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Table of Content

    25 June 2014, Volume 30 Issue 6
    Review and editorial
    Comprehensive Overview of JAZ Proteins in Plants
    Sun Cheng, Zhou Xiaojin, Chen Rumei, Fan Yunliu, Wang Lei
    2014, 30(6):  1-8. 
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    Jasmonates(JA)as an important phytohormone regulates many aspects of plant development, reproduction, and defence. Within the JA signaling cascades, jasmonate zim-domain(JAZ)proteins that are triggered by jasmonates play a central role. JAZ proteins regulate JA-responsive gene transcription by inhibiting DNA-binding transcription factors(TFs)in the absence of JA. However, in the presence of JA, JAZ proteins interact in a hormone-dependent manner with Coronatine Insensitive 1(COI1). After recognition component of the E3 ubiquitin ligase SCFCOI1, as a result, JAZ proteins are ubiquitinated and subsequently degraded in the 26S proteasome, releasing TFs from inhibition and activating JA-responsive gene transcription. This conclusion indicate that JAZ proteins, which function as transcriptional repressors of the JA signaling response, are not merely regulators of the JA signaling pathway, but, through interaction with other proteins, also serve as signaling hubs in the wider hormone regulatory network, plays important roles in plants..
    Advance of Studies on SVP Gene in Plants
    Liu Shinan,Lin Xinchun
    2014, 30(6):  9-13. 
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    SHORT VEGETATIVE PHASESVP)is an important repressor of flowering process which is expressed at the stage of vegetative development. SVP mediates the plants’ flowering time, by which participates in the formation of meristematic tissue of flowers and regulates the expression of some flowering integrator such as FLOWERING LOCUS TFT),SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1(SOC1)or FLOWERING LOCUS CFLC). The expression of SVP is affected by photoperiod, temperature and other factors. In the paper, review the present research progress of the SVP gene and homologous gene were reviewed, the prospects of future research activities were clisussed.
    Function and Regulation Mechanisms of Nitrate Transporters in Higher Plants
    Jia Hongfang, Zhang Hongying, Liu Weizhi, Cui Hong, Liu Guoshun
    2014, 30(6):  14-21. 
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    Nitrate(NO3-)is main nitrogen sources which plants absorb from soil. There are two different uptake systems in plants to cope with low or high NO3- concentrations in soil, the high affinity systems(HATS)and low affinity NO3- uptake systems(LATS). It is suggested that NRT1 and NRT2 families contribute to LATS and HATS for both NO3- uptake and distribution within the plant. Specific transport systems are essential for the uptake of NO3- and for its internal redistribution within plant. Over the past years, a significant advance has been obtained on the gene cloning, expression regulation and functional characterization of NRT1/2 transporters in higher plants. In this article, the research progresses on molecular level of known Nitrate transporters in higher plant in last decade were reviewed.
    Research Progress on Abscisic Acid Receptor and Signal Transduction Pathway
    Cao Jing, Lan Haiyan
    2014, 30(6):  22-28. 
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    Abscisic acid(ABA)is a multi-functional hormone widely existing in plant, by interaction with receptors and consequently with the complex signal network, it can play the physiological roles in regulation of plant growth and development, as well as mediating adaptive responses to diverse environmental stresses. The screening and identification of abscisic acid receptors have been controversial. Recently, there are some breakthroughs in the discovery of the ABA receptors, and its signal transduction has attracted great attention again. In this paper, the latest advances on identification of ABA receptors and ABA signal transduction were reviewed, and the future developing prospects were also discussed.
    Advances of NDR1 Gene Determined Broad-spectrum Disease Resistance in Arabidopsis
    Gong Qianyuan, Zhang Chao, Li Weimin, Zhang Yongqiang
    2014, 30(6):  29-33. 
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    The research of resistance genes is the basis of disease resistance breeding and plant diseases controlling. Arabidopsis NDR1(non-race-specific disease resistance 1)gene, encoding a plasma membrane protein, plays an important role in the R gene mediated disease resistance, by interacting with CC-NB-LRR(nucleic acid binding or coiled-coil leucine-rich repeat)class of antiviral proteins. Here, we summarized the latest progresses of Arabidopsis NDR1 gene and the broad-spectrum disease resistance of NDR1.
    Progress on Origins and Mechanism of Dwarfish Plants Mutant
    Bai Lijun, Yin Shuxia
    2014, 30(6):  34-39. 
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    Dwarf is one of essential agronomic features and the important research field in life science as well as the hot area in plant breeding. Based on dwarf origins, dwarf can be divided into mutated dwarf and spontaneous dwarf. The mutated dwarf includes physical mutation,  chemical mutation and  biological mutation. Much more researches about physical mutation have been done, especially in space mutation area. The study of dwarf mechanism mainly focused on plant endogenous hormone, including gibberellins,  brassinolides and auxins etc. Regarding the concrete dwarf mechanism of different plants, no clear classification and detailed researches are available now. This paper summarized the origins of dwarf mutant and their dwarf mechanisms, the dwarf origins and mechanism of the dwarfish plants mutant.
