25 August 2012, Volume 32 Issue 08

    

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  RIOK3 Promotes the Cleavage-activation of Caspase-10 on PAK2

  LIN Ying, LI Pu, SHAN Jing-xuan, CHEN Xiao-jing, SHI Hui-li, HUO Ke-ke

  China Biotechnology. 2012, 32(08): 1-8.

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  RIO3, a member of atypical protein kinase, is only discovered in multicellular eukaryotes. The human liver cDNA library was screened with pDBLeu-RIOK3 as bait plasmid by yeast two-hybrid system and PAK2 was identified as a RIOK3 interactive protein. The interaction was confirmed by co-immunoprecipitation assays and immunofluorescent localization experiments. Results of real-time quantitative PCR and Western blot showed that RIOK3 can reduce the amount of PAK2 at protein expression level. CCK-8 and apoptosis detection experiment showed that co-expression of RIOK3 and PAK2 can inhibit cell proliferation and promote apoptosis,and the apoptotic effect can be inhibited by caspase-10G, which is the minimum isoform of caspase-10 and had no enzyme activity. The experimental results showed that RIOK3 may promote cleavage of caspase-10 on PAK2 and play an important role in the PAK2 cleavage-activation pathway.
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  Expression and Identification of Recombinant Human Apolipoprotein C-I in Pichia pastoris

  SU Man-man, CHEN Yue, WANG Ding-ding, XU Tian-min, YAN Wei-qun

  China Biotechnology. 2012, 32(08): 9-13.

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  Apolipoprotein C-I (ApoC-I) is a small, basic apolipoprotein which is mainly secreted by the liver as a component of triglyceride-rich lipoproteins and high density lipoproteins whose importance in plasma lipoprotein metabolism is increasingly evident. Therefore, a Pichia pastoris expression system was first used to obtain a high level expression of secreted, recombinant human ApoC-I (rhApoC-I). The full-length ApoC-I encoding sequence, gained by RT-PCR, was inserted into the pPICZαC vector and transform Pichia pastoris strain X33 after sequencing. High expression transformants with zeocin resistance were detected by SDS-PAGE and Western blot analysis. The antiproliferation effects of rhApoC-I on rat coronary artery smooth muscle cells was analysed by the methylthiazoletetrazolium (MTT) assay. It was showed that there were rhApoC-I(6.6kDa) in the culture supernatant induced by methanol. The expression level of rhApoC-I was 80mg/L. RhApoC-I can inhibit the proliferation which induced by PDGF remarkably. Ground on this, the composition and functions of rhApoC-I can be studied.
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  PES1 Interacts with Estrogen Receptor

  LI Jie-ping, ZHUANG Qin-ren, LAN Xiao-peng, ZENG Guo-bin, LUO Xiao-feng

  China Biotechnology. 2012, 32(08): 14-18.

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  Estrogen(E₂) and estrogen receptors (ER) play a vital role in the E₂-induced neoplasms. The coregulators of ERs modulate their biological functions by binding these receptor. PES1, as a potential regulator, is mainly expressed in the target organs such as breast and ovarian, and also, is highly expressed in the breast cancer cells. In current work, the HA-tagged recombinant plasmids of full-length PES1, and its different function regions(1~322aa, 312~588aa and 414~588aa) were constructed by PCR. To test whether PES1 can interact with ERs and their interaction regions, different recombinant plasmids constructed were co-transfected with FLAG-ERα or/and FLAGC-ERβ in 293T cells before co-immunoPrecipitation (co-IP). The co-IP results showed that PES1 could interact with ERα and ERβ by binding their 1~322aa region. Further experiments demonstrated that PES1 specifically inhibited the transactivation activity of ERβ in E₂-independent manner by analyzing estrogen receptor element luciferase (ERE-LUC). These results suggested that PES1 may act as a new ER coregulator. Further mechanisms and roles in the ERβ signaling pathway and E₂-induced neoplasms remain to be determined.
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  Construction and Expression of Rat Proprotein Convertase 2 Gene

  WU Shuang, ZHOU Dong, ZHANG Juan-hui, TANG Song-shan

  China Biotechnology. 2012, 32(08): 19-23.

