Screening, mutagenesis and protoplast fusion of Aspergillus niger for the enhancement of extracellular glucose oxidase production

Various strains of Aspergillus niger were screened for extracellular glucose oxidase (GOD) activity. The most effective producer, strain FS-3 (15.9 U mL^–1), was mutagenized using UV-irradiation or ethyl methane sulfonate. Of the 400 mutants obtained, 32 were found to be resistant to 2-deoxy d-glucose, and 17 of these exhibited higher GOD activities (from 114.5 to 332.1%) than the original FS-3 strain. Following determination of antifungal resistance of the highest producing mutants, four mutants were selected and used in protoplast fusions in three different intraspecific crosses. All fusants showed higher activities (from 285.5 to 394.2%) than the original strain. Moreover, of the 30 fusants isolated, 19 showed higher GOD activity than their corresponding higher-producing parent strain.     

Production and seeding of protoplasts of <Emphasis Type="Italic">Porphyra okhaensis</Emphasis> (Bangiales,Rhodophyta) in laboratory culture

High yields of viable protoplasts were produced from Porphyra okhaensis H. Joshi, Oza & Tewari following two-step enzymatic digestion (protease pretreatment and cell wall polysaccharides-degrading enzyme treatment) of the thallus. Pretreatment of the tissues with 1% Protease P6 at 20± 1 ^°C for 30 min prior to digestion with cell wall polysaccharide-degrading enzymes increased the protoplast yield two fold compared to tissues that were digested with polysaccharide-degrading enzyme mixture. The polysaccharide-degrading enzymes employed for protoplast isolation from P. okhaensis were Cellulase Onozuka R-10, Macerozyme R-10, abalone acetone powder and agarase. Suitable pH, temperature and duration of enzyme treatment for optimal production of viable protoplasts were pH 6, 20± 1 ^°C and 3 h, respectively. Mannitol (0.8 M) was found to be an excellent osmotic stabilizer. When the tissue of P. okhaensis pretreated with 1% protease solution was digested with commercial enzyme mixture consisting of 2% Cellulase Onozuka R-10, 2% Macerozyme R-10, 1% abalone acetone powder, 50 units of agarase and 0.8 M mannitol in 1% NaCl (adjusted to pH 6.0 with 25 mM MES buffer) with gentle agitation for 3 h at 20± 1 ^°C, 23.2± 0.24× 10⁶ protoplasts g⁻¹ fresh wt. were obtained. The regeneration rate of protoplasts isolated in the present study was found to be 79%. Protoplasts that regenerated cell walls underwent regular cell divisions and developed into leafy gametophytic thallus in the laboratory cultures. Further, the seeding of nylon threads with partially developed protoplasts of P. okhaensis was successful in the laboratory conditions and germlings as long as 3–4 cm were obtained from such seeded threads in one month period in aerated cultures.     

Analysis of amino acids in individual wheat embryonic protoplast

Summary. Amino acids analysis in single wheat embryonic protoplast was performed using capillary electrophoresis equipped with laser-induced fluorescence (CE-LIF), combination with tissue culture technique. Reagent fluorescein isothiocyanate (FITC) was introduced into living protoplasts by electroporation for intracellular derivatization. A special osmotic buffer (0.6 mol/L mannitol, 5 mmol/L CaCl₂) was used to keep the osmotic balance of embryonic protoplasts during the protoplasts derivatization. After completion of the derivatization reaction in the protoplasts, a single protoplast was drawn into the capillary tip by electroosmotic flow. Then a 0.1 M NaOH lysing solution was injected by diffusion. The derivatized amino acids were separated by capillary electrophoresis and detected by laser-induced fluorescence detection after the protoplast was lysed Nine amino acids were quantitatively and qualitatively determined and compared in lysate and single protoplast of wheat embryonic cells respectively, with mean concentrations of amino acids ranging from 2.68×10⁻⁵ mol/L to 18.18×10⁻⁵ mol/L in single protoplast.     

