Table of Content

14 October 2013, Volume 0 Issue 10

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Review

Advance on JMJC Proteins in Arabidopsis and Oryza sativa

Qin Qiao Zhang Haiwen Huang Rongfeng

2013, 0(10):  1-5. 

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Epigenetics is to study heritable changes in gene expression and function that occur without a change in DNA sequence.
Jumonji C（jmjC）domain-containing proteins have been identified as histone lysine demethylases, which regulate gene expression and plant development though influencing chromatin remodeling, genomic imprinting or other approaches. In this review, we described the research advance of JMJC proteins during recent years, which could provide a reference for further exploring how JMJC histone demethylases influence the growth and development processes in Arabidopsis and rice.

Research of Reactive Oxygen Species in Plants and Its Application on Stress Tolerance

Xue Xin, Zhang Qian, Wu Jinxia,

2013, 0(10):  6-11. 

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The ROS（Reactive oxygen species）is a plant byproduct of aerobic metabolism，environmental stress also makes a large amount of a ROS accumulate in plant cells. Low concentrations of ROS can exist as a signaling molecule to induce the expression of defense genes and plant adaptation to the environment reaction. When the stress force in plant cells produce large amounts of reactive oxygen species, it will cause damage to intracellular macromolecules and other components, to impede normal plant metabolism and growth, and even death. Plants existence of active oxygen scavenging mechanism, the balance can be maintained within a certain range of reactive oxygen species. Studies have shown that the plants active oxygen scavenging mechanism can improve the resistance of plants. In this paper, an overview of the current dynamic of Reactive Oxygen Species, at the same time explore the application of plant active oxygen scavenging mechanism in improving plant stress resistance.

Review on Crosstalk Regulation Involving in Sugar Signal in Plant

Bai Hua, Wang Peiyun, Tian Xiaowei, Yao Xinling

2013, 0(10):  12-17. 

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Sugar is not only metabolite, but also as a signal molecule to regulate metabolic process to adopt diverse environments. Among the sugar, trehalose-6-phosphate（T6P）is one of signal molecules highlighted. Based on recently progress of study on T6P through molecular biological approach, This review discussed pathways of metabolism regulation of T6P as a molecular signal involving in, including diurnal cycle and phytohormone as well as response of enviroment stress. Function, behavior, questions remaining to answer and methods of study relevant to T6P as a molecular signal were evaluated in the pathways.

Research Progress of Molecular Markers for Forage Germplasm Resources Important Traits in China

Xu Chunbo, Wang Yong, Zhao Laixi, Zhao Haixia

2013, 0(10):  18-23. 

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With the rapid development of molecular markers, the technology is becoming more and more mature and has been an important means of studying forage germplasm resources. This article provides an overview of research progress on molecular markers, which used in yield, quality, stress resistance, insect resistance and other important traits of forage, analyze the problems in molecular markers with critical traits of forage and looks forward to the application prospect in order to provide references for further research on the forage germplasm resources.

Calreticulin and Apoptosis Mediated by Endoplasmic Reticulum Stress

Wu Wei Liu Chaoqi

2013, 0(10):  24-27. 

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Calreticulin（CRT）is a 46 kD endoplasmic reticulum luminal protein and has various biological functions including chaperone activity, regulation of Ca2+ homeostasis, cell apoptosis. Endoplasmic reticulum stress（ERS）is pathological changes of subcellular organelles, early time of ERS can be reversed, as the time go, cellular damage is more and more serious result in apoptosis. New research shows that CRT plays an important role in apoptosis mediated by endoplasmic reticulum stress. This review explained clearly the interrelation between them and was expected to provide some information for the study.

Application of SUMO Protein in Fusion Expression System

Wang Xiaojie Mao Ruoyu Zhang Yong Teng Da Wang Xiumin Wang Jianhua

2013, 0(10):  28-33. 

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The expression of toxic protein is more challenging than common protein. Fusion expression strategy may be a good choice to reslove this problem. Traditional fusion tags including thioredoxin, green fluorescent protein, maltose binding protein, ketosteroid isomerase, NusA, glutathione-s-transferase have been widely used. Besides the characteristics of the traditional fusion tags above, SUMO protein has other advantages, such as small molecules, promoting folding and specific cleaved by SUMO protease 1. As a result, it has been widely used recently.
This article depicted the characteristics of SUMO and SUMO pretease. Additonally, the application of SUMO fusion expression system was elaborated.

