Table of Content

26 March 2019, Volume 35 Issue 3

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Analysis of the Integration Site of Exogenous Gene in Transgenic Maize

WANG Cui-yun, LIU Yan, LIU Yun-jun

2019, 35(3):  1-5.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0756

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Maize（Zea mays）is the largest crop in China and plays a key role in ensuring food security. Development of transgenic maize varieties with resistance to diseases and insects may effectively reduce the loss of maize yield. In the process of transgenic maize development,it is necessary to analyze the integration sites of exogenous genes,which will provide important basis for the safety evaluation of transgenic maize. In this study,the integration sites and flanking sequences of exogenous genes in a transgenic maize event IE34were analyzed using thermal asymmetric PCR（TAIL-PCR）and genetic mapping. A segment of maize genomic sequence with 776 bp was obtained using TAIL-PCR,and an event-specific PCR method for the transgenic maize was established with the specific primers in the flanking sequences and upstream sequences of exogenous genes. Sequence BLAST in MaizeGDB showed that the flanking sequence was repeat sequence and hit the locus in several chromosomes. To further confirm the integration chromosome of exogenous gene,a F₂ population was generated by crossing IE34 with maize inbred line B73. BSR-Seq analysis results demonstrated that the exogenous gene was located in the interval of 2.32-2.70 Mb on the short arm of chromosome 5. Further,fine mapping results reduced the integration sites of exogenous genes to a 260 kb region of 2.35-2.61 Mb on chromosome 5. The results of this study suggest that,for the transgenic event with complex integration site,combined TAIL-PCR and genetic mapping may effectively identify the integration sites of exogenous genes.

Integration Analysis of Exogenous Gene and Line-Specific Detection in the Insect-Resistant cry1C-Transgenic Rice Jishengjing3

JIN Yong-mei, MA Rui, YU Zhi-jing, LIN Xiu-feng

2019, 35(3):  6-12.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0788

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The flanking sequences of exogenous gene conferring insect-resistant cry1C-transgenic rice line Jishengjing3 and its inserted sites were analyzed by genome walking PCR,and a line-specific PCR detection method was established. T-DNA was found to be in nucleotides 2 790 685 to 2 790 589（GenBank accession number：NC_029257.1）in the chromosome 2 of Oryza Sativa（Japonica）genome after aligning the flanking sequences in left and right borders of Jishengjing3’s exogenous gene with the rice’s genome and T-DNA sequences. Based on the T-DNA insertion site,specific primers in the 2-sides region of the inserted site and the left border of T-DNA region were designed,and a event-specific PCR detection method for Jishengjing3 was established,which may provide accurate and rapid detection approach for identifying Jishengjing3.

Suspension Culture of Somatic Cells of Pinus elliottii Against Brown Spot Needle Blight of Pine

FANG Luo, WU Xiao-qin

2019, 35(3):  13-18.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0694

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In order to obtain a large number of embryogenic calluses of Pinus elliottii being resistant to brown spot needle blight of pine and to improve the technique of somatic embryogenesis from the resistant embryogenic callus,the suitable conditions of somatic cell suspension culture of P. elliottii were studied,and the suspension culture system of somatic cells of P. elliottii resistant to brown spot needle blight of pine was established. The results showed that suspension culture of embryogenic cells was more dispersed,which was conducive for embryonic cells to have full nutrition,and 153 mature somatic embryos per gram was produced,the embryogenic cells produced in solid culture per gram were only 56. The optimal culture period of suspension cells of resistant P. elliottii was 7-9 d,the suitable basic medium was 1/4 DCR,the dosage of maltose was 15 g/L,NAA was 0.5 mg/L,and the optimal subculture addition ratio was 1/2. Meanwhile under the same culture conditions,the viabilities of suspension cells of resistant P. elliottii in different genotypes varied,and that of clone 13#-9 was the best in this experiment.

Effect of Warming After Toping on the Growth and Development of the Upper Leaves of Flue-cured Tobacco

HE Wen-jun, YANG Tie-zhao, TIAN Pei, DUAN Du-wei, LI Xiao-hui, HE Jia, ZHOU Jian-fei

2019, 35(3):  19-23.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0776

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By studying the effects of warming after topping on the amino acid content and enzyme activity related to growth and development of the upper leaves of flue-cured tobacco,we aim to provide a reference for promoting the growth and development of upper leaves and increasing the yield of tobacco leaves. Having flue-cured tobacco variety Yunyan 116 as an experimental materials,two treatments was set：the normal temperature（T1）and warming treatment（T2）. The hydroxyproline content and POD activity in cell wall as well as IAAO activity in the upper leaves of the two treatments were measured. The results showed that warming after toping effectively reduced the content of hydroxyproline,cell wall POD activity and IAAO activity in the upper leaves of tobacco leaves. The length,leaf width,leaf area,the yield and production value of the upper leaves for T2 treatment increased by 8.6%,3.9%,12.9%,7.0%,and 13.8%,compared with T1. In sum,the upper leaves development of flue-cured tobacco can be promoted significantly through warming properly after toping.

