米曲霉两株营养缺陷型原生质体的形成和再生的条件实验*

采用1%溶壁酶加1%玛瑙螺酶(褐云玛瑙螺消化液的冷冻干粉)的混合酶,自米曲霉(Aspergillus oryzae)的两株营养缺陷型中获得了大量的原生质体,并比较了渗透压稳定剂、温度、菌丝体的培养基成分等因素对原生质体形成和再生的作用。无机盐类稳定剂(NaCl、KCl)获得了高产量的原生质体,而有机类(蔗糖、甘露醇、山梨醇)做为稳定剂不甚理想。对120和720菌株的原生质体在高渗再生培养基上进行再生试验,再生率分别为52%和65%。     

Regeneration and molecular characterization of an intergeneric hybrid between Graphium putredinis and Trichoderma harzianum by protoplasmic fusion

The fungal strains Graphium putredinis and Trichoderma harzianum were selected as parents for fusant development. Protoplasts were isolated using the combination of lysing enzymes Novozym 234 and cellulase with 0.6 M KCl as osmotic stabilizer. The optimum conditions for release of viable protoplasts from the fungal mycelium viz. age of the mycelium, lytic enzymes, osmotic stabilizers, pH, incubation period and regeneration medium were determined. Intergeneric protoplast fusion was carried out using 50% polyethylene glycol with calcium chloride (CaCl₂) and glycine buffer and the conditions for effective protoplast fusion, viz. fusogen, osmotic stabilizer, pH, incubation period and regeneration medium were optimized. At optimum conditions, the regeneration frequency of the fused protoplasts on potato dextrose agar (PDA) medium and fusion frequency were calculated. The regeneration frequency on non-selective (PDA) and selective media (PDA amended with starch) was determined for the parental and fusant strains in which, fusant showed a higher rate of regeneration. Fusant formation was confirmed by morphological markers (colony morphology and spore size and shape) and genetical markers like, mycelial protein pattern, restriction digestion pattern and random amplified polymorphic DNA (RAPD) analysis. The efficiency of these parental strains and their intergeneric fusant in the production of hydrolytic enzymes – amylases (treatment plant for sago factory effluent), cellulases (bioethanol), xylanases (bleaching agents for waste paper pulp) and proteases (additives in commercial detergents) – have probable applications in various industrial processes.     

疏绵状嗜热丝孢菌原生质体的制备与再生

以疏绵状嗜热丝孢菌(Thermomyces lanuginosus)为供试菌株,研究了菌龄、酶的种类及浓度、酶解时间、酶解温度和稳渗剂对原生质体制备的影响及稳渗剂对原生质体再生的影响。结果表明,制备嗜热丝孢菌原生质体比较适宜的条件为:PDB液体培养基培养28 h,以0.7 mol/L NaCl为稳渗剂,0.15 mol/L的溶壁酶,30℃酶解4 h。原生质体再生以0.7 mol/L蔗糖作稳渗剂为最佳。     

产琥珀酸放线杆菌的原生质体制备与再生

基因组重组技术是一项重要的菌种改造技术,原生质体制备和再生是进行基因组重组的前提和基础。目前少有关于产琥珀酸放线杆菌(Actinobacillus succinogenes)CGMCC2650原生质体研究的报道。为了优化该菌的原生质体制备和再生条件,及利用基因组重组技术构建优良菌种提供参考,研究了甘氨酸预处理,菌龄,酶浓度,作用时间,温度对产琥珀酸放线杆菌原生质体制备和再生的影响,并考察了不同渗透压稳定剂对其再生的影响。结果表明,菌体在添加了0.6mg/ml甘氨酸的TSB培养基中培养5h后收集,用SMM稀释到OD660=1.0,用0.025mg/ml溶菌酶在37℃下酶解45min制备原生质体,将原生质体涂布于含0.3mol/L蔗糖的再生培养基中,再生率最大,达到40.9%。确定了产琥珀酸放线杆菌原生质体制备和再生的最佳条件,所用的原生质体制备的方法对琥珀酸的产生没有影响,这为进一步开展该菌的原生质体诱变及基因组重组等研究奠定了基础。     

