25 June 2012, Volume 32 Issue 6

    

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RESEARCH PAPERS
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  Prokaryotic Expression and Immunogenicity Analysis of HPV16 L2 Peptide Coupled to AP205VLP

  JIANG Bo, BI Yan-wei, ZHANG Ying-ying, CAI Lu-kui, JI Qiu-yan, LI Zhi-hua, XU Wei-ming

  China Biotechnology. 2012, 32(6): 1-6.

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  Objective: To connect the residues 65~71 and 112~120 of HPV minor capsid protein L2 to the coat protein of RNA bacteriophage AP205. The fusion protein was self-assembled into virus-like-particles (VLPs) after expressing in E.coli,then the protein was purified and the immunogenicity was analyzed. Methods: After being synthesized artificially,the AP205 coat protein gene and HPV16L2 gene were connected by PCR and cloned into vector pET-30a(+).The constructed plasmid pET30-AP205-HPV16ΔL2 was transformed to E.coli BL21(DH3), the recombinant protein was expressed under induction of IPTG and purified by Gel chromatography, then identified through SDS-PAGE and Western blot. The immunogenicity of purified product was measured by indirect ELISA after ICR mouse was immunized. Results: Recombinant plasmid pET30-AP205-HPV16ΔL2 was constructed correctly and the protein was expressed solubly in E.coli BL21(DH3). The typical VLPs can be observed through Transmission electron microscopy which Showed good immunogenicity in animal experiments. Conclusion: The HPV16L2 epitope was fused successfully to the N-terminus of AP205 coat protein,and The VLPs was self-assembled in E. coli.
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  Influence of Mouse Embryonic Stem Cells on Melanoma Cells in vitro

  ZHANG Xiao-mei, FANG Liao-qiong, WANG Zhi-biao

  China Biotechnology. 2012, 32(6): 7-12.

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  To investigate the impacts of mouse embryonic stem cells on mouse melanoma B16 cells in vitro co-cultured environment. C57BL / 6 mouse embryonic stem cell lines were established.The effects of mouse embryonic stem cells on tumor cell morphology and growth behavior were observed with establishing a co-culture system in vitro.MTT assay and transwell chamber assay were used to detect change of the co-cultured tumor cell adhesion, migration and invasiveness. Mouse embryonic stem cells could invade and push the mouse melanoma cells to form their own room for growth in co-culturing. The adhesion, migration and invasion ability of the tumor cells after co-cultured with mouse embryonic stem cells significantly declined(P<0.05,P<0.01). The results implied that the co-culture mouse embryonic stem cells could invade tumor cells and inhibit their related malignant behaviors like adhesion,migration and invasion ability.
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  Preparation, Characterization and Application of Monoclonal Antibodies Against Human Y box-binding Protein 1

  LI Pu, SHI Jing, CHENG Fen, LIANG Qin-dong, KUANG Wen-bin, WANG Qin, DONG Jin-yu, TU Zhi-guang

  China Biotechnology. 2012, 32(6): 13-19.

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  The purpose is to prepare and characterize the monoclonal antibodies (mAbs) of anti-human Y-box binding protein 1 (anti-YB-1) and identify its application of immunoassay. Hybridoma cells were generated by fusing the Sp2/0 myeloma cells with spleen cel1s, which were obtained from BALB/c mice immunized by recombinant YB-1 protein. Indirect ELISA was used to screen positive clones, and to detect titers, subtypes, and relative affinity of the mAbs. Protein G affinity chromatography system was applied to prepare highly-purified mAbs. B-cell epitope prediction method was applied to assay the corresponding epitopes of mAbs. Western blot and immunohistochemisty were adopted to identify immunoassay application of the mAbs. The results showed that two positive hybridoma-cell clones were obtained (1-D9, 3-E8). The titers of mAbs were ≥ 1 :1×10⁶, and the subtype of the two mAbs were IgG_l. The recognized epitope of the two mAbs might harbor in 134-160aa and 266-303aa of the YB-1 protein, respectively. The results of Western blot and immunohistochemisty showed that mAbs (1-D9) could specifically react with endogenous and recombinant YB-1 protein. All these results indicate that two kinds of anti-YB-1 mAbs which could recognize different epitope of the YB-1 protein are successfully prepared; it would provide foundations for quantitative and qualitative detection of YB-1, diagnostic immunoassay, anti-cancer targeted-therapy, and investigation of YB-1 function.
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  Effect of Melatonin on Genes Including FGF5 That Related to Hair Growth in Cashmere Goat Skin

