Alternaria alternata ļ ļżƑhǬʯʓʘˁ

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Alternaria alternata ļ ļżƑhǬʯʓʘˁ

Ņǝ˗˯̀˔˞˅ƗʬʝʤƑhèƒƃŅŀʰȌİ

(Studies on molecular mechanisms of pathogenicity of Alternaria alternata plant pathogens based on functional genomics)

ɻ»ɿ ~¬

Kazumi Takao

(2016)

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1 ƘƘɿ Ň

Ƶ1Ƴɿ ǑtÏȟ ---2

Ƶ2Ƴɿ ĬėʓʾʳĚŘ --- 4

Ƶ3Ƴɿ ǫĵƑǬʯʓʘˁƑhèɗȼɇ ¥ʰĄǅ --- 26

ɿ Ƶ1ƹɿ Ïȟ --- 26

ɿ Ƶ2ƹɿ ĬėʓʾʳĚŘ --- 29

ɿ Ƶ3ƹɿ Ǌĳ --- 31

ɿ Ƶ4ƹɿ Ǚ³ --- 66

Ƶ4Ƴɿɿ °ŽƏƕAALŏǄƇtñɇ ¥˔̈˞ˣ̐ʰ ɿ ɿ ɿ ɿ ɿ ɿ ɿ ɨƑhèA. alternataǬʴʰ·> --- 72

ɿ Ƶ1ƹɿ Ïȟ --- 72

ɿ Ƶ2ƹɿ ĬėʓʾʳĚŘ --- 74

ɿ Ƶ3ƹɿ Ǌĳ --- 76

ɿ Ƶ4ƹɿ Ǚ³ --- 88

Ƶ5Ƴɿ Ċȅ ---90

Ƶ6Ƴɿ Summury ---92

Ƶ7Ƴɿ ÔƉĕſ ---94

¨ȗ@ǽȟĕ̉˞˪ ---104

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2 Ƶ

Ƶ1Ƴɿ ǑtÏȟ

ɿ ƍoʰZʽęǦʰɵʰ_ʯ!ʐʊɵƾʰƢ.ʱsʰǐʰȝɱʬ ʮˀʨʨʏˁʋʚʰɱ˅ȌŖʟˁʤʻʊ©«ʝʤ)żƇƈʕȅŕʜ˂ʪʐˁʋĚʊ

²řʽËʲʨʮʭʰŒ3_ʽƑ®ǴʯʾˁǾ®ʯʾˀʊʗʰ)żʱŨl˅(9 ʮʗʜ˂ʪʐˁʋʚ˂ʿǾ®˅ĥ¸əʯɵʐňʻˁʤʻʊļżʯŁʌʮù÷èʕ ʜ˂ʪʐˁʋʝʔʝʮʕʿʊʚ˂ʿù÷èʰʑʦƇżʯ´ʟˁʼʰʱʊ´ȤƇżʰ

Əʯʾˀôơʜ˂ˁqǝèʕʏˁʋ

ɿ ļżʰƑŒʰ80%˅eʻˁǬɳʱʊƃʸʫʯ 10ƭʕzʜ˂ʪʓ ˀʊʢʰʑʦʰǁ 7,000 ƭʕʊļżƑhǬʬʝʪzʜ˂ʪʐˁʋʚʰʾʑʯēʗʰ ƑhǬʕKɡʊu«ʜ˂ʪʐˁʕʊʢ˂ʿʰʗʱƑhèƒƃŅŀʯʨʐʪȖǆʕ ĝʿʔʬʮʧʪʐʮʐʋĚʊļżʴʰù÷èʰʽęȈȲǲʰɕƒ˅ǹʑtʊ ƑhèǬʬɨƑhèǬʬʰɄʐʽʊƑhèʰȾ_ʊƀÞʊţ¢ʮʭǬʰéʰ ʫʼʊʬˀ˄ʘƑhèƒƃŅŀʯɗʟˁéʕäȅʯʮˁʚʬʱȍʑʸʫʼʮʐʋ ɿ ȵÍʊ˜̐˔ˍ̎˞öǺʰǯʝʐƒ½ʯʾˀʊʗʰƇżƭʰ˗˯́éʕǱƮʜ

˂ʨʨʏˁʋ˳˪ʽļżʯŐʵʊ˗˯́˚ˊ˟ʰ¸ʜʐǬɳʱʊ˫̈˶˪Ȍİ˅xʻˁ ʬʊÍɖē`ʰ˗˯́éʕ@ɕʜ˂ʪʓˀʊʚ˂ʿ˅QƉʝʤƑhèɇ ¥ʰĄǅ ʮʭʼǹ˄˂ʨʨʏˁʋʚʰʾʑʯʊ˘̎˵̅̐ˣ̐ʫɑʰ˗˯́éʔʿ3ȁʬ ʮˁƑhèɇ ¥˅ȇJʟʚʬʕ¯ĞʯʮˁĚʫʊ¬ɞʯʢʰ3ȁɇ ¥ʰŅ ǝ˅u«ʟˁʤʻʯʱʊɇ ¥ơʽɇ ¥˚ˊ̋̎˜̎˕ŘʯʾˀŅǝȌİ˅ǹ ʑäȅʕʏˁʋʢʰʤʻʊ˗˯̀˔˞Ơư˺̐˞ʫʗʰƑhè3ȁɇ ¥ʱĆ«ʜ

˂ˁʼʰʰʊ¬ɞʰŅǝʱĝʫʏˁtʕʐʋ

ɿ Alternaria alternata ʱƍ ʯKÆʟˁǬʫʏˀʊīƕʯǟèƇŝ˅ȷʧʪʐ ˁ (Rotem 1994)ʋʢʰ ʫʊ°ŽƏƕŏǄ (host-specific toxinʊʊHST) Ƈ ƈǝ˅ƀÞʝʤʼʰʰʹʕʊƑhǬʬʮˁʋʚ˂ʸʫʯʊ¹ʮʗʬʼ7ƭʰƑhʕ§

ʟˁʚʬʕĝʿʔʬʮʧʪʐˁ (Kohmoto et al. 1995, Thomma 2003)ʋȵÍʊʚ˂ʿ HSTʰƇtñʯɗ˄ˁɇ ¥ʕȇJʜ˂ʊʢʰƇtñɇ ¥ʕ˔̈˞ˣ̐_ʝʪʐ ˁʚʬʼzʜ˂ʪʐˁ (Akagi et al. 2005, Akamatsu et al. 2003, Harimoto et al. 2007, Hatta et al. 2002, Johnson et al. 2001, Miyamoto et al. 2009)ʋʜʿʯƑhèʯäɮʫ ʏˁʚ˂ʿ HST Ƈtñɇ ¥ʱʊ¹ʮʗʬʼˊ˥˙ʊ̉̎˙ʊ˪˿˪Ƒhʯʓʐʪ conditionally dispensable chromosome (CDC) (Covert 1998) ʬ|ʲ˂ˁ1.8 Mb ʰĶǩ&ʯÑʝʪʐˁʚʬʕĝʿʔʬʮʧʪʐˁ (Akagi et al. 2009, Akamatsu et al. 2003, Hatta et al. 2002, Harimoto et al. 2007, Johnson et al. 2001)ʋ

ɿ īƠưʫʱʊHSTƇƈA. alternataƑhǬ˅´Ȥʬʝʪʊˮ˔̌˪̌˶ļżƑhǬʯ

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3

ʓʘˁƑhèɇ ¥˅˗˯́éʯʩʖŅǝȌİʝʊʢʰƑhèƒƃŅŀ˅^

ýƕʯȌĝʟˁʚʬ˅ƘĀʝʤʋƑhèɗȼɇ ¥ʬʝʪʊŏǄƇtñɇ ¥ʊ˜

˕ˬ̉̎˕ɗȼɇ ¥ʓʾʳˍ˵˝ˌˮ˨ˉ˦˔Rßʯɗ˄ˁ˕̌̐˱̊̋˓̅̋̐

ˣ̐ɇ ¥˅ʮȌİ´Ȥʬʝʤʋ

ɿ ¨$ȟĕĈJʯʏʤˀʊʛĀ·ʊʛbXɫʖʸʝʤʊĀ·Ēʰɼm¨¨

ɚȼtȲ¨ƠưƨļżƑƅ¨Ơưɿ =ƂħĒĂʊvp¼¨ɿ ķļºå ĒĂʊÁÀ¨ɿ ÀīÎdGĒĂʊɼm¨ļżƑƅ¨Ơưɿ ȪĨɦCdʊ ȪĨƛǗőʮʿʳʯƠưȠőʊʸʤʊɼm¨ɿ TÃ ɉĒĂʊƟhɿ ĒĂʊ Ào¨ɿ ǳƛĒĂʊʓʾʳÂĺ¨ɿ ɐɿ țGĒĂʯgʗßƤƋʝʙʸ ʟʋ

