Table of Content

25 May 2017, Volume 33 Issue 5

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Research Advances on RNAi Mechanism and Its Application

FENG Xiao-yan ZHANG Shu-zhen

2017, 33(5):  1-8.  doi:10.13560/j.cnki.biotech.bull.1985.2017.05.001

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RNA interference（RNAi）is a gene silencing phenomenon induced by small non-coding RNA（sncRNA）,which is widely found in eukaryotes. RNAi has become the focus of biological research with some achievements since its discovery. In order to provide some references for the further study of RNAi,the mechanism of RNAi,the key factors involved in RNAi and the application of RNAi are reviewed in this paper.

Research Progress on the Radiation-resistant Mechanisms of Deinococcus radiodurans

CHEN Xiao-fei NING Meng FENG Fei XIANG Ling-yun ZHOU Fu-zhong

2017, 33(5):  9-18.  doi:10.13560/j.cnki.biotech.bull.1985.2017.05.002

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Deinococcus radiodurans is one of the most radiation-resistant organisms that was found on the earth,and has very high resistance to UV,drought,and mutagenic agents. D. radiodurans has drawn attentions from scientists around the world,and there are many reports about its radiation-resistant mechanisms. In this paper the recent progresses on radiation-resistant mechanisms of D. radiodurans are summarized from DNA-repair pathway,antioxidative defense system,special survival approaches,and cellular purification system. The development trends and future prospects of radiation-resistant mechanisms of D. radiodurans are also discussed,the aim of this paper is to provide some foundations for further study of radiation-resistant mechanisms of the bacteria.

Research Progress on the Role of COP9 Signalosome in Growth,Development and Secondary Metabolism of Fungus

QIAO Yu, YANG Shui-ying, LI Zhen-lun, WANG Fang, XU Yi

2017, 33(5):  19-25.  doi:10.13560/j.cnki.biotech.bull.1985.2017.05.003

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The COP9 signalosome（CSN）is a highly conserved protein complex in eukaryotes,and is involved in the control of the whole life. At present,the researches on COP9 signalosome are mainly concentrated in humans,animals and plants. The research on fungal COP9 signalosome is mainly focused on several patterns of fungi,relatively few on other fungi,and even less in China. In this paper,we summarized the composition and structure traits,the related regulation mechanisms for growth and development,and the coordination of secondary metabolism of fungal COP9 signalosome. Further,we analyzed the existing doubts about COP9 signalosome,aiming at providing references for further studying the functions of COP9 signalosome in fungus.

Research Progress on Cysteine Participation in Heavy Metal Resistance in Organism

ZHANG Li, SUN Dui, WANG Xiao, ZHENG Chun-li

2017, 33(5):  26-33.  doi:10.13560/j.cnki.biotech.bull.1985.2017.05.004

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Cysteine（Cys）,the final product of the sulfate assimilation pathway,widely exists in the organism. The oxidation state sulfur is absorbed and restored,and then integrated into the molecules skeletal structure of cysteine by organisms,participating in the other metabolic pathways of organisms. Because the structure of Cys contains thiol,which makes it being able to specifically bind with heavy metal ion,thus involving in the heavy metal resistance of organism,directly or indirectly. Considering a series of physiological and biochemical process in biological cells are orderly conducted under the heterogeneous condition,the investigation of Cys and the structure characteristics of Cys-M（M represents metal ion）complexes has significant reference value for studying Cys and the physiological behavior of Cys-M at the molecular level. In recent years,the application of new technique in the biological science,for example atomic force microscope（AFM）,makes this research feasible. In this paper,the basic characteristics and the biosynthetic pathway of Cys are introduced,concurrently,the research progress of Cys involved in heavy metal resistance are summarized,which aims to provide certain scientific theoretical basis for studying the detoxification mechanism of Cys in heavy metal resistance of organism.

Drug Research in Cancer Therapy Based on Polyamine Transport System

LI Jian-ming DING Jin-song

2017, 33(5):  34-39.  doi:10.13560/j.cnki.biotech.bull.1985.2017.05.005

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Polyamine is an essential substance in cells growth,differentiation and other life activities. The proliferation of tumor is closely related to the level of intracellular polyamine. Delivering polyamine analogs and conjugates into cells,mediated by polyamine transport system overexpressed on tumor cell surface,leads to the exhaustion of intracellular polyamine and then induces tumor cells apoptosis. In addition,using the positive structure characteristics of polyamine to deliver therapeutic genes may increase the transfection efficiency of exogenous genes. In order to provide reference for the research of anticancer agents based on polyamine in the future,the research progress on polyamine analogues and conjugates for treating cancer and using polyamine as a gene deliver vector are summarized here.

Applications of Isothermal Titration Calorimetry in Protein-ligand Interactions

QI Xin-jie, WANG Yue, WANG Yan-sheng, FANG Guo-kang, HUANG Ying-chun

2017, 33(5):  40-49.  doi:10.13560/j.cnki.biotech.bull.1985.2017.05.006

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Isothermal titration calorimetry（ITC）is a biophysical technique widely used to study molecular interactions in molecular biology and these related fields,it is the sole direct method to measure the heat change during complex formation at constant temperature. We may obtain much important information about molecular interactions（such as binding constant,number of binding sites,free energy,enthalpy,and entropy）simply by measuring the heat absorbed or released during an interaction between two liquid solutions. This review concentrates on the principle and features of ITC,the new applications of ITC in protein-ligand interactions,and potential development direction,it has been shown that it plays an important role in the elucidation of binding mechanisms and the validity of the data.