  ZHAO De-chao, FU Shao-yin, LV Xiao-man, WU Jiang-hong, ZHANG Yan-jun, LI Jin-quan, WANG Zhi-xin, ZHANG Yong-bin, ZHANG Wen-guang

  China Biotechnology. 2012, 32(6): 20-26.

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  The aim is to study the effect of melatonin on genes which related to hair growth on skin, and explore the mechanism of melatonin in cashmere growth. Using the Inner Mongolia white cashmere goats as the research object, 10 lambs on 4 months age with similar body condition were selected, and divided into 2 groups randomly. Following testing requirements, the treatment group was implanted melatonin in subcutaneous behind the ear every two months according to 2mg / kg BW (body weight) dose, and nothing to the control. The experiment time was last for one year. Then the cashmere goat skin and hair in the body side were gathered, using real-time PCR to detect the expression changes of 9 genes related to hair growth. The results showed that (1)melatonin implanting can up-regulate FGF5’s expression significantly (P<0.0001), but has no significantly effect to β-catenin, MSX-2, Wnt10b, BMP2, BMPRⅠB, NTRK3, Delta1and hairless (P>0.05); (2) melatonin implanting obviously improved the correlation between FGF5 and hairless, and make the negative correlation turn to positive correlation on β-catenin and NTRK3, the correlation with Wnt10b was reduced, while the other 4 were little changed. These results indicated that Melatonin up-regulate the expression of FGF5 in cashmere goat skin, impact the correlation of FGF5 with hairless, β-catenin and NTRK3; the impact of melatonin on hair is different with other animals; the effect of melatonin on complex organizational and individual cells are different. MAPK signaling pathway is the greatest impacted by melatonin. It is putative that melatonin can regulate RORα in cashmere goat skin to impact the correlation of FGF5 with hairless, β-catenin, NTRK3 and Wnt10b, and inhibit the function of MSX2 and BMP2 for promoting hair growth.
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  Purification and Characterization of Pyrroline-5-carboxylate Reductase from Drosophila melanogaster

  FENG Chang-zeng, XIE Yue-hui, CAO Yi, MENG Zhao-hui

  China Biotechnology. 2012, 32(6): 27-34.

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  P5CRs exist in every domain of life, including eukarya, bacteria and archea. They catalyze the conversion from Δ1-pyrroline-5-carboxylate (P5C) to proline with concomitant oxidation of NAD(P)H to NAD(P)⁺ in the final step of proline biosynthesis. In order to provide the theory basis of the function and three-dimensional structure of P5CR, it was performed to research the aggregation form, enzymatic properties and optimal crystallization conditions of P5CR from Drosophila melanogaster. Total RNA from Drosophila melanogaster was isolated, and reverse transcripted into cDNA with random primers. The full-length sequence of the P5CR gene was amplified from the cDNA and comfirmed by sequencing. The coding sequence of P5CR was subcloned into Novagen pET-28a (+) vector. The target protein was over-expressed in Escherichia coli and purified to homogeneity by chromatography. The homopolymer was characterized by EGS cross-linking and size exclusion gel filtration chromatography. The thioproline dehydrogenase activity of P5CR was assayed at room temperature using a sensitive spectrophotometric method. The crystallization conditions of P5CR using hanging-drop vapor diffusion method were screened and optimized.
  The main results were as follows: (1) The method of overexpression and purificaiton of Drosophila melanogaster P5CR was obtained, and the purity of P5CR is high. (2) Drosophila melanogaster P5CR mainly exists in the form of decamer in solution, on this basis, forms homopolymer. (3) P5CR can be involved in the metabolism of thioproline which was considered as a kind of anticancer drug. This enzyme has an optimal alkaline pH in this reversible reaction and is thermo stable with a half-life of approximately 15 min at 45℃. (4) One remarkable crystallization condition was obtained (0.2mol/L Ammonium phosphate dibasic, 20% PEG3350, and pH 8.0).
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  Optimization of Conditions for Soluble Expression and Purification of the Cytosolic Region of Arabidopsis thaliana PDV1

  HAN Cui-xiao, LI Feng, YAN Xiao-jin, FENG Duo, LI Qian-qian, GAO Hong-bo, GAO Wei

  China Biotechnology. 2012, 32(6): 35-42.