Prohibitin and Oxidative Stress

Li Tiesong Gao Yang Wang Ying Li Qingwei

2013, 0(10):  34-39. 

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Prohibitin（PHB）, a highly-conserved protein, widely distributed and functional diversity, is encoded by the nuclear genes and located in nucleus membranes, mitochondrial membrane substrates and matrixes. It has been paid high attention because of its participation in a variety of biological processes. Now it is becoming a new research hotpot, because the special position on the mitochondrial and its special structure and function on it. In accordance with the studies of the relation of PHB and mitochondrial, PHB's important role was systematically elaborated in oxidative stress and the related diseases and the process of ageing in this paper, and the PHB's prospect of application was also looked into the future.

SarA Family Proteins and Its Post-Translational Modification in Staphylococcus aureus

Wu Ruirui Zhu Fuxing

2013, 0(10):  40-45. 

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A class of family proteins found in staphylococcus aureus genome is similar to SarA, it contributes to the main physiological and virulence of staphylococcus aureus. protein cysteine phosphorylation of MgA/SarA/SarZ family or Oxidation and reduction modification can caused the change of protein conformation, which regulates the virulence factor of S. aureus and to vancomycin-resistant. SarA family proteins are homologous and interacted, and its post-translational modification is similar.

Development of Heavy Metal Ions Detection Biosensors

Wang Minghua, Zhao Erlao, Li Dujuan

2013, 0(10):  46-51. 

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This review discussesd on the detection methods of heavy metal ions pollution, including enzymatic biosensors, cell biosensors, microbial biosensors. The flexibilities of different transducers for biological elements was analyzed, and finally, it point out that the automated and miniaturized biosensors based-on bio-technologies and the detailed relations between structure and function would be potential meet to the need for heavy metal ions monitoring.

Normalisation Strategies for Plasma/Serum MicroRNAs Quantitative Analysis

Wang Yan Tang Naping Qiu Yunliang Nan Yaping Ma Jing

2013, 0(10):  52-57. 

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In recent years, the researchs on assossication between the circulating microRNAs from blood and various disease or tissue damage have attracted much attention. However, the imprecision of microRNAs concentrations may be caused by cellular contamination or variance attributable to extract and process RNA. Several strategies have been proposed for normalising these causes. In this review we discuss the relative advantages and disadvantages of different normalisation strategies and instruct researchers to choose a valid normalisation strategy that will enable the measurement of biologically meaningful results.

Application of BAC-FISH in Plant Genomics Research

Gan Yimei, Zeng Fanyun, Zhang Shuzhen, Yang Benpeng

2013, 0(10):  58-65. 

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Fluorescence in situ hybridization of bacterial artificial chromosome（BAC-FISH）is a new technology that BAC clones with different characteristics can be targeted directly to chromosomes, which plays an irreplaceable role in researches of plant genomics and molecular cytogenetics. We reviewed the application of the identification and karyotype analysis of chromosomes, the construction of genetic and physical maps, comparative mapping analysis, the origin and evolution of plants, the physical location of genes as well as the resolution and sensitivity analysis of FISH.

Report

Specific Detection of GM Maize Events Using Aldehyde PCR Chip

Zhang Mingzhe, Zhang Xiaofeng, Chen Xi, Wu Shan, Chen Xiaomei, Zhou Yuan

2013, 0(10):  66-70. 

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In this study, 9 kinds of genetically modified maize events Bt11, TC1507, Bt176, MON810, MON863, GA21, NK603, Mon88017, MIR604 were under high-throughput detection and condition optimization with aldehyde PCR chips. According to the specificity and sensitivity experiments, it showed that the specificity of the aldehyde PCR chip is good enough to be applied in the identification of genetically modified maize events. But the sensitivity of this method was 5%, if it is applied to the transgenic detection. It calls for further optimization in the follow-up experiments.

Transient Expression of ha FGF in Pea Plants by New Agroinoculation Biotechnology

Yang Liping Jin Taicheng Zhou Xiaofu

2013, 0(10):  71-75. 