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4 Ƶ

Ƶ 2Ƴɿ ĬėʓʾʳĚŘ

ɿ ʚʰƳʫʱʊɘʰ¬ɹʫ*Ɖʜ˂ˁAȹʰĬėʬĚŘ˅ȑʝʤʋ

1. +ȔǬʓʾʳ+Ȕļż

+ȔǬʬʝʪʊ*ƉʝʤǬĸʰ̉˞˪ʱǽ 2 ʯƣʝʤʋǬĸʱ˝̄ˑˊ̃²

(PDA) ʫɷʝʤʋǬĸʰ.§ʱ˃ǃʯʾˁ.§ʬ˕̉ˠ̌̐̊ʯʾˁ.§ʰ 2

ƭɳʰĚŘʫǹʧʤʋ

̏˃ǃʯʾˁ.§: ǁ1 cmȋʯLʧʤ˃ǃ (Whatman, 3MM CHR̓ ˅ēĲʊʰ ¡ʔʿɡʝʪʰʡʤÜʊǬ&˅ ¡ʯļǬʝʤʋǬ&ʕ˃ǃʯ"ʳʤŌɜ ʫʔʿÔʖʱʕʝʊ˔̉̐̎˺̎˥ ʫɴÜʊ5ʀ5 cm ʰ¸µƷʯ>˂

-20ʇʫ.§ʝʤʋ

̏˕̉ˠ̌̐̊ʯʾˁ.§: ʫñɔʝʤǬ&˅5 mmȋʯLˀmˀʊ20%˕̉ˠ̌

̐̊ʰ>ʧʤˍ˦˻̎˫̊˶˥̅̐˷ʯƫʝʊ-80ʇʫ.§ʝʤʋ

ɿ +ȔļżʱǫĵƑǬʯínèʫʏˁ˪˿˪ (Solanum lycopersicum) ƭʊìƝ˶

ˇ̐˞˪˅Ɖʐʤʋ

2. Ȕǲʓʾʳ

īƠưʫ*ƉʝʤȔǲʓʾʳʰsǈñʱǽ3ʯƣʝʤʋ

3. ˗˯́ DNAʰúJ

ɿ sǬʔʿʰ˗˯́ DNA ʰúJʓʾʳ.§ʱȪĨ (ȪĨ 2010) ʰĚŘ 3 ƭ (Yoder ʿ (Yoder et al. 1994) ʰ Miniprep Ř˅đʝʤ Miniprep ŘʊSuzuki ʿ (Suzuki et al. 2006) ʰĚŘ˅đʝʤɣ¥̋̎˝ƼĞúJŘʓʾʳŤ&ƱǄƼĞ úJŘʊʊǽȑ˅ʢ˂ʣ˂MiniprepŘʊɣ¥̋̎˝ŘʓʾʳŤ&ƱǄŘʬʟˁ) ʯÝʐǹʧʤʋ

4. ? RNAʰúJ

4-1. RNeasy plant kit˅Ɖʐʤ RNAʰúJ

Ǭ&˅ PDAʯļǬʝʊ3 ĜƬÐñɔʜʡʤÜʊŘRichards Ť&ʯ 5 mm ȋŻ˅201ƬÐļǬʝʤʋēĜɖāʬʑɷ (120 rpm) ÜʊyÔŴɁʝʊǬ&˅

lʝʤʋʚʰÜʰĎ)ʱRNeasy plant kit (Qiagen) ʰ˸̌˪˘̊ʯÝʐǹʧʤʋÞʿ

˂ʤRNAʯxʸ˂ˁDNAʱDNaseʈʫIƅÜʊ˶ˌ˯̐̊/˔̌̌˼̊́úJʫɛ iʓʾʳǂ_ʝʤʋÞʿ˂ʤRNAʱPCRʯʾˀʊDNAʰ˘̎ˣ̀ˮ̐˜̇̎ʰĦŶ˅

ƢȘʝʤʋʢʰÜʊGeneQuant pro RNA/DNA Calculator (Amersham pharmacia biotechʊƃGE˹̊˞˖ˈ) ʫųÐʬǂÐ˅ū«ʝʤʋ

(6)

5

ǽ1 ALT˔̈˞ˣ̐ɇ ¥ʰAALŏǄƇƈʬĆ«Ņǝ

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6 ǽ2ɿ īƠưʫ*ƉʝʤǬĸ

Ǭĸ ƭv ° ăɠ

As-27 A. alternata ˪˿˪ ˈ̂̉ː

O-94 A. alternata ĩu« Ĝīʊɼm

O-276 A. alternata ˭˼̎ˬ˜ Ĝīʊɼm

AC320 A. alternata ˣ̎ˡ̉̎ ˈ̂̉ː

AC325 A. alternata ̈˶̋̃̎ ˈ̂̉ː

NAF8 A. alternata ˊ˥˙ ĜīʊìƝ

M-30 A. alternata ˊ˥˙ Ĝīʊɼm

M-71 A. alternata ̉̎˙ Ĝīʊɔɐ

IFO8984 A. alternata ̉̎˙ ĜīʊBÒ

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7

ǽ3ɿ īƠưʫ*ƉʝʤȔǲʓʾʳʰǈñ

LB medium 1 l

Tryptone peptone 10 g

Yeast extract 5 g

NaCl 10 g

Agar powder 15 g

Regeneration medium 1 l

1 M Sucrose 342.3 g

0.1 % Casein hydrolysate enzymatic 1 g 0.1 % Yeast extract 1 g

Crossing medium 1 l

Ca(NO3)2̏4H2O 1 g

K2HPO4̏3H2O 0.25 g

MgSO4̏7H2O 0.25 g

CaCO3 0.85 g

Agar 20 g

PDA medium 1 l

potato 200 g

glucose 20 g

agar 15 g

PDB medium 1 l

potato 200 g

glucose 20 g

V8 medium 1 l

V8 juice 200 ml

CaCO3 3 g

agar ɿ 15 g

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8

ǎʖ ɿ ɿ

Buffer1 (pH 7.5) 3 l

0.1 M Maleic acid 29.42 g

0.15 M NaCl 26.3 g

Buffer2 200 ml

Blocking reagent 2 g

Buffer1 200 ml

Buffer3 (pH 9.5) 200 ml

0.1 M Tris-Cl 2.42 g

0.1 M NaCl 1.16 g

50 mM MgCl2̏6H2O 2.03 g

Buffer4 1 l

10 mM Tris-Cl (pH 8.0) 10 ml (1 M Tris-Cl) 1.0 mM EDTA (pH 8.0) 1 ml (1 M EDTA)

20xSSC (pH 7.0) 1 l

3 M NaCl 175.32 g

0.3 M Na-citrate 88.23 g

Transfer buffer 2 l

0.4 M NaOH 32 g

1.5 M NaCl 174 g

Probe stripping buffer 1 l

0.2 M NaOH 8 g

0.1 % SDS 10 ml (10 % SDS)

Neutralizing buffer (pH 7.2) 1 l

0.5 M Tris-Cl 60.5 g

1.0 M NaCl ɿ 58 g

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9 ǎʖ

Richards medium (reviced) ɿ 1 l

KNO3 10 g

KH2PO4 5 g

MgSO4̏7H2O 2.5 g

FeCl3 0.02 g

glucose 25 g

Yeast extract 1 g

OM buffer (pH 5.8) 1 l

1.2 M MgSO4̏7H2O 296 g

10 mM Na2HPO4 100 ml (100 mM Na2HPO4) pH 5.8 with NaH2PO4̏2H2O

ST buffer 100 ml

1.0 M Sorbitol 18.2 g

0.1 M Tris-Cl (pH 8.0) 10 ml (1 M Tris-Cl)

STC buffer 500 ml

1.0 M Sorbitol 91 g

50 mM Tris-Cl (pH 8.0) 25 ml

50 mM CaCl2̏2H2O 3.65 g

0.5xTBE 2, 3 l

0.45 mM Tris-Cl 10.8, 16.2 g 0.45 mM Boric acid 5.5, 8.25 g

1.0 mM EDTA 0.75, 1.125 g

Hybridization buffer 200 ml

5xSSC 50 ml (20×SSC)

1 % Blocking reagent 2 g

0.1 % SLS 2 ml (10 % SLS)

0.02 % SDS 400 μl (10 % SDS)

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10

ǎʖ ɿ

Solution I 100 ml

50 mM glucose 0.9 g

25 mM Tris-Cl (pH 8.0) 2.5 ml (1 M Tris-Cl) 10 mM EDTA (pH 8.0) 1 ml (1 M EDTA)

Solution II 100 ml

0.2 M NaOH 0.8 g

1 % SDS 10 ml (10 % SDS)

Solution III 100 ml

3 M potassium acetate 29.45 g

acetic acid 11.5 ml (Add after autclaved)

TE buffer 100 ml

10 mM Tris-Cl (pH 7.5) 1 ml (1 M Tris-Cl) 1 mM EDTA (pH 8.0) 0.1 ml (1 M EDTA)

1xBromophenol Blue (BPB) 10 ml

0.25% BPB 25 mg

30% glycerol 3 ml

Yoder's buffer 100 ml

100 mM LiCl 10 ml (1 M LiCl) 10 mM EDTA (pH 8.0) 1 ml (1 M EDTA) 10 mM Tris-Cl (pH 7.4) 1 ml (1 M Tris-Cl)

0.5 % SDS 0.5 g

x % PEG 100 ml

50 mM CaCl2̏2H2O 0.73 g

50 mM Tris-Cl (pH 8.0) 5 ml (1 M Tris-Cl)

1 M Sorbitol 18.2 g

PEG4000 ɿ x g

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11 4-2. ȸȸȯFkæ

ȸȯFkæʱʊPrimeScript RT reagent Kit (Takara Bio) ŦʰȜĝģʯÝʐǹ ʧʤʋȑ4-1ʫÞʤRNAʱȪĨ (ȪĨ 2010) ʰĚŘʫkæŤ˅ȞȂʝʤʋÞʿ

