PCR-BASED RESTRICTION ANALYSIS OF INTERNAL TRANSCRIBED SPACERS OF NUCLEAR RIBOSOMAL DNA IN THE GENUS PLEUROTUS

Abstract：The internal transcribed spacers of the nuclear ribosomal DNA of eighteen Pleurotus taxa (52 isolates), together with outgroups A. bisporus, L. edodes, H. serotina, were amplified using PCR and then digested with seven restriction endonucleases. PCR-RFLP results showed that AluI, Hae III, HinfI, TaqI, HhaI, MspI could divide 52 isolates into 6, 5, 5, 4, 2, 2 groups respectively and distinguish P. citrinopileatus from the other Pleurotus. No restriction site was observed for BamHI in all isolates. Dendrogram based on PCR-RFLP suggested that seven groups were distinguishable for Pleurotus on 93% similarity coefficient, i.e. P. ostreatus complex (including P. ostreatus, P. florida, P. sapidus, P. corticatus, P. cornucopiae, P. columbinus, P. spodoleucus, P. ferulae, P. nebrodensis and P. sp.), P. eryngii, P. pulmonarius-P. sajor-caju, P. tuber-regium, P. abalonus-P. cystiodisus, P. djamor- P. salmoneostramineus and P. citrinopileatus. The phylogenetic relationships between P.citrinopileatus and the other Pleurotus were more distant than L. edodes, A. bisporus and the other Pleurotus.

Keywords：Oyster mushroom, phylogeny, polymerase chain reaction, RFLP

側(cè)耳屬核rDNA轉(zhuǎn)錄間區(qū)擴增產(chǎn)物的限制性酶切分析

馬富英　 羅信昌　

摘　要：對側(cè)耳屬18個形態(tài)種52個菌株和蘑菇屬、香菇屬、亞側(cè)耳屬各1個菌株的核糖體DNA轉(zhuǎn)錄間區(qū)進行PCR特異性擴增后,用7種限制性內(nèi)切酶進行酶切分析.結(jié)果表明,BamHⅠ對所有的菌株均無酶切位點;AluⅠ, Hae Ⅲ,HinfⅠ, TaqⅠ, HhaⅠ, MspⅠ可分別將52個菌株分為6, 5, 5, 4, 2, 2組,且可將金頂側(cè)耳與其它側(cè)耳區(qū)分開.基于限制性酶切多態(tài)性構(gòu)建的聚類圖表明,在93%相似水平,可將所有的側(cè)耳分為七組,第一組:糙皮側(cè)耳、佛羅里達側(cè)耳、美味側(cè)耳、裂皮側(cè)耳、黃白側(cè)耳、哥倫比亞側(cè)耳、灰白側(cè)耳、白阿魏蘑、阿魏蘑及一些未定名的側(cè)耳;第二組:刺芹側(cè)耳;第三組:肺形側(cè)耳和鳳尾菇;第四組:具核側(cè)耳;第五組:鮑魚菇和囊蓋側(cè)耳;第六組:紅平菇和桃紅側(cè)耳;第七組:金頂側(cè)耳.聚類圖結(jié)果進一步表明,金頂側(cè)耳與其它側(cè)耳的關(guān)系較雙孢蘑菇和香菇與其它側(cè)耳的關(guān)系要遠.

關(guān)鍵詞：側(cè)耳,系統(tǒng)發(fā)育,聚合酶鏈式反應(yīng), 限制性片段長度多態(tài)性

CLC Number：Q939.96 　Document ID：A

Article ID：1007-3515(2002)03-0356-0362

Foundation Item：Supported by the National Natural Science Foundation of China (國家自然科學基金資助項目)

Author Unit：馬富英（農(nóng)業(yè)部農(nóng)業(yè)微生物重點實驗室,華中農(nóng)業(yè)大學植物保護系,武漢,430070）　

　　　　　羅信昌（農(nóng)業(yè)部農(nóng)業(yè)微生物重點實驗室,華中農(nóng)業(yè)大學植物保護系,武漢,430070）　

References：

［1］Bunyard B A, Nicholson M S, Royse D J, 1996. Phylogeny of the genus Agaricus inferred from restricition analysis of enzymatically amplified ribosomal DNA. Fungal Genetics and Biology, 20: 243～253

［2］Gardes M,White T J,Fortin J A et al., 1991. Identification of indigenous and introduced symbiotic fungi in ectomycorrhizae by amplification of nuclear and mitochondrial ribosomal DNA. Can J Bot, 69: 180～190

［3］Gonzalez P, Labarère J, 2000. Phylogenetic relationships of Pleurotus species according to the sequence and secondary structure of the mitochodrial small-subunit rRNA V4, V6 and V9 domains. Microbiology, 146: 209～221

［4］Iracabal B, Zervakis G, Labarère J, 1995. Molecular systematics of the genus Pleurotus: analysis of restriction polymorphisms in ribosomal DNA. Microbiology, 141: 1479～1490

［5］Lee H K, Shin C S, Min K B et al., 2000. Molecular systematics of the genus Pleurotus using sequence-specific oligonucleotide probes. Van Griensven (ed.) Science and Cultivation of Edible Fungi. 207～213

［6］Molina F I, Shen P, Jong S C, 1992. Molecular evidence supports the separation of Lentinula edodes from Lentinus and related genera. Can J Bot 70: 2446～2452

［7］Moncalvo J M, Wang H H, Hseu R S, 1995. Phylogenetic relationships in Ganoderma inferred from the internal transcribed spacers and 25S ribosomal DNA sequences. Mycologia 87(2): 223～238

［8］Nicholson M S, Bunyard B A, Royse D J, 1997. Phylogeny of the genus Lentinula based on ribosomal DNA restriction fragment length polymorphism analysis. Mycologia 89(3): 400～407

［9］Vilgays R, Sun B, 1994. Ancient and recent patterns of geographic speciation in the oyster mushroom Pleurotus revealed by phylogenetic analysis of the ribosomal DNA sequence. Proc Natl Acad Sci USA 91: 4599～4603