Screening of Multi-function Nitrogen-fixing Bacteria in Peanut Rhizosphere and Their Tolerances to Saline

JIANG Huan-huan, QI Pei-shi, WANG Tong, CHI Xiao-yuan, CHEN Ming-na

2019, 35(3):  24-30.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0770

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This study aims to acquire the saline-tolerant root-promoting rhizobacteria（PGPR）with multi-function of fixing nitrogen,dissolving phosphorus and synthesizing the IAA for increasing the peanut yield in saline soil. Spread plate was used to isolate the peanut rhizosphere nitrogen-fixing bacteria in saline soil,16S rDNA was to identify the sequence,and acetylene reduction method to determine the nitrogenase activity. Total 22 nitrogen-fixing strains were obtained,and their nitrogenase activities were between 125.82-346.32 nmol C₂H₄/（h·mL）. All strains had the activities of ACC deaminase and dissolving insoluble phosphorus source;their ACC deaminase activity were among 0.12-1.26 U/mg,the amount of dissolving tricalcium phosphate was between 1.45-53.58 mg/L,the that of dissolving ferric phosphate was between 1.45-8.27 mg/L,and that of dissolving aluminum phosphate was between 1.1-22.82 mg/L. Ten strains of them secreted IAA with the amount ranging in 1.36-1.36 g/mL,accounting for the 55% of the total strains. The ten strains were also tested for their survival abilities in different concentrations of NaCl and pH,results showed that the maximum tolerance to pH was 9 and the maximum tolerance to NaCl was 1.5 mol/L. In conclusion,the 10 strains demonstrate strong tolerance to salinity and showed their potential of applying in saline soil.

Growth-promoting and Adverse-resistant Characteristics of JYZ-SD5,a Tree Rhizobacterium and Its Species Identification

XU Xiu-qian, WU Xiao-qin, WU Tian-yu, ZENG Meng-man

2019, 35(3):  31-38.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0844

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This work aims to explore the performance of promoting growth and stress resistance of JYZ-SD5,a tree rhizobacterium,and to reveal the value of this bacterium in the field. Specific culture medium was used to detect its ability of nitrogen fixation and decomposition of phosphorus and potassium,also to measure its capacity of producing IAA. Then plate confrontation was employed to check the antagonistic activity to pathogenic fungi,and growth was observed to determine its tolerance to heavy metals. Further,the tree rhizobacterium JYZ-SD5 was applied to the seedlings of Metasequoia glyptostroboides Hu et Cheng in plots to test its effects on the growth of the seedlings. Morphology observation,physiological and biochemical tests,and 16S rRNA gene sequence analysis were combined to identify the strain JYZ-SD5. Results showed that JYZ-SD5 had the ability of fixing nitrogen and decomposing phosphorus and potassium. The production of IAA without adding tryptophan reached 6.818 6 μg/mL. JYZ-SD5 presented the antagonistic activity to Alternaria sp and Pestalotiopsis vesicolor,and protease and cellulose were also detected. This strain also demonstrated strong tolerance to heavy metals Mn⁷⁺ and Cr⁶⁺,to the highest content of 400 mg/L. The greenhouse plot experiment revealed that JYZ-SD5 resulted in the increase of relative content of chlorophyll in the seedlings of M. glyptostroboides Hu et Cheng,and it promoted the growth and ground diameter of M.glyptostroboides Hu et Cheng. Finally,the rhizobacterium was identified as Bacillus paramycoides by morphological,physiological,biochemical,and molecular approaches. In sum,the rhizobacterium JYZ-SD5 shows favorable performance on promoting growth and stress resistance,and thus it has the potential to be a novel biological fertilizer and meets the requirement of applying it in field.

Physiological Response of Elodea nuttallii to Uranium Stress and Its Enrichment Effect

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

2019, 35(3):  39-46.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0773

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In order to detect whether or not E. nuttallii has uranium enrichment capacity,hydroponic solution culture experiments were conducted to study the growth and enrichment characteristics of E. nutallii under different initial uranium concentrations. The experiment results showed that the enrichment of uranium in E. nuttallii increased with the increase of initial uranium concentration,and the uranium enrichment content reached the maximum of 14 mg/kg（DW）while the initial uranium concentration reached 30 mg/L. Meanwhile,the antioxidant enzyme activity was affected significantly,SOD decreased by 86.3%,POD increased by 202.4%,and CAT increased by 291.6%,compared with the control group. The cell membrane of E. nutallii was seriously damaged,MDA content increased by 117.8%. The photosynthetic pigments were affected significantly,chlorophyll-a,chlorophyll-b,and carotenoids decreased respectively by 27.7%,26.7%,and 35.6%;the PSII of E. nutallii was most affected,with fluorescence kinetics index F_v/F_m,PI_ABS,ABS/CS₀,RC/CS₀,TR₀/CS_0,and ET₀/CS₀ decreased by 47.7%,83.8%,68.7%,90.0%,87.7% and 87.3%,respectively;photosynthetic index P_n,G_a,and C_i decreased by 82.1%,20.5%,and 6.4%,respectively,compared with the control group. Therefore,it is concluded that E. nutallii has certain enrichment on uranium in waters and a significant physiological response to uranium.

