荧光标记香菇原生质体融合菌株的鉴定

以异硫氰基荧光素（FITC）标记和香菇单核L4菌株的原生质体和未经标记的香菇单核B14菌株的原生质体为亲本,在聚乙二醇（PEG）的促融下进行融合,选取一个带有荧光,另一个不带荧光的原生质体粘合对进行再生培养,通过对利用“锁状联合”筛选得到的菌株进行鉴定后表明：融合菌株为双核菌丝,与双亲产生明显拮抗,其酯酶同工酶及可溶性蛋白质凝胶电泳图谱分析表明融合菌株均与双亲有区别,经琼脂平板快速出菇及出菇实验证明融合菌株出菇早,产量有所提高。     

香菇原生质体融合及融合子的鉴定

以香菇（Ｌｅｎｔｉｎｕｓｅｄｏｄｅｓ）种内不同株一对亲和的单核菌丝（７４０２〈２〉和９１０１〈１２〉）为亲本,以碘乙酰胺失活７４０２〈２〉作为筛选标记,经过原生质体制备、ＰＥＧ融合及融合子再生等步骤,选育得融合子。融合子与双亲无拮抗性,在菌丝形态,核数目及可溶性蛋白质图谱、酯酶同工酶谱和过氧化物酶谱上均与双亲单核菌丝及担孢子杂交子有区别。将Ｋｎｏｗｌｔｏｎ等于１９８４年建立的一种分离高频再生衍生株的方法首次运用至食用菌中,使原生质体再生率提高２－３倍,为融合操作提供了方便。     

金针菇褐化病毒(FvBV)脱毒方法

【目的】评价5种不同脱毒方法对金针菇(Flammulina velutipes)菌株的脱毒效果,筛选出脱毒率高和脱毒后金针菇菌株菌丝生长速度、生物量、漆酶活力等性状改善明显的脱毒方法。【方法】以栽培金针菇菌株F-4889为研究材料,从菌丝体中提取大小约2.0 kb的病毒dsRNA,经RT-PCR鉴定该病毒为金针菇褐化病毒(FvBV)。采用菌丝尖端分离、原基组织分离、原生质体单核化、有性生殖和核迁移5种脱毒方法对金针菇菌株进行脱毒处理,利用dsRNA技术和RT-PCR检测脱毒效果。【结果】菌丝尖端分离脱毒后得到1株脱毒菌株;原基组织分离法未能脱毒;原生质体单核化脱毒法得到3株脱毒单核菌株和2株原单杂交脱毒菌株;有性生殖脱毒法获得脱毒孢子单核菌株23株和单孢杂交脱毒菌株8株;核迁移脱毒后得到5株核迁移脱毒菌株。脱毒率依次为25.0%、0、7.5%、57.5%和100%。脱毒菌株的菌丝生长速度、生物量、漆酶活力等均优于出发菌株、菌丝尖端和原基组织分离菌株。【结论】这5种方法中原生质体单核化、有性生殖和核迁移脱毒法脱毒效果较佳,均能有效脱除FvBV,脱毒率高,脱毒后菌株菌丝生长速度、生物量、漆酶活力等均明显提高。     

基于等位基因InDel快速检测黑木耳原生质体单核体

以黑木耳 Auricularia auricula-judae栽培菌株Au916三磷酸甘油醛脱氢酶（GAPDH）两个等位基因中的InDel为基础,以原生质体再生菌株为研究材料,建立了基于InDel的黑木耳原生质体单核体检测技术,并采用荧光显微镜检和配对试验对此方法进行了验证。应用InDel能清晰地区分黑木耳双核体和两种原生质体单核体,且不同的单核体之间可以进行交配,其杂交双核体能正常出耳,结果表明基于等位基因InDel标记可以快速准确地检测黑木耳原生质体单核体。在大规模测序获得真菌基因组信息的基础上,InDel标记在原生质体单核体鉴定中的应用将更加普遍。     

茯苓与凤尾菇目间原生质体融合研究初报

以无锁状联合的茯苓次级菌丝和具锁状联合的凤尾菇次级菌丝为亲本制备原生质体,以PEG为促融剂,在高钙、高pH条件下,将茯苓(或凤尾菇)原生质体热灭活,与凤尾菇(或茯苓)原生质体融合,从再生菌株中均能选择选择出能够再形成双核的单核菌株。栽培实验结果显示,单核菌株经诱导形成双核菌株后可以形成子实体或原基,若单核菌株不经诱导,则始终维持稳定的单核状态,且难以形成子实体。     