Research Progress on the Biological Conversion of Energy Grass to Cellulosic Ethanol

BAI Long LI Chun-mei LÜ Tu DU Ying YANG Yue TIAN Shen

2017, 33(5):  50-56.  doi:10.13560/j.cnki.biotech.bull.1985.2017.05.007

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With the increasing depletion of fossil fuels and the deterioration of environmental pollution,seeking a green energy to replace fossil energy is an urgent task for the world. Bio-ethanol as a clean energy presents the value for vehicle,and can be used as a clean substitute of fossil energy,which is widely concerned by the researchers. And bio-conversion of grass energy is recognized as the most efficient measure for the industrialization of biomass energy. Energy grass as one of lignocellulose materials,because of its fast growth,high yield,strong resistance and other advantages,has been attracted much attention. This paper discusses the recent progresses on the bio-conversion of energy grass as substrate for cellulosic ethanol,i.e.,advances and existing problems in varied aspects from the pretreatment of raw cellulose to the fermentation process of ethanol,bioconversion efficiency in the bioenergy production from lignocellulose,and conprehensive utilizations of all components of lignocellulose are briefly discussed,aiming at finding out an optimal production mode for the industrial production of cellulosic ethanol.

Subcellular Localization of Profilin-1 from Arabidopsis Utilizing Two Transient Expression Systems

ZHANG Xiao-hui HAN Rong

2017, 33(5):  57-62.  doi:10.13560/j.cnki.biotech.bull.1985.2017.05.008

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Here we study the subcellular localization of profilin-1（PRF1）using two transient expression methods and compare the advantages and disadvantages of two transient expression methods in the subcellular localization. Using the leaves of Arabidopsis thaliana as material,the total RNA from the leaves was extracted,and PRF1 gene was cloned using specific primers by RT-PCR and then ligated to pCAMBIA1300-GFP vectors,finally one plant expression vector pCAMBIA1300- GFP-PRF1 was constructed successfully. Two transient expression systems,agroinfiltration of tobacco leaves,and PEG transformation of protoplasts isolated from the leaves of Arabidopsis thaliana were adopted. The expression of the fusion proteins was observed by a confocal laser scanning microscopy. The results showed that the green fluorescence of the fusion protein was observed when the PRF1 gene was introduced into the protoplasts of A. thaliana and tobacco epidermal cells,the product of PRF1 gene and GFP fusion protein was mainly localized in the cytoplasm of tobacco epidermal cells,and there was also an expression in the nuclear cell organelle. PRF1 was localized in the nucleus and cytoplasm of Arabidopsis thaliana. The localization of PRF1 protein was different in two different transient expression systems,which may be related to the characteristics of the plants that are homologous or heterologous expression.

Determination and Purification of Total Flavonoids from Seeds of Ammopiptanthus mongolica

TAO Bo FANG Mei ZHANG Jia-nan OU Yun-wen JIA Ning

2017, 33(5):  63-70.  doi:10.13560/j.cnki.biotech.bull.1985.2017.05.009

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This work is to establish the determination and purification method of total flavonoids from seeds of Ammopiptanthus mongolica. Four chromogenic methods were chosen for the determination of total flavonoids,the best absorption wavelengths were selected by UV-wavelength scanning of quercetin standard and total flavonoids from seeds of A. mongolica. Standard curves for quercetin by four chromogenic methods were built,and each method was verified via methodology,and the new experimental condition and method after verification were used to determine the content of total flavonoids from seeds of A. mongolica. In this experiment,3 kinds of macroporous resins of AB-8,D101,and S-8 were selected for static adsorption and elution test,and AB-8 was chosen for dynamic test,with which the process condition for purifying total flavonoids from seeds of A. mongolica was established,and the extraction was conducted with ethyl acetate to improve the purity of total flavonoid. The results showed that the determination of total flavonoids from seeds of A. mongolica with AlCl₃-CH₄O chromogenic method at 335 nm and quercetin as the standard substance was reliable and effective. Under the optimal condition,adsorption with AB-8 macroporous resins was 5 BV of 6 mg/mL sample solution at pH 5.5 at rate of 3.5 BV/h,and then elution was 6 BV of 70% ethanol at rate of 1.5 BV/h,the purity of the product was 41.07% from 5.69% before purification. The total flavonoids after purified with AB-8 resin were prepared into a certain concentration solution,and then extracted with ethyl acetate,the total flavonoids content reached 76.15%.

Development of SSR Molecular Markers Based on Transcriptome Data of Pinus kesiya var. langbianensis

ZHAO Neng, YUAN Xiao-long, MIAO Fu-jun, WANG Yi, CHEN Wei, LI Jiang, WU Tao, WANG Juan