Isolation and Identification of Flavonoids-producing Endophytic Fungi in Papaya

KE Shu-wei, LÜ Jin-hui, CHEN Ping, ZHANG Rong-ping, YU Yue

2019, 35(3):  47-52.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0857

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In order to develop the microbial resources of endophytic fungi,endophytic fungi were isolated from the leaves,fruits and stems of papaya plants by tissue separation. The secondary metabolites of endophytic fungi from papaya were preliminarily analyzed by color reaction of flavonoids,thin layer chromatography,and UV spectroscopy. One strain of endophytic fungus producing flavonoids was screened,and the strain was analyzed and identified by using the conidial morphological characteristics and the internal transcribed spacer. The results showed that the endophytic fungus strain G41 isolated from papaya fruit could be classified as Penicillium citrinum. This study confirms that there are endophytic fungi strains producing flavonoids in papaya fruit,which demonstrates potential value for further development and utilization.

Isolation of Endophytic Fungi from Cinnamomum camphora Leaves,Screening and Identification of Biologically Active Strains

JIN Hong-jie, CAO Hong, LIU Hong, ZHENG Shuang, JIANG Chao

2019, 35(3):  53-58.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0750

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This work aims to isolate endophytic fungi from leaves of Cinnamomum camphora,to determine its anti-bacterial activity,anti-tumor activity and anti-oxidation activity in vitro,and to screen and identify strains with strong biological activity. On the isolated endophytic fungi,K-b disk method was used to determine its anti-bacterial activity,MTT assay to determine its anti-tumor activity,ultraviolet spectrophotometry to determine its anti-oxidation activity,and ITS（Internal transcribed spacer）to analyze and determine the species of active strains. Results showed that total 39 endophytic fungi were isolated from the leaves of C. camphora.Among them,there were 7 strains highly inhibiting Escherichia coli,Staphylococcus aureus,Bacillus subtilis and Pseudomonas aeruginosa. Further,the strain ZS14 with anti-tumor and anti-oxidation activities from the 7 strains was screened,its inhibition rate on human breast cancer cell MCF-7 reached 73.00%,clearance rate of DPPH was 70.92%,scavenging rate of hydroxyl radical was 78.83%,and clearance rate of hydrogen peroxide was 81.36%. With ITS sequencing results it was designated as Diaporthales sp. ZS14. In sum,the isolated strain Diaporthales sp. ZS14 had strong anti-bacterial,anti-tumor and anti-oxidation activities,and it can be used as potential sources for drug development in further study.

Isolation,Identification,and Antibacterial Activity of Fungi Associated with Marine Organisms

ZHANG Sheng-liang, CHU Xiao-xiao, ZHAO You-xing, KONG Fan-dong, HUANG Xiao-long

2019, 35(3):  59-64.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0856

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Isolation,identification and antibacterial activity analysis of fungus associated with sea grasses,sponges and stony corals from Wenchang sea area of Hainan were conducted. Three media were used to isolate and purify the fungi associated with these marine organisms,the isolated fungi were identified according to their ITS sequences,and their antibacterial activities were tested using standard disk diffusion. Total 113 strains of fungi were isolated from 10 sea grasses,9 sponges,and 11 stony corals. The 32 strains of independent fungi,belonging to 10 genera within 8 orders of Ascomycota,Basidiomycotina and Deutemycotina,were identified by ITS sequences analysis. The percentage of Ascomycota was as high as 58.82%,that of Deutemycotina was 32.35% and Basidiomycotina was 8.83%,respectively. The results of antibacterial activity showed that the fermentation products of 12 strains of fungi had strong antibacterial activity against Bacillus subtilis and Staphylococcus aureus. In sum,the diversity and antibacterial activity of fungi associated with sea grasses,sponges and stony corals from Wenchang sea area of Hainan are revealed initially.

Cloning and Identification of Keratinase Gene from Deinococcus gobiensis I-0

GENG Xiu-xiu, ZHOU Zheng-fu, LIU Ying-ying, PING Shu-zhen, WANG Jin

2019, 35(3):  65-70.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0976

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In order to further tap the important gene resources of keratinase,improve its hydrolytic activity,and provide theoretical basis for industrial production,this experiment cloned a gene encoding keratinase from Deinococcus gobiensis I-0 isolated from Gobi desert of Xinjiang and named it as Dgker. Prokaryotic expression vector pET-22B-Dgker was constructed and then induced,expressed and purified in vitro,the optimal temperature and pH of the crude enzyme solution were determined through the hydrolysis activity to feathers. Results showed that the first 50 amino acids of N terminal had a great influence on the expression and purification of protein Dgker. The crude enzyme solution of recombinant strain completely decomposed feathers in three days. The transparent circle on milk powder plate appeared more notable in crude enzyme solution of recombinant strain than that of empty strain. Dgker adapted to a wide range of temperatures and pH,among which the optimal temperature was 60℃ and the optimal pH was 5.0. Dgker can degrade feathers and thus will have great application space in the future industrial production and treatment of waste feathers.