    The Research Progress of EPSP Synthase
    Xu Jie, Jiang Shiyun, Fu Fengming, Geng Pengfei, Huang Kai
    2014, 30(6):  40-50. 
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    5-Enolpyruvylshikimate-3-phosphate synthase(EPSP synthase for short), is the sixth enzyme of shikimic acid pathway and participates in the synthesis of aromatic amino acids and some of secondary metabolites. Meanwhile, EPSP synthase are not only targets of the herbicide(glyphosate), antibiotics, anti-parasitic drugs, but also is important regulatory site of promoting the accumulation of shikimic acid in the organism. In recent years, with the rapid development of molecular biology technology and the in-depth study of EPSP synthase, EPSP synthase genes have been widely used in resistance to glyphosate genetically modified crops, medicine and health, etc. The research progress of EPSP synthase were reviewed and prospects in this paper.
    Characteristics and Application Prospects of Bacterial Exopolysaccharides
    Li Mingyuan,Wang Jilian,Wei Yunlin,Ji Xiuling,
    2014, 30(6):  51-56. 
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    The majority of bacteria are packaged by extracellular polysaccharides,which play an important role in their survival and growth,whether in nature or pathological conditions.The unique bioactivities of bacterial expolysaccharides and their application prospects were paid great attention by the concerned people.The structure,physical and chemical characteristics,biological activities of polysaccharides produced by bacterium were introduced in detail in this paper,especially the application of several bacterial expolysaccharides in industry.The future development of bacteria polysaccharides was envisaged.It provides theoretical basis for the further development of functional bacteria and expansion of application of bacterial exopolysaccharides in industrial.
    Heparanase and Its Applications in Cancer Therapy
    Wang Jing, Chao Getu, Li Bei, Wang Zhigang
    2014, 30(6):  57-61. 
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    Heparanase(Hpa)is the only endo-glycosidase which cleaves heparan sulfate(HS)in mammalian. The enzyme also can disrupt the integrity of the extracellular matrix(ECM)and basement membrane(BM), to release various growth factors from the ECM. Hpa is associated with migration and invasion of cancer cells. Recently, a series of studies have indicated that Hpa was expressed in most advanced cancers, especially in malignant cancer, while decreased expression of Hpa is helpful to inhibit the migration of cancer cells. Heparanase can be used as a potential therapeutic target for cancers. This paper reviews the structure and functions of Hpa, its role in promoting cancer metastasis and the application in cancer therapy.
    Technology and methods
    Applications of Liquid Chromatography-Mass Spectrometry in the Qualitative Analysis of Peptides and Proteins
    Fan Xuehai, Zhu Yishen, Chen Lanting, Li Wenhui, Wei Ping
    2014, 30(6):  62-66. 
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    With liquid chromatography-mass spectrometry(LC-MS)technology developing continuously, the system of LC-MS is widely used in pharmaceutical analysis, food analysis, environmental analysis and related fields. LC-MS provides powerful function of structure identification, which includes the ability of separation by LC and the advantages of high resolution, high sensitivity, high selectivity by MS. These advantages demonstrate that LC-MS will be applied in more related fields. In recent years, many researches were carried out in the identification of biological macromolecules by LC-MS. The analysis of peptide and protein qualitatively by LC-MS is reviewed.
    The Application of Uncultured Methods in the Study of Ruminal Methanogen Population
    Wang Xingwen,Wang Jiaqi,Zhao Shengguo,Li Fadi,Bu Dengpan
    2014, 30(6):  67-74. 
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    The ruminal methanogen are strictly anaerobic, which are hard to culture.The ruminal methanogen which have been cultured successfully are only 4 species.while hundreds of species of methanogen could not be cultured.The application and development of uncultured technology promotes the research of ruminal methanogen, which overcome the limitation of the tranditional technology of isolation and culturing. Several dominant culture-independent technologies used to study ruminal methanogens were introduced in this paper to provide references on methods for future ruminal methanogen study.
    Development of EvaGreen Real-time Fluorescence Quantitative Polymerase Chain Reaction to Detect Porcine Circovirus Type 2
    Cai Ye, Rao Pinbin, Wu Haigang, Jiang Yonghou
    2014, 30(6):  75-80. 
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    According to genome sequences of ORF4 of porcine circovirus type 2(PCV2)published in GenBank, a pair of primers was designed. The ORF4 gene was amplified with traditional PCR. The PCR product was cloned into pMD18-T vector and sequenced to construct positive recombinant plasmid. The recombinant plasmid was used as template for EvaGreen real-time PCR to generate standard curve and melt curve. The results showed that EvaGreen real-time PCR in the present study was highly specific while used to detect other nontarget viruses, and its sensitivity was proved to be 10 copies/μL and reproducibility for both intra-assay and inter-assay were less than 2.5%. Then the established method was utilized to test 118 clinical samples. The result indicated that the PCV2 positive rate was 25/118, which was 99.2% consistent with that of conventional PCR tests, except one sample that was positive for PCV2 by real-time PCR but negative by conventional PCR. Thus, this method in the current study showed the characteristics of specificity, sensitivity, reproducibility and simple operation, which could be used in subclinical diagnosis and epidemiological investigation of PCV2.