˂ʤʊcDNAʱƒƃȌİʰ˨̎˸̋̐˪ʬʝʪɘʰ¬ɹʯ*Ɖʝʤʋ

5. ɇ ¥ʰƒƃȌİ

5-1. Reverse transcription PCR (RT-PCR)

ɿ ɇ ¥ʰƒƃ˅ƢȘʟˁʤʻʊĽ«ʟˁǬĸʰRNAʱTȶʰĚŘʫúJʓʾʳ ȸȯFʝʤʋÞʿ˂ʤ cDNA ˅˨̎˸̋̐˪ʬʝʊëʰ˸̈ˊ˿̐˅*ƉʝʊRTʉ PCR ˅ǹʧʤʋPCR ÜʊɣŒś]˅ǹʐʊƘƕʰ˱̎˫˅ƢȘʟˁʚʬʫƒƃʰĦ Ŷ˅Ľȏʝʤʋ

5-2. ˊ̎ˣ̐ː̋̐˪Řʯʾˁ Real-time PCR

sɇ ¥ʰƒƃ˅«ɑʟˁʤʻʯʊReal-time PCR (Roche, Light Cycler 480) ˅ǹ ʧʤʋPCR ʰɞʰ mixture ʱǶ<ǩǄ SYBR Green I (Roche, Light Cycler 480 SYBR Green I Master) ʰȜĝģʯÝʐ)ñʝʤʋcDNAʱȑ4-2ʰʬʓˀkæɑ

˅20 μlʬʮˁʾʑʯʝʊRNAʕ0.8 μgʯʮˁʾʑZʒtñʝʤʋȸȯFÜʊmilliQ˅ Zʒ100 μlʯ̂˞ˈ˦˸ʝʤʋ

ɿ SYBR Green Iʰ˒˦˪ʯ¾ʝʪʐˁwater 4 μlʊPCR primer 2 μlʊmaster mix 9 μl

˅˥̅̐˷ʯ>˂ʊ˵˻˦˨ˉ̎˕ʯʾˀťtʝʊLight Cycler 480 Multiwell Plate 96 ʯ15 μlʠʨZʒʤʋʢʰÜʊ˨̎˸̋̐˪ʫʏˁcDNA˅5 μlʠʨZʒʤʋkæʱ Ȝĝģʰ˸̌˪˘̊ʯÝʐʊƒƃɑʱī&¾ʰˢ˶˪ˋˌˈʫ«ɑʝʤʋ˸̈ˊ˿

̐ʰųÐʱ10 µMʬʝʤʋ˸̈ˊ˿̐ʱǽ7ʯƣʝʤʋ

6. PCR

ɿ PCR (ˣː̈˱ˊˏ, TP650 ʓʾʳ TP350̏˱ˊˏ̈˦˫, 170-9703JȀeppendorf, 95004) ʯʾˁDNAʰÊʱʊsƭTaqʛʬʯŦʜ˂ʪʐˁ˸̌˪˘̊ʯÝʐǹʧ ʤʋ˸̈ˊ˿̐ʰųÐʱ10ʏˁʐʱ20 µMʯȞȂʝʤʋsƭTaqʛʬʰPCRğʰ ǈñʱǽ5ʯʊPCRʯ*Ɖʜ˂ʤ?˸̈ˊ˿̐ʱǽ4ʯƣʝʤʋ

7. ɣŒś]

ɿ Agarose S ʓʾʳAgarose HSʯ0.5xTBE buffer ˅Zʒʊɣ¥̋̎˝ʫŮȌʝʤ (ųÐʱƈż˚ˊ˟ʯ,§)ʋŮʘʤ˗̊˅ĴʯŞʝʻʊ0.5xTBE buffer ʫŭʤʝ ʤś]łʯ˗̊˅>˂ʊɣŒś] (Mupid-exu) ˅ǹʧʤʋ

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12 ǽ4ɿ īƠưʫƉʐʤ˸̈ˊ˿̐

˸̈ˊ˿̐ ɋM TM4

ALT10AF gcgctaaccgcagtgtgact 67.6

ALT10AR-SfiA cacggcctatatggcccagagccgtccttgacat 69.3

ALT10BR ctgcctctgataccgactca 63.0

ALT10BF-SfiB gtgggccacgcaggccagtgagatggcacccgac 74.1 Hph-SfiA-1-ra cacggccatataggccttacactttatgcttccg 77.3 Hph-SfiB-1-ra gtgggcctgcgtggcccttcgctattacgcca 71.2

hph-F acgtctgtcgagaagtttc 59.0

hph-F2 gacgtctgtcgagaagtttctg 62.9

hph-R gtattgaccgattccttgcg 64.2

ALT10inF tgcacatagcaatcatccg 63.2

ALT10inR gtggcatggagtctttggtg 65.5

ALT10homoF agatcactcgttgctgcttg 63.2

HphA-homoR cagagattacttcaagtcag 68.3

HphB-homoF ttacaacgtcgtgactggga 64.2

ALT10-wholeF atgacggccaacgtaaacgtc 67.9 ALT10-wholeR ctaactgcaacggaacaaggac 64.6 npt-F gacaatcggctgctctgatg

npt-R atgcgatgtttcgcttggtg

ALT5AF1 ggcaggcagtatagatcagaatctcc 67.6

ALT5FuAR1 atcaggtcgatgctagcatcagcaaaatctcccacatacg 81.7 ALT5FuBF1 atgcgagtgctaccagatgtcggaaggcgtagctatagtc 80.5

ALT5BR1 gccatggcagtcgaaactctgtatgc 73.7

ALT5inF ttgtaggtcctgtgggttcg 67.8

ALT5inR tgagggcccaaagatgtggc 71.5

ALT6FuAF ggaagactgggttggtgaag 63.5

ALT6FuAR20 atcaggtcgatgctagcatctaacgcaggccctgaatgag 83.4 ALT6FuBF20 atgcgagtgctaccagatgtcattgcttaaggctgaggag 81.5

ALT6FuBR caagcacggacactctagct 62.2

ALT6inF aactccctctgtccttcgag 62.3

ALT6inR tctccgggtgacttggccag 71.9

TF1AF cggcaaagcgtcgtcgtgttgttgc 79.7

TF1FuAR atcaggtcgatgctagcatcggcttaccacatgtctcaga 82.7 TRF1BF1 atgcgagtgctaccagatgtaccggagacgccaagacgac 85.4

TRF1BR1 gatgcgacgctgcaaccgcagaagg 80.7

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13 ǎʖ

TF1inF ccactggtcgagaagggtac 63.5

TF1inR gacctctgcctgcgatggac 69.5

TF1homoF gatcgctcacctgggtagac 63.6

TF2AF gcacgatccctgcacggataaggac 75.5

TF2FuAR atcaggtcgatgctagcatcgttgcaggccaagttgagct 85.0 TF2FuBF atgcgagtgctaccagatgttaagaggcatccagtccgac 82.6

TF2BR catgcgaccatgatcccttggcctctc 79.2

TF2inF tatgctcgcagccttcatcg 68.5

TF2inR acaaggacgcgttatcgtcg 67.1

TF2homoF gcgtttatcccggaactgtc 65.4

TF3AF ctcgaacatgtctcgcatgatgatcag 73.2

TF3FuAR atcaggtcgatgctagcatcctagcatccttgacccacgc 84.5 TF3FuBF atgcgagtgctaccagatgttatggtctgagcagccattg 82.7

TF3BR ctactacgtgcagcagatcctagcaac 68.5

TF3inF tctgtctgttccactggtgc 64.1

TF3inR cagatcgtactggaagcacg 63.4

TF3homoF gacctgcctcctgcagcaac 69.6

TF4AF caatggcagtcgccttactcgagac 72.3

TF4FuAR atcaggtcgatgctagcatcccttaacacagcacataacg 80.5 TF4FuBF atgcgagtgctaccagatgtgtagtccaggatctgcacgg 84.2

TF4BR gatgtcgggaaatacaagcacatgtg 70.7

TF4inF tctagagatgccggaaggcg 68.6

TF4inR cgaacggggttacattcgag 66.3

TF4homoF gattgcctgctggcgaccag 72.4

Ave1AFL aaggcgcagactgaggggat 69.3

Ave1ARL atcaggtcgatgctagcatcccggacatgtctgccatctg 86.2 Ave1BFL atcaggtcgatgctagcatcgcttgttaccgtcctccagc 81.8

Ave1BRL tggtaggggcatattgccga 70.9

Ave1inFL acttcatgcggacacgatgc 67.6

Ave1inRL ccgtcctccagctccagaag 65.8

Ave1homoF gcgatgctagaggctttgtc 63.9

LAE1AF ggtactcgtctcgcgtaagc 63.8

LAE1AR atcaggtcgatgctagcatcgcagcgcaggccattaacag 86.3 LAE1BF atgcgagtgctaccagatgtgtaccacgcgactacgagag 83.0

LAE1BR gtgacaggcagactcccttg 64.9

(15)

14 ǎʖ

LAE1inF catgtacccgtgcgacgagg 70.9

LAE1inR gagatccaatcccagcacct 65.3

LAE1homoF gtgctcttacaacgctgcag 63.7

Lae1comR ctgctgccatgccattgccg 76.1

AGAt1AF caggctagcactagcagcag 63.0

AGAt1AR atcaggtcgatgctagcatcgtacgtagtgatgttccgtc 80.9 AGAt1BF atgcgagtgctaccagatgtctccgagtatgaccagctgc 83.7

