25 August 2006, Volume 26 Issue 08

    

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专题
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  Study on the bombardment factors for Kentucky bluegrasss (Poa pratensis L.)

  China Biotechnology. 2006, 26(08): 1-4.

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  Embryonic callus of Kentucky bluegrass were used as material for genetic transformation with the DREB1A gene by particle bombardment. Parameters of biolistic bombardment were studied. The optimal methods showed as follows. Plasmid DNA are coated by Ca（NO3）2 and PEG4000. 1μm golds are the vectors of plasmid DNA. 6 cm bombardment height distance, 1 time and without osmotic treatment are favorable to transgenic efficiency. The concentration hygromycin (Hy) , which is the selection mark, for cultivar 'Baron' is 100mg/L.
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  Construction of CMO-BADH bivalent-expression vector and its expression in tobacco

  China Biotechnology. 2006, 26(08): 5-9.

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  The objective of this research is to perfect the approach for synthesizing bataine in plants in order to improve the resistance of drought and salt by transferred CMO and BADH genes into the target plants. A bivalent-expression vector, pC35SC35SB1303　were reconstructed based on PC1303 plasmid with CMO and BADH driven by 35S promoter and introduced into Agrobacterium tumefaciens strain LBA4404 by freezethaw method. Transgenic tobacco plants recovered from leaf discs applying Agrobacteriummediated gene were detected by PCR analysis and Northern blotting. The results showed that the foreign gene was consistent with the recipient plants' genome and expresses normally. The analysis of the content of bataine in transgenic plants and blank plants showed that it was higher in the bivalent-expression vector transgenic plants than those transferred with single gene BADH or CMO and blank plants.
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  Transformation of Kentucky bluegrasss (Poa pratensis L.) by particle bombardment

  China Biotechnology. 2006, 26(08): 10-14.

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  BADH-CMO double gene, CMO gene and DREB1A gene were transformed respectively to embryonic callus of Kentucky bluegrass by particle bombardment. The hygromycin-selected plants were obtained. The results of the PCR and Southern blot analysis indicated that the DREB1A gene，CMO gene and BADH-CMO double-gene were integrated into the genomic DNA of Kentucky bluegrass.
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  Advances of Genetic Transformation of Tall Fescue

  China Biotechnology. 2006, 26(08): 15-21.

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  Biotechnology has great application potential in varietals improvement of tall fescue, which is commonly grown as cool-season turf grass in the temperate region. The establishment of tall fescue regeneration and genetic transformation systems is summarized in this paper. At the same time，problems and prospects of genetic transformation of tall fescue are discussed.
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  The deveplment of dwarf mutants related gibberellin

  China Biotechnology. 2006, 26(08): 22-27.

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  Gibberellins(GAs) play an important role in many aspects of plant growth and development, such as seed germination, stem elongation and flower development. In recent years, with the development of research methods and technology, great progresses had been made in identifying many genes related gibberellin biosynthesis and gibberellin signal transduction pathway. There are two kinds dwarf mutants mainly related gibberellin, one is gibberellin deficient dwarf mutants, and the other is gibberellin insensitive dwarf mutants. We summarized the development of two kinds dwarf mutants involved in gibberellin biosynthesis and signal transduction pathway. Researching these dwarf mutants helped us understand gibberellin biosynthesis and sihnal transduction pathway, in the same time, the development can be used as science theory for taking advantage of gibberellin in production.
研究报告
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  Cloning、expression and characterization of pyruvate formate-lyase in Escherichia coli

  China Biotechnology. 2006, 26(08): 28-31.