    Rapid Detection of Vibrio vulnificus by Loop-mediated Isothermal Amplification Combined with Lateral Flow Dipstick Assay
    Wang Yaohuan, Wang Ruina, Zhou Qianjin, Chen Jiong
    2014, 30(6):  81-87. 
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    A novel and rapid loop-mediated isothermal amplification(LAMP)combined with chromatographic lateral flow dipstick(LFD)assay was developed to detect Vibrio vulnificus. A set of six primers and a fluorescein isothiocyanate(FITC)-labeled probe that recognized V. vulnificus outer membrane protein TolC gene were designed. Biotinylated LAMP amplicons were hybridized exclusively with the FITC-labeled probe and detected by LFD assay. The assay was optimized and could detect V. vulnificus by incubation at 63℃ for only 35 min, and the whole detection procedure from extraction of bacterial genomic DNA to the visualization of the amplicons by LFD last 80 min. V. vulnificus could be accurately detected by LAMP-LFD, and no amplification could be observed when another 9 bacterial genomic DNA were used. The sensitivity for V. vulnificus detection in pure culture was 3.7×102 CFU/mL or equivalent to 7.4 CFU per reaction, which is 100 times higher than that of PCR assay. The results indicate that LAMP-LFD is an accurate, rapid and sensitive tool for V. vulnificus detection and can be used for detection of V. vulnificus in contaminated aquatic foods.
    Report
    Label-free Quantitative Proteomics Analysis of a Wheat Near-isogenic Line Pair with Q Exactive Mass Spectrometer
    Huang Yu,Feng Jing,Rao Liqun,Xu Shichang,Pan Yinghong
    2014, 30(6):  88-95. 
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    In this study, the protein expression of a wheat near-isogenic line pair, Taichung 29*6/Yr10 and Taichung 29, have been compared with label-free quantitative proteomic approach based on liquid chromatography tandem Q Exactive hybrid quadrupole-Orbitrap mass spectrometer. By MASCOT database search, a total of 2 257 proteins were identified from both of the samples, and among of them 1 549 proteins were accurately quantitated and 102 differentially expressed proteins(>2 fold)were detected. Gene ontology(GO)analysis showed that these differential proteins were mainly localized to cell matrix and organelles such as ribosome, and mainly involved in binding and catalytic functions. Among of 102 proteins which mainly take part in metabolic, cellular and stress responses processes, Superoxide dismutase, Methionine aminopeptidase, Lysosomal-β-glucosidase, and Ferritin may play some important roles in Taichung 29*6/Yr10 resistance to Yellow rust.
    Genetic Analysis and Molecular Mapping of Stripe Rust Resistance Gene in Wheat Line CH5383
    Zhan Haixian,Chang Zhijian,Li Guangrong,Jia Juqing,Guo Huijuan,Zhang Xiaojun,Li Xin,Qiao Linyi,Yang Zujun
    2014, 30(6):  96-100. 
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    CH5383 is an introgression lines from Thinopyrum intermedium with high resistance to wheat stripe rust and powdery mildew. The evaluations of disease were tested using Pst races CYR31 and CYR32 in seedlings. The result showed that the stripe rust resistance came from Thinopyrum intermedium in CH5383. Genetic analysis revealed that the resistance was controlled by a single dominant gene inoculating Pst race CYR32 inadult stage. It was temporarily named YrCH5383. Two polymorphic SSR markers, Xgwm108, Xbarc206 and Xbarc77 were linked to the resistance gene with genetic distance 8.2 cM, 10.7 cM and 13.6 cM, respectively. Based on marker loci and the origination, YrCH5383 might be a new gene to wheat stripe rust on 3BL.
    Genetic Diversity and Relationship Analysis of Broccoli with Its Related Species by SRAP Markers
    Jing Zange,Pei Xuli,Tang Zheng,Zhang Xiaoling,Luo Tiankuan,Liu Qing,Zhu Shiyang
    2014, 30(6):  101-105. 
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    In this study, we analyzed genetic diversity and relationship between broccoli and its related species by SRAP molecular markers. Using 28 SRAP primer combinations, a total of 302 amplified fragments were detected and 203 were polymorphic, with polymorphism rate 67.22%, indicating high polymorphism among these germplasm. Similarity coefficient analysis showed that the variation ranged from 0.461 5 to 0.900 6, and the average genetic similarity coefficient was 0.693 6. ‘Lü di’ and ‘Ai kang qing’ had the farthest genetic relationship with genetic similarity coefficient 0.461 5. By contrast, the relationship was closest between ‘Wzvcst-09-224’ and ‘Wzvcst-09-225’, with genetic similarity coefficient 0.900 6. Cluster analysis divided 16 germplasm into two major clusters. Class I contained Brassica oleracea, and class II only had one member Chinese no heading cabbage. The result of cluster indicated that the genetic basis had more similarity between broccoli and its related species, with a closer relationship, comparing with Brassica campestris ssp. chinensis Makino. The results also showed that the same geographic or origin could make the germplasm had relatively similar genetic background and closer genetic relationship. The research could be helpful to germplasm classification and excellent genes utilization for broccoli and its relative species, to speed up the breeding process.