用灭活原生质体融合进行高温香菇育种I．融合产物的选出及其鉴定

用灭活的近裸香菇(Lentinus subnudus Berk.)双核菌株原生质体与香菇[L. edodes(Berk) Sing.] 双核菌株原生质体融合,在35℃的条件下选得融合子。融合频率为0—4.3x10^{-5。融合子与双亲有明显的拮抗性。融合子的菌丝形态、氨基酸含量,子实体的形态,以及酸性磷酸酶同功酶的测定都与双亲不同。}     

豌豆根瘤菌与新疆中华根瘤菌原生质体的属间融合研究

以青霉素和氯霉素分别作为RhizobiumleguminosorumUSDA2 370和SinorhizobiumxinjiangnesisCCBAU110的抗药性标记。利用原生质体融合技术 ,成功地获得了USDA2-370和CCBAU110的属间融合菌株。该融合菌株可分别在双亲寄主植物上结瘤。融合菌株在细胞形态、大小、菌落特征及蛋白质电泳图谱上与亲本菌株均有所不同。融合菌株与USDA2-370的DNA同源性为 5.66 % ,而与CCBAU110的DNA同源性为10.2 %。     

金针菇担孢子核相及遗传属性的研究

以3个不同的金针菇菌株为材料,研究了其担孢子的核相及遗传属性。荧光染色观察显示,担孢子核相以双核为主,双核孢子、单核孢子和无核孢子分别占80.2%、7.5%和12.3%。源于单孢分离物的菌丝为有隔膜、无锁状联合的多核菌丝。在交配试验中,源于不同菌株单孢分离物的菌丝原生质体的配对形成具锁状联合的菌落,而源于同一单孢分离物的菌丝原生质体的配对则形成无锁状联合的菌落,暗示担孢子中的两个核具有相同的交配型。RAPD分析显示,源于同一单孢分离物的菌丝原生质体为10个随机引物所扩增的图谱彼此完全相同,印证了担孢子中的双核是同质的。此外,观察表明,一个担子上着生有4个担孢子。因此,金针菇是一种具4个含同质双核担孢子的四极性蕈菌。     

玉米大斑病菌（Exserohilum turcicum）原生质体的分离及转化

玉米大斑病是严重危害玉米生产的一个世界性真菌病害。由于玉米大斑病菌（Exserohilum turcicum）在无性生长过程中迅速产生黑色素,致使原生质体难以分离。测试了包括Fungase、Funcelase、Novozyme、Glucanex、Driselase、Uskizyme、Kitalase在内的7种细胞壁降解酶及其组合、病原菌菌株和培养基对原生质体分离效果的影响。结果表明菌株的培养形态和菌丝的生长状态显著影响原生质体的分离效率;酶组合Kitalase+Glucanex+Driselase, Kitalase+Glucanex和Kitalase+Uskizyme能够有效地分离玉米大斑病菌的原生质体。初步的转化试验表明,质粒pAN71可以用于该病原菌的转化。这些结果将为E.turcicum和Exserohium属其它真菌的基因克隆提供一些有用的信息。     

米曲霉原生质体融合及杂合二倍体的形成

采用混合酶液处理纤维素酶高产苗株３０４２_Ｎ－２（天冬酰胺缺陷型“Ａｓｎ^－”）及蛋白酶高产菌株３０４２_Ｎ－１９（蛋氨酸缺陷型“Ｍｅｔ^－”）的营养菌丝,获得原生质体,以ＰＥＧ为助融剂进行融合处理,成功地获得了米曲霉（Ａｓｐｅｒｇｉｌｌｕｓｏｒｙｚａｅ）原生质体的营养互补融合。将异核体菌落的菌丝转接到合有０．１％樟脑的新鲜ＭＭ上,２５℃诱导培养７─１５天,挑取绿色角变菌落的孢子,将其转接到ＭＭ上能迅速生长,经孢     

Facilitated protoplasting in certain auxotrophic mutants of Corynebacterium glutamicum