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  Pyruvate formate-lyase(PFL) is one of the key enzymes in the metabolic pathway of anaerobic or facultative anaerobic microorganism. In order to further study the function of PFL, the pfl gene was amplified by PCR using the E. coli JM109 genomic DNA, and the amplified fraction was ligated into the vector pMD18-T for DNA sequencing. The identified pfl gene was cloned into the expression vector pET-22b and the recombinant expression vector was induced and expressed in E. coli BL21(DE3). A new protein band appeared by analysis of SDS-PAGE compared to the control, whose molecular weight was 85kD. The PFL with 6×His-Tag was purified by metal affinity chromatography. The characterization of purified PFL was carried out. The result showed that the temperature optimum and pH optimum were 35℃ and 7.5 , respectively. The Km value of the enzyme was 2.3mmol, and the value of melting temperature was 49.9℃.
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  Heterologous expression of Zygosacharomyces rouxii ZrGpd 1 in Pichia farinosa

  China Biotechnology. 2006, 26(08): 32-36.

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  To examine the effects of heterologous expression of ZrGPD1 (encoding glycerol 3-phosphate dehydrogenase ) cloned from osmotolerant yeast Zygosacharomyces rouxii on glycerol production in wild Pichia farinosa, the URA3 gene was amplified from P. farinosa as the homology integrative region. A recombinant plasmid (pUR-ZG) was constructed then transformed into P. farinosa by electroporation. The transformant pfa-gu was obtained by the selectable marker ZeocinTM .Primary results showed that the biomass of pfa-gu was higher than the wild type in the flask and after 72h fermentation the concentration of glycerol of pfa-gu was 37g/L enhanced 30% in comparison with the wild type. It is concluded that heterologous expression of ZrGPD1 is useful for increasing glycerol production and the ability of osmoregulation in P. farinosa.
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  Cloning and expression of a fragment of swine HEV ORF2 AG02 in E.coli and the Development of Indirect ELISA with

  China Biotechnology. 2006, 26(08): 37-41.

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  Nest RT-PCR was used to amplified swine hepatitis E virus(SHEV) ORF2-AG02 fragment.AG02 was inserted into pET32a vector and the recombinant expression plasmid PET-HEV was constructed. PET-HEV was transferred into BL21(DE3) and induced by 1mM IPTG, the SDS-PAGE result showed that the 33kDa recombinant protein was expressed. The Western blot analysis showed that the recombinant protein has a good antigenicity with positive SHEV serum. An indirected ELISA was established with the purified recombinant protein, after the square matrix titrate determination best density is 2.43μg/mL, the blood serum best dilution ratio is 1:40,Compares with ten reagents boxes, proved we establish the ELISA method has the high specificity and the sensitivity.
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  Two-Step MS-PCR combined with ELISA Method for the detection of Drug Resistance Mutations in HIV-1 RT Gene

  China Biotechnology. 2006, 26(08): 42-46.

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  Highly Active Anti-Retroviral Therapy (HAART) has effectively inhibited the prevalence of HIV-1 and reduced the death rate caused by AIDS. In recent years, the emergence of resistance-conferring RT gene mutations in HIV-1 strains has become the major reason for HAART failure. The detection of drug resistance is important for the HAART regimen choice and novel drug development. In this work, a novel assay for the detection of HIV-1 RT drug resistance mutations was developed. HIV-1 drug resistance and wild strains in B subtypes were investigated using Two-Step Mutagenically-Separated PCR (MS-PCR), and point mutations including M41L, K70R, K103N, Y181C, T215F were detected. We designed a longer mutant type primer, using microplates hybridization and ELISA technique to detect several point mutations within a mixed mutant-wild type population. The results indicate that the Two-Step MS-PCR is as sensitive and specific as that in the traditional MS-PCR and MS-PCR combined with ELISA can give a good P/N quotient with better sensitivity, low cost, relatively less time consumption and high-throughput screening. We suggest that it will be used in clinic usage for the detection of HIV-1 drug resistance mutations as well as other point mutations.
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  Studies of Escherichia coli accumulating lycopene and its culturing conditions

  China Biotechnology. 2006, 26(08): 47-51.