2017, 33(5):  71-77.  doi:10.13560/j.cnki.biotech.bull.1985.2017.05.010

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In Simao pine（Pinus kesiya var. langbianensis）breeding programs,lack of co-dominant genetic markers constrains the development of molecular marker assisted breeding. The aim of this study is to develop SSR molecular markers using transcriptome sequencing data. Searching the SSR loci from 59636 unigenes of P. kesiya var. langbianensis with MISA software,total 3745 SSRs were obtained,accounting for 6.28% of the total unigenes,averagely one SSR per 11.36 kb. Trinucleotide and dinucleotide repeats were dominant types among SSRs with the ratio of 49% and 24%,and others were only 27%. AGC/CTG was the most trinucleotide motif and AT/TA was the most dinucleotide motif. The primers were designed and synthetized based on randomly selected 224 SSR loci,verification by agarose gel electrophoresis revealed that 29 loci showed clear polymorphism. While only 12 of the 29 loci satisfied the multiple fragments of SSR repeat motifs in capillary electrophoresis and their amplification efficiencies were higher than 85%. Genetic diversity of 42 Simao pine samples from Jingdong and Puwen populations was investigated with these 12 fluorescently labeled primers. A total of 35 alleles were detected,the polymorphism information content（PIC）of all loci ranged from 0.0932-0.4809（the mean was 0.3069）. Among these,4 loci were classified as lowly polymorphic ones（0 < PIC <0.25）and 8 loci as moderately polymorphic ones（0.25 < PIC <0.5）. Conclusively,these 12 SSRs may be used in future studies on genetic diversity,linkage mapping,gene location and cloning,providing the technical support for molecular marker assisted breeding and mutation in P. kesiya var. langbianensis.

Accurate Detection Efficiency of SF2 Protein RIP Enriching RNA Using Exogenous RNA as Reference Gene with qPCR

LI Yu ZHAO Lei CHEN Li ZHOU Yu-xun LI Kai XIAO Jun-hua

2017, 33(5):  78-82.  doi:10.13560/j.cnki.biotech.bull.1985.2017.05.011

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Using real-time quantitative polymerase chain reaction（qPCR）to detect the efficiency of SF2 enriching RNA based on 0.1% formaldehyde cross-linking RNA immunoprecipitation（RIP）,we aim to provide an accurate detection method for studying the enriching efficiency of flexible splicing factor SF2 interacting with RNA. The RNA interacting with SF2 in HeLa cells was acquired with RIP,then RNA of exogenous yeast（BY474）in the same ratio was added,and the efficiency of RIP enriching RNA interacting with SF2 was measured via qPCR while using β-actin as reference gene. Results showed the RNA-SF2 protein complex was successfully obtained based on 0.1% formaldehyde cross-linking immunoprecipitation. The differences of RNA by positive genes（PABP,Srsf1）achieved 60 folds in SF2 and IgG samples via qPCR. Conclusively,qPCR combined with exogenous yeast（BY474）as reference gene may expeditiously and accurately quantify the RNA-enriched efficiency for SF2.

Improving the Accuracy of FMO_GS-OX1 Subcellular Localization Using Cycloheximide

XIA Hong-yu KONG Wen-wen XU Rui CANG Wei LI Jing

2017, 33(5):  83-88.  doi:10.13560/j.cnki.biotech.bull.1985.2017.05.012

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The conventional method of protein subcellular localization is to build the vector with the expression of fused target gene and green fluorescent protein gene（GFP）driven by 35S promoter. The subcellular localization of the target protein is then determined in the cells transiently expressing the fusion gene. Utilization of 35S promoter will lead to overexpression of the fusion gene and obtaining strong GFP signal. But sometimes the excessively synthesized protein will possibly remain in the transportation organelle or the areas exceeding the native protein location. The aim of this study is to solve this problem in the study of protein subcellular localization. To accurately determine the subcellular localization of Flavin-Containing Monooxygenase 1（FMO_GS-OX1）in model plant Arabidopsis,a protein inhibitor,cycloheximide was applied to repress the over-expression of FMO_GS-OX1-GFP fusion protein in tobacco epidermal cell. The results showed that before the treatment of cycloheximide,strong GFP signal was presented in both ER and cytosol. While after treated with cycloheximide,GFP signal disappeared in ER but remained in cytosol. This study demonstrated that proper treatment of cycloheximide may effectively avoid the excessive over-expression of the gene driven by 35S and thus is conducive to precisely determine the intercellular position where the protein facilitates its function.

Purification of Lysozyme from Egg White by Combination of Polyethylene Glycol Precipitation and Aqueous Two-phase Extraction

WEN Chong-wei ZHAO Ye-qing SHI Li OUYANG Zhen

2017, 33(5):  89-93.  doi:10.13560/j.cnki.biotech.bull.1985.2017.05.013

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This work aims to establish a new technology of purifying lysozyme from egg white based on the combination of polyethylene glycol（PEG）precipitation and aqueous two-phase extraction. The results showed that 98.1% of non-lysozyme proteins were removed by the precipitation when PEG 4000 was added into pretreated egg white until its mass fraction reached 16%. Then,the supernatant of PEG precipitation was collected and mixed with saturated（NH₄）₂SO₄ solution until the mass fraction of this salt reached 4.32%. Therefore,the aqueous two-phase system of PEG/（NH₄）₂SO₄ was established and most of lysozyme could be transferred into the top phase. It was found that 96.34% of the freeze-dried top phase was lysozyme,and the rest was PEG 4000,no any other proteins existed. The recovery rate and the specific activity of lysozyme were 70.2% and 25 000 U/mg,respectively. In conclusion,this purification technology was simple,accurate,safe and reliable,and could be used for large-scale production of lysozyme.