Summary Auxotrophic mutants of Corynebacterium glutamicum strain ATCC 13 059 showed considerable variation in their ability to form protoplasts when treated with lysozyme and ethylene-diaminetetraacetic acid (EDTA) following growth in nutrient medium containing up to 2% glycine. The proportion of protoplasts formed by the parent strain was normally 10 to 15% whereas certain amino-acid auxotrophs formed protoplasts at much higher frequencies (>95%). There was no obvious correlation between the presence of specific auxotrophic markers or the number of rounds of mutagenesis and protoplasting ability. Strains which were most readily protoplasted were morphologically distinct from other auxotrophs and the parent strain but were similar to the parent strain in their sensitivity to lysozyme. However, isoleucine auxotrophs were more sensitive to penicillin G. All strains produced osmotically sensitive cells (lysed by dilution in water) after growth in glycine-containing media and lysozyme-EDTA treatment. These damaged cells could be distinguished from true protoplasts by their ability to recover on osmotically non-protective media if diluted in high osmotic pressure buffers. Protoplasts were regenerated on an osmotically protective medium (ET) in 48 to 72 h, at frequencies averaging 50–60%.     

黑曲霉单宁酶高活性菌株的诱变选育*

以黑曲霉（Ａｓｐｅｒｇｉｌｌｕｓ ｎｈｉｇｅｒ）Ｎｏ．１３为出发菌株,经紫外线诱变处理,获得一株制备原生质体的起始菌,该菌株单宁酶活性比Ｎｏ．１３提高５５％,并对其制备原生质体的条件进行了研究,在优化方案基础上,紫外诱变原生质体,诱变株经筛选,最后得到一株具有稳定遗传性的单宁酶高活性菌株,在摇瓶培养基中进行生物转化实验,连续传代１０次,结果显示发酵液中没食子酸浓度始 维持在２２．８－２３．９ｍｇ／     

A Simple and Rapid Method of Transformation of Streptomyces rimosus R6 and Other Streptomycetes by Electroporation

Usually plasmid DNA is introduced into Streptomyces strains by polyethylene glycol-mediated transformation of protoplasts. However, many Streptomyces strains are only poorly or not at all transformable via protoplasts. Therefore, we have optimized the parameters critical for the application of electrotransformation of plasmid DNA into Streptomyces species. The most critical parameters evaluated for electrotransformation of the model strain Streptomyces rimosus R6 were the pretreatment of mycelia, buffer composition, and electric field strength. The electrocompetent mycelia were prepared from 24-h-old cultures, treated mildly with lysozyme, resuspended in sucrose-glycerol-polyethylene glycol buffer, and stored in aliquots at -70 deg C. The electric field strength of 10 kV/cm at 400 (Omega) and a capacitance of 25 (mu)F was applied. The method is simple and rapid, yielding transformant colonies in 48 to 72 h. Efficiencies of 10(sup5) to 10(sup6) transformants per (mu)g of plasmid DNA were reproducibly achieved for S. rimosus R6 and its mutants, and these numbers were 10(sup2) to 10(sup3) higher than those attained by polyethylene glycol-assisted transformation of protoplasts. In addition, we show that electroporation can be applied to other Streptomyces species, such as S. lividans 66, S. coelicolor A3(2), and an S. venezuelae strain. This last one could not be transformed by the standard protoplast procedure. Our data suggest that, because of the diversity of streptomycetes, the conditions have to be optimized for each strain.     

Characteristics of obtaining protoplasts from 2 strains of Tolypocladium inflatum subsp. Blastosporum]

The optimal conditions for preparing protoplasts with high yields by using the cells of two (low and high potent) isogenic cyclosporine-producing Tolypocladium strains were developed. A specific medium containing 0.5 per cent yeast autolysate (by dry weight) and 3 per cent glucose was used. When grown on this medium the cells of the highly potent strain 847 acquired a yeast-like shape. High yields of protoplasts prepared from the low potent strain 43 mycelium were obtained via prior incubation with 0.01 M dithiothreitol followed by treatment with a complex of enzymes from Helix pomatia for 1.5 to 2 hours was used. For preparation of the protoplasts with employing the highly potent strain 847 cells the prior incubation with dithiothreitol was not required, but it was necessary to employ a mixture of the enzyme complex (Helix pomatia), drizilase (Irpex lacteus) and chitinase (Streptomyces griseus) for 18 hours. The electron microscopic data on the two isogenic strains and their protoplasts are presented. The protoplasts proved to be a suitable initial material for investigating bioenergetic processes at the subcellular level and further genetic improvement of the strains.     