    The Research on the Influence of Trewia nudiflora Seed Chemical Components on Its Endophyte
    Wu Xin
    2014, 30(6):  106-110. 
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    Streptomyces sp. WXC was an endophytic strain isolated from the seed of Trewia nudiflora. Trewia nudiflora seed chemical components were extracted and classified. The results showed that the production of frenolicin B was induced by the addition of the Water ext., which was correlated to the upregulation of growth and secondary metabolites. Therefore, we speculated that the inducible production of the antifungal frenolicin B may be an important adaptation mechanism allowing the symbiont, S. sp. WXC, to affect its host, T. nudiflora, through the function of symbiotic chemical defense.
    FST-related Genes Expression in FST Gene Knock-down Pig Fetal Fibroblast Cells
    Sun Yameng,Zhang Dongjie,Wang Liang,Zhang Xu,Yin Xue,Liu Di
    2014, 30(6):  111-114. 
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    In order to obtain FST gene knock-down pig fetal fibroblast cells and investigate the effects of related gene’s expression, the short hairpin RNA eukaryotic expression vectors for FST was constructed and transfected into pig fetal fibroblast cells. After screened with G418, transfected stable cell clones were obtained, and Real-time PCR was used to detect FST-related genes change in gene’s expression. The results showed that in stably transfected cell lines, FST gene expression was significantly inhibited, then resulting in the expression of ActivinRⅡA, Myostatin and Bmp4 downward trend, and the expression of MyoD gene very significantly reduced.
    The Improvement of Chicken Alpha Interferon Expression by a Recombinant Pichia pastoris with Cultural Condition Optimization
    Jiang Zhengjun,Liu Shuhai,Wang Maochao,Cheng Quanming,Huang Jin
    2014, 30(6):  115-119. 
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    To achieve excellent performance of Pichia pastoris for chicken alpha interferon expression, pH value, temperature, culture volume and carbon source feeding pattern were optimized. The maximum anti-virus activity(3×106 IU/mL)was harvested in the conditions of pH5.5, 28℃, and 40% culture volume in 10 L fermentor, as well as the glycerol-methanol(20∶1, V/V)co-feeding pattern was conducted when carbon source exhausted. This process was successfully scaled up to 50 L fermentor and the fermentation performance was also steady, which could be taken advantage of for its industrial application.
    Expression Analysis of Immune Genes at Different Induction Conditions of Third Instar Larvae of Musca domestica
    Xiu Jiangfan, Wei Chuanchuan, Chen Mingming, Wu Jianwei
    2014, 30(6):  120-127. 
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    We studied on the innate immune gene expression at different induction conditions of third instar larvae of Musca domestica. We chose the cold, heat stimulation and gram negative bacteria, gram positive bacteria and fungi induced the third instar larvae, 12 hours after the total RNA was extracted from third instar larvae.We designed the primers according to the published GenBank glyceraldehyde phosphate dehydrogenase(GAPDH), Attacin, Cecropin, Defensin, Diptericin, Lysozyme, heat shock protein(HSP)and Musca antifungal peptide(MAF-1)gene sequences of housefly. We were using the GAPDH as reference gene. Analysed the gene expression in different inducing conditions of third instar larvae of housefly through RT-PCR reaction. Results showed that in different inducing conditions, the genes related innate immunity expression showed large differences. Microbial stimulation showed the higher levels of gene expression than physical stimulation. The gene expression induction of fungi and positive bacteria was the highest, the cold stimulation induced the lowest. The innate immune system of the housefly larvae could activate by both microbial and physical stimulation. In different conditions of stimulation, the body’s immune response to different stress.
    Construction and Preliminary Application of the Yeast Two-hybrid cDNA Library from Grass Carp CIK Cells
    Yan Xiuying,Xie Jiguo,Li Jie,Ding Yu,Wu Zaohe,Lu Yishan,Jian Jichang
    2014, 30(6):  128-133. 
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    In order to explore the interactions between grass carp reovirus and the host cell proteins, the yeast two-hybrid cDNA library from grass carp CIK(Ctenopharyngodon idellus kidney) was constructed by SMART technology. Total RNA of the CIK cells was extracted and mRNA was purified. Then, mRNA as the template, the first strand of cDNA was synthesized by reverse transcription. The double-stranded cDNA was amplified through the long-distance PCR with the DNA polymerase. The cDNA library from grass carp CIK cells was constructed in the yeast strain 187, using SMART technology and the homologous recombination method. After testing, the conversion rate and the capacity of the original library was 1.6×105 and 2.4×106, respectively. The length of the inserted double-stranded cDNA fragments were 250-2 000 bp. The titer of the library was 7×107 CFU/mL and the recombination rate was 98%. This library had the well polymorphism and the integrity of the cDNA fragments. Using the constructed yeast two-hybrid from the CIK cells, the VP7 and VP5 protein in grass carp reovirus were as the baits for the screening experiment. The positive colonies of the interacting proteins of VP7 had been acquired, without the positive colony of the interacting protein of VP5. The construction of the yeast two-hybrid cDNA library from the grass carp CIK cells provided an important research tool for the study on the interaction mechanism of grass carp reovirus with the host cell.