轮梗霉原生质体的制备*

本文比较了酶浓度、菌龄、渗透压稳定剂以及酶解温度和时间等因素对轮梗霉原生质体得率的影响。结果基本获得了制备原生质体的适宜条件:用0.6mol/L甘露醇稳渗剂配制成的4%纤维素酶和0.5%蜗牛酶混合酶,35℃酶解培养了30h的菌丝1.0h,即可得到较高产量的原生质体。对该原生质体进行了再生实验,其再生率约为23.8%。     

Genetic recombination by protoplast fusion inNocardia asteroides

Summary Fusion and regeneration of protoplasts ofNocardia asteroides strains ATCC 3318, IMRU W3599 and HIK B971 have been used to study genetic recombination in this species. Protoplasts were produced by treatment with lysozyme, following incubation with glycine. Mutants of ATCC 3318 were grown in peptone yeast extract medium at 32°C prior to protoplast production to maximize protoplast frequency, whereas mutants of IMRU W3599 and HIK B971 were grown in trypticase-soy broth. Glycine concentrations favoring protoplast formation varied from 1.5% to 5% depending on strain. For all strains, protoplast formation was complete 1 h after addition of 5 mg/ml lysozyme. Protoplasts were fused by addition of 50% polyethylene glycol-1000. In general, 25% of the protoplasts could be regenerated. The incidence of recombinant recovery was increased up to 750-fold. The distribution of recombinant phenotypes in matings was similar for protoplast fusion and conventional crosses.     

Optimization of protoplast formation,regeneration, and viability in Microsporum gypseum

Factors affecting high yields, regeneration frequencies, and viability of protoplasts from clonal cultures of Microsporum gypseum were investigated. Maximum yields of protoplasts were obtained after 6 hrs digestion of 2–4 days old mycelium with Novozyme 234 using CaCl₂ (0.4 M) as an osmotic stabilizer and glycine + HCl (pH 4.5) as the buffer system. Mercaptoethanol + dithiothreitol (0.01 M) proved to be the best pretreatment of mycelium prior to digestion with enzyme. A regeneration frequency of 94.4% was obtained using the top agar method with complete medium (pH 6.5) containing 0.5% agar and 0.4 M CaCl₂ as an osmoticum. Colonies from regenerated protoplasts on medium containing CaCl₂ were pigmented and completely powdery with high sporulation. Protoplast viability was studied in osmotic stabilizer supplemented with glucose or glutamine. After 24 hrs, glucose (2%) and glutamine (2%) enhanced protoplast viability by 22% and 23%, respectively. Protein synthesis, as measured by ³H-lysine uptake, matched the viability profile determined by fluorescence microscopy.     

Mycelial protoplast isolation and regeneration of Lentinus lepideus

Generation of fungal protoplast is essential for fusion and transformation systems. Protoplast fusion offers great potential for the improvement of industrially important microorganisms. To establish conditions for the protoplast isolation and regeneration of the mycelia of Lentinus lepideus, various enzymes and osmotic stabilizers were examined. To investigate suitable medium for the culture of L. lepideus, the mycelia were grown in ten different media at 28 degrees C for 10 days. Among them potato dextrose agar (PDA) medium was found to be the best for colony growth. When Novozym 234, cellulase and beta-glucuronidase were added to the mycelia in combination or alone, Novozym 234 alone at the concentration of 10 mg/ml was the most effective for the protoplast yield. Purified spherical protoplasts of the mycelia were osmotically hypersensitive and further incubation of the mycelia with the lytic enzyme resulted in the older parts of the hyphae swollen. When we applied various osmotic stabilizers at the fixed concentration of 0.6 M on the protoplasts, the yields of protoplasts were increased until 4-hr incubation. However application of sucrose or MgSO4 led to further protection of protoplasts after that time and reached a plateau on 5- and 7-hr incubations, respectively. The suitable incubation time and optimal pH with the lytic enzyme for the maximum release of protoplasts were 6 hrs of incubation and pH 5, respectively. When we examined various osmotic stabilizers for the regeneration of the protoplast, the complete medium containing 0.6 M sucrose induced highest hyphal growth with regeneration frequency of 3.28%.     

