紫孢侧耳、糙皮侧耳及其融合菌株的同工酶分析

采用聚丙烯酰胺凝胶垂直板电泳方法对紫孢侧耳、糙皮侧耳及其融合菌株的酯酶和过氧化物酶同工酶进行了分析。经多次试验,得到了两亲株和融合株恒定的同工酶谱, 在酶谱中融合菌株与亲株有明显差异,并表现出典型的双亲株互补酶带特征,从而证实融合菌株为两亲株基因重组之产物。 Abstract: After many repetitions,the patterns of esterase isozyme and peroxidase isozyme of Pleurotus sapidus,Pleurotus ostreatus and their fusant strains were obtained.There were differences among the parent strains and theirfusant strains in theirisozyme bands.Parental isozyme bands appeared in patterns of fusant strains.Itdemonstrated that fusant were recombinants of P.sapidus and P.ostreatus.     

凤尾菇和香菇原生质体非对称融合

The protoplasts of auxotrophic mutant monokaryon of Pleurotus. Sajor-caju was used as recipient parenL the protoplasts of wild dikareon of Lentinus edodes were heat-inactivated and used as nuclear donor parent. The fusants of two parents showed some their individual characteristics and physiological difference between themselves. Thrugh protoplast re-isolation and reversion of one randomly selective fusant, two parents had been recovered and the donor parent achieved some new physiological characteristics su…     

Formation and Growth of Bryopsis hypnoides Lamouroux Regenerated from Its Protoplasts

Tissue culture, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and spectra analysis were used for studying the aggregation mechanism of protoplasts from Bryopsis hypnoides Lamouroux and the discrepancy between the protoplast-regenerated plants and the wild type. The aggregation of protoplasts from B. hypnoides was observed in natural seawater and artificial seawater with different pH values, and the location and mechanism of the materials causing the aggregation were also studied. Results showed that the protoplasts could aggregate into some viable spheres in natural seawater and subsequently grow into mature individuals. Aggregation of the protoplasts depended exclusively upon the pH value (6-11), and the protoplasts aggregated best at pH 8-9. Some of the extruded protoplasts were separated into two parts by centrifugation: the pellet (PO) and the supernatant (PL). The PO could aggregate in artificial seawater (pH8.3) but not in PL. No aggregation was found in PO cultured in natural seawater containing nigericin, which can dissipate the proton gradients across the membrane. These experiments suggest that the aggregation of protoplasts is proton-gradient dependent and the materials causing the aggregation were not in the vacuolar sap, but located on the surface or inside the organelles. Furthermore, the transfer of the materials across the membrane was similar to △pH-based translocafion (△pH/TAT) pathway that occurs in the chloroplasts of higher plants and bacteria. Obvious discrepancies in both the total soluble proteins and the ratio of chlorophyll a to chlorophyll b between the regenerated B. hypnoides and the wild type were found, which may be related to the exchange of genetic material during aggregation of the organelles. In the process of development, diatom Amphora coffeaeformis Agardh attached to the protoplast aggregations, retarding their further development, and once they were removed, the aggregations immediately germinated, which showed that diatoms can affect the development of other algae.     

几种担子菌胞外漆酶同工酶谱的研究

从猴头(Hericium erinaceus)、亚侧耳(Hohenbuehelia serotina)、毛柄金钱菌(Co-llybia velutipes)、多脂鳞伞(Pholiota adiposa)、香菇(Lentinus edodes)、黑木耳(Auriculariaauricula)、草菇(Volvaria volpacea)二孢蘑菇(Agaricus bisporus)和紫孢侧耳(Pleurotussapidus)、侧耳(Pleurotus ostreatus)风尾菇(P.sajor-caju)、金顶侧耳(P.citrinopileatus)、鲍鱼菇(P.cystidiosus)、佛罗里达侧耳(P.florida)、爪哇漏斗状侧耳(P.sajor-caju var.javanicus)商品化菌种的培养滤液里所得到的胞外漆酶(LC),采用聚丙烯酰胺凝胶等电聚焦盘状电泳方法,分析比较了它们的胞外漆酶同工酶。经多批次比较试验,获得了侧耳属七个种和八个不同属间各自恒定的漆酶同工酶谱。在这些酶谱中,分别包含了 A 组(pH3—5)和 B 组(pH5—8)的谱带。而且,所有菌种间漆酶带的等电点值(pI)各有异同。没有测定菌种间各漆酶带含量差别。讨论了漆酶同工酶谱用于分类研究的可能性。     

