摘要
本文针对前人在侧耳属分类研究中未曾将中国的白灵菇和典型的Pleurotus nebrodensis进行比较分析的缺陷,对来自中国的白灵菇菌株和来自欧洲的Pleurotus nebrodensis, Pleurotus ferulae, Pleurotus eryngii, Pleurotus elaeoselini菌株进行DNA分子标记(ITS, IGS2, SRAP)分析,从分子水平为白灵菇分类地位的确定提供可靠依据,同时研究了各侧耳种的系统发育关系和遗传多样性。主要研究结果如下：
1、菌丝体形态和菌丝生长速度实验观察结果：PN802(1号)和PN803(2号)菌株在生长后期会产色素与其他供试菌株有明显的差异,但二者根据菌丝形态特征难以区分；菌株KH5(15号)、PF ds 264(17号)、PF 882(18号)的菌丝形态也与其它菌株明显不同,说明这些菌株与其他供试菌株的遗传背景有明显差异。其余供试菌株的菌丝体形态也有一定差异,但是组内各菌株无法区分。根据菌丝体生长速度数据,无法将供试菌株分类,但从数据中可以看出KH2(14号)和PF 882(18号)菌株的生长速度显著快于其他菌株,说明这两株菌株与其他供试菌株的遗传差异较大。中国的白灵菇菌株比欧洲的Pleurotus nebrodensis菌株生长速度快,Pleurotus eryngii和Pleurotus elaeoselini菌株的菌丝体生长速度比其他侧耳种快。
2、菌丝体拮抗反应试验结果：来自中国的绝大多数白灵菇菌株PN 2, PN sn 1, PN sn 2, PN sn 4, PN ts 1, PN ts 2, PN1 (8、9、10、11、12、13和33号)分在一组,PN802(1号)和PN803(2号)两株Pleurotus nebrodensis菌株分在一组,这与菌丝体形态特征观察的结果一致。PE ds 77, PE ds 359, PE4(27、28和32号)菌株分在一组,它们均为Pleurotus eryngii。3株Pleurotus elaeoselini菌株EL 759,EL 738,EL 720(22、23和24号)分在一组。其它菌株各自独立成组。结果表明拮抗试验可以作为侧耳初步分类的方法。
3、对ITS序列进行比对分析,结果显示全部供试菌株的ITS序列长度范围为534～589bp。从序列长度变异来看,ITS1区序列长度变化范围为184～255bp,稍大于ITS2区的180～200bp；5.8S序列相对保守,只有154bp和158bp两种长度。所有侧耳菌株无论是ITS1区还是ITS2区(G+C)%都比较低,ITS1平均为42.5%,ITS2平均为43.9%,G+C含量低说明其碱基置换具有很大的随机性。基于ITS序列信息,构建了34株供试菌株的系统发育树,结果显示：白灵菇与Pleurotus nebrodensis具有很近的亲缘关系,Pleurotus nebrodensis菌株的多态性更丰富；多数Pleurotus eryngii菌株聚在一组；3株Pleurotus elaeoselini聚在一起；Pleurotus ferulae菌株没有独立聚为一组。
4、用4种识别四个碱基的内切酶对供试菌株进行IGS2-RFLP分析,其中3种酶的酶切效果较好。结果显示：每个菌株均存在3种酶的酶切位点,电泳后产生不同的带型。HaeⅢ酶切后产生21条条带,HhaⅠ酶切后产生13条条带,RsaⅠ酶切后产生19条条带,共计53条,其中多态性条带为52条,多态性比率为98%。3个内切酶都不能单独将全部供试菌株区别开,3种酶共同使用时,PN802(1号)和PN803(2号)两个菌株不能区分开,PN sn 1, PN sn 2, PN sn 4 and PN ts 2(9、10、11和13号)菌株不能区分开。根据IGS2-RFLP的信息构建系统发育树,结果与基于ITS构建的系统发育树显示的结果基本一致,不同的是3株Pleurotus elaeoselini没有聚在一起。
5、用筛选出的7对扩增条带清晰、多态性丰富、稳定性好的引物组合对34株供试菌株进行SRAP扩增,共得到420条具有重复性的条带,且均为多态性条带,多态性比率为100%。每对引物的扩增效率不同,扩增条带数目为53-71条不等,平均每对引物扩增出60条条带,其中F1-R4扩增条带数最多,为71条条带。基于SRAP标记信息,构建了供试菌株的系统发育树,聚类分析结果与ITS基本一致,表明SRAP标记可以作为侧耳菌株分类和遗传多样性分析的有效技术手段。
6、三种DNA分子标记分析结果表明,在一定相似性水平上,白灵菇与Pleurotus nebrodensis有很近的亲缘关系,二者很可能为同一菌种；Pleurotus nebrodensis菌株的多态性较白灵菇菌株丰富,说明中国的白灵菇菌株的遗传背景较单一；供试的Pleurotus ferulae菌株不能聚在一组,各菌株之间遗传差异较大；SRAP标记技术可以作为侧耳菌株分类及遗传多样性研究的有效手段。
Because the reported Pleurotus taxonomical study didn't compare the strains of white king oyster mushroom with typical strains of Pleurotus nebrodensis, we conducted a comparative taxonomic study of white king oyster mushroom from China and Pleurotus nebrodensis, Pleurotus ferulae, Pleurotus eryngii, Pleurotus elaeoselini isolated from Europe using analysis of morphology and DNA markers. Three DNA marker systems, internally transcribed spacer (ITS) sequences analysis, intergenic spacer 2-restriction fragment length polymorphism (IGS2-RFLP) and sequence-related amplified polymorphism (SRAP) technique were used to assess the taxonomic position of white king oyster mushroom and detect genetic polymorphism among tested strains. The main results were as follows:
1、The results based on mycelial growth rate and morphology experiment indicated that the strains PN802 (NO.1) and PN803 (NO.2) were different from other tested strains visibly because they can produce pigment, but it was impossible to distinguish one from the other according to morphology character. The strains KH5 (NO.15), PF ds 264 (NO.17) and PF 882 (NO.18) were also different from other tested strains according to morphology character. Results suggested that the genetic characters of these strains had visible difference compared with other tested strains. The other tested strains also have different morphology characters, but none of them can distinguished from the strains which in the same groupe. Mycelial growth rate of KH2 (NO.14) and PF 882 (NO.18) were visibly faster than that of other strains, this phenomenon indicated that the two strains may have very different genetic characters compared with other tested strains.