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  Plastid Division1 (PDV1), an outer envelope membrane protein of chloroplasts, with its larger N-terminal portion facing the cytosol and its smaller C-terminal portion existing in the inter-membrane space, plays a key role in chloroplast division. To improve the soluble expression level of Arabidopsis thaliana PDV1, and get large-scale purified proteins, types of the expression vector, different regions of the gene, concentrations of IPTG, temperatures of induction were optimized. Additionally, the co-expression system of recombinant plastids and five molecular chaperone vectors (pG-KJE8、pGro7、pKJE7、pGTf2 and pTf16)were constructed respectively. The results are described as follows: (1) The target proteins expressed from the recombinant plastids, which were constructed in pET22b and pET28b with the N-terminal 1-206, 1-180, 38-206 and 38-180 amino acid residues of PDV1, formed into inclusion bodies, even with different expression conditions. (2) The soluble protein level was increased significantly when the N-terminal 180 amino acid residues of PDV1 was cloned into the expression vector pGEX6p-1, induced with 0.2 mmol/L IPTG and grown at 16℃ overnight. However, the proteins fused with GST expressed from pGEX6p-1-PDV1-206 formed into inclusion bodies. The soluble protein level was higher when a region of the N-terminal 180 amino acid residues was expressed with the expression vector pET44a. (3) Highly pure soluble NusA-PDV1-180 fusion protein was obtained 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 PDV1 in chloroplast division.
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  Biological Activities Analysis of the Nematocyst Venom from Jellyfish Stomolophus meleagris

  LI Rong-feng, YU Hua-hua, XING Rong-e, LIU Song, LI Peng-cheng

  China Biotechnology. 2012, 32(6): 43-47.

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  The nematocyst venom was extracted from the tentacle of jellyfish Stomolophus meleagris. Hemolytic activity, lethal activity, SOD activity and antineoplasmic activity of the nematocyst venom was studied and the results showed that approximately 10.5μg/ml of the venom resulted in 50% hemolysis of the erythrocyte suspension and the half lethal dose (LD₅₀) of the venom was about 50 μg venom/g fish. The total SOD activity was about 161 U/mg when the venom protein concentration was 18μg/ml and the inhibition ratio to the hepatoma Bel-7402 was up to 54.9% when the venom protein concentration was 1mg/ml. Therefore, it is of significance to study the bioactive components from the venom, which is useful for the application of the venom of jellyfish Stomolophus meleagris.
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  Effects of Different Nitrogen Nutrition Level on the Growth and Photosynthetic Physiology of Odontella aurita

  WANG Lu-yao, SANG Min, LI Ai-fen, ZHANG Cheng-wu

  China Biotechnology. 2012, 32(6): 48-56.

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  In order to analysis the effects of different nitrogen nutrition level on the growth and photosynthetic physiology of Odontella aurita, diatoms were cultured under different nitrogen conditions(17.6 mmol/L N, 8.8mmol/L N, 5.87 mmol/L N, 0 mmol/L N), using 6cm columnar light biological reactor. The results showed that the algae of different nitrogen groups reached the maximum growth at different time. In the early of culturing, nitrogen limitation(5.87mmol/L N, 8.8mmol/L N) promoted the growth of Odontella aurita comparing with the control, while nitrogen starvation significantly inhibited the growth of the algae(P<0.05).The carbohydrate content increased significantly (P<0.05) under nitrogen limitation conditon, while total protein content decreased significantly (P<0.05).The content of chlorophyll a, c, total carotenoids of Odontella aurit are positively related with nitrogen nutrition levels of culture medium. The maximum photosynthetic oxygen release rate P_m decreased with nitrogen concentration, the respiratory rate of R_d presented the opposite tendency. The maximum efficiency of light energy conversion of PSⅡ (Fv/Fm), the actual energy conversion efficiency(Yield), potential activity(Fv/Fo) and relative electron transfer efficiency (ETR) decreased significantly with the nitrogen limitation. Illuminating that algal cells of apparent photosynthetic physiological status are directly related to the level of nitrogen nutrition.
TECHNIQUES AND METHODS
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  A Novel Method for Constructing Small Fragment Gene of Expression Vector

  JIN Lei, ZHAO Wen-xiu, MA Lan

  China Biotechnology. 2012, 32(6): 57-63.

