Studies on protoplast formation and regeneration ofGanoderma lucidum

To obtain a new strain ofGanoderma lucidum by protoplast fusion technique, its protoplast formation and regeneration were studied. Several factors affecting the protoplast formation and regeneration were investigated to find their optimum conditions. The mycelium was grown for four days on the cellophane membrane placed onG. lucidum complete medium (GCM). When various commercial lytic enzymes were examined for protoplast isolation, the combination of Novozym 234 and β-glucuronidase was found to be effective. An osmotic stabilizer, 0.6 M sucrose in 20 mM phosphate buffer pH 5.8, gave the highest yield of protoplasts. Three-hour incubation in shaking incubator was most suitable for releasing protoplasts. To increase the protoplast yield, pretreatment with 2-mercaptoethanol was carried out. The regeneration frequency in GCM containing 0.6 M MgSO₄·7H₂O was shown to be 0.66%.     

诺西肽产生菌活跃链霉菌的原生质体制备与再生

目的研究诺西肽产生菌活跃链霉菌的原生质体制备与再生的最适条件。方法使用溶菌酶脱去细胞壁制备原生质体,并考察原生质体制备和再生的各种影响因素。结果确定了原生质体制备的条件:一级培养采用种子培养基,培养30 h,转种量的体积分数为5%;二级培养采用R2YE培养基,培养时间为32 h,最适甘氨酸质量浓度为6.0 g.L-1,最佳溶菌酶质量浓度为1.5 g.L-1,酶解时间为60 min,原生质体再生率达到5.3%。结论上述条件为活跃链霉菌原生质体制备与再生的最适条件,该条件的建立为活跃链霉菌原生质体的诱变育种奠定了基础。     

吸水链霉菌井冈变种原生质体的制备与再生

目的研究井冈霉素产生菌吸水链霉菌井冈变种的原生质体制备与再生的最佳条件。方法使用溶菌酶水解菌体细胞壁法,分别考察影响吸水链霉菌井冈变种原生质体的制备与再生的各种因素。结果在R2YE液体培养基中,一级菌丝体的最佳培养时间为24 h,二级菌丝体的最佳培养时间为16 h,最佳甘氨酸质量浓度为5 g.L-1,最佳溶菌酶质量浓度为2 g.L-1,最佳酶解时间为60 min,最佳再生培养基为R2YE,原生质体的再生率最高可达到15.79%。结论本实验确定了吸水链霉菌井冈变种的原生质体制备与再生的最佳条件,能够得到较高的再生率。     

黑暗链霉菌原生质体制备、再生及其DNA转化条件的研究

利用CP培养基培养黑暗链霉菌 (Streptomycestenebrarius) 990 4,采用二级培养即首先在37℃培养 48h ,然后按 10 %的转种量转种于新鲜的培养基中 ,同时补加 2 %甘氨酸 ,2 8℃培养 2 0h ,收获的菌丝体对溶菌酶敏感。在适宜的酶解条件下可形成 4 6 2× 10 9/mL原生质体 ,再生率为18%。利用经修饰的质粒DNA转化冻存的原生质体获得成功 ,转化率为 10 3～ 10 4 / μgDNA。     

Liquid culture enhances protoplast formation from the auxotroph (ser−) ofLentinula edodes

The optimal conditions for the production and regeneration of the protoplasts fromLentinula edodes were studied. Protoplast formation from the mycelia ofL. edodes which were cultured in liquid medium showed a significantly high yield compared with that of the mycelia which were cultured on cellophane covered agar media. A mixture of Novozyme 234 (15 mg/ml) and Cellulase Onozuka R10 (10 mg/ml) in 0.6 M mannitol (pH 4) was optimal lytic enzyme for the protoplast release. The optimal incubation time and mycelia age were 3.5-4 hours at 30 degrees C and 6-8 days, respectively. Regeneration frequency was 0.18% plated onto a medium containing 0.6 M sucrose, and 0.08% plated onto a medium containing mannitol. But hardly any regeneration was observed in the media containing NaCl, KCl, or MgSO(4). More than 90% of the protoplasts contianed nuclei and the nucleus number per protoplast was 1.1. The DNA content per nucleus was 5.1 pg. The diameter of the protoplast was 3-5 mum and it had a well defined cell structure.     