The effects of oligomycin on content of adenylates in mesophyll protoplasts,chloroplasts and mitochondria from Pb<Superscript>2+</Superscript> treated pea and barley leaves

The effects of oligomycin on photosynthesis and respiration in relation to ATP production in chloroplasts and mitochondria were investigated in protoplasts isolated from the detached pea (Pisum sativum L cv. Iłowiecki.) and barley (Hordeum vulgare L. cv. Gunilla) leaves treated 5 mM Pb(NO₃)₂. The oligomycin (OM), an inhibitor of oxidative phosphorylation at 0.1 μM concentration caused the inhibition of photosynthesis rate in the protoplasts from both the control and the Pb-treated pea leaves. The respiration rate and ATP/ADP ratio in the protoplasts and the activity of ATPase in mitochondria, were also diminished in the control protoplasts. These effects were not observed in the protoplasts and mitochondria isolated from the Pb-treated leaves. Oligomycin, an inhibitor of photophosphorylation at 10 μM concentration decreased ATPase activity in chloroplasts from both the control and the Pb- treated leaves. Using the method of rapid fractionation of barley protoplasts it was shown that the ATP/ADP ratio in the mitochondria from Pb-treated leaves was largely suppressed (from 1.8 to 0.4) by OM under nonphotorespiratory conditions (high CO₂), whereas under photorespiratory conditions (low CO₂) this ratio was high (5.3) and under OM decreased less (to 3.1). Our results indicate that oligomycin, in organelle isolated from Pb-treated leaves, had no inhibitory effect on the mitochondrial ATPase, whereas it inhibited chloroplasts ATPase. We suggest that Pb ions affected the catalytic cycle and/or conformational changes of ATPase in pea chloroplasts differently than in mitochondria. The differences in Pb responses may reflect fine mechanisms for the regulation of ATP production in the plant cells under stress conditions.     

基于ITS序列分析对我国主要栽培的侧耳品种的鉴定及评价

利用PCR产物克隆测序测定了20个我国主要栽培的侧耳品种的ITS序列,另外从GenBank获得侧耳属15个种25条ITS序列及亚侧耳属2个种的ITS序列。以Hohenbueheliagrisea和H.tremula为外群,运用PAUP软件中的简约分析法(parsimonyanalysis)构建的系统发育树表明:侧耳属Pleurotus是单起源的,20个主要栽培的侧耳品种分别聚在三个组,即Ostreatus-eryngii-populinus复合组、Pulmonarius组、Citrinopileatus-cornucopiae组。Ostreatus-eryngii-populinus组含刺芹侧耳Pleurotuseryngii、白灵侧耳P.nebrodensis、香侧耳Pleurotussp.、阿魏侧耳P.eryngiivar.ferulae、平963-1Pleurotussp.及糙皮侧耳P.ostreatus、日本秀珍Pleurotussp.、平802Pleurotussp.、姬菇Pleurotussp.、灰白侧耳P.spodoleucus、缘刺侧耳Pleurotussp.、凤尾菇P.sajor-caju;Pulmonarius组含肺形侧耳P.pulmonarius、小白平菇Pleurotussp.、平8804Pleurotussp.、平侧5Pleurotussp.、美味侧耳P.sapidus;Citrinopileatus-cornucopiae组含黄白侧耳P.cornucopiae、金顶侧耳P.citrinopileatus、鸡汁菌Pleurotussp.。系统树还显示黄白侧耳与金顶侧耳、白灵侧耳与刺芹侧耳亲缘关系密切,而凤尾菇与肺形侧耳分属于不同的组,属于两个不同的种。基于ITS序列分析,本文还针对目前我国栽培的主要侧耳品种在名称使用上的混淆和混乱进行了初步的评价和讨论。     

Comparative study of symmetric and asymmetric somatic hybridization between common wheat and Haynaldia villosa

Symmetric and asymmetric protoplast fusion between long term cell suspension-derived protoplasts of Triticum aestivum (cv. Jinan 177) and protoplasts of Haynaldia villosa prepared from one-year-old embryogeneric calli was performed by PEG method. In asymmetric fusion, donor calli were treated with gamma ray at a dose of 40, 60, 80 Gy (1.3 Gy/min) respectively and then used to isolate protoplasts. Results of morphological, cytological, biochemical (isozyme) and 5S rDNA spacer sequence analysis revealed that we obtained somatic hybrid lines at high frequency from both symmetric and asymmetric fusion. Hybrid plants were recovered from symmetric and low dose γ-fusion combinations. GISH (genomic in situ hybridization) analysis proved exactly the existence of both parental chromosomes and the common occurrence of several kinds of translocation between them in the hybrid clones regenerated from symmetric and asymmetric fusion. And the elimination of donor DNA in hybrid clones regenerated from asymmetric fusion     

Asymmetric somatic hybridization between wheat (Triticum aestivum) and Avena sativa L.

