木材腐朽菌定のために DNA バーコードとして 核の rDNA …

1 23 . DNA . rDNA ITS D1/D2 .. Use of ITS region and D1/D2 domain of nuclear rDNA as a two-locus DNA. barcode for identification of wood rot fungi.. 1 . 2 . 3 . 4 . 5 . 6 .. 1 .. DNA ITS DNA . DNA .. DNA ITS ITS . D1/D2 . D1/D2 PCR . 28 SrRNA D1/D2 600bp . ITS . D1/D2 . 23 55 . D1/D2 PCR . Neighbor Joining NJ .. DB D1/D2 .. PCR .. D1/D2 DB . D1/D2 . D1/D2 ITS .. PCR .. 2 .. RNA rRNA rDNA 1) . rDNA Internal transcribed spacer (ITS) ITS . DNA .. 5) . DNA DNA . DNA DNA . DNA DNA DNA .. rDNA .. rDNA . rDNA . rDNA .. rDNA NTS ( nontranscribed spacer ) ETS (external transcribed spacer ) 18S ITS ITS 28S 5S . 1 rDNA.. (60S ) 28S, , 5S. rRNA . (40S ) 18S rRNA . D1/D2 rDNA. (28S rRNA) . ITS rRNA 18S rRNA 28S. rRNA ITS ITS ITS 2) D1/D2 . 28S rRNA NL1 NL4 PCR.

2 D1/D2 28S rRNA .. D1/D2 . DNA ITS ITS D1/D2 .. D1/D2 .. D1/D2 . ITS1 ITS3 NL1. 18S 28S 5S. ITS4 NL4. ITS . 1 rDNA . D1/D2 1),5) . 3 . 3-1 D1/D2 . 3-1-1 . 23 55 ( 3-1) .. ( 3-2) . (PDA) 25 . 3-1-2 DNA . Isoplant PDA . cm 7 mm 1 mm . 1 25 Hz 2 Wash buffer 1ml 25 Hz 2 14000 g 5 Sol. ( . ) 300 l 25 Hz 2 Sol. ( ) 150 l 25 Hz 2 50 10 Sol. -A 100 l Sol. -B 150 l 10 15000 rpm 10 2 vol. 15000. rpm 10 70% wash . TE 50 ml . 3-1-3 D1/D2 . Takara Ex TaqTM TaKaRa DNA . NL1 (5'-GCA TAT CAA TAA GCG GAG GAA AAG-3') NL4 (5'-GGT CCG TGT. TTC AAG ACG G-3') . PCR ( ) GeneAmp PCR System 9700 (Applied Biosystems ) . PCR . 94 5 1 94 . 30 56 30 72 . 1 25 72 5 10 Takara Ex Taq Buffer 2 l 1 . 1 dNTP Mixture ( mM each) l mM. PCR PCR Takara Ex Taq 5 U/ l l U/ l 3 l 1% 10 mM NL1 1 l M.

3 10 mM NL4 1 l M. DNA 1 l UV DNA up to 20 l Total 20 l 3-1-4 PCR . pGEM Promega WI US PCR . 2 Ligation buffer l T4 DNA Ligase l T-Vector l . l PCR 1 l 5 l . 4 . JM109 Competent Cells TaKaRa PCR . 0 1 . 42 50 5 SOC . 37 1 LB X-gal IPTG . 100 ml 37 16 . Go Taq Promega WI US . PCR DNA M13 -20 . (5'-GTAAAACGACGGCCAGT-3') M13R (5'-AACAGCTATGACCATG-3') . LB 5 ml 37 16 180 rpm 16.. Promega Wizard Plus SV Minipreps DNA Purification System (Promega WI US) . DNA . BigDye Terminator Cycle Sequencig Ready Reaction Kit (ABi, CA, USA) ABI PRISM 3700 DNA (ABi, CA, USA) . (DB). DDBJ/EMBL/GenBank Basic Local Alignment Search Tool (BLAST) . 4) . 3-1-5 . DDBJ ClustalW6) . Pleurotus Gloeophyllum Trametes 3 9 D1/D2 Pleurotus . 6 11 Gloeophyllum 7 8.

