35(2):  109-115.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0611
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    This work aims to express the Musca domestica antibacterial peptide Domesticin gene domesticin in Pichia pastoris and determine the antibacterial activity of the expressed product. The cloned domesticin gene fragment was ligated to pPIC9K vector to construct a recombinant expression vector pPIC9K-domesticin,which was transformed into competent Pichia pastoris KM71. Recombinant fusion protein Domesticin was induced with methanol,the expressed product was identified by Tricine-SDS-PAGE and Western Blot,and antibacterial activity was determined by minimal inhibitory concentration. The results indicated that antibacterial peptide Domesticin was successfully expressed in P. pastoris,and Domesticin demonstrated antibacterial activity against many kinds of bacteria. Domesticin is a novel broad spectrum antibacterial peptide that has antibacterial activity against both Gram-positive bacteria and Gram-negative bacteria,which has the capacity to be a new generation antimicrobial agent.
    Molecular Cloning and Characterization of TGF-β and Its Response to Seawater Acidification in the Sea Urchin Meconocrotus nudus
    LI Ying-ying, CUI Dong-yao, CHANG Ya-qing, LIU Lin, ZHANG Bao-jing, JIANG Hui-ting, LIU Yin, ZHAN Yao-yao
    2019, 35(2):  116-128.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0470
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    This work aims to clarify the sequences and structure of transforming growth factor-β gene(TGF-β)and explore its expression mode under seawater acidification. We used homology-based cloning and rapid amplification of cDNA ends(RACE)to acquire the full-length cDNA sequence of novel gene TGF-β from Meconocrotus nudus(here designated MnTGF-β). Results are as such:(1)The full-length cDNA of MnTGF-β were 2 299 bp,the length of 5'noncoding region was 745 bp,the length of 3'noncoding region was 261 bp,the length of ORF was 1 290 bp,and it encoded 430 amino acid with the relative molecular weight of 48.31 kD,and the theoretical pI of 5.34. (2)Homology and phylogenetic analysis showed that MnTGF-β protein sequence was highly conserved with SpTGF-β2 protein sequence in Strongylocentrotus purpuratus(homology was 97.69%). (3)Quantitative real-time PCR analysis demonstrated that MnTGF-β was ubiquitously expressed in gonad,coelomic fluid,intestines,peristomial membrane and tube foot of healthy adult M. nudus,and the order of its expression was as tube foot > peristomial membrane > intestines > coelomic fluid >gonad.(4)Compared with the natural seawater group(pH 8.06 ±0. 01),when △pH was -0. 3,the relative expression of gene MnTGF-β in the gonad of sea urchin tended to increase first,then decrease significantly(P < 0. 01),and then increased significantly(P < 0. 01)with the increase of acidification time;the relative expression level in tube foot was significantly up-regulated(P < 0. 01),but that in intestines significantly decreased(P < 0. 01)and then increased(P < 0. 05). When the △pH in seawater were -0. 4 and -0. 5,the relative expression of gene MnTGF-β in the gonads and tube foot of sea urchins showed a tendency of first decreasing and then increasing with the prolongation of acidification time,while that in the intestines decreased significantly(P < 0.01).

    Cloning and Tissue Expression Analysis of MDHI Gene in Yak
    CHEN Lu-lu, WANG Hui, CHAI Zhi-xin, ZHONG Jin-cheng, WANG Ji-kun, CHEN Zhi-hua, JI Qiu-mei, XIN Jin-wei
    2019, 35(2):  129-136.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0712
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    Malate dehydrogenase has redox activity and plays a key role in energy metabolism of animal organisms. This study aims to clone the yak MDHI gene,combined with bioinformatics analysis and tissue expression analysis,which may lay a foundation for studying the physical and chemical properties and functions of the yak MDHI gene. The results showed that the cDNA sequence of this gene was 1 169 bp,the CDS region was 1 005 bp in length and it encoded 334 amino acids. The encoded protein was a hydrophilic and stable,which belonged to the conserved domain family. There were two transmembrane areas(located in 6-22 and 34-54 amino acids respectively)and 26 phosphorylation sites(11 Ser phosphorylation sites,11 Thr phosphorylation sites and 4 Tyr phosphorylation sites). The secondary structure was mainly composed of α-helix(152,accounting for 45.51%),random coil(110,accounting for 32.93%). The tertiary structure had a similarity of 98.50% with crystal structure of the ternary complex of cytoplasmic malate dehydrogenase,α-Ketomalonic acid and tetrahydro NAD in porcine. Subcellular localization analysis revealed that most of them were distributed in the endoplasmic reticulum,least in the Golgi and nucleus. Furthermore,homology and phylogenetic tree analysis indicated that its homology was the highest with common cattle,which can be speculate that the yak MDHI gene maybe highly conserved at the evolution level. Tissue expression analysis showed that the gene was expressed the highest in gluteal fat but lower in gluteal muscle,indicating that the gene may function in fat deposition. In sum,we cloned MDHI gene successfully and performed bioinformatics and tissue expression analysis.