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The paper described a simple system to product ha FGF protein in pea plants by seed absorbing the Agrobacterium suspension containing PEBV-based vectors, and the expression results was detected by semi-quantitative RT-PCR, Western blotting, and Coomassie brilliant blue method. It has been shown that GFP and ha FGF protein were successfully expressed in pea（Pisum sativum L.）plants. The optimization of Agrobacterium cell density dramatically improved expression efficiency of GFP protein. The amount of recombinant pharmaceutical ha FGF in plants reached up to 2.6% of the total soluble proteins. The new system has the special advantages as easiest way, high efficiency, stable expression, and has the wide application in production of pharmaceutical proteins in the future.

Cloning and Sequence Analysis of 1-aminocyclopropane-1-carboxylate （ACC）Oxidase Gene cDNA from Gynoecious Momordica charantia

Wang Risheng, Zhang Man, Huang Rukui, Liu Wenjun, Dong Wenbin, Che Jianglü, Fang Fengxue, Li Yangrui

2013, 0(10):  76-80. 

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The full cDNA sequence of 1-aminocyclopropane-1-carboxylic acid oxidase（ACO）gene was cloned by RT-PCR and RACE from floral buds of gynoecious bitter melon‘X-Hei-d-d’, which was named as Mc-ACO1（GenBank accession No. FJ459813）. The full length cDNA of Mc-ACO1 is 1 137 bp, which includes 1 005 bp complete CDS and encodes 334 amino acid residues with a putative molecular mass of 37.30 kD. The protein encoded by Mc-ACO1 gene lacks one typically conserved domain and two amino acid residues of Fe2+ ascorbate family of dioxygenases. The bioinformatics characterization indicate that the evolution of plant ACO has higher consistence with their genetic relationship ； and the different amino acid sequences of Mc-ACO1 are mainly at the N-terminal area compared with those of other plants ；Mc-ACO1 should belong to the same member as cucumber Cs-ACO2, and the presumed function of Mc-ACO1 is probably related with sex differentiation.

Identification of Rust Resistant Genes Based on SCAR Markers in Common Bean（Phaseolus vulgaris L.）

Wu Xingbo, Yue Huan, Hao Junjie, Zhang Xiaoyan, Li Hongwei, Wang Zhenqing, Lü Xianghua

2013, 0(10):  81-86. 

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Ten SCAR primer combinations（SK14 、SA14 、SI19 、SBC6 、SAD12 、SAE19 、UR11-GT2 、KB126 、SF10 and SBA8） of common bean rust resistant genes were used to check the genome DNA of 78 common bean accessions. The SCAR primer pairs, SA14 and KB126, amplified bands without difference among all the tested accessions ；SI19 and SAD12 were not amplified target bands at all ；SK14 marker appeared in 8 accessions, SBC6 marker in 23 accessions, SAE19 marker in 28 accessions, UR11-GT2 marker in 5 accessions, SF10 marker in 62 accessions, and SBA8 marker in 76 accessions. 2-5 SCAR markers appeared in 65 accessions respectively. The types of rust resistant genes in each of the 78 accessions have been identified, and we found common that several rust resistant genes aggregated in one common bean accession.

Extraction of High Quality Total RNA from Ammopiptanthus mongolicus with Improved Trizol Method

Chen Jing Gao Fei Zhou Yijun Zhang Zichen Li Zhanglei Cao Yuzhen Zhang Zhiwei

2013, 0(10):  87-92. 

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Total RNA was extracted using an improved Trizol method, with the leaves and roots of xylophyta, Ammopiptanthus mongolicus （Maxim）Cheng f. The result showed that compared to traditional Trizol method, the improved Trizol method can effectively remove residual guanidinium isothiocyanate, beta-mercaptoethanol in extraction process, as well as the sugar and polyphenols of the plant itself, and high-quality total RNA was obtained. The improved Trizol method has been successfully applied to transcriptome database constrction of A. mongolicus. It also provided a reference for xylophyta to extract high-quality RNA.

Influence of Trewia nudiflora Seed Extract on the Antifungal Compound of Endophyte

Wu Xin

2013, 0(10):  93-97. 

