利用原生质体融合研究凤尾菇中性菌株的交配反应_英文_

利用原生质体融合研究凤尾菇中性菌株的交配反应_英文_ 菌 物 系 统 20 2 279~282, 2001 Mycosystema STUDY ON MATING REACTION OF NEUTER STRAIN IN ×PLEUROTUS SAJOR-CAJU BY PROTOPLAST FUSION LIANG Zhi-Rong (Department of Rural Economy and Technology, Chinese Academy of Sciences, Beijing 100080) AN Mo-Ping TONG Zhan-Yuan (Hebei Agro-environmental Protection & Monitoring Station, Shijiazhuang 050011) The parasexual cycle has been already used for many years to map genes in asexually or sexually reproducting fungi (Finchan & Day, 1977). In addition to genetic mapping, the parasexual cycle has also been applied with some successes to the breeding of fungi for industrial use, but the lack of an efficient system to induce the paraxexual cycle was often the limiting step. The introduction of protoplast fusion as a method to obtain formation of heterokaryons at high frequencies has taken away this limiting factor (Anne, 1983). The protoplast fusion has played, and will continue to play , a significant role in fundamental and applied studies of fungal genetics and in breeding programs. So far, it has been applied in four areas of this field: 1). crosses between isogenic strains which may provide an opportunity for genetic mapping; 2). crosses between different strains or isolateds of a species primarily for breeding; 3). crosses between apparently incompatible strains of the same species; 4). crosses involving different species and genera (Peberdy, 1989). The neuter strain with auxotrophic marker was isolated from Pleurotus sajor-caju auct. non Sing. in primary study(Liang & Chao, 1999). The mating reaction of this strain with another double auxotrophic mutant was finally accomplished through protoplast fusion. Some interesting phenomena and peculiar characteristics were observed. The results showed the intraspecific protoplast fusion would provide a framework for future studies to examine genetic recombination between mutant forms of higher basidial fungi and also should provoke more interests and attentions of mycologists and morphologists to study the relationship between structure and its function in higher basidial fungi. 1 Materials and Methods 1.1 Fungal strains P27, Pleurotus sajor-caju auct.non Sing. wild dikaryotic stock from Department of Biology, Chinese University of Hong Kong. P377, neuter strain with lysine auxotrophic marker, see liang & Chao(1999). P102, mating type genes are A2B2, double auxotrophic mutant strain with proline and nicotinate markers, see Wang & Liang (2000a). Media and chemical solutions 1.2 See liang & Zhao(1999) 1.3 Nuclear staining See liang & Zhao(1999) Protoplast isolation and fusion 1.4 See Liang et al.(1999) 2 Results and Discussion 2.1 Fusion products 7 After protoplasts were purified and concentrated by centrifugation, 5X10protoplasts were collected from each parent strain. The mixture of protoplasts of the two parents were treated with PEG solution and then were spread onto various types of media(liang & Zhao, 1999). The plates were sealed with parafilm and incubated at 28 for 7 days. The plates were then checked daily for the next 20 days. The emerged colonies were counted and observed. The fusion products which grew on OMMA media were considered as fusion products and transferred to MMA and CMA plates respectively for further observation. Results of the fusion reactions are shown in Table 1. Table 1 Result of intraspecific protoplast fusionin P. sajor-caju between P102 and p377 Parents P102 P377 P102+P377 447No.of protoplasts 10 10 10 No. of revertants 5On OCMA 250 320 1.2X10 0 0 0 On CMA On OMMA 0 0 63 As shown in Table 1, there were no any regenerants grew on CMA. This demonstrated that protoplasts were purified and no active mycelial debris was present. There were 250 and 320 regenerants of P102 and P377 respectively which appeared on OCMA, but none of them were observed on OMMA, this showed that the two panrents were viable, and their mutant markers were stable. 63 colonies which appeared on OMMA from the fusion reaction between P102 and P377 were considered as the fusion products, and 13 from them were randomly selected and transferred to CMA and MMA respectively for further observation. 2.2 Characteristics of fusion products 2.2.1 Morphology of colonies and mycelial growth: Based on the criteria of growth rate, most of the fusion products could be divided into three groups (Table 2). The first group includes H311 and H312 which grew very fast, even faster than the P27 wild dikaryon, especially on MMA medium. The majority of the remaining fusion products formed the second group, all of which were generally comparable with wild dikaryon P27, but grew faster than their two parents P102 and P377. Several fusants grew comparable similar with their two parents such as H318, H3110, H3111, and H3113; only one fusant, H3113, grew slowly than its two parents. 