    Comparison of Biological Characteristics of Brown Adipose-derived Mesenchymal Stem Cells in Young and Elder Mice
    ZHANG Da-xiu, HE Shuang-li, WANG Qian, PU Shi-ming, WU Qiong
    2019, 35(2):  137-142.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0755
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    This work aims to compare the biological characteristics of brown adipose-derived mesenchymal stem cells(BADSCs)between young and elder mice. Cell flow was used to detect the third-generation BADSCs surface antigen expression,MTT test to determine cell proliferation,7-ADD-AnnexinV double staining to observe the apoptosis,β-glucoside enzyme staining method to detect BADSCs cell aging,and osteogenesis and adiposesis differentiation between 2 groups were compared. As results,the expression levels of surface antigen CD29,CD105 and CD44 of the 3rd generation BADSCs in the young and elder mice were all positive,while CD73 was in low expression. The MTT method showed that the proliferation capacity of BADSCs in the elder mice was not significantly different from that in the young ones. 7-ADD-AnnexinV double staining demonstrated that the proportion of BADSCs early apoptosis in the young mice was 4.63±0.87%,and that for the elder ones was 9.88±0.81%,i.e.,there was significant difference(P < 0.05). The dyed blue cell number in the young group was 2.33±0.3,and that in the elder group was 6.66±1.2,i.e.,difference was significant(P < 0.05).The OD value of osteogenetic differentiation in the young group was 1.26±0.066,and that in the elder group was 0.88±0.047,thus this was significantly different(P< 0.05). The OD value of adipogenic differentiation in the young group was 0.9808±0.066,and that in the elder group was 0.769±0.035,i.e.,difference was significant(P<0.05). In conclusion,BADSCs in the young group were more inclined to osteogenetic differentiation,while BADSCs in the elder group was more inclined to adipogenic differentiation;the proportion of apoptosis and senescence cells in the elder mice increased,while osteogenesis and adipogenesis decreased.
    The Latest Research Progress on the Stress Responses of bZIP Involved in Plants
    CUI Rong-xiu, ZHANG Yi-wen, CHEN Xiao-qian, GU Cai-hong, ZHANG Quan
    2019, 35(2):  143-155.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0562
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    bZIP(basic leucine zipper)protein contains two domains,a highly conserved basic region which binds to DNA,and a diversified leucine zipper,and is one of the largest transcription factor(TF)families in plants. The homo-dimers and hetero-dimers of bZIP regulates the expressions of downstream-related genes such as ERF5,KIN1,CORl5A,COR78,CYP707A1,CYP707A3,ADS,and CYP71AV1 through binding to cis-elements G-box,C-box,ABRE,LTRE,and BoxII. Plant bZIPs are involved in the regulation of flower development,seed maturation,dormancy and senescence,and play an important role in ABA signal response and abiotic stresses such as salt damage,drought,chilling injury,osmotic stress and mechanical damage. Furthermore,bZIP is involved in the responses and regulations to biological stresses such as insect infestation and pathogen infection via salicylic acid,jasmonate and ABA signaling pathways. This paper mainly summarizes the structure characteristics,classification features and the mechanisms of bZIP,as well as the latest research progress on the responses of bZIP to biotic and abiotic stresses in plants. This review provides a theoretical basis and research ideas for further exploring the stress-related function of bZIP transcription factor.