Sequence Characterization and Expression Analysis of OsRhoGAP1 Gene in Rice

YAN Xin-tian HUANG Jun-jun LOU Chen GE Hui-wen LIANG Wei-hong

2017, 33(5):  94-101.  doi:10.13560/j.cnki.biotech.bull.1985.2017.05.014

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A function-unknown gene was isolated from a cDNA library of young spike of rice and designated as OsRhoGAP1. Based on the bioinformatics and modern molecular biology,we systematically surveyed its expression patterns at different development stages and under different hormones treatments. Bioinformatics analysis showed that OsRhoGAP1 had typical structural feature of plant RhoGAP protein,including CRIB motif and RhoGAP domain. qRT-PCR analysis revealed that OsRhoGAP1 gene presented specifically spatio-temporal characteristics,i.e.,relative high expression levels in root and leave at the seedling stage,in young panicle and leave at the panicle stage,and in root at the mature stage. Furthermore,the transcription of OsRhoGAP1 was strongly induced by ABA,SA,GA,and MeJA,but suppressed by IAA and 6-BA. It was speculated that the expression of OsRhoGAP1 was regulated by hormones.

Effects of Nitric Oxide on Oxidative Damage Metabolism in Wheat Seedling Under Cadmium Stress

MA Xiao-li, JI Rui-ping, TIAN Bao-hua, HUO Jian-xin

2017, 33(5):  102-107.  doi:10.13560/j.cnki.biotech.bull.1985.2017.05.015

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This study is to further study the response mechanism of nitric oxide gas signal on oxidative damage of wheat seedlings under cadmium（Cd）stress. Through nutrient solution cultivation,Triticum aestivum ‘Jinmai 8’ was selected as the plant material,and nitric oxide（NO）-donor sodium nitroprusside（SNP）,NO-scavenger haemoglobin（Hb）,and sodium ferrocyanide（SNP analogue）were used to investigate the effects of NO on oxidative damage metabolism in seedlings under Cd stress. The results showed that SNP alleviated the growth inhibition in wheat seedlings,significantly increased the contents of soluble protein,chlorophyll and glutathion,decreased the contents of malondiadehyde（MDA）and hydrogen peroxide（H₂O₂）,as well as the production of O₂·^-,and the accumulations of soluble sugar and proline. However,the NO-scavenger hemoglobin treatment increased the toxicity of Cd stress to wheat seedlings,and the SNP analogue treatment presented no effect. Meanwhile,SNP decreased the activities of superoxide dismutase（SOD）,catalase（CAT）,glutathione reductase（GR）and ascorbate peroxidase（APX）,which eliminated the oxidative damage under Cd stress. Conclusively,NO alleviates Cd-damage by regulating the antioxidase system.

Cloning and Expression Analysis of AmMYB4-like in Ammopiptanthus mongolicus

XIAO Zi-hua GAO Fei SUN Hui-gai ZHOU Yi-jun

2017, 33(5):  108-116.  doi:10.13560/j.cnki.biotech.bull.1985.2017.05.016

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Based on our previous work,a gene encoding MYB transcription factor was cloned from Ammopiptanthus mongolicus,and it was designated as AmMYB4-like. The full length of the AmMYB4-like cDNA was 1,145 bp,including a 960 bp opening reading frame（ORF）which encoded a 319-amino acid peptide. AmMYB4-like contained R2 and R3 conserved domains and belonged to the R2R3-MYB subfamily. Quantitative real-time PCR（qRT-PCR）analyses revealed that AmMYB4-like was up-regulated in leaves under low temperature and drought stresses,but it was mainly induced by drought stress in roots. Plant expression vector pPZP212- AmMYB4-like was constructed for further research of AmMYB4-like.

Studies on the Interaction Between FD and 14-3-3/GRF7 in Arabidopsis thaliana

YUAN Min WANG Li GE Wei-na ZHANG Lan

2017, 33(5):  117-122.  doi:10.13560/j.cnki.biotech.bull.1985.2017.05.017

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In order to prove whether or not there is any interaction between FD and 14-3-3/GRF7 of 14-3-3/GRFs family,both yeast two hybrid and BiFC assays were performed. In yeast,the yeast colonies co-transformed with FD-BD and 14-3-3/GRF7-AD constructs grew well in -Leu/-Trp/-His/-Ade medium comparing with negative controls,indicating that FD directly interacted with 14-3-3/GRF7 in yeast. In tobacco,comparing with negative controls,the obvious YFP fluorescence signals were detected in the tobacco cells in which Agrobacterium co-transformed with FD-cYFP and 14-3-3/GRF7-nYFP,or FD-nYFP and 14-3-3/GRF7-cYFP vectors was injected,revealing that FD also directly interacted with 14-3-3/GRF7 in tobacco. The results by both assays confirm that there is direct interaction between FD and 14-3-3/GRF7 of 14-3-3/GRFs family.

Identification,Phylogenetic Relationship Analysis of Lycium Based on rbcL-a and ITS Sequence and the Discovery of ITS Pseudogene

CHEN Jin-jin, ZHAO Ming-xia, JIANG Shu, CAO Li-li, ZHAO Qing-sheng, ZHAO Bing, WANG Xiao-dong

2017, 33(5):  123-130.  doi:10.13560/j.cnki.biotech.bull.1985.2017.05.018

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Identification and phylogenetic relationship analysis of 14 species or cultivars of Lycium and 1 adulterant of Nitraria tangutorum were studied using rbcL-a and ITS as molecular markers. Total DNA was extracted by CTAB method. With universal primers,rbcL-a and ITS were amplified,cloned,sequenced and analyzed via PCR. T cloning technique was used to deal with the unsuccessful sequencing of ITS sequence of some samples. rbcL-a and ITS sequences with universal primers were amplified successfully in all samples. The lengths of rbcL-a in Lycium and N. tangutorum were 643 bp,and the sequences of rbcL-a were the same in all samples of Lycium,but existed 45 bp variation sites（7%）compared with N. tangutorum. The lengths of ITS in 14 species or cultivars of Lycium ranged from 513 - 692 bp. The UPGMA phylogenetic tree was constructed based on ITS sequence to distinguish samples of Lycium and N. tangutorum well. ITS sequences of L. ruthenicum and L. barbarum were clustered into two groups,respectively. L. yunnanense,L. chinense and L. barbarum var.auranticarpum were classed as the same branch of L. barbarum. The marker of rbcL-a can effectively identify Lycium from its adulterant. The molecular marker of ITS is applicable in variety identification and genetic relationship analysis of Lycium. This study revealed the polymorphism of ITS in L. barbarum L. varieties and individuals for the first time,and the presence of ITS pseudogenes in L. barbarum L.,which offers some new views for systematic evolution research of Lycium using ITS sequence.