Isolation and Biological Characteristics of a Stenotrophomonas maltophilia

XU Jing-zhao, CHEN Bei, DU Bing-hai, ZHAO Dong-ying, WANG Cheng-qiang, DING Yan-qin

2019, 35(3):  71-77.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0769

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In recent years,based on high-throughput sequencing technology,the determination of microbial flora structure in plant rhizosphere soil shows that most microorganisms in soil are difficult to culture and separate via traditional eutrophic medium. It is aimed to further explore an effective method for isolating oligotrophic microorganisms from soil. A variety of factors were combined to design an oligotrophic medium to isolate soil bacteria based on soil leaching solution. One of the strains was identified by cell morphology,physiological and biochemical characteristics,16S rRNA,and gyrB gene sequences;meanwhile,its function of proteases-producing and siderophore-producing was detected to assess the potential in promoting plant growth. Using soil leaching solution as a medium was a method for effectively isolating soil oligotrophic bacteria,and the strain numbered S35 was identified as Stenotrophomonas maltophilia,it had the ability to produce protease and siderophore. The oligotrophic medium used in this study can be used to obtain bacteria that are difficult to isolate through nutrient-rich medium. In addition,S. maltophilia S35 has certain application potential in plant growth promotion.

Investigation of Bacillus Resources in the Soil of Agroforestry Biome：Case Study in Some Parts of Sichuan and Chongqing

LIU Guo-hong, LIU Qin-ying, LIU Bo, CHE Jian-mei, CHEN Qian-qian

2019, 35(3):  78-86.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0795

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To provide a foundation for new resource mining of the Bacillus-like species and development of microbial agents,we investigated the species distribution and diversity of Bacillus-like isolates from soil samples in the agroforestry biome of Sichuan province and Chongqing city. Isolates Bacillus were obtained from the soil samples of agroforestry biome by cultural method,and identified based on 16S rRNA gene sequences. The community structure of the Bacillus-like bacteria was analyzed using the diversity index. A total of 95 Bacillus-like isolates were obtained from the 5 soil samples,and identified as 38 species belonging to 7 genera within Bacillaceae by 16S rRNA gene sequences. The species number of each genus was 22 for Bacillus,4 for Lysinibacillus,7 for Paenibacillus,2 for Brevibacillus,and 1 for Psychrobacillus,Rummeliibacillus and Fictibacillus respectively. And 16S rRNA similarities between 6 isolates and their most-closed type species were lower than 98.65 % of species defined threshold,indicating that these 6 isolates might be potential new Bacillus species. The order of Shannon index of Bacillus from the agroforestry biome was as forest land in Leshan > farmland in Yanting County > forest land in Zhong County > forest land Yanting County,respectively. According to Bacillus-like species and content,as Euclidean distance was about 15,Bacillus-like species could be divided into 2 clusters,i.e. high-content wide-distribution and low-content uneven-distribution. Thus,there are rich Bacillus-like species in the agroforestry biome,which offers the guarantees for the development and utilization of Bacillus-like resources.

Improving the Tolerance of Saccharomyces cerevisiae to Ethanol by the Over-expression of Inositol-3-phosphate Synthase Gene INO1

HUANG Zhen-jie, CHEN You-qiang, XUE Ting

2019, 35(3):  87-92.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0784

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In order to obtain the strains producing fuel ethanol,the engineering strain Saccharomyces cerevisiae,which may ferment using sugarcane juice to yield high ethanol,were constructed by genetic engineering. In other words,the inositol-3-phosphate synthase gene（ino1）was over-expressed in S. cerevisiae;the antibiotic resistance gene（kanMX）were knocked out,thus recombinant S. cerevisiae was acquired. Further,the tolerances（survivability）of the over-expressed strains to ethanol were analyzed. The sugarcane juice was used as a carbon source in the fermentation,and the ethanol content in the fermentation broth of strains was detected by gas chromatography. The experimental results showed that over-expressed strains YI2-1tolerated 19%（V/V）ethanol. The 20^oBx of sugarcane juice was employed in the anaerobic fermentation,which yielded 13.10%（V/V）of the ethanol,and this was 8.55% more than that by the initial strain. The biggest output of ethanol by the over-expressed strain YI2-1△KP was accumulated to 13.17%（V/V）,and this was 9.16% more than that by the original one. This study indicates that over-expression of ino1 gene efficiently enhances the cell viability and tolerance of S. cerevisiae to ethanol and the constructed strain S. cerevisiae is proved to highly yield ethanol by the fermentation of sugarcane juice.

Effects of Elevated Ammonia Concentration on Production and N-glycosylation of Antibody Fusion Protein in CHO Cell Cultures at Maintenance Phase

CHEN Xin-ning, ZHAO Liang, FAN Li, LIU Xu-ping, TAN Wen-song

2019, 35(3):  93-102.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0629

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In order to understand the effects of ammonia on production and N-glycosylation of antibody fusion protein（AFP）during fed-batch cultures of Chinese hamster ovary（CHO）cells and to reveal the N-glycosylation steps affected by ammonia,the CHO cell maintenance and metabolism,protein production,and N-glycan structure of AFP at different ammonia concentrations were investigated during cell maintenance phase（protein production phase）. It was found that cell growth profile,glucose and glutamine consumption,production level of lactate and ammonia during cell maintenance phase were insignificantly affected at 5-12 mmol/L of ammonia concentration. However,elevated ammonia concentration（>5 mmol/L）led to decreased sialylation and galactosylation but no effect on fucosylation and high mannose species of AFP. The productivity and final titer of AFP decreased while ammonia concentration was > 9 mmol/L. Thus,ammonia production should be carefully controlled < 5 mmol/L during the development of cell culture process to avoid impaired galactosylation and sialylation of the product and decreased product expression from ammonia accumulation.