    Research Progress on the Function of NO3- Transporters in the Adaptation of Plants to Adversity
    LI Xiao-ting, YUAN Jian-zhen, WANG Fang-zhen, LI Ren-hui, CUI Yan-nong, MA Qing
    2019, 35(2):  156-162.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0730
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    Nitrogen is an essential macronutrient for plants,and involved in various metabolic events. Nitrogen in nitrate(NO3-)is the major form of inorganic nitrogen source for plants. NO3-transporters not only play a vital role in nitrate uptake,transport and cyclic utilization during the whole growth and development of plants,but also participate in regulating the adversity adaptation in plants. In this review,we highlight recent progresses in studying the functions of NO3-transporters in the adaptation of plants to low NO3-,low K+,salt,drought and cadmium stresses,aiming at providing the reference for further exploring the stress resistance mechanisms in plants.
    Application of Chemical Regulating Technology in Sugarcane Production
    GAN Chong-kun, ZHOU Hui-wen, CHEN Rong-fa, FAN Ye-geng, QIU Li-hang, HUANG Xing, LI Yang-rui, LU Xing-gao, WU Jian-ming
    2019, 35(2):  163-170.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0723
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    Crop chemical regulation technology is one that applying endogenous plant hormones or those-like(plant growth regulators)regulate the gene expression,organs formation,function embodiment to reach a double regulations of exterior and inner physiological metabolism. Chemical regulation is not only an important technology for agricultural production but also a research focus for agricultural science because of its convenient,lower dosage,efficient and high benefit. Chemical regulation is widely applied in sugarcane production,and relevant researches have been carried out,including sugarcane tissue seedlings production,tiller and elongation regulation,flowering and defoliation regulation,resistance increasing,chemical ripening,and both yield and sugar increasing. This paper covered the whole sugarcane production from sowing to harvesting,reviewed the current application and research situation of chemical regulation in detailed,and discussed the promising research directions with application prospect. This work is expected to help us systematically understand the research progress on the chemical regulations and provide the guidelines for the application of chemical regulation technology in sugarcane production.
    Research Progress on Rearrangement Mechanism of Bunyaviruses
    SHI Kun, LIN Xiang-mei, LÜ Ji-zhou, DENG Jun-hua, WU Shao-qiang
    2019, 35(2):  171-175.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0706
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    In recent years,emerging diseases such as severe fever with throm-bocytopenia syndrome(commonly known as “tick biting disease”)and Schmallenberg disease have caused widespread attentions to Bunyavirus. Bunyavirus is a type of negative-strand RNA virus with high infectivity and high mortality. Due to the fragmented nature of its genome,it is prone to gene rearrangement among its members,resulting in new viruses with increased virulence and increased host range,thus posing a major threat to human health. This article reviews the recent studies on the rearranged phenomena,molecular mechanism and the potential pathogenic hazards of Bunyaviruses rearrangement. Keeping research on the Bunyavirus rearrangement mechanism and the vaccine development is prospected,aiming at providing theoretical basis for the prevention and control of this disease.
    Regulation of LKB1-AMPKα-SIRT1 Signal Pathway in Lipid Metabolism in the Adipose Tissue of Dairy Cows
    MING Peng-fei, HUANG Ying-ying, DONG Yan-li, NIE Xing-can, FENG Shi-bin, WANG Xi-chun, CHENG Jian-bo, LI Jin-chun, WU Jin-jie, LI Yu
    2019, 35(2):  176-181.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0707
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    The increasing volume and number of adipocyte leads to disorder of lipid metabolism in adipose tissue,which causes different chronic diseases such as type II diabetes,ketosis,fatty liver,and hyperlipidemia. The LKB1-AMPKα-SIRT1 signal pathway plays important regulating role in the lipid metabolism of dairy cows adipose tissue. AMPK as conservative serine/threonine kinase plays a pivotal role in the regulation of energy metabolism. LKB1(liver kinase B1)is a serine/threonine kinase,an upstream kinase of AMPKα,participates in various biological activities such as intracellular energy metabolism. Silencing information regulator factor 1(SIRT1),a kind of NAD+-dependent histone deacetylase,can increase AMPKα activity by deactivating LKB1. This article reviews the regulation mechanisms of LKB1-AMPKα-SIRT1 signal transduction pathways in lipid metabolism of adipose tissue in dairy cows,aiming at providing a basis for further studying the regulating mechanisms of related signal pathways in the lipid metabolism of dairy cows.