麻疯树MIPS基因启动子的分离及在烟草原生质体中瞬时表达活性分析

根据麻疯树MIPS基因序列,设计特异性的巢式引物,运用TAIL-PCR法两次步移得到MIPS基因5＇端侧翼序列,序列分析显示含有多个胁迫应答相关元件,如ABRE、HSE等。以该序列为基础,PCR扩增得到5个5＇端不同长度的缺失片段,分别插入pBI221载体置换CaMV35S启动子,构建的表达载体在PEG介导下转入烟草叶片原生质体进行瞬时表达,检测GUS报告基因的活性。经GUS活性荧光定量检测发现,分离到的MIPS基因侧翼序列5＇端不同缺失片段都能启动GUS报告基因表达,启动活性最高的是WQ1区（-565bp）,核心区位于-565～-449bp。在100μmol·L-1ABA诱导下启动活性增强,但不同区段的增长幅度不同。WQ1区增长幅度最大,比未处理时提高41.4%。     

Macrotubule-dependent protoplast volume regulation in plasmolysed root-tip cells of Triticum turgidum: involvement of phospholipase D

The probable involvement of phospholipase D (PLD)/phosphatidic acid (PA) signalling in the hyperosmotic stress response of Triticum turgidum root cells was investigated by examining the effects of butanol-1, butanol-2, phosphatidylbutanol (PtdBut), N-acylethanolamine (NAE) and PA on the hyperosmotic response, the organization of the tubulin cytoskeleton and the accumulation of a phosphorylated p38-like mitogen-activated protein (MAP) kinase (phospho-p46) in plasmolysed root cells. The effects of all the treatments were assessed by differential interference contrast (DIC) microscopy of living cells, tubulin immunofluorescence, conventional transmission electron microscopy (TEM), tubulin immunogold localization, protoplast volume measurements and western blot analysis. Butanol-1 and NAE compromised the viability of plasmolysed cells, induced a marked reduction in the plasmolysed protoplast volume, and inhibited hyperosmotically induced tubulin macrotubule formation and the accumulation of phospho-p46. Exogenous PA reinforced the hyperosmotic response of T. turgidum root cells and positively affected tubulin macrotubule formation. Additionally, PA reduced the effects of butanol-1 in plasmolysed cells. Taken together, the data suggest that PLD-mediated PA synthesis occurs upstream of the accumulation of phospho-p46 to regulate hyperosmotically induced macrotubule formation in plasmolysed T. turgidum root cells.     

结球甘蓝下胚轴原生质体培养再生植株体系的优化研究

以结球甘蓝‘新夏50’的无菌苗下胚轴为材料,对影响原生质体分离、纯化与培养的主要因素进行研究,建立适合结球甘蓝原生质体游离、纯化、收集、培养以至再生出完整植株的实用技术体系,为其非对称细胞融合及品种改良与创新等研究奠定基础。结果表明:2.5%纤维素酶R-10+0.05%果胶酶Y-23+9CPW+5mmol/L MES的混合酶液,从4d苗龄的下胚轴上分离出高产率的原生质体。在改良B5+0.5mg/L 2,4-D+0.2mg/L 6-BA+0.2mg/L NAA的液体培养基上,原生质体分裂旺盛。形成愈伤组织后经芽诱导和生根培养,获得了再生植株。倍性检测结果表明,不同原生质体所获得的24株再生植株中,19株为正常二倍体,4株为嵌合体,1株为四倍体。     

CONDITIONAL PRODUCTION OF A FUNCTIONAL FISH GROWTH HORMONE IN THE TRANSGENIC LINE OF NANNOCHLOROPSIS OCULATA (EUSTIGMATOPHYCEAE)1

Plasmid phr‐YPGHc, containing the fish growth hormone (GH) cDNA driven by a heat shock protein 70A promoter and a RUBISCO SSU 2 promoter, was transferred into the protoplast of marine microalga Nannochloropsis oculata (Droop) D. J. Hibberd by electroporation. Four transgenic clones were obtained in which the transferred phr‐YPGHc was integrated into the genome and existed stably at least until the 50th generation. When we treated these transgenic microalgae by heat shock, the heterologous fish GH was produced in the amount of 0.42 to 0.27 μg · mL^?1 from the 50 mL of medium. We incubated artemia with the wildtype and transgenic N. oculata for 6 h and then fed these microalgae‐treated artemia to red‐tilapia larvae. After feeding, the growth of larvae that were fed artemia incubated with transgenic microalgae was greater (i.e., statistically significant: P < 0.05) than that of larvae that were fed artemia incubated with nontransgenic microalgae: 316% versus 104% in weight gain, and 217% versus 146% in body length increase, respectively. Therefore, the N. oculata enables production of functional GH, and we propose that it might be an excellent bioreactor material.     