    Construction of Interference Vector of EsSox21b-like Gene from Chinese Mitten Crab(Eriocheir sinensis)and Preparation of dsRNA by Prokaryotic Expression
    Liu Zhiqiang, Chen Jie, Qiu Gaofeng
    2014, 30(6):  134-138. 
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    Sox(SRY-related HMG-box)gene is a kind of important transcription factor, which functions in sex determination, organogenesis, etc. The gonad-specific expression of this novel gene suggested that it might play an important role in the development of gonad. To determine the exact role of EsSox21b-like gene using RNA interference, a EsSox21b-like-L4440 interference vector was successfully constructed for production of large amount of dsRNA. The target sequence of EsSox21b-like gene was inserted into the L4440 vector containing two T7 promoters. Then the recombinant plasmid was transformed into HT115 bacteria, a RNaseⅢ deficient strain. A large amount of EsSox21b-like sense and antisense RNAs was simultaneously transcripted in vivo when induced by IPTG. After denatured and annealed, EsSox21b-like double strand RNAs(dsRNAs)were produced and the yield of EsSox21b-like-dsRNA was enough for future RNA interference experiment on the role of EsSox21b-like gene.
    Development and Characteristics of Tetranucleotide Repeat Microsatellite Loci in Hyriopsis cumingii
    Han Xuekai,Li Jiale,Wang Zhaoqi,Bai Zhiyi
    2014, 30(6):  139-144. 
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    A microsatellite-enriched library of the Hyriopsis cumingii was constructed using repeat-enrichment method with biotin-labeled oligo(ATAC)5 and streptavidin coated magnetic beads. Forty-three pairs of primers designed by software PrimerSelect according to the sequences were synthesized for the pre-screening. Twenty pairs of primers which could amplify constant and legible expected DNA products were labeled by fluorescence. Seventeen microsatellite loci showed high levels in genetic polymorphism testing on 36 individuals sampled from Dongting Lake. The number of alleles at each locus ranged from 6 to 28. The expected and observed heterozygosities varied from 0.677 6 to 0.946 4 and from 0.638 9 to 1.000, respectively. The PIC value ranged from 0.630 to 0.929. Twelve microsatellite loci fitted to Hardy-Weinberg equilibrium. Compared with the previous dinucleotide repeat microsatellite developed in our laboratory, these 17 tetranucleotide repeat microsatellite loci showed higher polymorphism. Also, these 17 microsatellite loci can make genotyping easier by conventional methods such as polyacrylamide gel electrophoresis and save cost. Therefore, it offered accurate and inexpensive microsatellite markers for population genetic analysis and paternity test in Hyriopsis cumingii.
    Isolation of Microsatellite in C. ectenes by Magnetic Beads
    Deng Pingping, Shi Yonghai, Zhang Genyu, Zhang Zhiwen, Zhang Haiming, Lu Genhai, Liu Yongshi
    2014, 30(6):  145-149. 
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    It was a study to develop microsatellite markers with the method of microsatellite enrichment by magnetic beads. Genomic DNA of C. ectenes was digested with restriction enzyme Mse I. The fragments of 400-1 000 bp were recycled and ligated with relevant adaptors, then the “complete genome PCR library” was constructed. The PCR products were hybridized by a biotin-labeled Oligo probe(CA)12, the microsatellites were enriched with magnetic beads. The eluted fragments from magnetic beads were amplified and cloned into pMD18-T vector. 118 positive clones were screened with PCR method from 170 clones. After sequenced, 97 microsatellite sequences were found, 59 pairs of SSR primers were designed, and 9 pairs of primers were polymorphism within 30 cultured Chang Jiang C. ectenes.
    Construction of Genetic Recombination for Sea Cucumber Lysozyme in Bacillus subtilis
    Sun Lu, Liu Zhiwen, Zou Dan, Li Dan, Pan Bo, Cong Lina
    2014, 30(6):  150-154. 
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    The expression of lysozyme from sea cucumber Stichopus japonicus in Bacillus subtilis were constructed by the method of recombinant DNA technique, and the expression plasmid pHT43-SjLys of growth curve and stability were analyzed. Results showed that a specific strip about 400 bp was visible in the result of agarose gel electrophoresis, which was consistent with expected size of sea cucumber lysozyme gene. Construction of the lysozyme was performed in a vector pHT43 and validated by double digestion with BamHⅠand SmaⅠ, witch indicated the fragment size was consistent with the expected result. The growth trend of engineering bacteria was consistent compared with the wild type strain WB600, and insertion of foreign genes did not affect the cell’s metabolism. In the absence of selection pressure, the recombinant plasmid was stability, and there was no gene rearrangement and lost. The results showed that the recombinant sea cucumber lysozyme genetic engineering bacteria pHT43-SjLys/WB600 is constructed successfully.