Comparative Proteomics Analysis of Escherichia coli in Response to Barofloxacin Stress

ZHANG Liang, CHEN Xiao-qing, SONG Jia-yu, MAO Ran-ran, JIANG Qian-wen, LIN Xiang-min

2019, 35(3):  103-109.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0848

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With the increasingly severe situation of bacterial drug resistance in the world,bacterial drug-resistant mechanism has become a hot topic. An iTRAQ-labeling-based quantitative proteomics method was performed to compare the differential expression of Escherichia coli K12 BW25113 in response to 1/4MIC balofloxacin stress. The result showed that a total of 118 differential proteins with 66 down-regulated and 52 up-regulated ones were identified. Bioinformatics analysis further showed that bacterial drug-resistances were regulated by altering some energy metabolism such as reducing TCA cycle,pyruvate cycle,and carbon,and bacterial survival increased by rising the expressions of proteins related to nucleic acid metabolism and then eliminating the stresses from blofloxacin. Meanwhile,the qPCR method was used to validate the differential expressions of genes related to the carbon metabolism and pyrimidine metabolism;consequently,most of the transcription levels of these genes were consistent with their protein levels. These findings provide the theoretical basis for further investigating the bacterial drug resistance process and action targets for better controlling and treating drug-resistant bacteria.

Effects of Amino Acid Mutation in the RnaseIII RncS of Listeria monocytogenes on RNA Degradation Activity

WANG Li-xia, MENG Qing-ling, QIAO Jun, CAI Kuo-jun, WANG Deng-feng, WU Ye-hui, GUO Jing, CAI Xue-peng

2019, 35(3):  110-116.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0861

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To investigate the effect of amino acid mutation in Listeria monocytogenes（LM）ribonuclease RnaseIII RncS on RNA degradation activity,bioinformatics software was used to analyze the domains of RnaseIII encoded by rncS gene of LM wild-type strain SB5,and gene overlap extension PCR（SOE-PCR）was to perform its gene mutations. Then the rncS mutant gene fragments D50A and E122A were cloned into the expression vector pET-32a（+）,and induced by IPTG in Escherichia coli to generate mutant RnaseⅢ. SDS-PAGE and Western Blot were adapted to detect the expression form and the antigen specificity of fusion protein,respectively. Finally,the enzyme activity assay in vitro was to determine the effect of amino acid mutation on RNA degradation activity. The domain analysis revealed that the LM-RnaseIII protein amino acid sequence included a nuclease domain（RIBOc）,in which there were 5 active sites,and a double-stranded RNA binding domain（DSRM）. The results of SDS-PAGE showed that the relative molecular masses of the recombinant mutants RnaseIII-D50A and RnaseIII-E122 A were 42.5 kD,which was consistent with the theoretical values. Western Blot showed that the recombinant mutant RnaseIII-D50A and RnaseIII-E122A reacted with positive serum against LM. Enzyme activity experiments demonstrated that the degradation activity of RnaseIII was dependent on Mn²⁺ or Mg²⁺. After the 50th aspartic acid was mutated,the degradation activity of RnaseIII RncS reduced（P <0.001）,while it significantly（P <0.0001）decreased after the mutation of the 122nd glutamate,suggesting that the 122nd glutamate was the key site for maintaining the activity of LM RnaseIII. This study provides a scientific basis for further revealing the characteristics and mechanism of LM RncS.

Construction and Identification of Lentiviral Vector for RNA Interference of USE1 Gene

WANG Jia-yue, LIU Xiang-nan, PENG Kang-li, ZHAO Bo

2019, 35(3):  117-122.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0659

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This work aims to construct a lentiviral vector for RNA interference of ubiquitin-conjugating enzyme USE1 gene and to detect the inhibition level of this gene in cells for identifying the downstream substrates corresponding to the specific ubiquitination pathway of Uba6-USE1. The specific shRNA sequence targeting USE1 gene were selected,synthesized,and cloned into pLL3.7 lentiviral vector,and the recombinant lentiviral plasmids inhibiting the expression of USE1 gene were constructed and identified. The identified recombinant plasmids were co-transfected into HEK-293 cell with psPAX2 and VSVG lentiviral packaging vectors. Then viral supernatants were collected and the titers as well as the optimal dilution factors of the virus were determined. The inhibition of the USE1 gene in viral infected HEK-293 cells was detected by qPCR and Western Blot. Results showed that 3 RNA-interfered lentiviral vectors targeting USE1 gene were constructed successfully,the supernatants of 3 lentiviral packaging vectors with satisfied titers were collected. Number 2 and 3 shRNA sequences presented obvious inhibitions to the expression of USE1 gene with an inhibition rate at 50%（^*P<0.05）. In sum,lentiviral vectors specifically inhibiting the expression of ubiquitin-conjugating enzyme USE1 gene are successfully constructed by RNA interference,and supernatants and shRNA sequences significantly inhibiting the expression of USE1 are obtained after mRNA and protein verification.