2、The results of antagonism reactions among 34 strains revealed that the most strains of white king oyster mushroom from China were classified into one group which contained PN 2, PN sn 1, PN sn 2, PN sn 4, PN ts 1, PN ts 2 and PN 1 (NO.8,9,10,11,12,13 and 33). The strains PN 802 (NO.1) and PN 803 (NO.2) also were classified into one group. These results were consistent with the results of analysis of morphology. PE ds 77, PE ds 359 and PE 4 (NO.27,28 and 32) were in one group, they were Pleurotus eryngii. The strains EL 759, EL 738 and EL 720 (NO.22,23 and 24) were Pleurotus elaeoselini, they were also in one group. These results indicated that antagonism was a method which could be used in preliminary taxonomic analysis of Pleurotus strains.
3、he results of ITS sequence analysis revealed that the size of the ITS region of tested strains was different, ranging from 534bp to 589bp. The length of the ITS1 and ITS2 genes among 34 strains was 184～255 bp and 180～200 bp, respectively. Most variation occurred in ITS1. The length of the 5.8S gene among 34 strains was relatively consistent, which was 154bp and 158bp. Both ITS1 and ITS2 region among 34 strains, the (G+C)% content was relatively low, the mean (G+C)% content of ITS1 and ITS2 was 42.5% and 43.9%, respectively. The low (G+C)% content indicated that the base substitution had large randomness. Based on the analysis of ITS sequence, we conducted the evolutionary trees about the 34 strains. The result revealed that the relationship between white king oyster mushroom and Pleurotus nebrodensis was very close; great mass of the strains of Pleurotus eryngii were grouped together; the strains of Pleurotus elaeoselini were grouped together; the strains of Pleurotus ferulae were not grouped together.
4、The IGS2-RFLP analysis of tested strains using four restriction enzyme, but three of them had efficacious result. All strains had the restriction sites of three enzyme and different band pattern. The number of bands digested by HaeⅢ、HhaⅠ、RsaⅠwas 21、13 and 19, respectively. In the 53 bands, the number of the polymorphic bands was 52, the polymorphic ratio was 98%. PN 802 (NO.1) and PN 803 (NO.2) were not distinguished from each other, PN sn 1, PN sn 2, PN sn 4 and PN ts 2 (NO.9, NO.10、NO. 11、NO.13) were also not distinguished. Based on the IGS2-RFLP analysis, we constructed the phylogenetic tree. Except for the phenomenon of the strains of Pleurotus elaeoselini weren't grouped together, the results of IGS2-RFLP were rather similar with the results showed by ITS.
5、The results of SRAP profile of DNAs from 34 strains of Pleurotus strains with seven combinations of primers showed none of all strains shared the same bands. Seven combinations produced a total of 420 bands, which were all polymorphic. The number of fragments amplified using each primer combination was ranging from 53 to 71, with an average of 60 polymorphic bands per combination of primers. Based on SRAP markers, phylogenetic dendrogram of 34 Pleurotus strains was constructed. And the results showed by SRAP were rather similar with the results showed by ITS.
6、The results of three markers showed that white king oyster mushroom closely related to Pleurotus nebrodensis, they may be the same species. However, Pleurotus nebrodensis had more genetic polymorphism than white king oyster mushroom. The strains of Pleurotus ferulae were not grouped together, but grouped with Pleurotus eryngii or with Pleurotus nebrodensis. This phenomenon indicated that the strains of Pleurotus ferulae had genetic diversity. SRAP marker is a suitable method which could be used in the taxonomic and polymorphic analysis of Pleurotus strains.
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