AGAt1BR gttgggttgagttggaaccg 66.5

AGAt1inF ggagggcaagcagaggaacg 70.4

AGAt1inR gtgctggctgcatgaagatg 67.0

AGAt1homoF gtgatgctagtcgtcgcaag 63.6

STE2AF cgattcgttgcgactgccgc 75.2

STE2AR atcaggtcgatgctagcatcgattgcttgggatagcggtg 84.7 STE2BF atgcgagtgctaccagatgtaacggctgatcaggagcgac 84.4

STE2BR gcgaagtggttgtagcagag 62.6

STE2inF ctgtcatggcctgcattgcg 72.2

STE2inR tggtcgttgacgacgccagc 74.2

STE2homoF ctggtgagctaacggtgacg 65.8

STE2homoR ccagatagaactgagtgcag 57.5

GPCR1-AF-re gtagtgctacagtagtaccacgaggtag 63.1 GPCR1-AR20 atcaggtcgatgctagcatccctgagctgaaccaaaactc 82.1 GPCR1-BF20 atgcgagtgctaccagatgtgagtaggtaatgcgctgaga 81.4

GPCR1-BR gacgcgatacgccatagtct 64.0

GPCR1-inF ggtagccggtaacaatgtag 58.6

GPCR1-inR cctatgcgagcttcagggac 66.0

GPCR1-homoF gacatgaaccacacagttgg 61.5

GPCR2AF gtgttgacgggccctctcag 69.0

GRCE2AR atcaggtcgatgctagcatcccaaggatgaaaacccagag 67.9 GRCR2BF atgcgagtgctaccagatgtcaacaagggtcatcagcgag 68.9

GPCR2BR gtatgtcgcgctatggcggc 71.6

GPCR2inF cgcttgctcgtctttgcctc 69.4

GPCR2inR gatcgtcgaccccttgtgtg 67.9

GPCRhomoF cgctcttgtcctctccccag 68.5

GPCR3AF ggcctgactcttggtccatg 67.0

GPCR3AR atcaggtcgatgctagcatccagaactgtcaccgcaacgc 70.0

(16)

15 ǎʖ

GPCR3BF atgcgagtgctaccagatgtcaacgagaagagccaaatcg 67.9

GPCR3BR cataccccaacctgtggatc 64.0

GPCR3inF gtgtgagcttgctagtgtcg 61.0

GPCR3inR caggagcgatatttcgctcg 66.8

GPCRhomoF acatgtcaaaggagctgcgg 67.5

GPCR3comF caacactcccctgattaccc 63.1

GPCR3comR cgcacatcagcacggcgag 73.9

HPHA20-fusion gatgctagcatcgacctgatttacactttatgcttccg 77.6 HPHB20-fusion acatctggtagcactcgcatcttcgctattacgcca 80.3

Btub1 tccgtcgtgccttcccccaaggtctccgac 85.2

Btub2 ggagcgaatccgaccatgaagaagtggaga 78.7

(17)

16 ǽ5ɿ PCRkæŤʰǈñ

KOD FX (TOYOBO) µl µl

2x PCR buffer for KOD FX 25 10

2 mM dNTPs 10 4

Primer F (10 µM) 1 0.4

Primer R (10 µM) 1 0.4

KOD plus (1 U/µl) 1 0.4

Sample DNA x x

MilliQ 12-x 4.8-x

Total 50 20

EX Taq (TOYOBO) µl µl

10x EX buffer 5 2

2.5 mM dNTPs 4 1.6

Primer F (10 µM) 1 1

Primer R (10 µM) 1 1

EX Taq (5 U/µl) 0.25 0.1

Sample DNA x x

MilliQ 38.75-x 14.3-x

Total 50 20

LA Taq (TAKARA) µl µl

10x LA buffer 5 2

2.5 mM dNTPs 8 3.2

25 mM MgCl2 5 2

Primer F (10 µM) 1 1

Primer R (10 µM) 1 1

LA Taq (5 U/µl) 0.5 0.2

Sample DNA x x

MilliQ 29.5-x 10.6-x

Total 50 20

(18)

17

ǎʖ ɿ ɿ

rTaq (TOYOBO) µl

10x buffer 2

2 mM dNTPs 2

Primer F (10 µM) 1

Primer R (10 µM) 1

Recombinant Taq (5 U/µl) 0.1-0.2

Sample DNA X

MilliQ 13.9 or 13.8-x

Total 20

2x Quick Taq HS DyeMix (TOYOBO) µl

2x Quick Taq HS DyeMix 25

Primer F (10 µM) 1

Primer R (10 µM) 1

MilliQ 23-x

Sample DNA x

Total 50

ɿ

(19)

18 8. ˗˗̊úJ

ɿ Ƙƕʰ˱̎˫˅ɣŒś]ʯʪKɡÜʊEtBr ʫĶǩʊǰƎœʫǞǩʝʊUV ̈̎˸

(300 nm) Ÿ¶ʫʊƘƕʬʟˁ˱̎˫˅LˀJʝʤʋ˗̊˅1.5 mlˍ˦˻̎˫̊˶˥

̅̐˷ʴƫʝʊQIAquick Gel Extraction Kit (Qiagen) Ŧʰ˿˭̅ˈ̊ʯƣʜ˂ʤ óɭʯÝʐ˗̊ʔʿDNAʰúJ˅ǹʧʤʋ

9. fusion PCRŘ˅Ɖʐʤɇ ¥˯˦˔ˈˋ˪ (KO) ˺˔ˣ̐ʰŀƻ

ɿ ˺˔ˣ̐ʰŀƻʱʊ ıʿ ( ı 2010) ʰĚŘ˅đʝʊǹʧʤʋʸʠʊĘŻAʰ 3’5˸̈ˊ˿̐ʯëʰɋMʊ20 mer˅Zʝʤ˸̈ˊ˿̐˅ƉʐʊĘŻA˅

ÊʝʤʋŇʯʊĘŻBʰ5’5˸̈ˊ˿̐ʯ;ʶʭʬʱƏʮˁëʰɋMʊ20 mer

˅Zʜʡʤ˸̈ˊ˿̐˅ƉʐʊĘŻB˅Êʝʤʋʸʤʊ5’5˸̈ˊ˿̐ʯĘŻAʫ Ɖʐʤëʰ20 merʰƚȁɋM˅Zʝʊ3’5˸̈ˊ˿̐ʯĘŻBʫƉʐʤëʰ 20 merʰƚȁɋM˅Zʜʡʤʋʚ˂ʿʰ˸̈ˊ˿̐˻ˈ˅Ɖʐʪhphːˠ˦˪˅

ÊʝʤʋĘŻAʓʾʳB˅ÊʟˁɞʱEX Taq˅Ɖʐʪtotal 20 μlʫǹʐʊhphːˠ˦

˪˅ÊʟˁɞʱʊKOD FX˅Ɖʐʪtotal 20 μlʫǹʧʤʋʚ˂ʿ3ʨʰPCRƈżs1 μl˅˨̎˸̋̐˪ʯĘŻAʰ5’5˸̈ˊ˿̐ʊĘŻBʰ3’5˸̈ˊ˿̐˅Ɖʐʪǽ6ʫ ƣʝʤĮʫfusion PCR˅ǹʐʊƘƕʰ˚ˊ˟˅˗̊úJÜʊKO˺˔ˣ̐ʬʝʤʋ

10. ˸̌˪˸̈˞˪ʰcɡ

˸̌˪˸̈˞˪ʰcɡʱʊYeltonʿʰĚŘ˅đʝʤAkamatsuʿʰĚŘʯÝʐǹʧ ʤ (Akamatsu et al. 1997, Tsuge et al. 1990, Yelton et al. 1984)ʋPDAʫɷʝʤ sǬĸ˅1 mmȋʯLˀʊǁ20ĘŻ˅50 mlʰPDBʯļǬʝʊ25ʇʫ36 hʊāʬʑ

ɷ (ˣˊ˨˦˔, Bio-Shaker BR-300L, 100 strokes/min) ʝʤʋÞʿ˂ʤǬƿ˅ůǬ œʊOM bufferʰɭʯŜşʝʤʋ5 mlʰɌǄŮŤ [10 mg/ml Lysing enzymes (Sigma), 5 mg/ml Kitalase (~<ǂǲÄľ) in OM buffer] ʯœK˅Jįˁʥʘɛʐ ʤǬ&˅ðŲʝʊ30ʇʫāʬʑɷ (ˣˊ˨˦˔, INCUBATOR Personal-10, 80 strokes/min, 4-6 h) ʝʤʋ˸̌˪˸̈˞˪˅ɌǄŤʛʬ˞˵˦˧Ƹʯƫʝʤʰʦʊ1 mlʰ ST buffer˅ʢʰʯƯʽʔʯɏ¿ʝʊɅãKɡ ̒2,000 rpm, 5 min̓ ʝʤʋOM bufferʬST bufferʰ ɖ¿ (ǬƿŻ˅xʺ˸̌˪˸̈˞˪) ˅Pʰęʝʐ˞˵˦˧Ƹ ʯƫʝʤʋʚʰ˞˵˦˧Ƹʰ8WƬʸʫSTC buffer˅Zʒ˵˻˦˨ˉ̎˕ʝʊɅãKɡ (700 rpm, 5 min) ʫĩŮȌʰǬƿŻ˅ŗŎʜʡʤʋŧ˅ęʝʐ˞˵˦˧Ƹʯ˵˻˦