利用紫外线、利福平、氯化锂诱变原生质体筛选L—亮氨酸高产菌株的研究

本实验是以黄色短杆菌T_(6—13)的诱变株L—亮氨酸产生菌D—R—4为出发菌株,经青霉素、甘氨酸、溶菌酶作用制备原生质体,形成率达91.30％,再生率达53.68％;然后对原生质体进行紫外线、利福平、氯化锂复合诱变处理;在再生培养基平皿上培养,获得再生突变株,从中挑取单独菌落,进行摇瓶发酵筛选,已选育出一株57—4S号高产稳定菌株;经氨基酸分析仪测定其发酵液L—亮氨酸产量由出发菌株的17.35mg/ml提高到23.45mg/ml提高了35％。发酵液中主要副酸——异亮氨酸含量很少。     

小麦白粉病生防菌G-1菌株原生质体诱变育种

以链霉菌G-1(Streptomyces sp.G-1)为出发菌株,通过研究菌株G-1原生质体形成与再生的条件,发现该菌株在含0.5%甘氨酸的菌丝体培养基中经过二次培养后,所得菌丝体用2 mg/mL溶菌酶在30℃下处理90 min,可获得大量原生质体,其再生率可达8.2%。菌株G-1的原生质体经紫外诱变和宁南霉素抗性筛选后,得到一高产突变株G-1-125,其有效组分A的产量达到794mg/L,较出发菌株提高了180%。     

Electron microscopic study of Bacillus subtilis protoplast fusion.

When protoplasts derived from sporulating cells of Bacillus subtilis were fused by exposure to polyethylene glycol (PEG) and fixed immediately thereafter, protoplasts with two enclosed prespores could be seen by electron microscope. The number of fusion events was greatly increased, and multiply fused protoplasts appeared, when the PEG-treated suspension was diluted in hypertonic broth and reincubated before fixation. This post-PEG incubation effect is taken to indicate a fusion mechanism of two steps: a short, PEG-dependent step of membrane activation, followed by a slow, metabolism-requiring step completing fusion. When prespore-bearing protoplasts from two genetically different strains were mixed and fused, the extent of fusion could also be followed by counting clones of recombinant bacteria. Maximal from the start, their number (1% of each parent type protoplast present) was unaffected by post-PEG incubation. Fusion in this case is apparently completed after plating on the wall-regeneration medium. After optimal post-PEG incubation, the majority of the protoplasts were seen to participate in fusion, and the cytological fusion observed, corrected for wall-regeneration frequency, accounted quantitatively for the prototrophic bacteria eventually recovered. These results are in good agreement with those obtained independently by Sanchez-Rivas and Garro (J. Bacteriol. 137:1340--1345, 1979).     

Water transport in intact yeast cells as assessed by fluorescence self-quenching

Intact yeast cells loaded with 5- and-6-carboxyfluorescein were used to assess water transport. The results were similar to those previously reported for protoplasts assessed by using either fluorescence or light scattering, and the activation energies were 8.0 and 15.1 kcal mol(-1) (33.4 and 63.2 kJ mol(-1)) for a strain overexpressing AQY1 aquaporin and a parental strain, respectively.     

凤尾菇和裂褶菌原生质体非对称融合研究

具有双重营养缺陷型的裂褶菌单核体突变菌株的原生质体经过灭活处理后作为供体与同样带有营养缺陷型标记的风尾菇单核体原生质体融合,得到大量生长速度和菌落形态差异较大的多核体和双核体融合子．这些融合子主要显示了供体菌株的特性,数次转接后,有的融合子从多核体转变成双核体,有的融合子完全恢复了供体亲本双核体的遗传特性,同时获得了一些有意义值得继续探讨的新的生理特性,如菌丝体在木屑 皮以及废棉培养料上生长速度较快,在平板培养基和木屑培养料上均较早较快地形成原基和子实体等．结果表明原生质体非对称融合是一种效率较高的融合方式,可以作为食用菌育种的一种有效途经．