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The chemical interactions between endophytes and plant hosts have been an interesting topic since the coining of the concept of symbiosis and co-evolution. Streptomyces sp. WXC was an endophytic strain isolated from the seed of Trewia nudiflora. Culture medium optimization revealed that supplemented with the aqueous extract of T. nudiflora seeds, S. sp. WXC grew faster, and the production of frenolicin B was increased by ca. 3-fold as well. Real-time PCR analysis revealed that the expression levels of frenolicin B biosynthetic genes were induced by the seed extract, which correlated to the increased production of frenolicin B indicated by HPLC analysis. These results indicated that host’s chemical components may induce the endophytic S. sp. WXC to increase the production of antifungal compound. This work shows, for the first time, that intimate chemical interaction exists between endophytic actinomycete and its host.

Study on Ethanol Extracting Conditions of Taxol from Leaves of Taxus Chinese var. maria

Yu Xianghua Dai Yongqiang Shao Jinhua

2013, 0(10):  98-102. 

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The best ethanol extraction technology of paclitaxel from leaves of Taxus chinensis var. mairei was established. By a serials of single-factor experiments and L16（43）orthographic experiments, the extraction conditions were determined .The main factors contained Zerkleinerungsgrad 、ethanol concentration 、solid-liquid ratio and extraction time, the results showed that the best extraction conditions as follow ：120 mesh, ethanol concentration was 95%, solid-liquid ratio was 1∶15, the extraction time was 4 hour, repeated 2 times, the leaching temperature was 40℃. Then elutioned with ethyl acetate-acetone（6∶4）through a small chromatographic column, and detected by HPLC.
Under these conditions, the total paclitaxel extraction yield can reach up to 0.012 5 mg/g（dry weight）.

The Correlation Between a Novel SNP on Chromosome 7 and the Trait of Sheep Tail or Rump

Liang Yaowei Zhang Wei Shen Min Li Huan Gao Lei Yang Jingquan Liu Shouren Gan Shangquan Wang Xinhua

2013, 0(10):  103-108. 

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The fat-tailed or fat-rumped is one of important trait of stress tolerance, however, the mechanism of fat deposition is still unclear. Therefore, the present study choose a novel SNP reported in recently literature as a candidate molecular marker and detect the polymorphism among Altay, Kazakh, Hu, Chinese Merino and Suffolk which has extreme differences in tail type by using PCR-RFLP method. In addition, a mathematical model was employed to analyze the correlation between the polymorphism and the trait of fat-tailed or fat-rumped. Our results showed that the frequency of G allele was highest in Altay which had higher phenotype score, and the frequency of A allele was rather higher in thin-tailed sheep breeds. The result of mathematical model showed that the ratio of G/A increased exponentially with the phenotype score increasing. Together, the results suggested that there was significant differences in the SNP distribution between fat-tailed and thin-tailed sheep populations, and the SNP could be used as an ideal molecular marker to help high and low fat sheep breeding.

Construction and Analysis of Luciferase Reporter Vectors of Mouse Dnmt1 Gene Promoter

Zhang Yuan, Yang Xunxun, Wu Fengrui, Liu Yong, Ding Biao, Wang Caihong, Huang Jichang, Li Wenyong

2013, 0(10):  109-112. 

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To study the different 5'-flanking regions activity of mouse Dnmt1 gene, 4 truncated Dnmt1 promoters were amplified by polymerase chain reaction（PCR）from mouse genomic DNA and subcloned into pMD19-T vector, and then cloned into the pGL3-Basic luciferase reporter gene vector to construct Dnmt1 promoter-pGL3-Basic recombinants. After the recombinants were transfected into NIH/3T3 cells with Lipofectamine, relative luciferase activity were obtained by using the Dual-Luciferase Reporter Assay System and Glomax. Results showed that the recombinants of 5'-flanking sequential deletion of mouse Dnmt1 promoter-pGL3-Basic reporter gene were successfully constructed. The relative luciferase activity of Dnmt1-1-pGL3-Basic was three times higher than the other three truncated constructs. Together, this study was preliminary confirmed that the mouse Dnmt1 promoter region（-1 866-+57 bp）had strong transcriptional activity.