˪ʫƫʝʊɅãKɡ (2,000 rpm, 5 min) Üʊŧ˅ɛiʝʤʋŗŎʝʤ˸̌˪˸̈˞˪

ʯ5 mlƬÐʰSTC buffer˅Zʒʊ2ÐŜş (Ʌã2,000 rpm, 5 min) ʝʤʋÞʿ˂ʤ

˸̌˪˸̈˞˪ʰŗŎ˅1 mlʰSTC bufferʯEðŲʝʊ˪̐˿őǸƄȎ˅ƉʐʪųÐ

(20)

19 ǽ6ɿ īƠưʫƉʐʤPCRʰĮ

kæ ŪÐ (^ʇ) ğɖ

Fusion PCR èkæ 94 2K

èkæ 94 15Ʃ 30˚ˊ˔̊

ˈ˭̐̉̎˕ 60 30Ʃ

"ɔkæ 72 5K

"ɔkæ 72 10K

Long PCR èkæ 94 2K

èkæ 98 10Ʃ 30˚ˊ˔̊

ˈ˭̐̉̎˕ʓʾʳ

"ɔkæ

68 1K/kb

ɿ "ɔkæ 72 10K ɿ

(21)

20

˅ū«ʝʤʋ

11. ˾˾̉ˍ˥̋̎˕̉˘̐̊ (PEG) ŘʯʾˁÙȩȯĉ

ÙȩȯĉʱʊVollmerʬYanofsky (Vollmer et al. 1986) ʰĚŘ˅đʝʤItohʿ (Itoh et al. 1994)ʊTsugeʿ (Tsuge et al. 1990) ʓʾʳȪĨ (ȪĨ 2010)ʰĚŘʯÝ ʐǹʧʤʋ

ɿ ȶʝʤ˸̌˪˸̈˞˪ʰcɡʯʾˀȞȂʝʤ˸̈˪˸̈˞˪ðŲŤ80 μl (1.25ʀ10⁸ 1/ml) ʯʊ20 μlʰ40% PEG˅xʺSTC bufferʬʊ1-10 μlʰƆž˸̈˞̀˫DNA (100 ng/μl) ʏˁʐʱ/ʜʿʯǒžDNA (10-50 ng/μl) ˅ZʒʊŔʫ30KɖɧǕʝ ʤʋ900 μlʰ40% PEG˅xʺSTC buffer˅ZʒŪʫ10KɖɧǕʝʤʋɧǕÜʊ

?kæŤ˅50ʇʯ.ʧʤ5 mlʰ0.8̑EƇʯZʒ`KʯĐüʝʤʋ10 mlʰ 1.5%EƇʯǾʡʊ_ʜʡʤʋǁ12ğɖÜʊ50ʇʯ.ʧʤ5 mlʰ1.0%²

ʯĥǇƕʯ?&ʕ100 ppmʯʮˁʾʑʊɆøƉʰ÷Ƈżȩ˅ZʒʤʋʢʰÜʊ ʢ˂ʣ˂ʰ˸̋̐˪ʯǾʡʊ_ʜʡʊ26ʇʫɷʝʤʋ1 ȽɖÜʊJƃʝʤ˘̌˭

̐˅100 ppmʰ÷ƇżȩxĦɆø (PDA̔÷Ƈżȩ) ʯļʒǍʐʥʋ

12. ˸̌̐˷ʰ)Ȃ

ɿ DIG DNA Labeling Kit (̌˜̗̅ˤˊˈ˕˯˞˨ˉ˦˔˞) ˅ƉʐʪʊDIGŃȢdUTP

˅ƉʐʤPCR˅ǹʑʚʬʫʊ˸̌̐˷˅)ȂʝʤʋóɭʱŦʰ˿˭̅ˈ̊ʯÝʐǹ ʧʤʋDIGʫ̈˺̊ʝʤPCRkæŤ˅ʊ0.5ʀTBEʫȞȂʝʤ2.0̑ˈˑ̌̐˞˗̊

ʫ̈˺̊ʜ˂ʪʐʮʐPCRkæŤʬʬʼʯɣŒś]ʝʊDIGʯʾˀ̈˺̊ʜ˂ʪʐ ˁʚʬ˅ƢȘʝʤʋʚʰDNA˅˗̊úJʝʊ˸̌̐˷ʬʝʪƉʐʤʋ

13. ˚˛̎˷̌˦˨ˉ̎˕

ɿ ˚˛̎˷̌˦˨ˉ̎˕ʱȪĨ (ȪĨ 2010) ʰĚŘʯÝʐǹʧʤʋ˗˯̀˦˔DNAʰ ˬˊ̌̎ǡ (Hybond N⁺, GE˹̊˞˖ˈ) ʴʰȯFʱʊˈ̊ː̉Řʯʾˀǹʧʤʋʸʠʊ

˗̊˅UV chamberʫ˭˦˒̎˕ʝʊTransfer bufferʫ30Kʊ2ÐÌǼ_ʟˁʚʬʯʾ ʧʪ˗̊ ʰDNA˅īɒʯʝʤʋǎʐʪubuffer˅Ɖʐʊcapillary transfer (ķļ ʿ 1993) ʯʾʧʪ˗̊ ʰDNA˅ˬˊ̌̎ǡʯ24ğɖȯFʝʤʋȯFÜʊˬˊ

̌̎ǡ˅Neutralizing buffer ʫ30Kʊ2ÐƯʽʔʯāʬʑ (35 strokes/min) ʝʪ

~ʝʤʋŇʯʊŪʫ30KźÜʊUV chamberʫDNA˅ˬˊ̌̎ǡʯ«ʝʤʋ DNA˅ȯFʝʤˬˊ̌̎ǡ˅˰ˊ˷̉ˤˊˡ̐˜̇̎Ťʫ65ʇʊǁ2-5ğɖ˸̋˰ˊ

˷̉ˤˊˡ̐˜̇̎ʝʤÜʊ˸̌̐˷˅xʺ 15 ml ʰ˰ˊ˷̉ˤˊˡ̐˜̇̎Ťʬ>˂

Ĥʒʊ65ʇʫǁ15ğɖ˰ˊ˷̉ˤˊˡ̐˜̇̎˅ǹʧʤʋʢʰÜʊˬˊ̌̎ǡ˅65ʇ

(22)

21

ʊ0.1% SDS˅xʺ0.1ʀSSC bufferʫ30Kʊ2ÐŜşʝʤʋˬˊ̌̎ǡ˅buffer 1 ʫ5KɖÌǼ_ʝʊʢʰÜbuffer 2 ʫʊ45Kāʬʑʝʤʋ

ɿ ÷& [Anti-digoxigenin-AP fragment (Roche)] ˅buffer 1ʫ12ʯÇɎʝʊ20 ml ʯˬˊ̌̎ǡ˅>˂ 25 KɖāʬʑÜʊ0.3% Tween20 (Polyoxyethylene Sorbitan Monolaurate) ˅xʺbuffer 1ʫ20Kʊ2ÐŜşʝʤʋʢʰÜʊˬˊ̌̎ǡ˅buffer 3 ʫ5KɖÌǼ_ʝʤÜʊ10 μlʰCSPD (Roche) ˅xʺ1 mlʰbuffer 3˅˸̈˞˥˦

˔˱˦˔ʯƫʝʤˬˊ̌̎ǡʯZʒʊ37ʇʊĢɾ_ʫ 10KɖɧǕʝʤʋĥÜʯʊˬˊ

̌̎ǡ˅Xǒ˶ˉ̊́ (FUJI MEDICAL X-RAY FILM, Fuji Film) ʬʬʼʯɧǕʊʏ ˁʐʱ̊̀˯ˊ̂̐˝ˈˬ̈ˊ˛̐ (±˶ˉ̊́ʊLAS4000EPUVmini-TU) ʫ˜˕

ˬ̊˅ĽJʝʤʋĽJÜʊˬˊ̌̎ǡ˅buffer 4ʫ20Kʊ2ÐŜşʝʤʋEÐ˰ˊ˷

̉ˤˊˡ̐˜̇̎˅ǹʑt˅ǙʒʊProbe stripping buffer ʊ37ʇʫ30Kɖāʬʑʟ ˁʚʬʫ˸̌̐˷˅ɛiʝʤʋǎʐʪˬˊ̌̎ǡ˅2ʀSSC ʫ 20Kʊ2 ÐŜşʝʊ

Ūʫźʜʡ.§ʝʤʋ

14. ƑƑhèĽ«

ɿ ƑhèĽ«ʯƉʐˁǜ¥ðŲŤʰȞĔʓʾʳǮʴʰąƭʱȪĨ (ȪĨ 2010) ʰĚŘʯÝʐǹʧʤʋ

14-1. ǜ¥ðŲŤʰȞȂ

PDAʯǬ˅ļǬʝʊ2-3ĜÜV8²ʯļǬʝʤʋˈ˦ˠˊʯ*Ɖʟˁʤʻ ʯ1ǬĸØʤˀ5-10 ĲļǬʝʤʋ˜̄̐̋ʯ8WƬÐʊǬƿʕȆʧʤğʯʊBLB̈ ˊ˪˅ĠʊŸ¶ʝʤʋʢʰÜʊēĜɖɧǕʝʊǽɩʯǜ¥ʕȇʒ£ʻʤɬʯ ǜ¥˅lʝʤʋʯ0.1% tween20˅xʺůǬœ˅5 mlƬÐʔʘʊ˙́˺̈ʫ ǽɩ˅ďˀʊǜ¥˅ȿɡʜʡʪ˴̐ː̐ʯɠʻʤʋ˴̐ː̐DʫʾʗčüʝʤÜʊ˒