Cloning and Activity Analysis of Promoter of GRAS Transcription Factor Gene HcSCL13 from Halostachys caspica

FAN Shou-de WANG Yan

2017, 33(5):  131-138.  doi:10.13560/j.cnki.biotech.bull.1985.2017.05.019

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In order to explore the expression characteristic and function of GRAS transcription factor gene HcSCL13 from Halostachys caspica,the 2 200 bp sequence of promoter was cloned using the genomic walking method. The analysis results using the PlantCARE software suggested that the promoter sequence contained not only the core elements such as CAAT-box and TATA-box,but also some cis-element related to the stress response. The 35S promoter sequence of the pBI121 expression vector was replaced by the HcSCL13promoter sequence to construct the fusion expression vector. Then,the fusion expression vector was transformed into Arabidopsis thaliana by flora method and the T1 seedlings were stained by GUS. The results of GUS staining showed that the whole transformed A. thalianaseedlings were stained,indicating that the HcSCL13 gene promoter had expression activity,and it might be constitutive promoter.

The Expression of LEPRb mRNA in the Estrus Cycle of Different Sheep Strains

LU Shou-liang LI Liang-yuan GAO Lei SHEN Min WAN Peng-cheng SHI Guo-qing DAI Rong

2017, 33(5):  139-144.  doi:10.13560/j.cnki.biotech.bull.1985.2017.05.020

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The aim of the study is to explore LEPR expression characteristics in seasonal estrus of sheep. Chinese merino（seasonal estrus sheep）and Duolang sheep（perennial estrus sheep）were selected to determine the expression changes of LEPRb mRNA in hypothalamus,ovaries and uterus in different stage of estrus cycle by quantitative real-time PCR. The results showed that during the whole estrus cycle,except the metaestrus,the LEPRb levels in the ovarian and uterus were found to be significantly lower than the levels in hypothalamus of Chinese Merino,but the results in metaestrus showed the reverse. Although the expression level of LEPRb varied in the same tissue of 2 cultivars during estrus stage,the trend was similar. Moreover,the LEPRb expression levels in different tissues of Chinese Merino were significantly higher than those in Duolang sheep. The results indicate that the expression variation of LEPRb is correlated with sheep estrus.

Cloning and Tissue Expression Analysis of NITR in Nile Tilapia（Oreochromis niloticus）

WANG Zhi-wen HUANG Yu ZHANG Hai-yan TANG Ju-fen JIAN Ji-chang LU Yi-shan

2017, 33(5):  145-152.  doi:10.13560/j.cnki.biotech.bull.1985.2017.05.021

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The encoding sequence of NITR（Novel immune-type receptor）gene（GenBank accession number：KX989509;designated as On-NITR）was cloned from the spleen of Nile tilapia（Oreochromis niloticus）. The complete cDNA sequence of On-NITR gene was 1 119 bp with an ORF of 1 026 bp encoding 341 amino acids. The deduced amino acid sequence of On-NITR had an estimated molecular weight of 37.38 kD and a theoretical pI of 8.28. Through NCBI BLAST,we found the amino acid sequence identities between On-NITR and previously reported fish NITRs were approximately 27% - 46%. Amino acid alignment indicated that On-NITR consisted of one typical signal peptide,two extracellular immunoglobulin（Ig）domains（V and V/C2）,one transmembrane domain,and one highly conserved the cytoplasmic region with an immunoreceptor tyrosine-based inhibitor motif（ITIM）and an ITIM-like motif（itim）. Moreover,the analysis by real time quantitative PCR revealed that the expression of On-NITR was detected in all examined tissues of a healthy Nile tilapia with the higher expression level in intestine,skin and liver,while lower expression in thymus,gill,spleen,heart,and brain as well as the lowest level in head and kidney.

The Annotation of Enhancer-trapping Mediated by the SB Transposon in Zebrafish

LIU Shuai-jun SHEN Dan ZHONG Ji-han CHEN Wei WANG Wei ZHANG Li CHEN Cai, YANG Kun-lun GAO Bo SONG Cheng-yi