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  Three genes crtE, crtB, crtI related to lycopene synthesis in Erwinia uredovora were cloned into pET-15b to construct expressing vector pET-15bEIB. The expressing vector was introduced into E.coli BL21(DE3) to construct engineering bacteria. The engineering bacteria could accumulate a red pigment when induced by IPTG. The red pigment was lycopene as judged by absorbance spectra analysis and HPLC analysis. The engineering bacteria could grow to 3.45gDW/L and accumulate lycopene to 5.8mg/gDW under culturing conditions. The culturing conditions used were as follows: the medium was modified LB broth（Trypton 10g/L,Yeast Extract 5g/L,Maltose 5g/L,MgSO4 0.1g/L，NaCl 10g/L）, the engineering bacteia were first shake-cultured at 37℃ to OD600 of 0.6, and then IPTG was added to a final concentration of 0.5mmol/L, the engineering bacteria was further cultured for 14h at 30℃.
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  Polyhydroxybutyrate Synthesis in Recombinant Zymonomas mobilis affected Ethanol Production

  China Biotechnology. 2006, 26(08): 52-56.

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  Zymonomas mobilis was transformed with a polyhydroxybutyrate synthesis operon phbCAB equipped with a pdc promoter from Z. mobilis. For the first time, PHB was produced in recombinant Z.mobilis. Shake flask studies indicated that accumulation of PHB in Zymomonas mobilis increased approximately 10% ethanol productivity for the first 48 h of anaerobic fermentation. After that, the PHB effect was observed as insignificant probably due to the exhaustion of the sugar.
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  Analyzing Role of △5 Desaturase in Biosynthesis of Arachidonic Acid Using Real time PCR

  China Biotechnology. 2006, 26(08): 57-61.

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  Arachidonic acid is an essential fatty acid in human nutrition and a biogenetic precursor of the biologically active prostaglandins and leukotrienes. Δ5 desaturase catalyzes the Δ5 dehydrogenation of di-homo-γ-linolenic acid to form arachidonic acid in biosynthetic pathway of arachidonic acid. Using real time PCR technology, the transcriptional expression levels of gene encoding Δ5 desaturase in three Mortierella alpina strains M10, M6 and M23, and in different growth phase of high arachidonic acid yielding strain M6, were determined. Results showed that there was a distinct corelationship between mRNA transcript level of Δ5 desaturase gene and biosynthesis of arachidonic acid. Results indicated that Δ5 desaturase plays an important role in arachidonic acid biosynthesis.
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  Experimental Study on the Effects of surfactants on Cellulase from Trichoderma viride

  China Biotechnology. 2006, 26(08): 62-66.

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  The effects of surfactants on the production of cellulase by Trichoderma viride in liquid substrate fermentation process were investigated in this study. Straw was used as the sole carbon source and the surfactants were biosurfactant rhamnolipid from Pseudomonas aeruginosa and Tween 80. The changes of FPA, CMCase, Avicelase and surface tension with time were analyzed under different concentrations of the two surfactants. The results showed that the surfactants can enhance the enzyme activity of Trichoderma viride. The FPA, CMCase, Avicelase were promoted 1.08, 1.6 and 1.03 times higher than the controls by rhamnolipid. The enhancement of the enzyme activity by rhamnolipid was much higher than that of Tween 80. At the same time, rhamnolipid was not degraded prior to other substrate.
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  Production, Extraction and Stability for Crude Phytotoxin Produced by Alternaria zinniae

  China Biotechnology. 2006, 26(08): 67-71.

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  Alternaria zinniae was a plant fungal pathogen isolated from a world-wide weed Xanthium occidentale, which could cause some weeds of Asteraceae disease. It was found that the fungus made disease spot on the leaf through producing secondary metabolite--phytotoxin. The toxin-producing capability of the fungus was studied. The optimal cultural conditions for producing phytotoxin were temperature 25℃, pH 6.5, cultured period 9-11d, enough darkness and dissolved O2, rotating speed. Crude toxin was obtained through large scale fermentation. Analysis on the influence of time, temperature, lightness for storing on the stability of Phytotoxin of Alternaria Zinniae showed that the Phytotoxin had the potential to develop as a herbicide originating from microorganism.
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  Effect of Temperature and Carbon Source on the N-acyl-homoserine lactones Production by Food-derived Pseudomonas

  China Biotechnology. 2006, 26(08): 72-76.