林可链霉菌与委内瑞拉链霉菌种间原生质体融合的研究

本文报道氯霉素产生菌委内瑞拉链霉菌A-186株与林可霉素产生菌林可链霉菌林可变种78-11株种间原生质体融合的结果。首先,研究了委内瑞拉链霉菌A-186的原生质体制备和再生条件,再生率可达86％。然后,采用氯霉素产生菌S.venezuelae A-186的原生质体用紫外线灭活后,与具有耐药标记的S.lincolnensis var.lincolnensis78-11原生质体融合的方法,种间融合频率达到3.2×10~(-5) 。并筛选到一株遗传稳定的重组子,经初步鉴定它能同时产生两亲株所产的两种抗生素。     

红霉素链霉菌原生质体的形成和再生及其对紫外光的敏感性

红霉素链霉菌的菌丝生长在含有0.8％甘氨酸的S培养基中,对溶菌酶的作用敏感,通过酶解,并利用一个包含10mM-MgCl_2和25mM-CaCl_2的高渗培养基能使其菌丝脱壁形成10~(10)/ml的原生质体。 原生质体能再生细胞壁,回复成为一个完整的细胞。最佳条件时再生频率达90％左右。 利用紫外光对红霉素链霉菌的孢子及原生质体分别照射3分钟,其致死率分别为97.95％和98.81％。     

Plant regeneration from mesophyll and suspension protoplasts of Silybum marianum

Mesophyll protoplasts of six lines of Silybum marianum were enzymatically isolated from young leaves, embedded in sodium alginate, and cultivated in KM-medium. Division frequencies observed after ten days were strongly influenced by the protoplast density. When 5 x 10 (4)/ml protoplasts were plated, division frequencies of about 35% were obtained, with a protoplast population density of 1 x 10 (5)/ml division frequencies of about 75% resulted. Plant regeneration experiments undertaken with the protocalluses on medium containing BAP led to shoot formation in only two lines with regeneration frequencies of less than 1% in one (M 24) and up to 7% in a second line (M 2), respectively. However, when the protocalluses from line M 2 were treated with thidiazuron (TDZ) in a first culture step, and with BAP in a second step, the shoot formation frequency rose to 22%. Shoots were rooted on hormone free MS agar medium and transferred into soil where plants grew to maturity. Similar results were obtained when protoplasts of the line M 2, isolated from a suspension culture, were cultivated.     

Microbial transformation of aniline to acetaminophen

In order to obtain acetaminophen, a popular analgesic-antipyretic, through microbial p-hydroxylation and N-acetylation of aniline, various fungi and bacteria were screened. Among them,Streptomyces species were chosen for strain improvement by the use of interspecific protoplast fusion technique. Two interspecific fused strains were developed betweenS. rimosus (N-cetylation function) andS. aureofaciens (p-hydroxylation function) and also betweenS. lividans andS. globisporus. For efficient protoplast fusion and cell wall regeneration, various conditions were examined. In a typical experiment of mixedS. rimosus (pro⁻ his⁻) andS. aureofaciens (ilv⁻) protoplasts with 40% (w/v) polyethylene glycol 3350 (PEG) for 3 min gave 8.3×10⁻⁷ of fusion frequency. Treatment of mixedS. lividans (pant⁻) andS. globisporus (leu⁻) protoplasts with 50% (w/v) PEG for 3 min at 30°C gave 1.2×10⁻⁶ of frequency. Among the fused strains, up to 40–50% increase in p-hydroxylation power was observed. To investigate the possibility of plasmid involvement in p-hydroxylation of acetanilide, plasmid curing was attempted. We found that cells treated with acriflavine (at the frequency of 100%) and cells regenerated from protoplsts ofS. aureofaciens (2% frequency) lost their p-hydroxylation function.     

Protoplast fusion betweenLentinula edodes andCoriolus versicolor

Protoplast fusion between isoleucine-, arginine- and thymidine-requiring auxotroph (lle, Arg, Thy) ofLentinula edodes and arginine-requiring auxotroph (Arg) ofCoriolus versicolor has been achieved using 30% polyethylene glycol (M.W. 4000) in 10 mMCaCl₂-glycine solution (pH 8.0). Fusion hybrids were selected in the 0.6 M sucrose supplemented minimal media on the basis of nutritional complementation with fusion frequency of 7.4×10⁻⁶. The hybrids included both parental and non-parental types in colony morphology, growth rate and isozyme patterns. We succeeded inter-order protoplast fusion between the auxotrophs ofLentinula edodes and Coriolus versicolor overcoming the natural barriers of incompatibility. We examined the characteristics of the hybrids and clarified the fusion process using electron microscopy.     