    Research Progress on Mesoporous Silica in Cancer Therapy
    YAO Lin-tong, LIU Ya-ting, LIU Ya-jing, CHEN Zhen-zhen
    2019, 35(2):  182-191.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0404
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    Mesoporous silica nanoparticles(MSNs)have attracted much attention for their potential biomedical applications due to its distinctive mesoporous structure,favorable biocompatibility,large specific surface area,tunable pore size,and well-defined surface properties. The drug delivery system based on mesoporous silica nanoparticle have become a research focus for many researchers. Here firstly,we discuss the limitations and challenges while applying the combinations of MSNs with peptides,antibodies and antibody fragments,aptamers,and immunotherapies in cancer therapy. Finally,we analyze the existing problems and outlook the future development trends of mesoporous-silica-based drug delivery systems in practical applications.
    Research Progress on Food Fraud Using Non-targeted Metabolomics Based on High-resolution Mass Spectrometry
    LAI Bo-wen, LIU Bin, LIANG Yong-kang
    2019, 35(2):  192-197.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0632
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    Food fraud is a type of economically motivated adulteration(EMA),one of the most important factors affecting and destroying food safety. With the rapid development of living conditions,people demand food with better quality,thus food fraud has been concerned seriously by the public. Targeted metabolomics techniques for quantitative detection of known target substances have been widely used in food inspection,and metabolomics technology has become an indispensable tool in food science research. Regarding food fraud occurring in recent years,another branch of metabolomics,based on high resolution mass spectrometry(HRMS),non-targeted metabolomics(NTM)was gradually applied and has made great development in the identification of food fraud. Here we introduced the workflow of identifying food fraud using HRMS-based NTM and summarized the data-progressing methods. Then we made a sum on the relevant applications for identifying frauds in food species/varieties,origin and quality respectively. Finally,we analyzed the advantages and disadvantages of HRMS-based NTM in food inspection and prospected other research directions. It is aimed that this work summarizes and describes the most recent metabolomics results in food inspection.
    Application of SSR Marker Technique in the Identification of Traditional Chinese Medicine
    LIU Pei-pei, LUO Guang-ming, CHAI Hua-wen, ZHANG Jun-yi, ZHU Zi-hao, WANG De-yun
    2019, 35(2):  198-203.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0818
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    Chinese medicine is a precious resource and plays an important role in disease treatment and health care in our daily life. However,in recent years,the safety incidents from using traditional Chinese medicines due to inappropriate identification methods have repeatedly occurred,which has brought huge challenges to the clinical application of traditional Chinese medicines. The traditional identification method is that traditional Chinese medicine pharmacists identify Chinese medicinal materials via applying their long-term accumulated experiences. Although the method is getting more and more perfect,it is still subjective and undeniably has its limitations. With the development of science and technology,new technologies and new methods promote the development of the identification of traditional Chinese medicines;the traditional identification methods can be reasonably combined with modern identification methods,and more accurate and scientific identification of Chinese herbal medicines can be achieved. The recent development of high-throughput sequencing technology has provided a favorable platform for the development of molecular marker technology and a more accurate and reliable means for the identification of Chinese herbal medicines. Currently,RAPD,AFLP,RFLP,and SSR are the several molecular markers most widely used,each of them has its own advantages and disadvantages. SSR refers to a piece of DNA consisting of multiple repeated units of 1-6 nucleotides in the genome. The SSR marker characterizes of high specificity,fine repeatability,reliable results,simple operation,and low DNA requirements,and thus plays an increasingly important role in the variety identification of Chinese medicinal materials,plant germplasm identification,genetic map construction,and genetic diversity analysis. The application of SSR marker technique in the identification of traditional Chinese medicine in recent years is briefly summarized here,aiming at providing a reference for the further application of this technique in the identification of traditional Chinese medicine.