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  Objective: Proprotein convertase 2 (PC2) is one major member of the pro-protein convertases that involve in the maturation of neuroendocrinopeptide precursor. To obtain rat pc2 gene for further studying its construction and function relation in rodent tumor model, rat pc2 gene was recombined. Methods: Total RNAs were extracted from the rat pituitary. The pc2 cDNA was cloned and amplified by using RT-PCR technique. The pc2 DNA was inserted into the pCDNA3.1/Zeo vector. The transfection of recombinant vector pCDNA3.1-pc2 or together with its chaperon partner 7b2 gene successfully expressed and activated the pc2 gene in breast adenocarcinoma cell line 4T1. Conclusion: By gene sequencing, transfection, activation, and Western blot analyses, the pc2 gene was confirmed to be inserted and expressed successfully in mammal cells.
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  Expression, Purification, Activity Detection and Crystal Growth Studies on Sf2523 Protein from Shigella flexneri 2a Strain 301

  FENG Duo, ZHANG Kuo, LI Qian-qian, HAN Cui-xiao, GAO Wei

  China Biotechnology. 2012, 32(08): 24-29.

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  Sf2523 protein is a member from the Thioredoxin Peroxidase (Prx) family. Peroxiredoxins are ubiquitous proteins that catalyze the reduction of hydroperoxides, thus conferring resistance to oxidative stress. Sf2523 protein can protect the large biological molecules from reactive oxygen specie, playing an important role during aerobic metabolism. After the construction of prokaryotic expression system, the Sf2523 protein was expressed in soluble state, and purified by Ni Affinity Chromatography and Size Exclusive Chromatography. The peroxidase activity experiment proved that it still has a natural enzyme activity and the core structure has not changed. Enzyme protein in vivo and in vitro has different state of aggregation. The purified monomeric protein of crystallization experiments, screening out the needle-like crystals. Through further resection of label protein to optimize the processing, finally, a homogeneous three-dimensional crystal was got.
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  Abiotic Stress Tolerance Analysis Two Alternatively Spliced Isoforms of LEA3 Gene from Pogonatherum paniceum in Yeast

  LI Rui, WANG Wen-guo, FAN Lin-hong, WANG Sheng-hua

  China Biotechnology. 2012, 32(08): 30-35.

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  It was to analyze the sequence of Pogonatherum paniceum group 3 LEA proteins alternatively spliced isoforms, and to detect the abiotic stresses tolerance of PpLEA3 spliced isoforms. The two spliced isoforms of PpLEA3 gene were amplified by PCR reaction using the plasmids pMD19-T- PpLEA3.a and pMD19-T- PpLEA3.b as the templates, respectively. The yeast expression plasmid of pYES2- PpLEA3.a and pYES2- PpLEA3.b was constructed and then transformed into yeast to create recombinant INV- PpLEA3.a and INV- PpLEA3.b . Stress tolerance tests showed that LEA3 yeast transformants exhibited a higher survival rates than the control transformants under salt (NaCl), NaHCO₃, freezing, drought and ultraviolet radiation. PpLEA3.a has stronger abiotic stresses tolerance than PpLEA3.b . The nucleic acid sequence of two splicing isoforms have different protein hydrophilicity and structure which leading to differences in the stress tolerance.
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  Study on Agrobacterium-mediated Transformation of psy Gene into Shoot Meristem of Maize

  WANG Ting-ting, JI Jing, WANG Gang, GUAN Chun-feng, ZHANG Lie, JIN Chao

  China Biotechnology. 2012, 32(08): 36-40.

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  The psy gene was transferred into the shoot meristems of two different maize inbred lines including maternal Tian tawu and 7922 by Agrobacterium-mediated transformation. Factors influencing transformation efficiency were optimized.The results indicated that the optimal transformation condition is infection time with vacuum at 20 min and co-cultivation time for 3 days.The transgenic plants were obtained after selected by 200mg/L PPT and 16 of them were confirmed positive by PCR amplification. RT-PCR and HPLC analysis results showed that psy gene had been integrated into the maize genome and transcribed normally, the total carotenoid content in transgenic maize was increased 25% compared to the wild type. This method eliminates the tedious process of tissue culture and proves to be a simple, strong operable transformation method.
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  Double-stranded RNA-mediated Gene Silencing and Over-expression of FmSTS in Polygonum multiflorum Thunb Hairy Roots

  ZHU Kuan-peng, ZHAO Shu-jin

  China Biotechnology. 2012, 32(08): 41-48.