4 Trametes 11 11 ITS Pleurotus . 5 13 Gloeophyllum 8 11 . Trametes 11 21 Neighbor Joining NJ.. 3-2 . PCR D1/D2 . DB D1/D2 ClustalW.. Primer3 (v. ) 7) . PCR ITS3 ( 4). PCR . 2 .. Bjerkandera adusta KUT0804 . Bjerkandera adusta NBRC 5307 . Coniophora puteana NBRC 6275 . Coniophora puteana M . Daedalea serialis NBRC 9286 . Fomitopsis palustris NBRC 30339 . Fonsecaea pedrosoi NBRC 6729 . Ganoderma gibbosum KUT0805 . Ganoderma lucidum NBRC 8346 . Ganoderma lucidum GAL-M . Gloeophyllum sepiarium NBRC 4944 . Gloeophyllum sepiarium NBRC 6267 . Gloeophyllum striatum NBRC 6429 . Gloeophyllum striatum NBRC 30341 . Gloeophyllum trabeum NBRC 6430 . Gloeophyllum trabeum NBRC 6509 . Gloeophyllum ungulatum NBRC 6431 . Lentinus lepideus NBRC 6645.

5 Lentinus lepideus NBRC 8719 . Lentinus lepideus NBRC 30750 . Pleurotus cystidiosus NBRC 30607 . Pleurotus cystidiosus NBRC 31074 . Pleurotus ostreatus NBRC 6515 . Pleurotus ostreatus NBRC 30160 . Pleurotus ostreatus NBRC 33211 . Pleurotus pulmonarius NBRC 30791 . Pleurotus pulmonarius NBRC 31345 . Pycnoporus cinnabarinus NBRC 8255 . Schizophyllum commune Otoha . Schizophyllum commune SCC-0749 . Serpula lacrymans NBRC 8697 . Serpula lacrymans NBRC 30955 . Stereum hirsutum NBRC 6520 . Trametes pubescens NBRC 7892 . Trametes versicolor NBRC 30340 . Trametes versicolor NBRC 4937 . Trametes versicolor NBRC 4940 . Trametes versicolor NBRC 6481 . Trametes versicolor NBRC 8754 . Trametes versicolor NBRC 30388 . Bjerkandera adusta NBRC 4983.

6 Bjerkandera adusta NBRC 104974 . Daedalea dickinsii NBRC 4979 . Daedalea dickinsii NBRC 6487 . Daedalea dickinsii NBRC 30766 . Daedalea dickinsii NBRC 31163 . Ganoderma lucidum NBRC 31863 . Schizophyllum commune NBRC 4928 . Schizophyllum commune NBRC 6502 . Schizophyllum commune NBRC 30496 . Schizophyllum commune NBRC 30749 . Trametes hirsuta NBRC 4917 . Trametes hirsuta NBRC 4920 . Trametes hirsuta NBRC 6477 . Trametes hirsuta NBRC 7038 . 3 . NBRC No.. Aspergillus niger 9455 .. Aspergillus niger 105650 . Aspergillus terreus 6346 .. Cladosporium 6348 . cladosporioides . Cladosporium 105947 . cladosporioides Alternaria alternata 31805 .. Penicillium pinophilum 33285 . Penicillium funiculosum 100958 . Penicillium citrinum 6352 . Fusarium proliferatum 107681.