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  A new method for rapid constructing small fragment of expression vector is introduced. This method eliminates the need for the target gene digested with restriction enzyme and recovery steps, and eliminates the vector gel purified steps after digestion. It is simple and fast, saving reagent costs, high connection efficiency. Four expression vectors of small fragment gene(67bp) have been completed with this method.
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  Construction and Verification of a Novel System for B-cell Epitope Display

  LIU Ying-fu, HUO Jing-rui, FAN Guo-cai, SUN Zhi-xian, WANG Qing-ming

  China Biotechnology. 2012, 32(6): 64-68.

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  B cell epitopes are the sites of the molecules that are recognized by antibodies or B cell receptors. Prediction and verification of B cell epitopes of antigen is useful for specific antibody generation and vaccine design. The immunoglobulin heavy chain variable region was used as the scaffold protein for B cell epitope display, and its CDR3 region was the display site. Three B cell epitopes were displayed in the protein scaffold, including (His)₆ tag and two epitopes derived from VEGF. The three recombinant proteins were used to immunize mice, and the immune serum was isolated and analyzed by ELISA, Western blot and immunofluorescence. The results demonstrated that the immune serum exhibited good specificity for the corresponding epitopes and the source protein. This means that the immunoglobulin heavy chain variable region is a good scaffold protein for B cell epitope display.
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  Generation of Human β-thalassemia Induced Pluripotent Stem Cells Under Feeder-free and Xeno-free Conditions

  FAN Yong, LUO Yu-mei, CHEN Xin-jie, SUN Xiao-fang

  China Biotechnology. 2012, 32(6): 69-73.

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  β-thalassemia is an inherited blood disorder that is characterized by reduced synthesis of hemoglobin subunit beta. Even with transfusion and chelation therapy treatments, the life span of patients with thalassemia major (also called Cooley’s anemia) can only be extended for a limited time. The derivation of β-thalassemia patient specific induced pluripotent stem cells opens a new approach of treating β-thalassemia. For therapeutic usage of induced pluripotent stem (iPS) cells, to accomplish feeder-free and xeno-free culture is critical. However, most reprogramming protocols used to date involve the use of products of animal origin and mouse feeder cells. Here, human β-thalassemia iPS cells weve derived from commercially available xeno-free medium TeSR^TM2 and StemAdhere^TM Defined Matrix. A total of 5 feeder-free and xeno-free human β-thalassemia iPSC lines were generated, which could be continuously passaged in xeno-free conditions and maintained characteristics indistinguishable from hESCs, including colony morphology and growth behavior, expression of pluripotency-associated markers, and pluripotent differentiation ability in vitro and in teratoma assays.
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  Location of EGFP Tag Affects the Binding Activity of Peptide Fusions

  WANG Hai, SHI Bi-zhi, YANG Sheng-li, LI Zong-hai, LUO Xiao-ying

  China Biotechnology. 2012, 32(6): 74-78.

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  Purpose: To investigate whether the location between targeting peptide and tag protein (eGFP) would influence the binding capacity of fusing protein to cell. Methods: To get Ge11 and LyP1 fusing eGFP recombination protein by expressing system in E coli. respectively, and then put the recombination protein into the medium of serum starving SMMC-7721 liver cancer cell line, after treating 3 hours, to detect the binding capacity by Fluorescence microscopy. Results: when eGFP at the Carboxyl terminal of targeting peptides, the recombination protein could bind to the cell; But when eGFP at the Nitro terminal, the recombination protein loss the binding capacity. On the basis of these researches, the other 3 targeting peptides were detected. Conclusion: These results indicate that an inappropriate fusion strategy would lead to decreased or even totally loss of function of targeting peptides. Thus, it should be cautious when used EGFP as a tracer to study the targeting activities of peptides.
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  Activity of a New Recombinant Insecticidal Crystal Protein Encoded by HJC Gene from Bacillus thuringiensis

  SU Xu, WANG Jing, LV Li-kun, LI Li, YANG Dong-jing, LIU Hong-liang

  China Biotechnology. 2012, 32(6): 79-83.