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  Objective: To construct a method to research the gene FM-STS’ function that the gene come by RACE amplified from the stilbene synthase gene’s consensus sequence in Polygonum multiflorum Thunb. Method: The over-expression vector pBIN-35S-STS-GFP was constructed from the blank plasmids pBIN-35S-GFP and the gene FM-STS sequence and also the interference expression vector pBIN-35S -forward-reverse-GFP was constructed from the blank plasmids and the forward/ reverse direction sequence on account of double stranded RNA.Sent the over-expression plasmid/the interference plasmid and the blank plasmid into wild-type Agrobacterium rhizogenes ATCC15834, then induced hairy roots and cultured them. In the end the hairy roots were analyzed by HPLC and Real-time PCR. Result: The PCR results showed that the gene rolB and gfp are both expressed in hairy roots, and the content of stilbene glucoside in blank group is 2.18mg/g,in the over-expression group is 4.67mg/g and in the interference group is 0.65mg/g, At the mRNA level to detect gene Fm-STS expression level, in the over-expression group is the highest, it’s 3.17 times in the blank group and 101.44 times in the interference group. Conclusion: The method binding both technique that double stranded RNA-mediated gene silencing and over-expression is made good use of researching the gene’ s function, and the gene FM-STS is the key enzyme gene in synthesis stilbene glucoside.
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  Optimizing Soluble Expression and Inclusion Body Renature Research of β-Propeller Phytase of Bacillus sp. HJB17 in E.coli

  LI Qian-qian, LI Zhong-yuan, FENG Duo, HUANG Huo-qing, HAN Cui-xiao, YANG Pei-long, YAO Bin, GAO Wei

  China Biotechnology. 2012, 32(08): 49-55.

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  PhyH,a novel phytase from Bacillus sp. HJB17,successfully clone and express the truncated phytase PhyHT(cancel the first 120 nucleotides which encode a 40 amino acid secreted signal peptide) in Escherichia coli. To improve the soluble expression level of PhyHT, and get large-scale purified and active proteins,the expression conditions were optimized,including concentrations of IPTG and temperatures of induction. The co-expression system of pET28b-PhyHT and four molecular chaperone vectors(pG-KJE8,pGro7,pKJE7, and pTf16)were constructed respectively.Additionally,it was successful in vitro refolding of Bacillus phytase from the inclusion bodies. The results were described as follows: (1) the PhyHT gene was cloned into vector pET28b successfully ; the expressed proteins were found in the insoluble cytoplasmic fraction as inclusion bodies, even with different expression conditions.(2) Molecular chaperone vectors pGro7 and pKJE7 improved significantly the soluble protein level.(3)PhyHT with biological activity was obtained after the recombinant vector pET28b-PhyHT was expressed and renatured. Furthermore,the protein was purified through Ni-NTA and gel filtration chromatography. The soluble proteins provide a potential value for the further mechanistic study of the structure and function of PhyHT.
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  Cloning and Expression crtI Gene in Methylobacterium sp MB200

  LIN Qiong-shan, TANG Xian-lai, LI Xian, JIANG Cheng-jian, LI Jun-fang, SHEN Pei-hong

  China Biotechnology. 2012, 32(08): 56-61.