    Research Progress on DNA Immunization Technology in the Development of Monoclonal Antibodies
    NUERTURE Yeernazhaer, QIU Li-fen, ZHANG Fu-chun, ZHANG Mao-xiang
    2019, 35(2):  204-211.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0643
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    DNA immunization refers to a series of immune response processes in which the recombinant expression vector encoding a specific antigen is transferred into animal bodies,and the specific gene sequence is expressed in the animal cells and then induces to produce its specific antibody. At present,DNA immunization has become the main technical means for the research and development of various protective and diagnostic monoclonal antibodies,especially for those antigen that are difficult to express and purify in vitro. DNA immunization is superior to traditional protein immunization. DNA immunization expresses antigen in host cells and its presented manner is similar to viral infection,which can produce both humoral and cellular immunity,thereby overcoming the shortcomings of traditional protein immunization and providing an ideal technology platform for preparing high quality monoclonal antibodies. However,many challenges have been encountered in practical applications of this technique;especially the traditional intramuscular injection method is used to immunize a single plasmid DNA,which results in low delivery efficiency and antibody expression level,then leading to the low immunogenicity of DNA immunization. This article reviews the application of DNA immunoassay in the development of monoclonal antibodies,and to further discusses the DNA immunization technology in terms of its concepts,induction mechanism,vector construction,delivery methods,effects of different immunological adjuvants and its advantages and development prospects. Our aim is to provide an effective theoretical knowledge in the application of DNA immunization technology in preparing high quality monoclonal antibodies and improving its immune efficiency.
    Application of a Loop-mediated Isothermal Amplification Method for Rapid Diagnosis of Francisella tularensis
    LI Zi-wei, DENG Zhong-liang
    2019, 35(2):  212-217.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0573
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    This study aims to apply the loop-mediated isothermal amplification(LAMP)to detect Francisella tularensis. LAMP primers were designed for amplifying FopA gene from F. tularensis,reaction conditions were optimized,and the specificity and sensitivity of the method were evaluated. Results showed that:(1)12 bacteria and tick’s DNA(Containing a large number of Pseudomonas bacteria)were amplified with LAMP reaction;however,only F. tularensis demonstrated positive reaction;(2)the detection limit of pure F. tularensis culture was 4.9×102 pg/mL,the minimum threshold for cloned plasmid was 10 copies/μL,which were below those of conventional PCR,1×103copies/μL;(3)the minimum threshold for simulated soil samples was 44 CFU/g;(4)24 soil samples(8 dilutions)contaminated by F. tularensis were compared with the imported fluorescence quantitative PCR reagent,and all of them were accurately detected in the sensitivity range. In conclusion,applying LAMP technology in the detection of F. tularensis shows superiority of efficiency,high specificity and sensitivity,thus this has practical value for etiological diagnosis of tularemia.
    Using Escherichia coli Light-regulated Gene Expression System to Degrade MBC
    YU Shu-qiao, GUAN Zhao-ying, CHEN Hong
    2019, 35(2):  218-224.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0742
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    A newly developed Escherichia coli light-regulated gene expression system was used to produce methyl-1h-benzimidazol-2-yl carbamate(MBC)hydrolyzing esterase for degrading residual pesticide MBC in environment. This light-regulated system is based on dimerization of photosensitive protein LEV1 upon light exposure to repress the expression of downstream gene. SDS-PAGE and ELISA results showed that the gene of MBC hydrolyzing esterase could be overexpressed in our light-regulated system,by which the MBC hydrolyzing esterase was produced and it had enzymatic activity of degrading pesticide MBC in both liquid solution and soil samples. Light as an inducer,given its many advantages such as cheap,easy to be obtained,non-toxic,and added/removed at any time,is a promising tool for the industrial production of enzymes.
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    Cover
    2019, 35(2):  300-300. 
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    2019, 35(2):  400-400. 
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    2019, 35(2):  500-500. 
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