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  The many Gram-negative bacteria regulated the expression of some traits by producing the small molecules N-acyl-homoserine lactones (AHLs), i.e., quorum sensing. Pseudomonas ,an important bacteria that caused spoilage of food produced AHLs. In this study, we tested the influence of carbon source, temperature on the AHLs profile produced by food-derived Pseudomonas. The results showed that the strains produced two types of AHLs at 25℃. However, the long chain AHLs were produced mainly, and the short chain AHLs disappeared when the strains were grown at 4℃. The strains produced the different types of AHLs when grown in the different carbon source(glucose, fructose, xylose, maltose). Also the AHLs signal molecules became unstable with increasing pH(>7.5). Overall, our research demonstrates the effect of environmental parameters (temperature and carbon source) on AHL profile production by food-derived Pseudomonas. This study lays the foundation for further investigation to elucidate the relationship between quorum sensing and food spoilage, and for the development of new food preservative by blocking the quorum sensing of food spoilage bacteria.
技术与方法
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  Construction of cartilage using injectable fibrin sealant with human embryo cartilages cells in vivo.

  China Biotechnology. 2006, 26(08): 77-83.

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  Objective: To investigate the feasibility of Fibrin Sealant (FS) as a carrier of human embryo chondrocytes to create new cartilage in vivo. Methods: Chondrocytes isolated by collagenase digestion from the human embryo cartilage were cultured and expanded in monolayer system. The biomechanical properties of the chondrocytes were investigated. 1×107、2×107、3×107 fourth passage chondrocytes were mixed with FS respectively. Then the FS-chondrocyte mixture was injected into the dorsum of nude mice，the ability of chondrogenesis in vivo was investigated 10 weeks later. Results: The proliferation and secretion of substance of the third-fourth passage were powerful. There are specimens like cartilage were harvested in all experimental groups. The average wet weight and GAG content has increased with the increase of cell numbers planted. The difference between any two groups is significant (p<0.05, n=3). There is no significant difference in GAG content in 3×107 group and the normal embryo cartilage (p>0.05, n=3). When the specimens were sectioned and stained, it was demonstrated that they hooked like normal cartilage tissue, with chondrocytes lying in a lacunae in basophilic ground-glass substance around it. The Alcian blue staining and Type II Collagen expressions were strong positive. Most cells appeared to contain several golgi zones and large areas of rough endoplasmic reticulum. Conclusion: This study demonstrates that FS and human embryo cartilage cell can be used as a constructing technique of tissue engineered injectable cartilage.
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  Two-step Tandem Chromatography Purification of Anti-human CD80 Monoclonal Antibody 4E5 from Mouse Ascites

  China Biotechnology. 2006, 26(08): 84-87.

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  In this work, a two-step method for the purification of blocking-type anti-human CD80 monoclonal antibody 4E5 from mouse ascites was developed using anion exchange and gel filtration in combination. The ascites was first purified by anion exchange after centrifugation and filtration. The experimental parameters of sample loading and elution were optimized. The optimized loading condition was pH 8.0, 50 mmol/L Tris-HCl and satisfactory results were obtained using a 0-0.5mol/L NaCl step elution. The fraction containing the protein of interest was directly loaded on gel filtration column and eluted using a 20 mmol/L phosphate buffer at pH 7.2. The purity of the obtained monoclonal antibody was up to 95% with a recovery of 61%. The purity of mAb could efficiently inhibit the growth of Daudi cells. The amplification of the method was also studied using a Bio-Scale Q5 column and the result was satisfied.
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  Separation and Purification of Coenzyme Q10 from microbial fermentation by High-speed Counter-Current Chromatography

  China Biotechnology. 2006, 26(08): 88-92.