The Role of Vitamin C in Regulation of Aging of Postovulatory Mouse Oocytes Under Different Temperature

Li Qian,Cui Longbo, Zhao Zhenjun

2013, 0(10):  113-119. 

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To study the role of Vitamin C in regulation of aging of postovulatory mouse oocytes and find a suitable way to prevent oocytes aging, newly ovulated mouse oocytes were cultured at various temperature for various time in HCZB containing various concentrations of Vitamin C before examining for parthenogenesis activation and developmental potential. To assess the quality of oocytes cultured under optimization scheme that could effectively inhibited oocytes aging, early developmental competence after in vitro fertilization, distribution of cortical granule and levels of protein BCL2 were evaluated. Oocytes were cultured in HCZB with 200 μmol/L Vitamin C at 15℃ for 24 h before a 6 h recovery culture at 37℃ , which was the optimization scheme for inhibiting oocytes aging. The results suggested appropriate concentration of Vitamin C incorporating appropriate low temperature could effectively inhibit oocytes aging.

Foundation of Platform for Zebrafish Transgenic Technology

Liu Lili Wang Jian Wang Haisheng Yu Kaimin Li Guochao Yan Yanchun

2013, 0(10):  120-126. 

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The aim was to provide biomaterial, technique flow and protocol standard of transgenic zebrafish study via founding a platform of zebrafish transgenic technology. A couple of Tübingen zebrafish were multiplicated whose zygotes were microinjected with high active constitute plasmid pEGFP-N3. Injected eggs were collected and cultured and further observation. Fluorescent microscopic observation was followed by PCR on these embryos to screen and certify GFP expressed transgenic zebrafish. Results showed that the present study developed an intact cultivation system of zebrafish including its cultivation, reproduction and breeding. We also dug out the common emergency and proposed possible solutions during zebrafish maintenance. We designed the molecular processes with zebrafish embryo as material in our lab. Especially a platform of zebrafish transgenic technology was founded for the regeneration of transgenic zebrafish lines and study of transgene integration mechanism.

Screening and Identification of Actinomyces Q14 Antagonistic Against Alternaria Alternata

Lian Lihui Gao Lijun Yang Na Wang Tao Zhang Xianzhong

2013, 0(10):  127-130. 

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It was to screen the new antagonistic actinomyces against Alternaria alternata. Totally 36 strains of actinomycetes were obtained from eggplant roots. By dual culture on PDA plates, it was found that 6 strains（16.67% of total isolates）of actinomycetes showed satisfactory antagonistic effects to Alternaria alternate. Among them, strain Q14 exhibited the strongest antibiosis capacity with an inhibitory zone of 14.5 mm, and also showed good antagonistic effects to Fusarium oxysporum f. sp. cucumerinum and Fusarium oxysporum f. sp. lycopersici.
Strain Q14 was identified as Streptomyces polychromogenes according to its 16S rDNA sequence（GenBank acc. No. KC675186）analysis, and the morphological, physiological and biochemical characteristics.

Colonization of Bacillus subtilis BS24 on the Apple Leaf Surface and Their Effects on the Leaf Microbial Flora

Ran Ganqiao, Wang Nan, Dai Jiakun, Zhao Wenjuan, Ren Ping, Qin Tao

2013, 0(10):  131-136. 

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To confirm the biocontrol efficacy of the Bacillus subtilis BS24 strain against apple early defoliation disease, the colonization of BS24 strains on apple leaves and their effects on the other microbial species and populations were studied respectively. The results showed that under field conditions BS24 strains could colonize on apple leaves, but the population size declined with prolonging time. In the absence of rain and other inclement weather situations, the population size of BS24 strain increased from the initial 2.5×107 cfu/g to 2.1×105cfu/g after spraying the microbial agents for 15 days. Compared with the apple leaf microorganisms without spraying agents, the microbial populations on apple leaves declined after 20 days of spraying agents, meanwhile the bacteria of Pseudomonas oryzihabitans, Acinetobacter lwoffii, Bacillus lehensis and Kocuria rosea and the fungi of the Alternaria alternate, Eupenicillium janthinellum and Verticillium dahliae were not detected.