γ-亚麻酸生产菌深黄被孢霉原生质作的形成和再生

采用2％的溶壁酶加4％的蜗牛酶,从深黄被孢霉的菌丝中获得了大量的原生质体。同时对菌丝的培养时间、渗透压稳定剂、酶解系统和酶解温度等因素进行了系统的观察,从而获得了制备深黄被孢霉原生质体的最适条件。并对该原生质体在高渗培养基上进行了再生实验,其再生率为32％。     

Production and Regeneration of Lactobacillus casei Protoplasts

Methods for the production and regeneration of Lactobacillus casei protoplasts are described. Protoplasts of L. casei strains were obtained by treatment with mutanolysin or with mutanolysin and lysozyme together in a protoplast formation buffer containing 0.02 M HEPES (N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid) (pH 7.0), 1 mM MgCl₂, 0.5% gelatin, and 0.3 M raffinose. Cells were regenerated on a complex medium supplemented with bovine serum albumin, MgCl₂, CaCl₂, gelatin, and raffinose. Lengthy digestion with lytic enzymes inhibited the capacity of protoplasts to regenerate. The optimum conditions of protoplast formation varied from strain to strain. Using predetermined optimal conditions it was possible to prepare protoplasts of several L. casei strains and regenerate them with 10 to 40% efficiency. The methods were applicable to other species of lactobacilli as well.     

木耳属种间杂交技术研究 Ⅰ.木耳菌丝原生质体的形成与再生

本文比较了不同酶液、渗透压稳定剂、酶解温室及菌丝培养基成份等因素对木耳属(Auricularia)中木耳(Auricularia auricula)和毛木耳(Auricularia polytricha)菌丝释放原生质体的作用及影响。用0.5%纤维素酶加0.5%蜗牛酶的混合酶液,以0.6M的MgSO_4为稳定剂,在34℃下可自两种菌丝体获得大量原生质体。对原生质体再生条件的研究表明,纤维二糖和菌丝体培养物浸提物对再生有明显促进作用,再生率达20%左右。本文还用VBL型荧光增白剂观察了菌丝脱壁以及原生质体细胞壁再生的过程。     

蓝色犁头霉原生质体的制备与再生

研究了氢化可的松生产菌蓝色犁头霉原生质体的形成与再生。通过对溶解酶系统的选择,影响原生质体形成的因素如渗透压稳定剂、酶浓度、菌龄、菌丝培养基和培养方式等因素进行考察,发现以0．4mol/L NH4Cl做为稳定剂、2．5mg/mL溶壁酶和5mg/mL纤维素酶组成的混合酶液溶解菌丝,4h后原生质体量可达10^6cell/mL。通过显微镜观察原生质体的形成过程以及在高渗培养基上的再生情况,再生率为15．6％。     

Protoplast Formation and Regeneration of Streptococcus bovis Cells

The simultaneous use of hen egg lysozyme and mutanolysin, N-acetylmuramidase SG, showed synergistic effects on the lytic action and more effectiveness for protoplast formation of Streptococcus bovis IFO 12058. The frequency of regeneration reached 100% after 7 days of incubation under the improved conditions and by a method in which protoplasts were overlaid with the agar medium layer.     

红曲霉原生质体的制备、再生及其遗传转化系统

原生质体是研究和建立真菌遗传转化系统的重要工具。为了建立原生质体介导的红曲霉遗传转化系统,考察了各种细胞壁裂解酶和渗透压稳定剂等对红曲霉原生质体形成和再生的影响。将红曲霉分生孢子在铺有玻璃纸的平板上30℃培养30~40 h收获的菌丝体最有利于原生质体的形成和释放。红曲霉菌丝体形成和释放原生质体最适裂解酶和酶解时间分别为：0.3 % lysing enzyme、0.1 % cellulase和1 % snailase的酶组合,30℃作用2.5 h;最适渗透压稳定剂是：1mol /L MgSO4。最适合原生质体再生的培养基为含0.6 mol/L蔗糖的CM培养基。原生质体液涂布单层再生培养基的方法,再生率最高,菌株M34和N18分别为8.5 %和36.4 %。在PEG和CaCl2存在下,以潮霉素B为抗生素选择标记,用质粒pBC-Hygro和pNL1共转化菌株M34原生质体,每微克DNA克获得100个稳定转化子。     

Preparation of protoplasts from <Emphasis Type="Italic">Chlorella protothecoides</Emphasis>

This paper describes an enzymatic method for yielding protoplasts from the microalga Chlorella protothecoides. Four kinds of commercially available enzymes were tested. The enzymatic digestion was optimal with 2% cellulase R-10 and 1% snailase prepared in 25 mM Tris buffer (pH 6.0) containing 0.6 M D-mannitol, and the protoplast density could reach the peak after treatment at 30°C for 16 h. Nearly all liberated protoplasts were green in the presence of 0.01% phenosafranin, indicating their high viability. The regeneration rate was about 70% when 0.6 M D-mannitol was used as an osmotic stabilizer in the regeneration medium. This protocol will find useful applications in genetic studies of this algal species.     