Molecular Cloning and Tissue Expression of Gene HYOU1

JI Wen-bo, WANG Hui, CHAI Zhi-xin, WANG Ji-kun, XIN Jin-wei, ZHONG Jin-cheng

2019, 35(3):  123-131.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0852

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In order to understand the tissue expression profile of gene HYOU1,the CDS region of HYOU1 in Tibetan yak was cloned and sequenced,and the structure and function of gene HYOU1 were analyzed;furthermore,the mRNA expression profile of gene HYOU1 in the lung,heart,liver,mammary gland,brain and muscle of cattle and yak were detected by RT-PCR. The results were as followings.（1）The CDS region of gene HYOU1 was 3 006 bp,with the percentage of A,G,C and T being 25.0%,31.1%,26.7% and 17.1%,respectively,and there was base preference. One SNP site（ACG→ACA）was identified as a synonymous mutation.（2）Bioinformatics analysis showed that the protein was a neutral,relatively unstable and hydrophilic secretion signal one,and a transmembrane helix（TMhelix）region were obtained and located at the 13-35 amino acid sites. The second and tertiary structures analysis showed that the main spatial structure was random coil and α-helix.（3）Cluster analysis demonstrated that the gene HYOU1 of the Tibetan yak was the closest associated with wild yak,followed by zebu and cattle,and the most distant with buffalo.（4）RT-PCR results indicated that gene HYOU1 expressed in 6 tissues liver,mammary gland,lung,brain,heart,and muscle of cattle and yak at descending order. In addition,for the same tissue,the expression level of the cattle was significantly higher than that of the yak（P<0.05）,i.e.,the expression in the liver of the cattle was 4.4 times higher than that of yak.

Cloning of Gene TSHB,DIO2 and DIO3 and Their Expression Levels in Reproductive Axis of Female Yaks

WANG Qin, LU Jian-yuan, ZI Xiang-dong

2019, 35(3):  132-137.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0793

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In order to study the regulation mechanism of TSHB-DIO2/DIO3 system on seasonal breeding of the yak,RT-PCR was used to analyze the cDNA sequences of gene TSHB,DIO2,and DIO3,and the phylogenetic tree was constructed based on the sequences of bovine gene TSHB,DIO2,and DIO3 in the GenBank. Further,their mRNA expression levels in hypothalamus,pituitary,ovary,oviduct and uterus were analyzed by real-time PCR. These results indicated that there was very close evolutionary relationship in yak’s gene TSHB,DIO2,and DIO3 between a yak and a bovine. The coding region of gene TSHB was 417 bp,and its expression level in pituitary was significantly higher than that in other tissues（P < 0.01）. The cDNA sequence of DIO2 gene was 951 bp and expressed in all tissues,but it was significantly higher in the uterus than that in the other four tissues（P < 0.01）. The cDNA sequences of DIO3 gene was 873 bp and was not detected in the uterus,the expression of which in the ovary was significantly higher than that in the pituitary,hypothalamus and oviduct（P < 0.01）. This study suggests that the TSHB-DIO2/DIO3 may be involved in the regulation of yak reproduction,which may provide a reference for preliminarily revealing the molecular regulation mechanism of yak seasonal breeding.

Regulation of Genetically Modified Organisms in Australia and Its Enlightenment

WU Gang, LI Wen-long, SHI Jian-xin, ZHANG Li, WU Yu-hua, SONG Gui-wen

2019, 35(3):  138-143.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0711

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Australia is an important producer and exporter for agricultural products in the world. This paper reviewed the safety supervision system of the genetically modified organisms（GMOs）in Australia,including the legislative process of safety management on genetic engineering,the regulatory system and its responsibilities,the daily supervision and enforcement,the labeling policy of GMOs,and the studying and regulatory policies for the genetic breeding novel technology in Australia. Based on the safety management experience of GMOs in Australia and combined with the status quo of China's GMO safety management,we had the following suggestions：further improving the existing GMO management system,regulatory system and technical support system,conducting strict supervision and enforcement,implementing relevant responsibilities,raising the public participation,increasing the transparency of information in China,and clarifying regulation principles on new breeding technology such as genetic editing etc. as soon as possible.

Research Progress on Fibroblast Growth Factor 5

LEI Lei BAO, Peng-jia, LIANG Chun-nian, CHU Min, YAN Ping

2019, 35(3):  144-150.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0622

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FGF5（fibroblast growth factor 5，FGF5）is a member of fibroblast growth factors（FGFs），which possesses a variety of biological functions. Previous studies have found that FGF5 plays an important role in the cyclical growth of mammalian hair and is a key signal molecule from anagen to catagen. FGF5 also involves in animal limb development，human glioblastoma multiforme，melanoma，hepatocellular carcinoma and hypertension. In this paper，we review the biological functions of FGF5 and the related research results in disease formation in recent years，aiming at providing theoretical basis for its further research and rational utilization in the fields of human hair growth，animal villus resources utilization，limb development，and tumor diagnosis and treatment.