Protoplasts from cell suspensions of young-embryo-derived calli, which were nonregenerable for long-term subculture and protoplasts from embryogenic calli with the regeneration capacity of 75% of the same wheat Jinan 177, were mixed as recipient. Protoplasts from embryogenic calli of Avena sativa (with the regeneration capacity of less than 10%) irradiated with UV at an intensity of 300 μW/cm² for 30 s, 1 min, 2 min, 3 min, 5 min were used as the donor. Protoplasts of the recipient and the donor were fused by PEG method. Many calli and normal green plants were regenerated at high frequency, and were verified as somatic hybrids by chromosome counting, isozyme, 5S rDNA spacer sequence analysis and GISH (genomic in situ hybridization). Fusion combination between protoplasts either from the cell suspensions or from the calli and UV-treated Avena sativa protoplasts could not regenerate green plants.     

Intermediate fertile Triticum aestivum （＋）Agropyron elongatum somatic hybrids are generated by low doses of UV irradiation

We report the production and characterization of somatic hybrids between Triticum aestivum L. and Agropyron elongatum (Host) Nevishi (the synonym is Thinopyrum ponticum). Asymmetric protoplast fusion was performed between Agropyron elongatum protoplasts irradiated with a low UV dose and protoplasts of wheat taken from nonregenerable suspension cultures. More than 40 green plantlets were obtained from 15 regenerated clones and one of them produced seeds. The phenotypes of the hybrid plants and seeds were intermediate between wheat and Agropyron elongatum. All of the regenerated calli and plants were verified as intergeneric hybrids on the basis of morphological observation and analysis of isozyme, cytological, 5SrDNA spacer sequences and random amplified polymorphic DNA (RAPD). RFLP analysis of the mitochondrial genome revealed evidence of random segregation and recombination of mtDNA.     

Comparison of cell wall regeneration on maize protoplasts isolated from leaf tissue and suspension cultured cells

Summary The cell wall regeneration on protoplasts derived from maize mesophyll cells was compared with wall regeneration on protoplasts derived from suspension cultured cells using light microscopy, transmission electron microscopy, and mass spectrometry. The time course of cell wall regeneration has shown that the mesophyll protoplasts regenerated walls much slower than the protoplasts derived from cultured cells. Moreover, cell wall materials on the mesophyll protoplasts were often unevenly distributed. Electron microscopy has further demonstrated that the mesophyll protoplasts have less organized and compact walls than the protoplasts from cultured cells. Chemical analysis revealed that the mesophyll protoplasts had a lower ratio ofβ-(1–3)-glucan toβ-(1–4)-glucan than protoplasts from cultured cells. The significance of these results for the viability and development of protoplasts in culture is discussed. National Research Council of Canada paper no. 32458.     

Isolation of Intact and Functional Chloroplasts from Mesophyll and Bundle Sheath Protoplasts of the C(4) Plant Panicum miliaceum

A procedure is described for isolating and purifying mesophyll protoplasts and bundle sheath protoplasts of the C₄ plant Panicum miliaceum. Following enzymic digestion of leaf tissue, mesophyll protoplasts and bundle sheath protoplasts are released and purified by density centrifugation. The lower density of mesophyll protoplasts allowed rapid separation of the two protoplast types. Evidence for separation of mesophyll protoplasts and bundle sheath protoplasts (up to 95% purity) is provided from light microscopy (based on size difference in both chloroplasts and protoplasts), levels of marker enzymes in the preparations (i.e. pyruvate, Pi dikinase and phosphoenolpyruvate carboxylase for mesophyll and ribulose-1,5-bisphosphate carboxylase for bundle sheath), and differences in substrate-dependent O₂ evolution by chloroplasts isolated from protoplasts.     