大球盖菇原生质体再生及单核化特性的研究*

大球盖菇原生质体再生条件及单核化特性结果。原生质体再生速度极快,涂布平板３ｄ后肉眼即可见明显的再生菌落形成,在ＰＧＰＭ再生培养基上再生率为０．９７～２．０％,渗稳剂种类对再生率无明显影响,但可影响再生菌落形态,液体预培养１～２ｄ,再生率明显下降;大球盖菇原生质体单核化率高达７７．６％,且再生双核体和再生单核体在形成再生菌落时无时间差,其生长速度亦无快慢之分,液体预培养可显著减少单核化率,再生单核体中存在亲本两种交配型,但二者的比率不为１。     

Formation,regeneration, and fusion of protoplasts in aCellulomonas strain

The effect of different conditions on protoplast formation was studied in the streptomycin-resistant strainCellulomonas sp.M32Bo. The greatest efficiency (75% protoplasts) was achieved by use of 0.5M sodium succinate as osmotic stabilizer, supplemented with 20 mM MgCl₂, 200 µg/ml of lysozyme, and 0.01M EDTA at pH 7.4. Cells harvested at the midexponential growth phase were more suitable for protoplast formation than those of the stationary phase. Electron microscopy observations showed the presence of both protoplasts and spheroplasts in the treated samples, some of them still showing a rod shape. Two regeneration media were developed that showed similar regeneration frequencies (52%). StrainM32Bo was fused with a tetracycline-resistant strain (Cellulomonas sp. Sz). Segregation analysis of fusant colonies suggested the existence of a temporary diploid stage in which both parental genotypes were expressed.     

地衣芽孢杆菌原生质体的制备、再生及转化研究

目的：提高地衣芽孢杆菌原生质体的产量和形成率,为进一步提高原生质体转化率打下基础。方法：通过酶解法对地衣芽孢杆菌工业生产菌株Bacillus licheniformis303原生质体的制备及再生条件进行了研究。考察了菌体生长状态、溶菌酶浓度、处理时间、渗透压稳定剂和再生培养基等因素对地衣芽孢杆菌原生质体的制备及再生的影响。结果：对数生长后期的菌体,以SMMP作渗透压稳定剂,溶菌酶浓度为100mg/mL,37℃下酶解30min,原生质体生成量可达8&#215;109个/mL;再生培养基选用含1mol/L琥珀酸钠的DM3时,再生率最高可达17%。在此条件下,采用PEG法将游离型质粒pHY-P43-secQ转化宿主菌B.lichenifor-mis303,转化率可达10~15 CFU/μg DNA。     

The formation and regeneration of mycelial protoplasts in hyper lignolytic fungus,strain IZU-154

Over 2 × 10⁷/ml protoplasts were obtained from mycelia of hyper lignolytic fungus (nomenclatured as strain IZU-154) by treatment with the lytic enzyme NovoZym 234 in the presence of 0.05 M maleic acid buffer (pH 5.6) containing 0.6 M MgSO₄. The protoplasts regenerated at more than 10% of frequency on solid 2% agar medium containing 0.6 M sucrose as an osmotic stabilizer overlaid with 0.5% agar containing the stabilizer. In the determination of the lignolytic activities of 50 regenerants from protoplasts, 2 strains which degraded more than 56% of the lignin during incubation for 30 d and showed activity higher than the parent were found. The regeneration from protoplasts of this fungus was suggested to be useful for the breeding of strains having higher lignolytic activity than this fungus.     

Production and regeneration of protoplasts from Penicillium nalgiovense

The conditions for optimum protoplast formation from Penicillium nalgiovense were determined. Best results were achieved using Novozyme 234 in combination with β-glucuronidase. Chitinase or β-glucuronidase alone were not able to produce protoplasts under the given conditions. For the regeneration frequency the kind of osmotic stabilizer was important. The use of ionic stabilizers enhanced the regeneration efficiency up to 80% in contrast to only 30% regeneration with sorbitol.