    Molecular Cloning and Bioinformatics Analysis of T3SS Chaperone Escort Protein VscO from Vibrio alginolyticus
    Pang Huanying,Zhou Zejun,Ding Yu,Huan Yucong,Wu Zaohe,Jian Jichang
    2014, 30(6):  155-161. 
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    Primers for PCR cloning were designed according to the whole genome sequence of Vibrio alginolyticus published in GenBank. The type III secretion system(T3SS)chaperone escort protein vscO gene of V.alginolyticus strain ZJ03 was amplified by PCR and cloned into pMD18-T vector. Sequence analysis revealed that vscO gene is 462 bp and encodes a putative protein of 153 amino acids. The predicted molecular weight(MW)of VscO was 18.43 kD with an estimated pI of 9.22. Using SignalP 4.0 and TMHMM Server 2.0 software, it was predicted that the VscO protein was located in periplasm. It did not contain a signal peptide or a transmembranous region. The vscO gene with transcription initiation region at -35 and -10 region, and a translation recognition signal sequence(SD Shine-Dalgarno sequence). This protein had some active sites, such as phosphorylation sites. To further analyze the evolutionary relationship among VscO, a molecular phylogenetic tree was constructed using Mega 5.0 software. In this tree, the VscO protein showed high genetic relationship with Vibrio parahaemolyticus. The three-dimensional structure of VscO was determined using SWISS-MODEL work-space and it had a similar structure with YscO protein of V. parahaemolyticus. Signaling pathway analysis showed that VscO is located at the “needle” site of T3SS. Protein interaction map network found that the relation between VscO and 10 kinds of T3SS proteins was neighbourhood. These results can provide a basis for further studies on the chaperone escort machanism used by T3SS export pathway of Vibrio species.

    Colonization of Bacillus subtilis Y13 in Camellia oleifera Leaves and Its Effect on Native Bacteria
    Wang Ruiqin, Zhou Guoying, Liu Junang, Li Dongqin, Meng Qingmin
    2014, 30(6):  162-167. 
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    The colonization capacity of strain Y13 in the Camellia oleifera, was investigated by marking with rifampicin, periodical sampling, and recovering bacteria through diluting-plate method, as well as the effects of Y13 on native bacterial population were also studied by diluting-plate and 16S rDNA methods, which would provide a basis for ecological control of camellia anthracnose. The results showed strain Y13R displayed good colonization in camellia oleifera, 30 days after inoculation, 6.60×103CFU/g and 3.56×103CFU/g strains Y13R could be detected in healthy and diseased leaves, respectively. Endophytic bacteria isolated from Camellia oleifera belong to 5 genera of 7 species, Bacillus, Lysinibacillus, Klebsiella, Sphingomonas and Pseudomonas, Bacillus and Lysinibacillus was the most prevalent genera. The group and amount of predominant species had evident distinction in healthy and diseased leaves. The diversities of bacteria in the Camellia oleifera leveas were improved by Y13 treatment and suppressed the pathogen propagation.
    Construction of a Mangrove Soil Metagenome Library and Identification of Two Novel β-Glucosidase Genes
    Mai Zhimao, Su Hongfei, Li Lizhen, Zhang Si
    2014, 30(6):  168-172. 
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    Metagenomics provides a means of exploring new enzymes in microorganisms. A mangrove soil metagenome library with 100 000 clones was constructed. The average length of inserted DNA fragment is about 30 kb, and the total capacity of inserted DNA reached about 3 Gb. Seventeen clones with β-glucosidase activity were detected by screening 10 000 fosmid clones. The subcloning and sequencing revealed two novel β-glucosidase genes(MhGH3 and bgl66). Bioinformatics analysis indicated that MhGH3 and bgl66 composed of 1 992 bp and 2 025 bp in length, respectively. The deduced amino acid sequence of MhGH3 and bgl66 showed the highest sequence identity of 64% and 55% with the known β-glucosidase in GenBank datebase, respectively.
    Secretory Expression in Pichia pastoris and Characterization Study of Xylanase from Thermophilic Saccharomonospora viridis
    Yu Wangning, Liu Weina, Zheng Fei, Hou Lingyu, Wang Xiaoyu, Liang Di, Wang Weixuan, Zhang Shuo, Liu Jingyan, Jin Yi, Xie Xiangming
    2014, 30(6):  173-180. 
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    Abstract: Xylanase has attracted extensive attention because of the application potential in feed, food and paper area, capable of degrading hemicellulose which is prevalent in nature. This research is to construct a recombinant engineered bacteria with efficient secretory expression for thermophilic Saccharomonospora viridis xylanase SviXyN10A using molecular biology techniques, and then study the characterization of the gene product expressed in Pichia pastoris. The results indicate that the gene product of Svixyn10A was successfully expressed in Pichia pastoris, showing the highest enzymatic activity of 7.53 IU/mL with methanol induction for 2 days. Recombinant xylanase SviXyN10A showed good activity at wide range of pH effect(pH5.0-9.0), and the optimum reaction temperature is 70℃, pH7.0. What is more, the xylanase showed excellent thermo-alkali-stability and almost no cellulase activity. Thus, it is a potential candidate for future use in industrial areas particularly in the pulp and paper industry.