Co-electroporation of rice protoplasts with RNAs of cucumber mosaic and tobacco mosaic viruses

Cucumber mosaic virus (CMV) RNA was used to study electroporation conditions suitable for protoplasts from rice suspension cultures. Rice protoplasts required a stronger and shorter electric pulse than tobacco protoplasts for introduction of viral RNA. Under optimized conditions, CMV infection was established in 65 % of electroporated protoplasts. In contrast, electroporation with tobacco mosaic virus (TMV) RNA did not result in infection of rice protoplasts. However, when TMV RNA was electroporated into rice protoplasts together with CMV RNA, TMV production was demonstrated in 15 % of protoplasts. Differential staining with fluorescent antibodies against the two viruses showed that the protoplasts producing TMV were without exception also infected by CMV. The results show that CMV replicates in rice protoplasts by itself, whereas TMV does so only with the aid of CMV.Abbreviations CMV cucumber mosaiv virus - PBS phosphate buffered saline - TMV tobacco mosaic virus.     

1-Aminocyclopropane-1-carboxylic-acid-dependent ethylene production during re-formation of vacuoles in evacuolated protoplasts of Petunia hybrida

Summary Ethylene formation from 1-aminocycloprane-1-carboxylic acid (ACC) was studied in whole protoplasts, evaluolated protoplasts and isolated vacuoles from mesophyll cells of Petunia hybrida L. cv. Pink Magic. The re-formation of the large, central vacuole in evacuolated protoplasts and morphological characteristics of both types of protoplasts were examined by electron microscopy. Both the normal, whole protoplasts and vacuoles isolated from them produced ethylene from ACC at similar rates. Freshly-prepared evacuolated protoplasts had lost the capacity to produce ethylene. Re-formation of the central vacuole in these evacuolated protoplasts occurred between 14 to 17 h of incubation in the recovery medium and was followed by the development of ethyleneforming activity. Both these processes were inhibited by cycloheximide, indicating a requirement for new protein synthesis. Light stimulated the conversion of ACC to ethylene in both the regenerating, whole protoplasts and the evacuolated protoplasts that had re-formed the central vacuole.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglycine - CHI cycloheximide     

Surface Charge Density of Hetero-Fused Plant Protoplasts: an Electrophoretic Study

Electrophoretic mobilities of hetero-fused plant protoplasts,which were obtained by electrofusion of barley mesophyll cellprotoplasts and Rauwolfia serpentina cultured cell protoplasts,and those of the unfused parent protoplasts were measured invarious media of different pH values. At pH 5.2, the zeta potentialof the fused protoplasts was intermediate between those of thebarley and R. serpentina protoplasts and the average surfacecharge density of the fused protoplasts was closer to that ofR. serpentina than to that of barley. The distribution of thesurface charge density of fused protoplast obtained at pH 5.2is discussed in terms of the surface charge densities and thesizes of parent protoplasts. These results revealed that thesurface charge density of fused protoplasts was determined bythe surface charge densities and the ratio of the surface areasof the respective parent protoplasts. (Received December 28, 1989; Accepted August 10, 1990)     

小麦原生质体分离过程中生理状态的变化

酶解处理使小麦对肉原生质体膜流动性降低,膜脂过氧化产物丙二醛（ＭＤＡ）积累,说明脱璧过程对细胞有伤害作用,损伤位点可能发生在膜上。胚性愈伤组织的具有分裂能力的原生质体,不表现上述变化。酶解脱壁还使超氧化物歧化酶（ＳＯＤ）和过氧化氢酶（ＣＡＴ）活性上升;过氧化物酶（ＰＯＸ）在叶肉原生质体中活性下降,在胚性愈伤组织来源的原生质体中活性上升。以上结果表明：在原生质体分离过程中,细胞的生理特性发生了变化;膜损伤的发生可能与原生质体能否进入正常分裂状态有关。     