2017, 33(5):  153-158.  doi:10.13560/j.cnki.biotech.bull.1985.2017-0035

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In order to establish zebrafish enhancer-trapping mutant library and study the expression and regulation of functional genes,we prepared SB（Sleeping Beauty）transposon-mediated enhancer-trapping zebrafish F0,and built the line F1 with tissue- or organ-specific GFP（Green Fluorescent Protein）expression patterns through breeding. We selected SK-3 line of F1（head-specific expressing GFP）to breed with wild type TU zebrafish,and collected fertilized eggs. The GFP expression patterns of early embryos at 24 hpf（Hour post fertilization）,2 dpf（Day post fertilization）,3 dpf,4 dpf,5 dpf and 7 dpf were analyzed. SP-PCR（Splinkerette PCR）was used to detect the inserted position of SB transposon in the zebrafish genome to determine the captured enhancer and functional gene. The expression patterns of the functional gene were verified by in situ hybridization. The results showed that the expression of GFP had obvious temporal and spatial characteristics in SK-3 embryos at different developmental stages and highly expressed in the forebrain,midbrain and hindbrain at earlier stages,and was in a trend of backward at later stages. There was no variation in the expression level of each developmental stage,indicating that the captured enhancer was specifically expressed in the brain. SP-PCR results showed that the enhancer was located in the chromosome 1：35,914,498-35,914,and 621 near the ednraa. The results of in situ hybridization showed that the gene had transcription activity at the 24 hpf. Above data suggested that the SB transposon-mediated enhancer-trapping technique can be efficiently used to acquire the inserted mutant zebrafish, which has a great significance on the study of gene function and the acquisition of new genes with independent intellectual property rights.

Effects of Polysaccharides from Lu Dangshen（Codonopsis pilosula）on Proliferation and Migration of Human Cervical SiHa

HU Jian-ran LI Ping LEI Hai-ying LIU Xian-rui

2017, 33(5):  159-163.  doi:10.13560/j.cnki.biotech.bull.1985.2016-0953

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The effects of polysaccharides from Lu Dangshen（Codonopsis pilosula）（LDP）on the proliferation and migration of human cervical cancer cell SiHa was investigated. MTT method was used to determine the effects of LDP on SiHa cell proliferation,and cell adhesion assay,cell spreading assay and cell wound scratch assay were used to analyze the effect of LDP on SiHa cell migration. LDP significantly repressed SiHa cell proliferation（IC₅₀ = 0.68 mg/mL）,and inhibited adhesion between cells and extracellular matrix,and delayed cell spreading,and then restrained SiHa cell migration. LDP presents solid inhibition effects on the proliferation,adhesion,spreading and migration of human cervical cancer cell SiHa,and thus possesses potential value for developing auxiliary medicine for tumor treatment and dietary supplement.

Isolation and Purification of His-CYP6J1 Fusion Protein from Aphis gossypii and Preparation of Its Polyclonal Antibody

WANG Ya-mei WEI Yuan-jie AI Xin-yu LIU Xiao-ning

2017, 33(5):  164-169.  doi:10.13560/j.cnki.biotech.bull.1985.2016-0920

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This work aims to purify His-CYP6J1 fusion protein through affinity chromatography,and prepare and identify its polyclonal antibody. The fusion protein was purified by Ni-NTA chelating column,the purified product was detected by SDS-PAGE. Then the acquired fusion protein His-CYP6J1 was used to immune mice for preparing its polyclonal antibody against Aphis gossypii. Finally,ELISA was used to test antibody’s titer,and immunohistochemistry to identify its specificity. As results,antiserum titer was 1：200 000,the polyclonal antibody specifically bound to natural P450 CYP6J1 protein from A. gossypii,but not to the one from cotton bollworm. These results provide a foundation for studying the structure and function of a single P450 protein and its role in the insecticide- resistance of A. gossypii.

Eukaryotic Expression of Human Interleukin 10 Receptor α and Detection of Interactions in Protein JAK1s

GUO Xin-xin WANG Gang DENG Qiao-ting WU Han-tao LI Kun WU Ying-song LIU Tian-cai

2017, 33(5):  170-175.  doi:10.13560/j.cnki.biotech.bull.1985.2016-0985

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This work is to construct a eukaryotic express vector pENTER-IL-10RA-His that expresses human interleukin 10 receptor α（IL-10RA）in HEK293 cells,and then to detect the intracellular interaction between JAK1 and IL-10RA by co-immunoprecipitation. Human total RNA was extracted from Hela cells,then IL-10RA gene was amplified by RT-PCR and inserted into eukaryotic vector pENTER-His. After validation by PCR,enzymatic digestion,and sequencing,the HEK293 cells were transfected with the recombined plasmid pENTER-IL-10RA-His. The expression level of IL-10RA at 48 h was determined by Western blotting. The results revealed that plasmid was cloned correctly,and the target protein of 63 kD was observed. When HEK293 cells were simultaneously transfected with the plasmid of both JAK1 and IL-10RA,the target protein band in 133 kD and 63 kD were identified. Co-immunoprecipitation validated the interaction between JAK1 and IL-10RA. In conclusion,IL-10RA gene was successfully constructed in recombined plasmid pENTER-His and expressed effectively in HEK293 cells. The interaction between JAK1 and IL-10RA was validated co-immunoprecipitation,which lays a foundation for further understanding the mechanism of interaction between JAK1 and IL-10RA.

Prokaryotic Expression,Purification and Preparation of Polyclonal Antibody of C19orf18 Protein

TONG Xiao-Xia, YANG Yuan-Qin, CHEN Mian, WANG Huai-Yuan, HU Hai-Jun, ZHANG Kang-Jian

2017, 33(5):  176-182.  doi:10.13560/j.cnki.biotech.bull.1985.2016-1031

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The purpose of this study is to clone the human C19orf18 gene,construct the prokaryotic expression system of C19orf18,and prepare the recombinant human C19orf18 protein and its corresponding rabbit polyclonal antibody. According to the sequence reported in GenBank database,we used PCR to amplify intracellular and extracellular segments,and ligated them by flexible peptide,and inserted the target segment into prokaryotic expression vectors pET28a and pET32a. Then the vectors of high expression were selected by inducing a little expression of target protein. Further the highly expressing vector was employed for expressing the great amount of target protein. The C19orf18 protein was purified by Ni-NTA affinity column,and then identified by SDS-PAGE. Following that,New Zealand white rabbits were immunized with the purified proteins four times to produce polyclonal antibody. Specificity of the product polyclonal antibody was affirmed by Western blotting. Our results showed that pET28a was a better vector than pET32a for the expression of C19orf18 protein. More importantly,C19orf18 protein with truncated amino acids was expressed in prokaryotic cells,and its corresponding rabbit polyclonal antibody with high specificity was prepared successfully.