    Cloning,Expression of Thermostable β-mannanase and the Preparation of Mannooligosaccharide
    Ni Yujia,Zhou Minyu,Ouyang Jia,Zheng Zhaojuan,Yong Qiang
    2014, 30(6):  181-186. 
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    According to the Pichia pastoris’s codon preference, a DNA sequence encoding Aspergillus niger BK01 thermophilic β-mannanase gene was designed and synthesized. Firstly, it was inserted into pPICZαA and resulted in recombinant expression vector pPICZαA-man. Then, pPICZαA-man was linearized and transformed into different hosts by electrotransformation. An optimal recombinant stain KM71-MAN was obtained by screening activity. Using recombinant strain KM71-MAN, recombinant mannanase was overexpressed and its activity in the culture medium reached 2 318.85 IU/mL in a 3 L fermentor. Recombinant enzyme had an apparent molecular size of about 40 kD by SDS-PAGE, and optimal activity at pH 5.0 and 80℃. It was highly thermostable, retaining 43% of enzyme activity after 44 h of exposure at 70℃ and pH 5.0. Moreover, it remained over 85% activity from pH 3.0 to pH 7.0 after treating at 50℃ for 70 h. Using this crude enzyme, the main hydrolysis products yielded from konjak gum were mannobiose and mannohexaose and the yield of mannooligosaccharides was 55.6%. The recombinant enzyme exhibited good thermal and pH stability, which indicated that the recombinant yeast has potential value in preparation of konjac gum mannooligosaccharides.
    Study on Purification of Cellulase from Aspergillus niger AS0006
    Zhang Huan,Cao Yanxin,Jiang Lin,Wang Xiaoming,Liu Qi,Dong Xiaoying,Kou Wei
    2014, 30(6):  187-191. 
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    Two endoglucanases(EG)components and a cellobiohydrolase(CBH)from Aspergillus niger AS0006 were separated and purified by(NH42SO4 fractional precipitation, Sephadex G-25 and Sephadex G-100 chromatography. According to cellulase activity and electrophoresis detection found that the molecular weight of two EG components were 29.63 kD and 37.49 kD, respectively, the molecular weight of CBH component was 56.51 kD. Compared to the crude enzyme, the recovery rate of EG components could reach 18.07%, the purification fold was 3.46, the recovery rate of CBH component could reach 12.96%, the purification fold was 4.14, the recovery rate of β-glucosidase(CB)could reach 22.37% and the purification fold was 3.58.
    Display of Functionally Active Lipases on the Escherichia coli Cell Surface
    Xu Lixiang, Wang Zuozhen, Shu Zhengyu, Wu Hailong, Liu Yanru, Li Xin, Huang Jianzhong
    2014, 30(6):  192-198. 
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    Abstract: Cell-surface display technology was used widely in the filed of high throughput screening of a mutant library, which promoted the development of protein engineering. The carboxyl terminal domain of the EstA from Pseudomonas aeruginosa was used as carrier protein and the lipA gene was fused to the estA’ gene by overlap extension PCR. The fusion gene lipA-estA’ was then inserted the genetically modified plasmid pACYC-Duet, which promoter was changed into lacZ promoter. The resulting plasmid pBCMB-X1 was transformed into E. coli JK321 and E. coli UT5600, respectively. The lipA gene was induced expression by IPTG and the recombinant LipA was functionally displayed on the cell surface of E. coli JK321 and E. coli.UT5600, respectively. The hydrolysis activity of the LipA was(2.8±0.1)U/OD and(2.6±0.06)U/OD, respectively.
    Establishment of Agrobacterium tumefaciens-mediated Transformation System of Aspergillus ochraceus
    Cui Qian, Li Jie, Liu Xiaoguang
    2014, 30(6):  199-204. 
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    Filamentous fungus Aspergillus ochraceus TCCC41060is an industrial strain used for microbial steroid C11-α hydroxylation. In order to create genetic tools for A. ochraceus, the transformation system of A. ochraceus was establishedusing Agrobacterium tumefaciens- mediated system(ATMT). A. tumefaciens LBA4404 was used as the infective strain and hygromycin B gene as the selection marker. The major factors affecting the transformation efficiency including the concentrations of A. tumefaciens celland A. ochraceus spores, co-culture time, and co-culture temperature were investigated. Under the best conditions, the transformation efficiency of 57 transformants/107 spores was obtained. A. ochraceus transformants were confirmed by PCR amplification . Randomly selected 8 transformants were shown to be genetically stable after 10 rounds of successive cultivation. Results indicated this ATMT transformation system would provide important means for the directed genetic manipulation of this important industrial strain A. ochraceus TCCC41060.