́̍ˊ˸ʫŴɁʟˁʚʬʫ(KʮǬƿ˅ɛiʝʤʋēɅãʯʾˀŜşʝʤÜʊǜ¥

˅˪̐˿őǸƄȎʫȎūʝʊ0.1% tween20˅xʺůǬœʫ10⁶1/mlʯȞĔʝʤʋ

14-2. ąƭ¬ɹ

ǜ¥ąƭʱÙȩȯĉ&ʮʭ˅õʑʤʻʊ50 ml˥̅̐˷˅*ʐʊǹʧʤʋ˚̎˸̊

ļżʱLmˀǮ˅*ƉʝʊǨʗœʫŜşʝʤÜʊ˒́ˣˏ̊ʫœK˅þʖmʧʤʋʚ ʰLmˀǮ˅ʊȑ1ʫÞʿ˂ʤǜ¥ðŲŤ˅5-10 ml>˂ʤ50 ml˥̅̐˷ʰ ʯŢʝʤʋǜ¥ðŲŤʕǮʰǽɩʯǹʖũˁʾʑʯǓʽʔʯ˥̅̐˷˅ȯʜʡʤʋ ʢʰÜʊǮȀ˅ǽʯʝʪŪʓʾʳŬÐ 100%ʰ˥̄̎˱̐Dʫ 3-5 ĜɧǕÜʊǮ ʰžî˅˩˝ˣ̊ː̂̈ʫČÚʝʤʋ

(23)

22 15. AALŏŏǄĽ«

ɿ AALŏǄʰúJʊŏǄʰˈ˦ˠˊʓʾʳHPLCȌİʱȪĨ (ȪĨ 2010) ʰĚŘ ʯÝʐǹʧʤʋ

15-1. AALŏǄʰúJ

ɿ PDAʫƇǛʜʡʤǬ&˅̂˞ʫ5 mmȋʯǆĘʝʊ20ʰĘŻ˅50 mlʰŘ

̉˥̄̐˟ʯąƭʝʊ1-2ȽɖɧǕɷʝʤʋɷ˃Ť˅10-20 ml˘̐˭̎˕˥

̅̐˷ʯƫʝʊʉ80ʇʫHǊʜʡʤʋHǊʝʤ˥̅̐˷˅1-2ĜʶʭHǊźŅʯʔ ʘʤʋHǊźÜʊ˥̅̐˷ʯ1 mlʰ50%ˈˠ˪˭˪̉̊˅Zʒʪʊ1ğɖāʬʑʝʤ (ˣˊ˨˦˔, INCUBATOR Personal-10, 80 strokes/min)ʋāʬʑÜʊ13,000 rpmʊ1K Ʌãʝʊŧ˅ Sep-Pac C18 ː̈́ (Waters) ʯȹʝʪǂ_ʝʤʋː̈́ʱĥNʯʊ 100%ˈˠ˪˭˪̉̊˅ȹʝʊʢʰÜ milliQ ˅ȹʝʪː̈́D˅ŜşʝʤʋŇʯ˚̎˸

̊ (ŧ)ʊmilliQ (2 ml)ʊ15%ˈˠ˪˭˪̉̊ (2 ml) ˅ɭʯ>˂ʤʋĥÜʯ70%ˈˠ

˪˭˪̉̊ (2 ml) ʫúJʝʤʋúJʝʤ AALŏǄʱ-4ʇʫ.§ʝʤʋʚ˂˅Ǯʴʰ ˈ˦ˠˊʓʾʳHPLCʯ*Ɖʝʤʋ

15-2. Ǯʴʰˈ˦ˠˊ

ɿ ˜̄̐̋Dʯ5 mmȋƬÐʰ˃ǃ (Whatman, 3MM CHR) ˅Ǖʖʊȑ15-1ʫÞ ʤúJż˅25 μlŰʝʊʝʹȴʸʡʤʋ˔̉̐̎˺̎˥Dʫɴʜʡʊˈˠ˪˭˪̉̊

˅ǰƒʜʡʤʋĽ«ƉʰǮʱLmˀǮ˅*ƉʝʊǮʰȀʯ˒́̍ˊ˸˅Ȱʗďˀʨʘ ˁʚʬʫ7˅ʘʤʋ7ɊKʯúJż˅ʝʹȴʸʡʪʓʐʤ˃ǃ˅ʰʡʊ25 µl ʰœ

˅ŰʝʊŪʊŬÐ100%ʰ˥̄̎˱̐DʫɧǕʝʤʋ3-5 ĜÜʰǮʰžî˅˩˝

ˣ̊ː̂̈ʫČÚʝʤʋ

15-3. HPLC

ɿ o-phthalaldehyde (OPA) ʯʾˀǶ<Ț·&_ʝʤɷ˃Ť ʰAALŏǄʰHPLC ʯʾˁĽJʱʊ=ƂʿʰĚŘ (Kodama et al. 1995) ˅ɊđʝǹʧʤʋOPAȔǲ ʱʊ1 mlʰ100%̂ˣ˯̐̊ʯŮʔʝʤ40 mgʰOPAʬ50 μlʰ2-mercaptoethanol ʬ5 mlʰ0.1 M˼ˋɍˬ˪̉ˋ́ǓǻŤ (pH 9.5) ˅ťtʝʪ)ñʝʤʋȔėŮŤ25 μlʯOPAȔǲ125 μl˅ťtʝʤÜʊ30Ʃɖ˽̊˨˦˔˞ʝʊ2K30ƩɖʊŪʫ ɧǕʝʤÜʊƙʦʯHPLCː̈́ʯŚ>ʝʤʋHPLCKİʱʊ655 LC˘̎˪̌̐̈̐ʊ F-1050 Ƕ<ĽJʊD-2500 ˔̌˿˪ˊ̎˨˕̋̐ˣ̐ (ĜƲ) ʯʾʧʪǹʧʤʋː̈

́ʱʊDevelosil ODS-UG-5 (4.6ʀ250 mmʊɐĭ_¨) ʓʾʳDevelosi l ODS-UG-5 ˑ̐˫ː̐˪̉˦˝ː̈́ (4.0ʀ10mmʊɐĭ_¨) ˅Ɖʐʊ̂ˣ˯̐̊/0.1 M ̉̎ɍ œǄˬ˪̉ˋ́ǓǻŤ (pH 3.35)ʊ75 : 25 (v/v) ʰŮ¤ǀʫʊ0.7 ml/minʰŞȺʫ

(24)

23

ŮJʝʤʋʮʓʊĽJʱ[ȫřɔ335 nmʓʾʳĽJřɔ440 nmʫǹʧʤʋ

16. KOĸĸʴʰƚȁ¬ɹʊɆøʓʾʳŅǝȌİ

ɿ KO ĸʴʰɇ ¥ʰƚȁʱʊʰóɭʫǹʧʤʋʸʠʊƚȁʟˁɇ ¥ʰɕ£

˘˫̎ʰ1 kb ƬÐŞʊǇň˘˫̎ʰ 0.5 kbƬÐŞʯ˸̈ˊ˿̐˅ȒȎʝʊlong PCRʯʾˀʊǒžʰ PCRƈż˅ÊʝʤʋʚʰPCRƈżʬ˝ˌˮ˨ˉ˜̎ǚèɇ

¥ (nptII) ˅xʺ˸̈˞̀˫ʊpII99˅˘˪̈̎˞˶ˎ̐̂̐˜̇̎ʟˁʚʬʫÙȩȯĉʝ ʤʋÙȩȯĉʱǒžʰPCRƈż˅50 ngƬÐZʒˁʚʬʬʊ˸̈˞̀˫DNA˅100 ng uğʯZʒˁʱĬėʓʾʳĚŘʰ 11ʊPEG ŘʯʾˁÙȩȯĉʯÝʐǹʧʤʋ ʸʤʊƚȁĸʰɆøʊƑhèʊŏǄĽ«ʓʾʳƒƃȌİʱȑĚŘʯʾˀǹʧʤʋ

17: ˲̊˞˶ˉ̐̊˫˗̊ɣŒś] (PFGE) 17-1Ķǩ& DNAʰȞȂ

ɿ Ķǩ&DNAʰȞȂʱʊBrodyʬCarbonʰĚŘ (Brody et al. 1989) ˅đʝʤȪ Ĩ (ȪĨ 2010)ʰĚŘʫǹʧʤʋȑ 2-10ʫcɡʝʤ˸̌˪˸̈˞˪ðŲŤ˅GMP bufferʫEðŲʝʊ2.0×10^̖1/mlʯʮˁʾʑʯȞĔʝʤʋʚʰðŲŤʯ55ʇʫ.Ūʝ ʪʓʐʤ1.4%%Ƿŵˈˑ̌̐˞ (Agarose low melt preparative grade (BIO-RAD) in GMP buffer) ˅ƶɑZʒʊʾʗĐüʝʤʋʚ˂˅˸̈˕)ȂƉmold (BIO-RAD) ʯ KŚʝʊ5 Kɖ4ʇʫɧǕʝʊ_ʝʤʰ˅ƢȘʝʪ˸̈˕˅ʔʿmˀJʝʤʋ)Ȃ ʝʤ˸̈˕ʱSE buffer ʫ55ʇʊ24ğɖIƅʝʤʋˣ̎˲˔ȩɛiʰʤʻʊ1%