7 Fusarium oxysporum 31631 .. Aureobasidium 6353 . pullulans . Mucor racemosus 4555 . Rhizopus oryzae 31005 . Chaetomium globosum 6347 . Trichoderma virens 6355 .. Eurotium tonophilum 8157 . Paecilomyces variotii 30539 .. Paecilomyces variotii 33284 . 4 . (5' - 3'). NL2B-1 AGCATTGTTAGTCCGTGTCA. NL2B-2 CAAAGCATTGGTAGTCCGTGT. NL2B-3 AGAGCGCATCACAAAGCAT. NL2B-4 GAGAGCGCATCACAAAGCAT. 4.. 55 19 D1/D2 . GenBank/DDBJ/EMBL BLAST .. D1/D2 .. Pleurotus . Gloeophyllum Trametes D1/D2 ITS ( 2, 3) . D1/D2 ITS . ClustalW .. D1/D2 ITS D1/D2 . Pleurotus Pleurotus ostreatus Pleurotus pulmonarius . Pleurotus cystidiosus Trametes Trametes versicolor Trametes pubescens D1/D2 2 1 Trametes hirsuta D1/D2 Gloeophyllum Gloeophyllum ungulatum D1/D2 ITS . Gloeophyllum striatum Gloeophyllum trabeum ITS.

8 D1/D2 ITS Pleurotus ostreatus Pleurotus pulmonarius Trametes versicolor Trametes pubescens .. D1/D2 . ITS ITS . D1/D2 . ITS .. 989 Pleurotus_cystidiosus EU365636. Pleurotus cystidiosus EU365638. Pleurotus cystidiosus EU365639. Pleurotus pulmonarius GQ142040. 853. Pleurotus ostreatus 33211. Pleurotus pulmonarius AM269869. Pleurotus pulmonarius EU365669. Pleurotus ostreatus AY524786. Pleurotus ostreatus AM269868. Pleurotus ostreatus EU365665. Pleurotus ostreatus NG027634. Pleurotus ostreatus DQ071722. Pleurotus pulmonarius 31345. Pleurotus pulmonarius 30791. Pleurotus ostreatus 30160. Pleurotus ostreatus 6515. Trametes versicolor AM269878. Trametes versicolor AY333793. Trametes versicolor HM595617. Trametes versicolor AY684159. Trametes pubescens 7892.

9 Trametes versicolor 30340. Trametes versicolor 4937. Trametes versicolor 4940. Trametes versicolor 8754. Trametes versicolor 6481. 881 Trametes pubescens AY515341. Trametes versicolor 30388. 437 Trametes versicolor AY855914. Trametes pubescens AY855906. Trametes hirsuta AJ488129. Trametes hirsuta 4917. 1000 Trametes hirsuta AY855910. Trametes hirsuta 7038. Trametes hirsuta AY351923. 912 Trametes hirsuta AY351922. Trametes hirsuta 6477. Trametes hirsuta 4920. Gloeophyllum ungulatum 6431. 999 Gloeophyllum trabeum HM536067. Gloeophyllum trabeum AY333804. 598 Gloeophyllum trabeum EU232301. Gloeophyllum trabeum 6509. 999 Gloeophyllum trabeum 6430. Gloeophyllum striatum HM536063. Gloeophyllum sepiarium AY333805. 612 Gloeophyllum sepiarium AY333806.

10 Gloeophyllum sepiarium HM536061. 858 Gloeophyllum striatum 30341. Gloeophyllum striatum 6429. 833 Gloeophyllum sepiarium AJ583432. Gloeophyllum sepiarium 6267. Gloeophyllum sepiarium 4944. 2 Pleurotus Gloeophyllum Trametes D1/D2 . ClustalW N-J . 1000 .. 963 Pleurotus pulmonarius HM590446. Pleurotus pulmonarius JN043318. 1000 Pleurotus pulmonarius JN043319. Pleurotus pulmonarius GU722282. Pleurotus pulmonarius 30791. Pleurotus ostreatus 33211. Pleurotus ostreatus HQ286595. 563 Pleurotus ostreatus GU722280. Pleurotus ostreatus JF758887. Pleurotus ostreatus FR686578. Pleurotus ostreatus 30160. Pleurotus ostreatus 6515. 988 Pleurotus cystidiosus DQ882572. Pleurotus cystidiosus AY540320. Pleurotus cystidiosus EU424279. Pleurotus cystidiosus JF758880.