Enzymatic Characteristics of a Novel Protease and Its Application for Dehairing

ZHENG Xiang, YANG He-bao, HU Mei-rong, LIU Chun-mao, WU Fang-tong, CAO Qian-rong

2017, 33(5):  183-189.  doi:10.13560/j.cnki.biotech.bull.1985.2016-1098

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Protease can be applied in dehairing process of leather industry as an eco-friendly product. We aim to clear the dehairing performance and the process of enzymatic dehairing of JW-3 protease produced by recombinant strain WB800N/pHT43-npr,and to provide technical support for clean technology of leather manufacture. The thermostability and pH value tolerance of JW-3,as well as the effects of metal ions and chemicals on its enzyme activity were investigated. Meanwhile,the ability of enzymatic hydrolysis on collagen,and the comparison between enzymatic dehairing and lime-sulfide dehairing process on cowhide were evaluated. Results showed that JW-3 protease was in poor thermal resistance that enzyme activity began to decay after remaining 1 h at 40℃,and presented solid stability at pH 6 and 7,and was stable in the surfactant of Triton X-100,Tween20,Tween80;moreover,metal ions of Ca²⁺,Mn²⁺ activated its activity. Furthermore,JW-3 protease was incapable of hydrolyzing collagen,and showed the equivalent dehairing performance with lime-sulfide process,also by which nondestructive depilated hair could be recycled. Thus,JW-3 protease can complete cowhide hair removal process,but not hydrolyze the collagen of cowhide,and can be used as an environmentally friendly alternative to the conventional chemical process.

Screening and Identification of an Antagonistic Strain Against Fusarium moniliforme in Rice and Its Antifungal Activity

LI Yu-yang, XIN Han-xiao, FAN Xue-ming, LIU Li-ying, SUN Zhong-tao

2017, 33(5):  190-196.  doi:10.13560/j.cnki.biotech.bull.1985.2016-0826

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This work is to select an antagonistic strain against Fusarium moniliforme in rice from the rhizosphere soil of rice,and study its primary inhibitory effect on plant pathogens and biocontrol effect. The strain was separated from the rhizosphere soil of rice by the plate dilution method,using the F. moniliforme as the indicator strain for the antagonistic strain being screened by plate confrontation method. It was identified based on morphological,physiological and biochemical characteristics and 16S rDNA phylogenetic analysis. The effects of the sterile fermented liquor of the strain on the hypha growth of F. moniliforme were analyzed,and its antagonistic spectrum was measured,also pot experiment was conducted. Six strains were isolated,and one SH15 presented highly antagonistic effect on F. moniliforme,and was identified as Paenibacillus polymyxa. Sterile fermented liquor of strain SH15 showed significant inhibitory effect on hypha growth of F. moniliforme. Strain SH15 had broad antimicrobial spectrum,antibacterial activity on F. moniliforme,Fusarium proliferatum,Fusarium oxysporum,Phytophthora capsici,Phytopthora infestans,and Alternaria cucumerina. Rice pot experiments revealed that inoculation of the P. polymyxa SH15 significantly reduced the disease index of Fusarium moniliforme in rice with the average efficiency 65.68%. Therefore,there is certain application value for P. polymyxa SH15 in biological control of rice bakanae disease.

Degradation of Sulfamethazine by Co-culturing of White-rot Fungi

GUO Xiaodanna GUO Xia-li

2017, 33(5):  197-202.  doi:10.13560/j.cnki.biotech.bull.1985.2016-0905

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Sulfonamides are a class of antimicrobial agents that have been detected in China at high-frequency. Biological method has the advantages of no secondary pollution and green environmental protection,but the researches on biodegradation of sulfonamides is still scarce. This experiment was to investigate degradation of sulfamethazine（SM2）by co-culturing Trametes versicolor and Phanerochaete chrysosporium. Antibiotic concentration and enzyme activity were determined using high performance liquid chromatography（HPLC）and UV spectrophotometer. A deadlock between the two mycelia was observed on plate confrontation method of an agar medium. The laccase activity at the interaction zone was higher than that at the other regions in the incubation of the two white-rot fungi on an agar plate. The crude enzyme preparation with high laccase concentration was obtained at the co-culturing time of 4 days in liquid medium. At 48 h after incubation,the degradation percentage of SM2 by crude enzyme solution from co-culturing was 25.4%,which was higher than that by crude enzyme preparations from Trametes versicolor（9.6%）. When natural mediators and artificial mediator 2,2’-azinobis（3-ethylbenzthiazoline-6-sulfonic acid ammonium salt）（ABTS）were added to the reaction mixture,the degradation percentages of SM2 by the crude enzyme from mixed culture significantly increased to 49% and 93.1%,respectively. The results showed that the interspecific interactions involving P. chrysosporium and T. versicolor enhanced the SM2 oxidation through increasing laccase activity and producing potential mediators. Therefore,the co-culturing of different white-rot fungi has potential for application in antibiotic-contaminated soils and wastes.