一种改进卡特利链霉菌358原生质体再生的方法

以卡特利链霉菌（Ｓｔｒｅｐｔｏｍｙｃｅｓ ｃａｔｔｌｅｙａ）３５８为原始菌株,研究了影响原生质体形成和再生的各种因素,证实了再生培养基中含ＨＥＰＥＳ缓冲剂以及Ｐ缓冲液中加入１％的牛血清白蛋白（ＢＳＡ）后能有效地促进再生,在ＳＲ再生培养基上原生质体再生率由０．１％提高到２９．５％。     

原生质体诱变及高通量筛选选育链霉素高产菌株

目的 通过诱变和筛选方法的研究，提高灰色链霉菌(Streptomyces griseus)生产链霉素的水平。方法 优化灰色链霉 菌的原生质体的生成和再生条件，并对得到的原生质体进行紫外诱变，然后利用微孔板高通量方式对获得菌株进行筛选。结果 经过诱变选育获得一株菌NP-11703，其链霉素产量在100L罐上比出发菌株提高了21.8%。结论 用紫外诱变原生质体，可以有 效提高灰色链霉菌产链霉素的能力。结合高通量筛选模型的应用，可大大加快高产菌株的筛选效率。     

刺糖多孢菌原生质体制备与再生条件优化

目的研究多杀菌素产生菌刺糖多孢菌(Saccharopolyspora spinosa)原生质体制备与再生的最佳条件。方法利用数理统计的方法研究了不同制备培养基、菌龄、甘氨酸浓度、溶菌酶处理条件以及再生培养基对原生质体制备和再生的影响,并考察了原生质体的适宜保藏温度。结果菌体在添加0.3%甘氨酸的EHC培养基中培养72h,用2mg/mL溶菌酶32℃酶解40min后,涂布在再生培养基R6上再生,原生质体制备率超过99%,再生数可达到107cfu/mL。刺糖多孢菌原生质体可置于4℃短期保存72h,长期保存需要放置于-80℃条件下。结论优化的结果为刺糖多孢菌原生质体融合育种和遗传转化体系建立奠定了基础。     

Basic techniques for DNA cloning and conditions required for streptomycetes as a host

To develop a host-vector system in streptomycetes for DNA cloning, we examined the technical problems encountered and the conditions required for use of Streptomyces kasugaensis MB273 as the host. Basic techniques, such as plasmid DNA isolation, regeneration of mycelia from protoplasts and elimination of plasmids from cells were investigated. These techniques were found to be useful for many streptomycetes. Strain M518, a derivative of S. kasugaensis MB273, was found to have the following useful characteristics as a host. The plasmids of MB273 were easily cured by regeneration of mycelia from protoplasts. The protoplasts prepared from M518 regenerated mycelia at high frequency using an improved method and were efficiently transformed by plasmid DNA. The extra and intra cellular DNase activities were very weak, and no restriction endonuclease activity was detected. The sensitivity to various antibiotics was determined. This strain did not show any pathogenicity in mice nor suvival in the digestive organs of rats. MB273 and its derivatives died rather quickly in natural soil. M518 still forms aerial mycelial conidia. These results indicate that S. kasugaensis M518, derived from MB273, has useful characteristics as a host for DNA cloning. The techniques thus developed were found to be useful in other streptomycetes.     

Molecular cloning of a xylanase gene from Streptomyces sp. No. 36a and its expression in streptomycetes

The gene for an extracellular xylanase from Streptomyces sp. No. 36a was cloned into Streptomyces lividans TK21 using pIJ702 as a vector plasmid. The smallest DNA fragment encoding the xylanase gene and its possible promotor was determined to be a 1.04 kb Sph I-Sac I fragment by sub-cloning studies. This xylanase gene fragment was transferred into the pSK2 series of plasmids and introduced into Streptomyces kasugaensis G3 protoplasts. The cloned xylanase gene was expressed in both S. lividans TK21 and S. kasugaensis G3, and these clones produced and secreted high yields of xylanase into the culture medium. The xylanase production was not detected when a foreign DNA fragment was inserted into the Bcl I site locating in the xylanase gene fragment.     