Fusion characteristics of plant protoplasts in electric fields

The electrical parameters important in the fusion of plant protoplasts aligned dielectrophoretically in high-frequency alternating electric fields have been established. Protoplasts were aligned in an alternating electric field between two relatively distant (1 mm) electrodes, by dielectrophoresis induced by field inhomogeneities caused by the protoplasts themselves. This arrangement allowed ease of manipulations, large throughput and low loss of protoplasts. In analytical experiments, sufficiently large samples could be used to study pulse duration-fusion response relations at different pulse voltages for protoplasts of different species, tissues and size (mesophyll protoplasts of Solanum brevidens, Triticum aestivum, Hordeum vulgare; suspension-culture protoplasts of Nicotiana sylvestris, N. rustica, Datura innoxia and S. brevidens; root-tip protoplasts of Vicia faba, hypocotyl protoplasts of Brassica napus). The percentage of aligned protoplasts that fused increased with increasing pulse parameters (pulse duration; voltage) above a threshold that was dependant on pulse voltage. The maximum fusion values obtained depended on a number of factors including protoplast origin, size and chain length. Leaf mesophyll protoplasts fused much more readily than suspension-culture protoplasts. For both types, there was a correlation of size with fusion yield: large protoplasts tended to fuse more readily than small protoplasts. In short chains (five protoplasts), fusion frequency was lower, but the proportion of one-to-one products was greater than in long chains (ten protoplasts). In formation by electrofusion of heterokaryons between mesophyll and suspension-culture protoplasts, the fusion-frequency response curves reflected those of homofusion of mesophyll protoplasts rather than suspension-culture protoplasts. There was no apparent limitation to the fusion of the smallest mesophyll protoplast with the largest suspension-culture protoplasts. Based on these observations, it is possible to direct fusion towards a higher frequency of one-to-one (mesophyll/suspension) products by incorporating low densities of mesophyll protoplasts in high densities of suspensionculture protoplasts and by using a short fusion pulse. The viability of fusion products, assessed by staining with fluorescein diacetate, was not impaired by standard fusion conditions. On a preparative scale, heterokaryons (S. brevidens mesophyll-N. sylvestris or D. innoxia suspension-culture) were produced by electrofusion and cultured in liquid or embedded in agar, and were capable of wall formation, division and growth. It is concluded that the electrode arrangement described is more suitable for carrying out directed fusions of plant protoplasts than that employing closer electrodes.     

甘蔗和烟草叶原生质体分离期间的膜损伤及有关酶活性变化

甘蔗和烟草叶原生质体分离期间的膜损伤及有关酶活性变化何若天,覃伟,李任强（广西农业大学实验中心,南宁５３０００５）关键词：原生质体,超氧阴离子自由基（Ｏ_２~－）,膜损伤,甘蔗,烟草植物原生质体分离期间,所用细胞壁降解酶和高渗介质等对细胞生理有深刻影响...     

Reduced activity of antioxidant machinery is correlated with suppression of totipotency in plant protoplasts

We previously showed that during protoplast isolation, an oxidative burst occurred and the generation of active oxygen species was differentially mediated in tobacco (Nicotiana tabacum) and grapevine (Vitis vinifera), accompanied by significant quantitative differences (A.K. Papadakis, K.A. Roubelakis-Angelakis [1999] Plant Physiol 127: 197-205). We have now further tested if the expression of totipotency in protoplasts is related to the activity of cellular antioxidant machinery during protoplast culture. Totipotent (T) tobacco protoplasts had 2-fold lower contents of intracellular O2*- and H2O2 and 7-fold lower levels of O2*- and H2O2 in the culture medium, compared with non-totipotent (NT) tobacco protoplasts. Addition of alkaline dimethylsulfoxide, known to generate O2*-, resulted in isolation of tobacco protoplasts with reduced viability and cell division potential during subsequent culture. Active oxygen species levels decreased in tobacco and grapevine protoplasts during culturing, although higher contents of O2*- and H2O2 were still found in NT- compared with T-tobacco protoplasts, after 8 d in culture. In T-tobacco protoplasts, the reduced forms of ascorbate and glutathione predominated, whereas in NT-tobacco and grapevine protoplasts, the oxidized forms predominated. In addition, T-tobacco protoplasts exhibited severalfold lower lipid peroxidation than NT-tobacco and grapevine protoplasts. Furthermore, several antioxidant enzyme activities were increased in T-tobacco protoplasts. Superoxide dismutase activity increased in tobacco, but not in grapevine protoplasts during culturing due to the increased expression of cytoplasmic Cu/Zn-superoxide dismutase. The increase was only sustained in T-tobacco protoplasts for d 8. Together, these results suggest that suppressed expression of totipotency in protoplasts is correlated with reduced activity of the cellular antioxidant machinery.     

Extensor and Flexor Protoplasts from Samanea Pulvini : II. X-Ray Analysis of Potassium, Chlorine, Sulfur, Phosphorus, and Calcium

Concentrations of K, Cl, P, S, and Ca in extensor and flexor protoplasts from open pulvini of the nyctinastic tree Samanea saman were estimated using x-ray microanalysis. This technique is particularly suitable when absolute numbers of protoplasts are low, because less than 100 protoplasts are required to obtain statistically significant data. Flexor protoplasts contain similar concentrations of P and S but almost twice as much K and Cl as extensor protoplasts. Low levels of total measurable osmoticum suggest that extensive leakage has occurred during protoplast isolation. Both extensor and flexor protoplasts appear to contain some unidentified osmoticum not detectable by x-ray analysis. Extensor protoplasts must have more unidentified osmoticum to compensate for their lower levels of K and Cl.