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  The partial mutants library of M. sp MB200 was constructed by transposon mutagenesis and 11552 mutants were obtained which could survived in medium with methanol as the sole carbon source. Rescreening results showed that 33 strains were colorless mutants. The crtI gene was got from a colorless mutant by molecular cloning techniques. It shared 93% identity with the crtI gene of M. populi BJ001, M. chloromethanicum CM4 and M. extorquens AM1 at the nucleotide level reported in Genbank. The gene size is 1539 bp, encoding the phytoene desaturase (CrtI) that catalyzes phytoene to form lycopene with four desaturations, or catalyzes phytoene to form neurosporene with three desaturations, therefore it plays an important role in the biosynthetic pathway of carotenoid. Here, a recombinant plasmid pCM80-crtI was constructed by ligating the crtI to the vector pCM80, then imported into M. sp MB200 to obtain recombinant strain MB200/pCM80-crtI. The activity of enzyme from MB200/pCM80-crtI was more by 40% than M. sp MB200. The experimental results provided a theoretical reference to improve the biosynthetic and metabolic pathways of carotenoid in Methylobacterium.
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  Expression, Purification and Characterization of Non-taged Recombinant Human Thioredoxin

  GUO Yun-ping, SUN Lu, ZHANG Li-jian, WANG Zeng-lu, GAO Chao, YANG Qiang, LIU Yi, ZHANG Ying-qi, QU Yan, TAO Ling

  China Biotechnology. 2012, 32(08): 62-67.

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  Objective:To construct prokaryotic expression system for mass production of recombinant human thioredoxin and establish the purification process of thioredoxin. Methods: Total RNA was extracted from HEK293 (human embryonic kidney cells). The thioredoxin coding sequence was subcloned into the pET-22b(+) vector after amplified by PCR.The recombinant plasmids were transformed into E.coli BL21(DE3), and the thioredoxin was expressed with IPTG induction. The expressed thioredoxin was purified by two-step ion exchange chromatography and tested by SDS-PAGE, Western blotting, MALDI-TOF-M, HPLC, and insulin disulfide reduction assay for identification, purity assay and activity determination, respectively. Results: Gene sequencing demonstrated that thioredoxin coding sequence was cloned into pET-22b(+) vector successfully. The prokaryotic expression system achieved high yield of thioredoxin (180 mg/5L of fermentation broth), which was identified by Western blotting and MALDI-TOF-MS, with an estimated the molecular weight of 12 000. The purity of thioredoxin is more than 95%. The activity of purified thioredoxin had the same activity as the standard control. Conclusion: The prokaryotic expression system could achieve mass production of recombinant human thioredoxin, which can be highly purified by two-steps ion exchange chromatography. This preliminary study provides the foundation for the large-scale industrial production of thioredoxin.
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  Study on Generation of Chimeric Porcine Embryos Derived from Lentivirus Mediated Green Fluorescent Protein(GFP) Transgene Embryos

  SHI Yong-qian, HE Wen-teng, ZHOU Yang, JIAO Ming-xia, XIE Bing-teng, HU Kui, KONG Qing-ran, LIU Zhong-hua

  China Biotechnology. 2012, 32(08): 68-74.

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  Objective: Through the establishment system of the lentiviral vector infected porcine embryo, studied chimeric ability of porcine parthenogenetic embryos of the different development stages, made a foundation for further studying early embryonic development and cell differentiation. Method: The lentivirus with GFP report gene was injected into the perivitelline space of porcine embryos at 1-cell and 2-cell stage, and the cleavage rate, GFP-positive embryo rate, blastocyst rate, GFP-positive blastocyst rate and cell number of blastocyst were examined in fluorescence microscope. Well of well (WOW) method was employed to make chimeric embryos by aggregating two different embryos at the same developmental stage of 2-cell, 4-cell and 8-cell or 2-cell stage embryo with 2-cell, 4-cell and 8-cell stage embryos, and blastomere exchanging at 2-cell stage was also used for making chimeric embryo.Then the chimerism of porcine embryos were checked. Result: In groups of 2?10⁹I.U./ml lentivirous injecteion in the perivitelline of the 2-cell stage embryos, the infection rate and GFP-positive blastocyst rate of IVF(80.00%, 90.74%) and parthenogenetic embryos (76.36%, 89.56%) were significantly higher than other groups (P<0.05), and there is no significant difference in cleavage rate, blastocysts rate, blastocysts cell number compared with the control group. The chimeric rate of embryos derived from aggregating of two 2-cell stage embryos (53.85%) and embryos from blastomere exchanging of 2-cell stage embryos (62.50%) were significantly higher than that of aggregating of 2-cell with 4-cell or 8-cell embryos (18.60%, P<0.05), and the chimeric rate of embryos derived from aggregating of two 8-cell embryos (75.00%) were significantly higher than that of these embryos derived from aggregating of two 4-cell embryos (65.00%)or 2-cell embryos (53.80%). Conclusion: 2?10⁹I.U./ml of lentivirous injection in the perivitelline of two-cell stage embryos shows the highest infection efficiency,in addition,lentivirus infection has no significant effect on developmental of porcine embryos. The chimeric rate of embryos derived from aggregating two 8-cell embryos is higher than the others.The chimerism rate of the synchronized developmental embryos is higher than that of the non-synchronized embryos.
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  Optimization Fermentation Conditions of the Nitrile Hydratase Fusant Strain Tolerance to the Substrate