紫外线和亚硫酸氢钠对龟裂链霉菌原生质体的诱变作用

用紫外线和亚硫酸氢钠分别处理龟裂链霉菌的原生质体或孢子,结果表明:在相同的条件下,原生质体比孢子对诱变剂敏感;用亚硫酸氢钠处理,原生质体的正变幅度大于孢子。无论用紫外线还是用亚硫酸氢钠处理原生质体,产孢子能力都大大下降。     

抗炎化合物EP产生菌的原生质体制备和再生工艺研究

目的系统研究 6 ,2 2 二烯 5 ,8 过氧麦角甾 3 醇 (EP)产生菌粘帚霉属真菌F菌的原生质体制备、分离与再生的条件。方法通过研究不同的酶解系统、酶解时间、再生培养基等多种因素 ,考察它们对该菌原生质体得率及再生率的影响 ;同时 ,采用高效液相色谱法测定原生质体再生菌株中EP化合物的含量。结果F菌菌龄为 6 0h时 ,以 0 .5mol/L的甘露醇配制成 2 %的蜗牛酶和纤维素酶混合溶液 ,2 8℃酶解 4h ,原生质体得量较高。而以0 .5mol/L的甘露醇为稳渗剂的再生培养基时 ,该菌的原生质体再生率较高。结论从代谢产物的角度对该菌原生质体制备和再生方法的可行性进行分析 ,并探讨此过程对F菌生物合成EP的影响 ,为进一步对该菌的分子遗传及高产菌株的选育创造条件。     

阿卡波糖产生菌原生质体的制备与再生

目的研究阿卡波糖产生菌原生质体制备与再生的条件。方法通过溶菌酶破壁的方法制备原生质体,考察影响原生质体制备与再生的因素。结果和结论确认原生质体的制备条件为:一级培养采用SM2液体培养基,培养时间为33 h,转二级培养的转种体积分数为15%;二级培养采用R2YE培养基,培养时间为20 h,甘氨酸质量分数为0.7%;溶菌酶作用质量浓度为3 g.L-1,作用时间100 min,最大原生质体制备量达到8×1010个.L-1。考察了原生质体再生培养基的组成,在优化的再生培养基上,再生率达到9.2%。     

顶头孢霉229与12的分生孢子及原生质体的形成和再生

头孢菌素产生菌顶头孢霉菌株229的沉没培养或斜面培养都可形成分生孢子,并可用普通滤纸将它们与菌丝及节孢子分开,但是它们成活率极低.这种成活的分生孢子的数量与培养基成分有关.菌丝培养基成分对制备顶头孢霉原生质体有显著影响.用一种MM培养基培养的菌丝,不经巯基化合物预处理,酶解(1%纤维素酶)3小时后,可得到大量原生质体.原生质体的再生频率为1.8～4.6%.与分生孢子形成的菌落相比,原生质体再生菌落的产抗生素能力显示出较大的变异性.本文还讨论了山梨醇与Nikkomycin对菌丝生长形态及原生质体形成的影响.     

Anti-Helicobacter pylori effect of costunolide isolated from the stem bark ofMagnolia sieboldii

Helicobacter pylori (H. pylori) infection is now established as the major pathogenic factor in chronic gastritis and peptic ulcer disease. In addition, there is accumulating evidence thatH. pylori plays an important role in the process of gastric carcinogenesis. On the other hand, oriental traditional medicines have been used for stomach disease for thousands of years. In the present study, methanol extract from the stem bark ofMagnolia sieboldii (M. sieboldii) and its components were investigated on their inhibitory effects against urease activity and growth ofH. pylori in vitro. The methanol extract ofM. sieboldii significantly inhibited the growth ofH. pylori ATCC 43504 at 5 mg/ml. From the further fractionation, the chloroform fraction inhibited the bacterial growth dose-dependently. Among four fractions separated from the chloroform fraction by silica gel column chromatography, MS-C-2 was the most potent. Costunolide was isolated from the MS-C-2 subfraction by preparative TLC and recrystallization using n-hexane. Anti-H. pylori effect of costunolide was investigated using one commercial strain (H. pylori ATCC 43504) and three clinical strains (H. pylori 4, 43, 82548). Costunolide exhibited potent anti-H. pylori activity, and the MIC was around 100–200 μg/ml. However, costunolide had no inhibitory effect ofH. pylori urease activity at the concentration used for the growth inhibition assay. From these results, we conclude that costunolide inhibits the growth ofH. pylori by the independent manner ofH. pylori urease inhibition.