Research Progress on Molecular Genetics and Multi-omics of Dendrobium officinale

QIAO Yu-chen, ZHOU Si-jing, SONG Mei-fang, WANG Ping, LIU Gui-jun

2019, 35(3):  151-163.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0759

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Dendrobium officinale,alternatively named as “Heijiecao” in China,belongs to genus Dendrobium of Orchidaceae in the Angiosperm,Monocotyledonous. It is a perennial herb with combined edible,medicinal and ornamental properties and great economic value. The wild resource of D. officinale has been near extinct due to low germination efficiency of seeds under the natural conditions,habitat destruction and excessive picking. In 1991,it was listed as endangered plants by the state. The "Chinese Pharmacopoeia" records that it has the effect of relieving stomach upsets,promoting body fluid production,nourishing “yin” and enhancing antipyresis,thus called as “pharmaceutical panda” and possesses high medicinal value. Moreover,because the comprehensive health industry has developed vigorously in recent years and the market demand of D. officinale has increased year by year,this ancient dual-use plant as food and medicine has become a hotspot. With the maturing of genome sequencing and bioinformatics,a series of gratifying achievements in molecular biology for D. officinale has been achieved. This paper reviews the recent advances in molecular genetics,genome,transcriptome,proteome and metabolome on D. officinale at home and abroad,and discusses the significance of these works,and also prospects the work that needs to be carried out,aiming to provide the theoretical basis for molecular breeding,functional gene mining and biosynthesis mechanism of active components,and to promote the scientific and sustainable development of D. officinale industry.

Preparation of Cross-Linked Enzyme Aggregates and Its Application in Laccase Immobilization

HAN Shu-ran, LU Lei

2019, 35(3):  164-170.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0935

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Laccase is a copper-containing polyphenol oxidase with high catalytic efficiency. Due to its broad substrate range,laccase has been widely used in many industrial fields. Cross-linked enzyme aggregates（CLEAs）technology is a novel carrier-free enzyme immobilization approach that could be simply performed using crude enzyme at low cost. The CLEAs usually have high stability and can be reused in multiple cycles. In this paper,we first analyzed the factors affecting enzyme activity during the CLEAs preparation and the related improving strategies,and then summarized the application of cross-linked laccase aggregates in pollutant degradation,with the aim of laying a foundation for further research and application of immobilized laccase.

Research Advances in Genes Involved in Ethylene Biosynthesis and Signal Transduction During Flower Senescence

LIU Chang-yu, CHEN Xun, LONG Yu-qing, CHEN Ya, LIU Xiang-dan, ZHOU Ri-bao

2019, 35(3):  171-182.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0923

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Ethylene,a key endogenous phytohormone,is involved in multiple plants’ life activities,and plays an important role in regulating floral development and senescence. In flowers,researchers can affect the production of ethylene by regulating related genes of ethylene biosynthesis and its signal transduction,and further regulate the floral development process and aging process. Currently,studies on prolonging florescence by regulating endogenous ethylene are mainly applied to ornamental flowers,but few to medicinal or other flowers. This paper summarizes the models of ethylene biosynthesis and ethylene signal transduction pathway,interaction patterns of its related genes,and researches of cloning and regulating related genes involved in ethylene response mediating floral development and senescence in recent years. It is expected to apply studies which prolong plants’ florescence by regulating related genes of ethylene biosynthesis and signal transduction pathway to ornamental,medicinal and other flowers with short flowering period,and provide references for cultivating fine breeding of ornamental and medicinal flowers by regulating endogenous ethylene generation and transduction at genetic level.

A Review of Carbon-based Electrochemical Sensor for the Detection of Lead Ion

LIN Shao-hua, XIE Yin-xia, LUO Hong-xia, XU Wen-tao, SHEN Hui-jie

2019, 35(3):  183-193.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0599

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Lead ion commonly exists in air，water，soil and food，and in which the concentration of lead is increasing. Lead ion is a toxic pollutant and is extremely harmful to human health. A lot of strategies have been proposed for detecting lead ion，carbon-based electrochemical sensors have gained growingly attentions owing to simplicity，rapidity and sensitivity. From the point of view of modified material of composite electrode，this paper reviews the research progress of composite electrodes based on inorganic materials，organic materials and functional nucleic acids in the detection of lead ion. The development，construction approaches and sensing performances of carbon-based electrodes are mainly introduced. At last，the trends of the carbon-based lead-ion electrochemical sensor are also prospected.

Research Progress in Quantitative and Unitive Detecting Technologies of Functional Nucleic Acid and Label-Free Fluorescence

XIAO Bing, LIU Bang, LUO Yun-bo, HUANG Kun-lun, ZHANG Yuan, LI Xia-ying, ZHANG Xiu-jie, XU Wen-tao, ZHOU Xiang

2019, 35(3):  194-202.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0809

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Functional nucleic acid is a natural or an artificial nucleic acid sequence with specific spatial conformation and biological function. It has advantages such as easy method to modify,low cost,high stability and strong specificity. After combined with label-free fluorescence biosensors,the functional nucleic acid performs the function of transferring all different kinds of targets to stable signals of nucleic acid and amplifying signals by the amplification of nucleic acid. In addition,the complexity of process and cost from labeling and screening of fluorescence,quenching groups in label-fluorescence can be avoided by the technology of label-free fluorescence,while the fluorescence signal transfer can be ensured by the specific or unspecific combination of nucleic acid and fluorescence. Combining the advantages of 2 technologies,the sensitivity and real-time capability can be enhanced,and the functional nucleic acid-based label-free fluorescence biosensors have been widely applied in the detection of environmental pollutants,risk factors of food safety,disease diagnosis,etc. Firstly,we define the concepts of functional nucleic acid and quantitative fluorescence detecting technology,and elaborate in detail the features of key fluorescent materials,as well as their biological molecular recognitions,reaction principles,and luminous models with functional nucleic acid. Then,we describe,assesse,and compare the quantitative and unitive detecting technologies of functional nucleic acid and label-free fluorescence and their applications according to their features. Finally,we discuss the research significance and issues of the quantitative and unitive detecting technologies of functional nucleic acid and label-free fluorescence in detections,and prospect the future development and application.