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  For a better management of insect cross-resistance against Bt toxins, a fusion gene derived from cry1Ab and vip3 genes was created and named HJC. The gene was expressed in E. coli strain BL21 upon transformation with plasmid pET28a-HJC. The his-tagged HJC protein mainly existed as inclusion body in E. coli, and was purified by Ni-NTA resin affinity chromatography under denatured condition. After refolding upon gradient urea dialysis, recombinant HJC protein was analyzed for its immune recognition and insecticidal activity. The results showed that the HJC protein expressed from E.coli was substantially equivalent to that of a transgenic rice carrying an HJC gene in terms of immune recognition. The refolded HJC protein exhibited high insecticidal activity against Hyphantria cunea. To the end, this protein can be used to substitute extrinsic plant protein in food-safty assessment of genetically modified products with HJC gene.
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  Comparison and Evaluation of Nine Methods in Detecting Listeria monocytogenes

  LIU Ya-li, LIU Fang, HAN Shun-yu, LIU Qing, SUN Zhi-bo, XIA Jun-fang, ZHU Xiao-qing

  China Biotechnology. 2012, 32(6): 84-92.

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  Nine methods were used to detect Listeria monocytogenes in bacterial suspensions and artificial contaminated food extracts, including traditional biological methods, chromogenic medium method, TAS-ELISA, DNA Direct-PCR, Bacterium Direct-PCR, IC-PCR, Bio-PCR, SYBR Green Real time-PCR and TaqMan Real time-PCR, and the results were analyzed and compared. The result showed that Traditional biological methods had false positive. The lowest detectable limits of TAS-ELISA, DNA Direct-PCR, Bacterium Direct-PCR and IC-PCR were 10⁶, 10², 10⁵, 10⁴CFU/ml. The detection sensitivity of chromogenic medium method, SYBR Green Real time-PCR was 10²CFU/ml. Bio-PCR, TaqMan Real time-PCR had the highest sensitivity which could reach 10¹CFU/ml. Chromogenic medium and TAS-ELISA had the advantages of simple operation, and they were suitable for preliminary screening of large samples. IC-PCR was a sensitive, specific, rapid, economic technology, suitable for detecting micro-pathogens in large volume samples particularly. Bio-PCR and TaqMan Real time-PCR had high sensitivity and specificity, which adapted to recheck and research.
REVIEWS
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  Current Development on RNA-based Anti-HIV-1 Gene Therapy

  CHEN Feng, YANG Yi-shu, ZENG Yi

  China Biotechnology. 2012, 32(6): 93-97.

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  Acquired immune deficiency syndrome (AIDS) is a high-risk disease which spreads rapidly all over the world since it has been discovered. Although the highly active antiretroviral therapy (HAART) that are widely used in present can improve the quality of life of HIV-1 infected patient dramatically, treatment interruptions have often occurred because of the high cost, drug resistance and side effects. As a result, the new antiretroviral drugs and approaches are demanded for sustained antiretrovial effects. With the development of molecular biology, stem cell, nanotechnology and other related technology, gene therapy for HIV-1 infection has attracted considerable attention in recent years. RNA-based gene therapy for treatment of HIV-1 infection, including antisense RNA, ribozymes, RNA decoys and the RNA interference are focused on. These studies demonstrated that the RNA-based anti-HIV-1 gene therapy may serve as an effective adjuvant to traditional treatments.
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  Recent Advances in Separation and Purification of Antibiotics

  ZHANG Zheng-yu, WU Mian-bin

  China Biotechnology. 2012, 32(6): 98-103.