  ZHANG Li-ling, WANG Xiao-lan

  China Biotechnology. 2012, 32(08): 75-80.

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  Orthogonal design was used to optimized the fermentation conditions of the nitrile hydratase(NHase) fusant strain tolerance to the substrate, Fermented liquid starting pH, fermentation period, inoculation quantity, pack liquid coefficient as the investigation factors,Finally determined the best fermentation conditions for: start pH8.0, fermentation period 54 h, Inoculation quantity 12%, pack fluid coefficient 12%. Under the optimized conditions, activity of nitriles hydratase of the fusant strain increase to 11 millionU/ml, is up 83.3% before optimization. Response surface methodology was used to optimize the fermentation medium. A Plackett-Burman design was used to evaluate the influence of eight factors firstly. Results showed that the concentration of glucose, urea, K₂HPO₄ and KH₂PO₄ played important roles in influencing the activity of nitrile hydratase. Then, the path of steepest ascent and the Central composite design were adopted for further optimization, and the optimum concentration levels and the relationships among these factors was found out by quadratic regression model equation with Design-Expert statistic methods, the optimal concentration of the variables were determined as: glucose 22.62 g/L, urea 9.76g/L, K₂HPO₄ 1.22g/L,KH₂PO₄ 1.268g/L. Under such conditions, the activity of nitrile hydratase was increased to 12.80 millionU/ml, which was 16.4% higher than old medium composition.
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  Advances of the Technology of Spermatogonial Stem Cell Transplantation and Recipient Preparation

  WANG Chen, ZHU Hua-bin, LIU Ling, HAO Hai-sheng, ZHAO Xue-ming, FEN Rong, DU Wei-hua, QIN Tong, LIU Yan, LI Zong-qiang, WANG Dong

  China Biotechnology. 2012, 32(08): 81-86.

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  Spermatogonial stem cell (SSCs) transplantation laid the foundation for the study on spermatogenesis, male fertility and the new transgenic technology. Mature technology system of SSCs transplantation has been established in mice. The receptor preparation method of busulfan treatment, and the transplantation methods of seminiferous tubule injection and rete testis injection have been widely used in this scientific area. However, busulfan treatment can lead to a high mortality ; It costs much to prepare receptor animal using local irradiation and the animal emptied from endogenous stem cells; The receptor preparation method of heat shock is limited for application and its effect is unstable. All three transplantation methods require high operating techniques. Inject seminiferous tubule and efferent ducts need apparatus for microinjecting testis, while the catheter should be inserted into the rete testis under ultrasound guidance.The transplantation results are quite distinct among different experiments and species, and the efficiency needs to be further improved. The immunological rejection after transplantation also should be explored. A deep study on testicular structure and biological characteristics of SSCs will help to establish novel, simple and efficient methods for receptor preparation and transplantation of SSCs.
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  Advance in Studies of Animal-borne Lysozyme

  ZHANG Peng, JIANG Ming-feng, WANG Yong

  China Biotechnology. 2012, 32(08): 87-93.