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  High-speed counter-current chromatography (HSCCC) is applied for the first time in purification of Coenzyme Q10 (CoQ10) from a fermentation broth extract. A non-aqueous two-phase solvent system composed of heptane-acetonitrile- dichloromethane (12:7:3.5, v/v/v) is selected by analytical HSCCC and used for preparative purification of CoQ10 from 500 mg of the crude extract. The separation yielded 130 mg of CoQ10 at high purity of over 98%. The overall results of the present studies show advantages of HSCCC in the purity, recovery and yield of CoQ10 over the silica gel chromatography with subsequent crystallization.
研究简报
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  Gene Coloning, Expression and Enzymatic Assay of Human sPLA2-IIA

  China Biotechnology. 2006, 26(08): 93-97.

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  Objective To clone the cDNA of human sPLA2-IIA, construct the engineered Escherischia coli expressing human sPLA2-IIA and Identify the expressed human sPLA2-IIA. Methods: Total RNAs were purified from human fetal spleen. The cDNA of human sPLA2-IIA was cloned by RT-PCR and inserted into plasmid pET32a(+) between NcoI and EcoRI sites for expressing the recombinant human sPLA2-IIA in Escherischia coli BL21(DE3). The recombinants were screened by SDS-PAGE. The engineered Escherischia coli expressing trxA-human sPLA2-IIA fusion protein was established. The expressed human sPLA2-IIA exists in the form of inclusion body and accounts for about 25% of the total soluble proteins of Escherischia coli BL21(DE3). Conclusion: the engineered E. coli methods are suitable for preparing plenty of human sPLA2-IIA which has laid base for the large-scale expression, purification and basic studies of human sPLA2-IIA.
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  The Influence of Peptidoglycan of Lactobacillus on immune function of Mouse

  China Biotechnology. 2006, 26(08): 98-102.

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  Mice was stimulated by peptidoglycan from Lactobacillus sp. Affymetrix MOE430A genechip was used to analyze changed gene expression of immune cells. It was found that expression of cytokines and related genes were changed under peptidoglycan administration. This might induced by activation of TLR-NF-κB signal pathway.
综述
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  Structure of Wheat High Molecular Weight Glutenin Subunits and Their Role in Determining Processing Properties

  China Biotechnology. 2006, 26(08): 103-110.

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  The high molecular weight glutenin subunits (HMW-GS) are the main components of storage proteins of wheat, and play a critical role in determining the visco-elastic properties of gluten. There are both quantitative and qualitative effects of HMW-GS on the processing properties of wheat. The recent advances in this field were summarized in details, and the relationship between HMW-GS and wheat quality was discussed systematically in this paper.
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  Reviews on Foodborne Fibrinolytic Enzyme

  China Biotechnology. 2006, 26(08): 111-114.

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  As thromolytic therapy is a safe and effective way to cure thrombotic diseases, it is important to develop safe, effective and cheap therapeutic thromolytic agent, such as food-sourced fibrinolytic enzyme, to prevent and cure thrombotic diseases. In recent years, several fibrinolytic enzymes resources have been found in some Asian traditional fermented foods, such as Japanese natto, Korea Chungkook-Jang, Chinese Douchi and fermented shrimp paste. In this paper, the research and development of fibrinolytic enzymes of Asian fermented food is reviewed.
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  Study Progress on Monitoring of the Complex Compost System by Immunosensor

  China Biotechnology. 2006, 26(08): 115-122.

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  With the development of immunoassay and sensing technologies and the solid waste compost technologies being paid more and more attention, it is of great significance to apply the immunosensor technologies in monitoring, and real-time, online measurement during compost process. In this paper, the components of the complex compost system were divided into solid phase, liquid phase and gas phase. The development and application of immunosensor in compost is introduced. The latest progress in immunosensor for determination of trace toxicants is reviewed. The application of immunosensor in environmental monitoring and its future development are also discussed.
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  Study Progress of Roles for Interferons during Mammalian Early Embryonic Development

  China Biotechnology. 2006, 26(08): 123-126.

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  Interferons（IFNs）is glycoprotein secreted by many kinds of cells. It has multiple roles in antivirus, anti-tumour and immunity regulation. More and more evidences have proved that IFNs may play important roles not only in implantation and maintenance of pregnancy, but also during early embryo development. This presentation mainly summarizes mechanism IFNs and progress of its role in early embryonic development combining with our research.