Screening and Identification of Interspecies Microorganisms Inducing Overproduction of Epothiones in Sorangium cellulosum

Li Yawei Zhao Lin Hao Yongwei Tang Xiuli Yang Jikun Gao Yuzhen Liu Xinli

2013, 0(10):  137-141. 

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Introducing other microorganisms generally can induce microorganisms to regulate their metabolism which is based on interspecies interactions. This research screened a strain of fungus named as Aspergillus fumigatus M03 that can induce Sorangium cellulosum to overproduce epthilones. The yields of epothilone A and B can be enhanced 72.3% and 59.7% respectively by the organic crude extracts from A.
fumigatus M03. Appropriate amount of its intracellular aqueous crude extracts also can improve the yields of epothilone A and B each by 25.6% and 24.7%, whereas the capability of improving disappears when the crude extracts are dealt with high-temperature, and inhibition of epothilones biosynthesis will show when the concentration of the crude extracts is high in fermentation broth of Sorangium cellulosum. The fermentation broth of A. fumigatus M03 does not promote biosyntheses of epothilones, but tends to inhibit them as the concentration increases.

Investigation on Diversity of Endophytic Bacterial Community in Xisha Wild Noni（Morinda citrifolia L.）Seed

Liu Yang, Li Hui, Li Jinxia, Cao Yanhua, Yao Su, Bai Feirong, Tan Wangqiao, Cheng Chi

2013, 0(10):  142-147. 

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Using the 16S rDNA clone library technique, a priliminary study was conducted on the community diversity of endophytic bacteria in seeds of Xisha wild Noni. The results indicated that the endophytic bacterial communities contained 42 Operational Taxonomic Units （OTUs）in their clone libraries respectively, involving 33 genus from 4 groups of Proteobacteria, Firmicutes, Bacteroidetes and Actinobacteria.
The dominant bacterium（abundance）were Enterobacter sp.（17.55%）, Bacillus sp.（16.79%）, Acinetobacter sp.（6.11%）, Pseudomonas sp.（6.11%）and Piscinibacter sp.（6.11%）. This was the first report of the study on endophytic bacterial communities in the Noni seeds with culture-independent method.

Constuction the Pichia pastoris Expression System of Neuritin and Its Impacts on PC12 Cell

Zhang Shujun, Zhao Chen, Di Jianjun, Mushamoli, Xian Lingling, Yu Na, Huang Jin

2013, 0(10):  148-152. 

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It was to intend to construct Pichia pastoris expression system of neuritin, to obtain large amount of active Neuritin protein and study its neurobiological function. Encoding cDNA sequence of neuritin was amplified by PCR, then the fragment was digested and ligated into shuttle vector pPIC9K. The neuritin-pPIC9K recombinant was transformed into E. coli DH5α, and was confirmed by PCR and sequencing. The recombinant was linearized by restriction enzyme Sal Ⅰ , and transformed into yeast GS115 by electric shock. After screening and identification, the Pichia pastoris expression system of neuritin was successfully constructed. Using methanol to induce the expression of neuritin, through SDSPAGE and Western blot analysis, we obtained Neuritin protein about 11 kD. The purified Neuritin protein was added into PC12 cell culture medium and it can promote the neurite growth of PC12 cell.

Saturation Mutagenesis and the Enzyme Properties of the Alpha-Amylase from Geobacillus sp.

Xue Bei, Pei Jianxin, Wang Zhibin, Luo Zhang, Wei Yutuo

2013, 0(10):  153-159. 

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In this study, manipulation of half rational design was performed. Firstly, the dimensional structure of AMY（alpha-amylase of Geobacillus sp. GXS1）was built via homology modeling by using a close-related（33% homology in sequence）maltogenic amylase（BSMA） from B. stearothermophilus as a template. Secondly, three amino acid positions at the active site of AMY（Asp192, Glu221 and Asp289）were selected by structural superposition onto AMY combined with energy calculation by the computer-aided design software ProSa2003. These three putative amino acids for the active site of AMY, Asp192, Glu221 and Asp289, were subjected to saturation mutagenesis using degenerate primers. Four mutants（Asp192Ala, Glu221Asn, Glu221Leu and Asp289Leu）were obtained from the saturation mutant library, and studied the enzymatic properties of mutation enzyme simply.