A Rapid Method of Detecting Viable Legionella pneumophila in the Water Environment of Public Places

GUO Pei, ZHAO Long, HU He

2019, 35(3):  203-209.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0791

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This work aims to establish a molecular detection method for the rapid detection of viable Legionella pneumophila based on 16S rRNA precursor,and the contamination level and status of L. pneumophila in the water environment of public places may be detected by this method and ISO method. The established method is based on the synthesis of 16S rRNA precursor in L. pneumophila via a nutritional stimulation,which is an indicator for cells’ viability. Pre-stimulated RT-qPCR method and ISO method were used to detect L. pneumophila and non-Legionella pneumophila,as well as other non-Legionella,and the specificity and sensitivity of these two methods were validated.Further,the pre-stimulated RT-qPCR method and ISO method were employed to detect L. pneumophila in the water environment of public places,and the consistency of the results between the 2 methods were compared.The 16S rRNA precursor of L. pneumophila increased slowly when the pre-stimulation time was > 3 h,thus the optimal pre-stimulation time was set to be 3 h. L. pneumophila were well detected by both pre-stimulated RT-qPCR method and ISO method,and the specificity for both was 100%. The Limit of Detection（LOD）by pre-stimulated RT-qPCR method was 10² cells/L,while LOD by ISO method was 10⁴ cells/L. The positive rate of L. pneumophila in the water environment of public places was 43.5%（30/69）by pre-stimulated RT-qPCR method and 40.6%（28/69）by ISO method,and the difference of detection by both methods was of no statistical significance（C²=0.119,P=0.730）. In conclusion,pre-stimulated RT-qPCR method is a rapid and effective method for detecting L. pneumophila with high sensitivity and specificity,and it can be used as a potential alternative to the ISO method.

Identification of a Bacillus Strain BS-2 with Anti-phages and Optimization of Glucose Feeding Strategy

QI Jia-ming, YANG Na, SUN Shan-shan, MING Yan-chao, GUO Liang, ZHANG Dong-xu, XU zhi-wen

2019, 35(3):  210-216.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0670

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This work aims to screen anti-phage strain for solving the problem of phage infection in Bacillus subtilis production and increase fermentation of anti-phage strain. Double-layer-agar plating method was used to screen and purify phages with Bacillus subtilis as host from abnormal fermentation broth,and anti-phage strains were obtained by screening the Bacillus library in our laboratory using the purified anti-phages. Phylogenetic trees were constructed by 16S rDNA and gyrA gene sequences analysis to identify the anti-phages strain. Based on glucose content curve and growth curve of anti-phages strain batch fermentation,initial glucose concentration in the medium and glucose feeding strategy were optimized for improving the fermentation of the screened anti-phage strains. As results,phages S01 and S02 were screened from the abnormal fermentation broth,and a strain Bacillus sp. BS-2,which was not infected by the phages,was screened from the Bacilluslibrary in our laboratory. According to the 16S rDNA and the phylogenetic trees of gene gyrA,the strain BS-2 was characterized as Bacillus subtilis. The biomass was up to 2.43×10¹⁰ CFU/mL,while suitable initial concentration of glucose was 15 g/L,and glucose was fed into the broth at the speed of 2 g/L·h when the glucose concentration in the broth was lower than 5 g/L,and the total supplying quantity was 10 g/L. B. subtilis BS-2 shows strong ability of anti-phages and high titer under optimized fermentation conditions,which has promising potential for large-scale industrialization.

Optimization of Glycosylation Distribution in the Production of Monoclonal Antibody Bio-Medicine by Full Factorial Design

ZHANG Lei, YANG Mu-di, DONG Shun, LIU Ming-qiu

2019, 35(3):  217-224.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0782

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This work aims to regulate the glycosylation distribution of monoclonal antibody bio-medicine,and thus which allows them to be in accord with original medicine. Full factorial design was applied to investigate the effects of 4 additives of galactose,uridine,manganese chloride and hydrolysate on the glycosylation of monoclonal antibody（mAb）. The results revealed that combined supplements of galactose,uridine and manganese chloride increased galactosylation of mAb;moreover,there was no significant impact on cell growth and antibody production. Furthermore,uridine and manganese chloride inhibited Man5 formation while excessive uridine led to a high Man5 level. Besides,protolysate not only decreased Man5 level and increased fucosylation,also enhanced cell growth. In summary,a desired target glycosylation profile was achieved after the validation was conducted through bioreactor cultivation under the optimized condition predicted by a model,thus production strategy of regulating the glycosylation of mAb during CHO cell culture process is successfully established.