    Optimization of the Synthesis of Alkyl Glucoside Catalyzed by Aspergillus aculeatus β-glucosidase-displaying Pichia pastoris Whole-cells
    Hu Xiayan, Liu Duanyu, Zheng Suiping
    2014, 30(6):  205-210. 
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    Aspergillus aculeatus β-glucosidase displayed Pichia pastoris cell surface was used as whole-cell biocatalyst, glucose as substrate, catalyzed synthesis alkyl glucosides of C6-C10 with reverse hydrolysis in water-alcohol two-phase system. The effects of various reaction factors, including water content, whole-cell biocatalyst dose, glucose concentration, pH and temperature were optimized. The catalytic effect of whole-cell biocatalyst were compared with commercial enzyme almond β-glucosidase and Novozym188 . The results showed that Novozym188 was not suitable for synthesis of APG, almond β-glucosidase need shorter time than whole-cell biocatalyst, but the final conversion of whole-cell biocatalyst was higher. In 5 mL total reaction volume, the optimal reaction conditions of HG:0.1 g glucose, 0.05 g whole-cells biocatalyst, 10% pH3.0 buffer;the optimal reaction conditions of OG:0.2 g glucose, 0.05 g whole-cells biocatalyst, 15% pH3.0 buffer;the optimal reaction conditions of DG:0.2 g glucose, 0.2 g whole-cells biocatalyst, 20% pH3.0 buffer, temperature 55℃, 200 r/min. After 72 h the maximum HG and DG yield could be 11.69% and 3.58%. The highest yield of OG was 6.34% after 96 h.
    High-error-rate Random Mutagenesis of GAP Promoter in Pichia pastoris Using an Optimited Error Prone PCR
    Qin Xiulin,Qian Jiangchao,Chu Ju
    2014, 30(6):  211-217. 
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    The first important step toward a successful preparation of large and diverse promoter library with desired complexity, is to select a suitable mutagenesis strategy. To generate a promoter library of GAP promoter(pGAP)variants, mutations were introduced using error-prone PCR. After optimization of the conditions for EP-PCR random mutagenes, high mutation(error rate 1.1%)frequence was obtained using 1 ng/μL template and 10 mmol/L Mg2+, in combination with 25 thermal cycles. To increase mutational diversity and reach an appropriate error rate, three consecutive rounds of EP-PCR were carried out under the same conditions. After random sequencing of 10 clones from each round, an overall range of mutation rates from 1.1% to 4.0% was observed. Then, 250 clones containing pGAP variants were screened using the highthroughput screening approach in 48-deep-well plates. Among them, 5 mutants exhibited higher fluorescent intensity compared to the wild-type promoter.
    Expression,Purification and DNA Binding Activity of Human Transcription Factor hASH4
    Su Zhuolei, Lou Tiantian, Wang Yuandong, Ji Chaoneng
    2014, 30(6):  218-224. 
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    hASH4 is a member of Helix-Loop-Helix(HLH)proteins which are an important group of transcription factors that exert such a determinative influence on a variety of cell proliferation, determination and differentiation from yeast to human. hASH4 has been reported closely related to skin differentiation and development, but the exact mechanism is unknown. In this study, the expression plasmid of pET28b- his- hASH4 was restructured and successfully expressed in BL21(DE3). After the optimization of temperature, time, IPTG concentration of expression, we ascertain that 1mmol/L IPTG expressed 4 hours at 37℃ can get the best expression. and we got the electrophoretic purity of the target protein by Ni-NTA affinity chromatography and ion cation exchange chromatography. The non-radioactive EMSA experiment between DNA and protein showed that the hASH4 protein only has the non-sepcific DNA binding activity without specific DNA binding activity. The play of transcription factors by hASH4 in the body may be need to form a heterodimer or multimer to further specific binding to DNA and act on the downstream genes. This study provided clues for the really function in vivo of hASH4 and laid the foundation for the further crystallization conditions screening, structural analysis and functional studies.
    Effect of Gene Ring Finger Protein 6 on the Expression of Insulin Receptor Substrate-2 in Hepatoma Cells
    Gong Jian, Song Jian
    2014, 30(6):  225-228. 
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    It was to construct an eukaryotic expression vector of ring finger protein 6(RNF6)gene and investigate the effect of RNF6 on the expression of insulin receptor substrate-2(IRS-2). The coding sequence of hRNF6 gene was amplified by PCR with human cDNA as template. The pcDNA3.1-CHA-RNF6 was constructed and transfected into hepatocarcinoma cells(HepG2)by routine molecular biology technology. The total RNA was extracted from HepG2 cells 72 hours post-transfection, the expression levels of IRS-2 was detected by real-time quantitative PCR. Western blotting was applied to detect the protein levels of IRS-2. Result showed that the mRNA level of IRS-2 gene in transfected HepG2 was 37% of the control. The expression level of IRS-2 was lower than the control group significantly (P﹤0.01). The expression of IRS-2 was down-regulated in HepG2 significantly, and the disorder in insulin signal transduction pathway, which may result from enhanced ubiquitylation level of IRS-2.