Expression of Fructose Valine Oxidase in Sf9 Cells

Xu Ying Zhao Xue Yu Yuanhua

2013, 0(10):  160-164. 

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It was to express the Fructose Valine Oxidase Gene with baculovirus expression system. FVO gene was artificially synthesized, and ligated to the vector pFastBac1 to obtain the recombinant vector, which was then transformed into E.coli DH10Bac competent cells to transposase with bacmid. After screening, the recombinant baculovirus Bacmid-pFastBac1-FVO was got, which appeared a single band after PCR identification. sf9 cells was transfected with the recombinant vector, and harvested the virus and expressed the protein. Results showed that the baculovirus expression vector was successfully constructed and expressed in the sf9 cells which appeared a 40 kD band with Western blot.

Effect of Overexpressed Exogenous Necdin on the Cell Proliferation of P19 Embryonal Carcinoma Cells

Hui Huandong, Liu Yong, Liu Shaojun

2013, 0(10):  165-169. 

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It was to construct eukaryotic express vector PcDNA3.1/necdin and obtain the monoclonal P19 cell highly expressing necdin, and then detect the effect of overexpressed necdin on the cell proliferation of P19 cell. Total RNA was extracted from normal P19 cells. RT-PCR was used to amplify the aimed segments necdin which was then digested with EcoR Ⅰ and Xho Ⅰ and inserted into a eukaryotic expression plasmid PcDNA3.1 to construct PcDNA3.1/necdin. The constructed vector was transfected into P19 cells through lipofectamine2000-mediated transfer method. The transfected cells were treated with G418 until monoclonal cells appeared. Expression level of Necdin in monoclonal P19 cells was assayed by Western blot. CCK-8 method was utilized to detect the effect of overexpressed necdin on the cell proliferation of P19 cell.
Results showed that eukaryotic express vector PcDNA3.1/necdin was successfully constructed and obtained monoclonal P19 cells stably and highly-expressing necdin. Detection of cell proliferation found no obvious change in P19 cells highly expressing necdin. The study showed that overexpressed exogenous necdin have no obvious effect on the cell proliferation of P19 cells.

The Overexpression of PELO in Human Embryonic Stem Cells

Hu Xulin Kee Kehkooi

2013, 0(10):  170-176. 

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Using full length human PELO cDNA as template, TOPO cloned PELO gene to pENTR-D-TOPO vector to form the pENTR-D-PELO vector, then performed the MultiSite Gateway LR recombination reaction between pENTR-5’-EF1α and pENTR-D-PELO to get the EF1α-PELO-p2k7 expression vector. PELO overexpression lentivirus was produced in 293FT cells and ransducing human embryonic stem cells to overexpress PELO.

The Prokaryotic Expression，Purification and Identification of Single Chain Antibodies 2F5 and 4E10

Liu Xiuxia Yang Xiong Chen Hongying

2013, 0(10):  177-183. 

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For further study of 2F5 and 4E10 antibody, 2F5-scFv and 4E10-scFv were expressed and purified from E.coli cells. The 2F5scFv and 4E10-scFv genes were amplified by overlapping PCR. Recombinant vectors pET28a/2F5-scFv and pET28a/4E1-scFv were constructed and transformed into E. coli BL21（DE3）and Rosetta-gami2（DE3）pLysS, and protein expression was induced with IPTG. 2F5-scFv and 4E10-scFv were expressed, with the molecular weight of 27 kD and 29 kD, respectively. The formed inclusion bodies in E. coli. Denatured single chain antibody proteins, which were purified by nickel chelating chromatography and refolded by dialysis, showed specific reactivates to both MPER antigens prepared from prokaryotic and eukaryotic expression systems.

Communication

Topic Evolution Analysis of the Field of Zebrafish Genetic from 2003 to 2012

Bai Yunxiao Sun Wei Zhang Xuefu

2013, 0(10):  184-188. 

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In this paper，we analyze the process of topic evolution of the field of zebrafish genetic，through cluster analysis，burst detection and drawing critical path. We focus on topic areas，hot topics and key nodes of the topic network. This paper provides an overview of zebrafish genetic area for researchers，and methods for reference.