N-Lauroyl sarcosine sodium-salt (SLS) ʓʾʳ2 mg Proteinase K (Merck) ˅xʺ1 mlʰ10×ET buffer ʯ˸̈˕˅ƫʝʊ50ʇʫ20ğɖIƅʝʤʋ˸̈˕ʱ1×ET bufferʫ2ÐŜşʝʤÜʊubuffer ʯ4ʇʫ.§ʝʤʋ

17-2˲̊˞˶ˉ̐̊˫˗̊ɣŒś]Ř (PFGE)

ɿ Ķǩ&ʰɣŒś]ʱȪĨ (ȪĨ 2010) ʰĚŘʯÝʐǹʧʤʋ˗̊ʱ Pulsed Field Certified Agaroseʸʤʱ%Ƿŵˈˑ̌̐˞ (BIO-RAD) ˅ʊbufferʱ0.5×TBE buffer ˅Ɖʐʤʋʸʤś]ǿǕʱ Contour-clamped Homogenous Electric Field Dynamically Regulated IIʏˁʐʱIII (CHEF-DRIII) system (BIO-RAD) ˅*Ɖʝʤʋ ǹʧʤś]Įʱǽ7ʯƣʝʤʋś]ǇÜʊˈˑ̌̐˞˗̊˅0.5 μg/mlǣ_

ˍ˥˝ˋ́ (EtBr) ʫĶǩʊǰƎœʫǞǩÜʊUV˪̈̎˞ˊ̊̀ˮ̐ˣ̐ʫȊ³ʝ ʤ ʋ ʸ ʤ ˚ ˊ ˟ ˿ ̐ ː ̐ ʬ ʝ ʪ ʊSaccharomyces cerevisiaeʊShizosaccharomyces pombeʓʾʳHansenula wingei (BIO-RAD) ˅Ɖʐʤʋ

(25)

24 ǽ 7 CHEFʰĮ

LĤğɖ ɣ ğɖ ˚ˊ˟ ŪÐ ˗̊ųÐ

A 1 0.5 s 6 V/cm 0.5 h 50-150 kb 10 1.0%

2 8 s 0.5 h

3 1 s 3 h

4 2 s 3 h

5 4 s 6 h

6 8 s 6 h

B 1 5-25 s 5 V/cm 22 h 100-400 kb 8 1.0%

2 1-40 s 18 h

C 1 3600-1800 s 1.5 V/cm 115 h 0.5-5.0 Mb 8 0.8%

2 1800-1300 s 1.5 V/cm 24 h 3 1300-800 s 1.8 V/cm 28 h 4 800-600 s 2.4 V/cm 28 h

D 1 120 s 5.5 V/cm 12 h 0.5-2.0 Mb 8 0.8%

2 180 s 12 h

E 1 60 s 6 V/cm 15 h 1.0-2.8 Mb 14 0.8%

2 90 s 6-8 h

F 1 1500-3000 s 1.5 V/cm 48 h 1-5 Mb> 10 0.8%

2 900-1500 s 1.8 V/cm 40 h 3 480-900 s 2.4 V/cm 57 h

G 1 1-6 s 6 V/cm 11 h 5-75 kb 14 1.0%

H 1 120-180 s 6 V/cm 24 h 0.5-1.2 Mb 14

I 1 120 s 4 V/cm 13 h 0.5-3 Mb 14 0.8%

2 240 s 13 h

3 360 s 13 h

J 1 50-90 s 6 V/cm 12 h 0.5-1.0 Mb 14 0.8%

?ʪ0.5xTBE ˱˦˶ˇ̐˅Ɖʐʪǹʧʤʋ

(26)

25 18: ɇɇ ¥˼̃̌˕ʰ˞˔̉̐˭̎˕

ɿ ǫĵƑǬ As-27 ʓʾʳ AL12 ʰ˫̈˶˪˗˯́˜̐˔ˍ̎˞Ȍİ˅ǹʐʊʢ˂ʣ˂

5847 ʓ ʾ ʳ 2016 ʰ ˘ ̎ ˨ ˉ ˕ ʕ Þ ʿ ˂ ʤ ʋ ʚ ʰ ˩ ̐ ˣ ˅ ʯ BioEdit (http://www.mbio.ncsu.edu/bioedit/bioedit.html) ˅*ʐʊĽǅʝʤʐɇ ¥ɋM˅˔

ˍ̉̐ʯʝʊLocal BLASTĽǅ˅ǹʧʤʋ˔ˍ̉̐ʯʱA. nidulansʰɇ ¥˅Ɖʐʤʋ ˈ̀˯ɍʰūʯʱSoftberry FGENESH (http://linux1.softberry.com/berry.phtml) ˅Ɖʐʤʋ

(27)

26 Ƶ

Ƶ3Ƴɿ ǫĵƑǬʯʓʘˁƑhèɗȼɇ ¥ʰĄǅ Ƶ1ƹɿ Ïȟ

ɿ ˪˿˪Ƒhʫʏˁ˪˿˪ˈ̊ˣ̐ˬ̉ˈǫĵƑǬ (A. alternata tomato pathotypeʊ ʊǫĵƑǬ) ʱ°ŽƏƕAALŏǄ˅Ƈƈʝʊínè˪˿˪ƭʯʰʹǯʝʐ

Ŋ˅ÔʖȫʚʟʋAAL ŏǄʱʊɅǔʰ Fusarium ƭʕƇƈʟˁ˿ˊ˘˪˒˜̎ʊ˶

̃˭˜̎ʰŀȻɳǔ&ʫʏˀʊʢ˂ʣ˂ʰŏǄƇtñʯäɮʫʏˁ˾̉˖˥˫tñ ɌǄ (PKS) ɇ ¥ (ǫĵƑǬʯʓʐʪʱ ALT1ʊFusarium ƭʯʓʐʪʱ FUM1) ʕ ʊ ˩ ˉ ˝ ˌ ˮ ̋ ̐ ˪ ˸ ̈ ˊ ˿ ̐ ˅ * ʧ ʤ ˔ ̌ ̐ ˭ ̎ ˕ ʯ ʾ ˀ c ɡ ʜ ˂ ʪ ʐ ˁ (Akamatsu et al. 2003, Proctor et al. 1999) (1)ʋʚ˂ʿɇ ¥ʱˈ̀˯ɍ̋˺̊ʫɺ ʐƚuèʕʏˀʊʸʤ{ȳʰ˜̐˔ˍ̎˞Ȍİʯʾˀʊʐʯƚuèʰɺʐɇ ¥˔

̈˞ˣ̐ʕȇJʜ˂ʤʋǫĵƑǬʫʱʊCDC ʰ?ɋMȌİʯʾˀʊ13 ɇ ¥ʔʿʮ ˁAALŏǄƇtñɇ ¥˔̈˞ˣ̐ (ALT˔̈˞ˣ̐) ʕʊCDCʯ2ˠ˦˪Ñʝ ʪʐˁʚʬʕƣʜ˂ʪʐˁʋʸʤʊØƠưʫʱʊCDC ʰ?ɋMʰȌİʬʱPʯʊ Ň˜̐˔ˍ̎˚̐ʊ̌˜̅454 FLX ʓʾʳˊ̊̀ˬHiSeq˅*ƉʝʤǫĵƑǬ

˫̈˶˪˗˯́Ȍİ˅ǹʐʊʶʷ?˗˯́ʰ˜̐˔ˍ̎˞é˅Þʪʐˁʋ

ɿ īƠưʫʱʊǫĵƑǬʰ˗˯́é˅QƉʝʤƑhèɗȼèɇ ¥ʰĄǅʓʾ ʳŅǝȌİ˅ȔʹʤʋǫĵƑǬʱʊȶʰʾʑʯ?˗˯́ʰ˫̈˶˪˗˯́éʬʬʼ ʯʊAALŏǄƇtñʯɗ˄ˁALT˔̈˞ˣ̐ʕÑʟˁCDCʰª?ʮɋMéʕ QƉJįˁʋʸʤʊʚ˂ʸʫʯǱƮʜ˂ʤȼʰɇ ¥KO¬ɹʰ˸̌˪˘̊ʯʾˀʊ

¯ĞʯƘƕɇ ¥ʰŅǝȌİʕqǝʫʏˁʋ

ɿ 13ɇ ¥ʬĆ«ʜ˂ˁALTɇ ¥ʱʊʚ˂ʸʫʯALT1-4ʊ7-9ʓʾʳ13ʰ8ɇ

¥ʯʓʐʪŅǝȌİʕǹ˄˂ʪʓˀʊʚ˂ʿʰʑʦALT1-4ʓʾʳALT13ʱAALŏ ǄƇtñʯɗʝʪʐˁʚʬʕƣʜ˂ʪʐˁ (ȪĨ 2010, ı 2010, ɂ 2010, Š 2011)(ǽ1)ʋĚʊABC transporterʊshort chain dehydrogenase/reductaseʊ fatty acyl-CoAʊphytanoyl-CoA synthetaseʓʾʳpeptide synthetase˅ʢ˂ʣ˂˘̐

˫ʟˁʬĆ«ʜ˂ˁALT5ʊ6ʓʾʳ10-12ʰ5ɇ ¥ʱŅǝĩƝʫʏˁʋʚ˂ʿALT ɇ ¥ʰŅǝȌİ˅ǹʑʚʬʯʾˀ AAL ŏǄƇtñǉȮʕȖǆʯȜĝJįˁʬǙ ʒʿ˂ˁʋ