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  Lysozyme is a kind of muramidases that widespread in the animal in vivo, and can catalyze the hydrolysis of β-1,4-glycosidic bonds between the N-acetylglucosamine and N-acetylmuramic acid in the peptidoglycan layer of bacterial cell walls. It has functions of the digestion and decomposition of bacteria, and inhibition of exogenous microbial growth, and enhancing immunity; and lysozyme has been a model protein for research in the function and the nature of the enzyme, and molecular evolution. Firstly, the lysozyme and its crystal structure and the advance in studies of lysozyme gene and its protein were introduced. Secondly, the functions of animal-borne lysozyme, including the biological functions of lysozyme and functional activities of recombinant proteins were introduced too,then the applied researches in lysozyme gene in transgenic engineering were focused on. Finally, the perspectives of animal-borne lysozyme were suggested. It’s very significant to research the animal-borne lysozyme because it’s helpful to understand the basic knowledge and to use it in the production.
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  Progress on Plant Anthocyanin Biosynthesis and Regulation

  GAO Yan-hui, HUANG Chun-hong, ZHU Yu-qiu, TONG Zai-kang

  China Biotechnology. 2012, 32(08): 94-99.

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  Anthocyanin with important nutritional and medical usages is flavonoids in plant metabolism, and decides the color of flowers, fruit, testa, stem leaves and roots and so on in angiosperm. Recently studies on the biosynthetic pathway were undertaken continuously. The research status and development tendency of plant anthyocyanin were summarized, including the anthocyanin biosynthetic pathway, structural gene and regulator genes and their functional study participating in the biosynthetic pathway and environment factors affecting the biosynthetic pathway.
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  The Antibiotic Metabolites Genes of Pseudomonas fluorescens

  YANG Yi, LI Zhi, GAO Ling-xia, SUN Yan

  China Biotechnology. 2012, 32(08): 100-106.

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  Pseudomonas fluorescens is an important kind of plant growth-promoting rhizobacteria (PGPR). It can produce many secondary metabolites, such as pyoluteorin, 2, 4-diacetylphloroglucinol, pyrrolnitrin and phenazine-1-carboxyl acid. These antibiotics play a major role in suppression of many pathogens.The synthesis mechanisms of the secondary metabolites in Pseudomonas fluorescens, especially the structures and functions of the related genes were summarized. At the same time, the applications of Pseudomonas fluorescens in biological control are presented.
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  Cell Surface Engineering of Yeast Application in Biofuel

  LI Xiao-dong, YANG Na, WAN Yong-hu, WU Jia, JIA Dong-chen, QIAO Min

  China Biotechnology. 2012, 32(08): 107-110.

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  The research of surface display has become a powerful and active topic, which has played a critical role from combinatorial library screening and protein engineering to biofuels production and bioremediation. Surface display technology and its application in biofuel has been described, research on the mechanism about the enzymes of fermentation process and application prospect of whole-cell biocatalysts were also reviewed.
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  The New Development of the Research Method for Molecular Microbial Ecology

  LV Chang-yong, CHEN Chao-yin, GE Feng, LIU Di-qiu, KONG Xiang-jun

  China Biotechnology. 2012, 32(08): 111-118.

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  Microbial community structure and functional metabolism are the research hotspots of microbial ecology. However, the research method of microbial community structure and functional metabolism has been limited by technique for a long time. With the development of new techniques, the research approaches for molecular microbial ecology have being changed. High-throughput sequencing technology has ameliorated the research method of microbial diversity, metagenomics and metatranscriptomics. Meanwhile GeoChip which covered large amount of known functional oligonucleotide probes in single chip could determine the presence or absence of microbes and functional genes quickly. The newest research approaches for molecular microbial ecology study were reviewed and compared, and the applicability, advantages and disadvantages of those approaches were discussed.
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  Biocircle Project Strategy Analysis and Suggestions

  ZHANG Da-lu

  China Biotechnology. 2012, 32(08): 119-122.

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  The EU 7^th Framework Programme is the world’s largest official major scientific and technological cooperation programs. Its research is related to the international forefront, and competitive technological difficult problems as the main content, with a high level of research covers a wide field, large investment involved in countries and other characteristics. Biocircle Project is part of the EU 7^th Framework Programme, with 24 participating countries, which is a national focal points for construction projects. By introducing Biocircle project, and the advantages of international cooperation projects in the EU,the purpose is to point out that China’s international cooperation program should be more active more open, and promote more and better opportunities for Chinese science and technology.