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  The separation and purification technique is the technical difficulty and key link of the antibiotics industry. Some usual separation and purification methods of the antibiotics were summarized, and it would give a theoretical support to the purification of the antibiotics. In the end, the novel separation and purification techniques were discussed.
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  Gene Strategies of Enhancing the Anti-apoptosis Ability of Mammalian Cell in Engineering

  XU Hong-ji, ZHANG Bing-bing

  China Biotechnology. 2012, 32(6): 104-108.

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  Mammalian cells have significant advantages in producing recombinant protein drugs, which make it widely used in industry. In the process of production, however, environmental stress, which can result from nutrient depletion and by-product accumulation, activates apoptosis through signalling cascades regulators. It is the burden of improving the protein production that have the cell more susceptible to apoptosis, decreasing bioreactor performance and hindering large-scale production. Therefore,apoptosis, programed cell death, seriously limits the recombinant protein production, and how to improve the anti-apoptotic ability of engineering cells has accordingly become a hot point that attracted the attention of biologists all over the world. Apoptosis, as well as proliferation and differentiation, is one of the biological processes that cell must experience. With the understanding of the mechanism of apoptosis and revealing of many apoptosis genes in previous studies, a variety of anti-apoptotic approaches were found which is helpful to inhibit apoptosis and to enhance the robustness, survival and productivity of production cell lines. The anti-apoptotic approaches commonly used in engineering include: lowering apoptotic proteins, increasing anti-apoptotic proteins, over-expressing growth factor and insuin-like growth factor and modifying the intracellular enzyme related to metabolism in order to prevent the toxic metabolites from accumulating. Although the resistance of cell to apoptosis were, to some extent, improved by the strategies mentioned above, it is very essential for biotechnologists to search for more effective anti-apoptotic approaches that could lead to the greatest improvements in cell survival, provide unequalled bioreactor performance and enhance biopharmaceutical productivities.
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  Advances on Sucrose Phosphate Synthase in Plants

  HUANG Dong-liang, LI Shuang-xi, LIAO Qing, QIN Cui-xian, LIN Li, FANG Feng-xue, LI Yang-rui

  China Biotechnology. 2012, 32(6): 109-119.

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  Sucrose phosphate synthase (SPS) is the key enzyme that controls the sucrose biosynthesis in plants. Accumulation of sucrose is significantly correlated with SPS activity. Moreover, SPS is involved in plant growth and production, and play an important role in stress-tolerance. There are at least SPS families in higher plant, named A, B and C, respectively, but there are at least five SPS families, named A, B, C, D_III and D_IV, respectively, in the monocotyledonous plants from Poaceae. Each family of SPS gene in different plant shows divergent expression pattern, implying the complicated functional significance of each SPS family in plant. SPS activity is regulated by plant development, light, metabolites, exogenous substance such as auxin, glucide etc. in two ways: gene expression and reversible protein phosphorylation. It’s worth for further studty on transgenetic researches because it’s an effective way to improve production, quality and stress-tolerance of plants by transfer of SPS gene. The comprehensive researches on sucrose phosphate synthase in plants were summarized, and some questions and research views were brought forward.
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  Advances in Engineering of Trichoderma for Improvement of Adaption to Adverse Environment and Efficiency of Biological Control Against Plant Pathogen

  CHEN Yong, ZHU Ting-heng, WANG Kun, CUI Zhi-feng

  China Biotechnology. 2012, 32(6): 120-124.

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  Trichoderma can survive in various environments and grow rapidly, it also can inhibit many phytopathogens, promote plant growth and induce the resistance of plants to the disease. Trichoderma has been the most successful biocontrol agent in plant disease control. Great efforts have been taken to improve their survival abilities and antifungal activities by genetic engineering. The recent achievements in the study of genetic engineering for Trichoderma were discussed.
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  Analysis of Molecular Diagnostics and Individuation Treatment Aiming at Serious Diseases Topics in "Eleventh Five-Year Plan" National High Technology Research and Development Program

  YU Zhen-hang, WANG De-ping

  China Biotechnology. 2012, 32(6): 125-130.

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  A brief analysis about National High Technology Research and Development Program(863)molecular diagnostics and individuation treatment aiming at serious diseases topics in "Eleventh Five-Year Plan" was given. The details about topics plan, institutions carrying out the topics and representative research results were presented,maybe these informations are useful to scientific and technical workers.