芜菁原生质体的分离及绿色荧光蛋白的瞬时表达

目的：分离芜菁叶片原生质体,建立蛋白质在芜菁原生质体的瞬时表达系统。方法：以津田芜菁成叶为试材,酶解分离原生质体;通过PEG介导的转化,将编码绿色荧光蛋白（GFP）的瞬时表达载体转入原生质体中,用激光扫描共聚焦显微镜检测原生质体中GFP的表达情况。结果：分离出大量的津田芜菁原生质体,并获得了较高的转化效率,GFP在整个原生质体中都有表达。结论：建立了津田芜菁原生质体瞬时表达系统。     

Assembly of the protoplasm of Codium fragile (Bryopsidales, Chlorophyta) into new protoplasts

The cell organelles of the coenocytic alga Codium fragile (Sun) Hariot aggregated rapidly and protoplasts were formed when its protoplasm was extruded out in seawater. Continuous observation showed that there were long and gelatinous threads connecting the cell organelles. The threads contracted, and thus the cell organelles aggregated into protoplasmic masses. The enzyme digestion experiments and Coomassie Brilliant Blue and Anthrone stainings showed that the long and gelatinous threads involved in the formation of the protoplasts might Include protein and saccharides as structure components. Nile Red staining Indicated that the protoplast primary envelope was non-lipid at first, and then lipid materials Integrated Into its surface gradually. The fluorescent brightener staining Indicated that the cell wall did not regenerate in the newly formed protoplasts and they all disintegrated within 72 h after formation. Transmission electron microscopy of the cell wall of wild C. fragile showed electron-dense material embedded in the whole cell wall at regular intervals. The experiments indicated that C. fragile would be a suitable model alga for studying the formation of protoplasts.     

Topography and functional information of plasma membrane

By using atomic force microscope (AFM), the topography and function of the plasmalemma surface of the isolated protoplasts from winter wheat mesophyll cells were observed, and compared with dead protoplasts induced by dehydrating stress. The observational results revealed that the plasma membrane of living protoplasts was in a state of polarization. Lipid layers of different cells and membrane areas exhibited distinct active states. The surfaces of plasma membranes were unequal, and were characterized of regionalisation. In addition, lattice structures were visualized in some regions of the membrane surface. These typical structures were assumed to be lipid molecular complexes, which were measured to be 15.8±0.09 nm in diameter and 1.9±0.3 nm in height. Both two-dimensional and three-dimensional imaging showed that the plasmalemma surfaces of winter wheat protoplasts were covered with numerous protruding particles. In order to determine the chemical nature of the protruding particles, living protoplasts were treated by proteolytic enzyme. Under the effect of enzyme, large particles became relatively looser, resulting that their width was increased and their height decreased. The results demonstrated that these particles were likely to be of protein nature. These protein particles at plasmalemma surface were different in size and unequal in distribution. The diameter of large protein particles ranged from 200 to 440 nm, with a central micropore, and the apparent height of them was found to vary from 12 to 40 nm. The diameter of mid-sized protein particles was between 40―60 nm, and a range of 1.8―5 nm was given for the apparent height of them. As for small protein particles, obtained values were 12―40 nm for their diameter and 0.7―2.2 nm for height. Some invaginated pits were also observed at the plasma membrane. They were formed by the endocytosis of protoplast. Distribution density of them at plasmalemma was about 16 pits per 15 μm2. According to their size, we classified the invaginated pits into two types―larger pits measuring 139 nm in diameter and 7.2 nm in depth, and smaller pits measuring 96 nm in diameter and 2.3 nm in depth. On dehydration-induced dead pro-toplasts, the degree of polarization of plasma membranes decreased. Lipid molecular layers appeared relatively smooth, and the quantity of integral proteins reduced a lot. Invaginated pits were still de-tectable at the membrane surface, but due to dehydration-induced protoplast contraction, the orifice diameter of pits reduced, and their depth increased. Larger pits averagely measuring 47.4 nm in di-ameter and 31.9 nm in depth, and smaller pits measuring 26.5 nm in diameter and 43 nm in depth at average. The measured thickness of plasma membranes of mesophyll cells from winter wheat examined by AFM was 6.6―9.8 nm, thicker in regions covered with proteins.