2 期 梁枝荣等:利用原生质体融合研究凤尾菇中性菌株的交配反应 281 2.2.2 Cytological observation:Further Table 2 Comparison of mycelial growth between cytological observation showed that all of the parents and fusion products observed fusion products were multinucleated. On CMA* On MMA* One interesting phenomenon observed was that Wild dikaryon P27 5.3 3.1 the nuclei of the fusion product seem to be more Parent strain P102 0.9 0 active than their parents. The higher percentages P377 1.0 0 of nuclei of the fusion products could be stained than in the parent strains. This phenomenon has Fusion products H311 5.4 6.1 also been observed in other studies of protoplast H312 6.6 6.5 fusion(Liang & Zhao, 1999; Wang & Liang, H313 2.2 2.2 2000b; Liang et al, 2001). H314 1.9 1.7 2.2.3 Segregation: The mycelia of fusion product, H315 3.0 4.0 H316 2.5 2.6 H315, were multinucleated and grew very fast in H317 1.2 1.1 contrast to the mononucleated and slow growing H318 0.7 0.6 mycelia of the two parents. The mycelia of sector H319 1.1 0.8 from its colony grew faster than the original H3110 0.8 0.3 mycelia, especially on MMA. The further H3111 0.7 0.8 H3112 1.4 1.4 observation showed that the mycelia of the sector H3113 0.1 0.1 were different from their original mycelia in that they contained twin nuclei per cell. The more * Colony dia. in 7 days at 28 peculiar phenomenon was that such binucleated mycelia produced the special cylinder like structures which seem to be involved in nuclear migration instead of normal clamp connections(Fig. 1,2,3). The structure is different from the pseudo-clamp connection in which the nuclei migration could not be observed. This phenomena seem to be showed that the artificially mating reaction between incompatable nuclei through protoplast fusion was not naturally taking place so a special structure produced to satisfy the migration of nuclei during the mycelia growth. Fig. 1 Typical clamp connections of P. sajor-caju P27; Fig. 2 Cylinder-like structure producted by fusant mycelia; Fig. 3 Several nuclei in cylinder-like structure. 2.2.4 Genetic analysis: After several trials, the fusion product H3110 in the bag of cotton waste was seccessfully induced to fruit. Spores were collected from the fruiting body and 30 single spore cultures were prepared. At first these 30 single spore cultures were subcultured on to CMA and MMA respectively and incubated at 28 for 7 days. It was observed that al l these cultures could grow normally on CMA, and only 4 showed slow leaky growth on MMA medium. These 30 cultures were then subcultured onto MMA + Lys and MMA + Prol + Nic, then incubated as above. 14 cultures among them could grow on the first medium, 6 on the second, 4 on the both, and the other 6 failed to grow on either media. The result reconfirmed that H3110 was a fusion product of H102 and H377 but the possible genetic recombination between two nuclei seems to be more complex and needs more experiments to analysis. [REFERENCES] Anne j, 1983. Protoplasts of filamentous fungi in genetics and metabolite production In: Protoplasts. 167-178, Basle:Birkhauser Verlag. Fincham JRS, Day PR, 1977. Fungal Genetics (3rd ed.). Black Well Scientific, Oxford. Liang ZR, Chao SF, 1999. Induction and selection of auxotrophic mutants in Pleurotus sajor-caju and the detection of their mating type genes. Edible Fungi 19(3): 9 (in Chinese). Liang ZR, Zhao LQ, 1999. A study of polarized protoplast fusion between Pleurotus sajor-caju and Pleurotus cystidiosus. Mycosystema 18(4): 440~444 (in Chinese). [梁枝荣等 1999. 菌物系统., 18(4): 440~444] Liang ZR, An MP, Tong ZH, 2001. Polarized protoplast fusion between Pleurotus sajor-caju and Schizophyllum commune. Mycosystema 20(1): 111~115 [梁枝荣等 2001. 菌物系统, 20(1): 111~115] Liang ZR. Zhang XQ, Wang CC, 1999. Application of inactivated protoplast as a dead donor in protoplast fusion of edible fungi. Mycosystema 18(3): 341~342 (in Chinese). [梁枝荣等 1999 菌物系统, 18(3): 341~342] Peberdy, JF, 1989. Genetic manipulation. In: Berry D R ed. Physiology of industrial Fungi 187~218. Oxford: Blackwell Scientific Publications. Wang CC, Liang ZR, 2000a. Selection and isolation of double auxotrophic mutants in higher basidiomycetes. Microbiology 27(4): 272~274 (in Chinese). Wang CC, Liang ZR, 2000b. A study on polarized protoplast fusion between Pleurotus sajor-caju and Lentinus edodes. Mycosystema 19 (3): 413~415 (in Chinese). [王澄澈等 2000b. 菌物系统] 19 (3): 413~415] 利用原生质体融合研究凤尾菇中性菌株的交配反应 梁枝荣安沫平通占元 (中国科学院农村经济技术发展部 北京 100080) (河北省农业环境保护监测站 石家庄 050011) 摘 要:凤尾菇营养缺陷型突变型中性菌株与另一株营养缺陷型的正常交配型菌株进行了原生质体 融合反应.融合子之间以及与亲本之间在菌落形态和菌丝生长速度上表现出了较大差异 融合子菌落 角变的菌丝体从多核体转变为双核体 并且在菌丝体上产生了圆桶状的特殊结构而不是通常的锁状 联合 初步观察表明这些结构似乎与细胞核的迁移有关.对融合子子实体的担孢子进行了遗传分析 结 果确证子实体是由两个亲本菌株的融合子产生 同时也表明要对中性菌株交配反应中基因重组进 行 深入分析还需要进行更多的研究 关键词: 凤尾菇, 中性菌株, 原生质体融合, 圆桶状结构, 遗传分析