Isolation and Characterization of Three Bacterial Strains Capable of Phosphate-solubilizing from Soil

LI Bai LI Jun SHEN Ya-qiang

2017, 33(5):  203-209.  doi:10.13560/j.cnki.biotech.bull.1985.2016-0915

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This work is to isolate and identify the phosphate-solubilizing microorganisms and develop microbial complex agents targeting for solving the problems of agricultural non-point source pollution. The organic phosphorus pesticide “chlorpyrifos”,“trichlorfon” and insoluble inorganic phosphorus Ca₃（PO₄）₂ were used as phosphorus source to screen and identify the phosphate-solubilizing microbe from soil,as well as analyze their phosphate-solubilizing capacity. The result showed that three phosphate-solubilizing bacterial strains（W,Y and B）were isolated from the soil. All three strains were gram-negative strains. The degradation results of organophosphorus pesticides showed that strain W had the strongest degradation rate of trichlorfon with 17.39%,and strain B had the strongest degradation rate of chlorpyrifos with 23.06%. All three strains solubilized significantly solid insoluble phosphate,and the B strain had the highest phosphorus-solubilizing capacity of 96.31 mg/L. The solubilizing phosphate results of single strain and compound strains showed that the effect of compound strains was significantly better than single strain. When W,Y,and B strains mixed with EM,the phosphorus-solubilizing capacity rose to 119.46 mg/L,and the phosphorus-solubilizing capacity in paddy fields and greenhouse soil was improved to 18.38 mg/L and 14.08 mg/L,respectively. In conclusion,three strains isolated from soil have the abilities of degrading organic phosphorus pesticide and solubilizing inorganic phosphorus Ca₃（PO₄）₂. The compound strains significantly improved the phosphorus-solubilizing capacity of soil significantly.

Isolation,Identification and Characterization of Arsenic-tolerant Bacteria from Lead-zinc Mine Tailing

WU Dan ZHANG Zhi-peng MA Yu-chao

2017, 33(5):  210-218.  doi:10.13560/j.cnki.biotech.bull.1985.2016-0983

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The aim of this study is to isolate highly arsenic-tolerant bacterial strains from heavy metal contaminated soils from the Kangjiawan lead-zinc mine,Hunan Province. Serial dilution and plating method was used to isolate arsenic-tolerant bacteria,and the strains were identified by 16S rRNA sequence analysis. Then the detection of arsenic tolerance-related genes was carried out. The arsenic molybdenum blue experiment was conducted to evaluate arsenic oxidation-reduction ability of arsenic-tolerant bacteria. Furthermore,the indole acetic acid（IAA）produced by the dominant bacterial strains was quantitatively determined. A total of 152 arsenic-tolerant bacterial strains were isolated from soils samples,and six strains were resistant to As⁵⁺ and As³⁺ up to 800 mmol/L and 20 mmol/L,respectively. The six arsenic-tolerant bacterial strains belonged to five different genera,Pseudomonas,Ochrobactrum,Bacillus,Williamsia,and Arthrobacter. In arsenic tolerance-related gene detection experiments,arsenate reductase gene arsCexisted in strains Tw31,Tw133,Sw149,and Tw222,and strains Bw218 and Tw222 contained arsenic ion efflux permease gene arsB/ACR3（2）. Within 72 h,the As³⁺ oxidation rate and As⁵⁺ reduction rate of strains Tw133 and Tw222 were higher than those of other strains,which were about 17 % and 35 %,respectively. In particular,strain Tw133 had an As⁵⁺ reduction rate of 48.66 % at 144 h. Moreover,the synthetic quantities of IAA in the two strains were 42.86 μg/mL and 24.36 μg/mL,respectively. The isolated strains Tw133 and Tw222 showed obvious advantages in terms of arsenic tolerance,arsenic oxidation-reduction capacity and IAA production,providing experimental materials for the further study of arsenic-tolerant mechanism in bacteria.

Gene Cloning and Subcellular Localization of DmpA from Nocardia seriolae

XIA Li-qun, CHEN Rui-min, LIAO Bao-shan, XU Liang, SU Ze-jie, TONG Bang-zhuo

2017, 33(5):  219-227.  doi:10.13560/j.cnki.biotech.bull.1985.2016-0968

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Nocardia seriolae,a facultative intracellular bacterium,is the main pathogen of fish nocardiosis. Bioinformatics analysis showed that the DmpA gene of N. seriolae encoded a secreted protein,and may be co-located with mitochondria of the host cell. In this study,the recombinant plasmids pEGFP-DmpA and pcDNA-DmpA were constructed,extracellular product of N. seriolae was identified,and the subcellular localization and over-expression of DmpA were carried out. The results showed that the protein DmpA was identified in the extracellular products of N. seriolae,and confirmed as a secreted protein. Subcellular localization of DmpA-GFP fusion proteins were evenly distributed in the whole cell of FHM cells,and were not coincided with the distribution of mitochondria,indicating that the protein DmpA was not co-localized with the mitochondria. The expression of DmpA changed the distribution of mitochondria into lumps in the FHM cell. The over-expression of DmpA in FHM cells had no significant effect on the nucleus,revealing that the gene had no function on inducing cell apoptosis. The cloning,subcellular localization and over-expression of DmpA from N. seriolae laid the foundation for further studying the function of the gene and promoting the understanding of the pathogenic mechanism of N. seriolae.