ɿ ǫĵƑǬʯəʿʠʊƑhèA. alternataʰʗʱʊʢ˂ʣ˂ʰHSTƇtñɇ ¥ ʓʾʳɇ ¥˔̈˞ˣ̐˅CDCʯ.ÿʝʪʐˁʚʬʕĝʿʔʬʮʧʪʐˁʕʊCDC ʰɇ ¥ʰʹʕƑhè˅ûʧʪʐˁʰʔʊCDCʰessential chromosome (EC) ʯʼƑhèɇ ¥˅.ÿʝʪʐˁʰʔʱKʔʧʪʐʮʐʋʰļżƑhǬʯʓ

(28)

27

ʐʪʢʰƑhèʯɏȅʫʏˁʚʬʕƣʜ˂ʪʐˁ̂̈˭̎Ƈtñʯɗ˄ˁɇ ¥

1ɿ ˶̃˭˜̎ʬAALŏǄʰŀȻ

(29)

28

(Kubo et al. 1982) ʽGˣ̎˲˔ȩα˚˷̆˭˦˪ (Wang et al. 2010) ʰŅǝȌİʕ

A. alternataʯʓʐʪǹ˄˂ʤʕʊʭ˂ʼƑhèʯäɮʫʱʮʐʚʬʕƣʜ˂ʪʐ

ˁ (Takano et al. 1995, Kheder et al. 2012, Yamagishi et al. 2006, Tsuge et al. 1990)ʋ ɿ ˗˯́éʕŝƉʫʖʮʔʧʤɬʱʊʏˁǬʫƑhèɇ ¥ʕȇJʜ˂ʤʬʝʪʼʊ ʢʰɋMʕƭ˅ȬʒʪɺÐʯ.§ʜ˂ʪʐˁʾʑʮtʱʊ˔̌̐˭̎˕ʱ

ɢʫʏʧʤʋØƠưʫʼʊGˣ̎˲˔ȩAÛn¯&(GPCR) ʰ˔̌̐˭̎˕˅Ȕ ʹʤʕʊ˔̌̐˭̎˕ʱJįʮʔʧʤʋĚʊ6¬ɹʯʾˀʊǫĵƑǬʰ?˗˯́

˫̈˶˪˩̐ˣʔʿʊGPCR ʰ˼̃̌˕˅Ľǅʝʤʬʚ˃ʊēɇ ¥ʕ3ȁɇ ¥ʬʝ ʪɆøʜ˂ʤʋʚʰʾʑʯʊTʱ˔̌̐˭̎˕ʕɨÉʯɢʝʔʧʤɇ ¥ʕ˗˯́

é˅ʯĽǅqǝʫʏˁʚʬʔʿʊěƝʰƑhèɗȼɇ ¥ʯʩʖʊǫĵƑǬ ʯʓʘˁƑhèɗȼɇ ¥ʰĄǅ˅Ȕʹʤʋ

(30)

29 Ƶ

Ƶ2ƹɿ ĬėʬĚŘ

1: +ȔǬ

ɿ +ȔǬʯʱǫĵƑǬAs-27Ǭĸʊ̉̎˙ĖŵǭǮƑǬIFO8984Ǭĸʓʾʳˊ˥˙

ɾĖƑhNAF8Ǭĸ˅˅*ƉʝʤʋǬĸʰɷʓʾʳ.§ʱ2-1ʯÝʧʤʋ

2: DNAĎ)

ɿ DNAʽRNAʰúJʊƼcʮĎ)ʱƵ2ƳʯÝʐǹʧʤʋ

3: KOƉ˘̎˞˪̈˔˪ʰ)ñʓʾʳ KOĸʰ)J

ɿ ˘̎˞˪̈˔˪ʰ)ȂʓʾʳKOĸʰ)Jʱ2-9, 10, 11ʯÝʐǹʧʤʋKOĸʰƢ ȘʱPCRʯʾˀʊhphʰDɊɯʊsɇ ¥ʰDɊɯʓʾʳëʰ$Ǖʫǈĉ ʒʕȫʖʤʚʬ˅ƢȘʟˁHomoʰɯ˅Êʝʤʋ

4: ˚˛̎˷̌˦˨ˉ̎˕

ɿ 2-14ʯÝʐʊsɇ ¥ʰKO3ȁĸʰ˚˛̎˷̌˦˨ˉ̎˕˅ǹʧʤʋ

5: ƒƃȌİ

ɿ ʚ˂ʿsɇ ¥KOĸʰ¬ɞʰƒƃ˅Ȟʵʤʋ2-4ʯÝʐRNA˅úJÜʊ2-5ʯ ÝʐʊcDNA ʰtñ˅ǹʧʤʋʚʰ cDNA ˅˨̎˸̋̐˪ʯɇ ¥ʰDɊɯ˅

Êʟˁ˸̈ˊ˿̐ʫPCR˅ǹʐʊʢʰ˱̎˫ʰĦŶʫƒƃ˅Ȟʵʤʋ

6: ƑhèʓʾʳŏǄƇƈǝĽ«

ɿ ƑhèĽ«ʓʾʳŏǄƇƈǝĽ«ʱ2-14, 15ʯÝʐǹʧʤʋʸʤʊHPLCȌİʼ uŁʯ2-15-3ʯÝʐǹʧʤʋƑĖɩƮʰū«ʱ˶̉̐ˢ˶˪ʰImage J˅Ɖʐʤʋ

7: KOĸʰƚȁ¬ɹ

ɿ ƚȁ¬ɹʱʊɐèĸʬŐȱʝĝʿʔʮ_ʕʏˁ KO ĸʯʰʹǹʧʤʋƚȁǬĸ ʰ)JʊɆøʓʾʳŅǝȌİʱ2-16ʯÝʐǹʧʤʋ

8: Ǭƿñɔʓʾʳǜ¥˚ˊ˟ʰŐȱ

ɿ ˘̌˭̐ŐȱʯʱPDA, V8, crossingʓʾʳcrossingʯʢ˂ʣ˂+5%ʰ sucrose, glucoseʓʾʳmannitol˅ŦZʝʤʰ˸̋̐˪˅*ƉʝʤʋŤ&ʫ ʰǬƿñɔŐȱʯʱPDBʓʾʳŘ̉˥̄̐˟˅Ɖʐʤʋǁ2ȽɖʊɧǕʸʤ

(31)

30 ʱɤʬʑɷʝʊɐèĸʬƏĸ˅Őȱʝʤʋ

ɿ ǜ¥˚ˊ˟ʱƑhèĽ«ʯƉʐʤǜ¥˅Ȋ³ʯƉʐʤʋɲáɓʓʾʳː̂̈ʱ Nikon, Eclipse TE300 Microscope˅Ɖʐʤʋ

9: Real-time PCR

ɿ AaLaeAɇ ¥ʰKOĸʯʓʘˁCDĶǩ&ʯÑʟˁɇ ¥ʰƒƃȌİʱ2-5 ʯÝʐǹʧʤʋŽʯRNAʰųÐ˅ɐèĸʬKOĸʫćʒˁʚʬʯƎëʝʤʋ

10: ɇɇ ¥ʰÑĶǩ&ʰŽ«

ɿ ˸̌˪˸̈˞˪ʱÙȩȯĉʬuŁʯ2-10ʯÝʐKɡʝʤʋ2-17ʯÝʐʊɣŒś]˅

ǹʧʤÜʊ2-13ʯÝʐʊ˷̌˦˨ˉ̎˕˅ǹʧʤʋ

(32)

31 Ƶ

Ƶ3ƹɿ Ǌĳ

ɿ 1. CD&7@)6;$

1-1. ALT@<=" KO'?!

ɿ ALT ˔̈˞ˣ̐ʯ§ʟˁ13ʰ ALTɇ ¥ʰʑʦʊʚ˂ʸʫʯŅǝʕzʜ˂ʪ

ʐʮʐ ALT5ʊ6ʊ10ʊ11 ʓʾʳ 12 ʰŅǝȌİ˅ǹʧʤʋʚ˂ʿɇ ¥ʱʢ˂ʣ˂

ABC transporterʊshort chain dehydrogenase/reductaseʊfatty acyl-CoA synthetaseʊ phytanoyl-CoA synthetaseʓʾʳpeptide synthetase˅˘̐˫ʝʪʐˁʬêʜ˂ʪʐ ˁ (ǽ1)ʋʸʤʊ˶̃˭˜̎Ƈtñʯɗ˄ˁFUMɇ ¥ʊFUM19ʊ13ʊ10ʊ3ʓʾʳ 14 ʬʢ˂ʣ˂ɺʐƚuèʕȘʻʿ˂ʪʐˁʋʜʿʯʊʚ˂ʿɇ ¥ʰʑʦʊALT5ʊ6 ʓʾʳ10ʱīǬʰ˗˯́ ʯ2˘˵̐§ʝʊALT11ʓʾʳ12ʱ4˘˵̐§ʟ ˁʚʬʕCDCʰŀȻȌİʔʿȇJʜ˂ʪʐˁʋ

ɿ ʚ˂ʿɇ ¥ʰKO¬ɹʱʊĬėʬĚŘʫȶʵʤʾʑʯPEGŘ˅*ʧʤÙȩȯĉ ʯʾˀǹʧʤʋÙȩȯĉ&ʔʭʑʔʰƢȘʱʊ·>ʝʤ÷Ƈżȩǚèɇ ¥ʯ´æ ʟˁ÷Ƈżȩʕ100 ppmŦZʜ˂ʤPDAʫ3ÐɆøʟˁʚʬʫǹʧʤʋɆøʝ ʤÙȩȯĉ&ʱʊĬėʬĚŘʫȶʵʤʾʑʯ PCR ʬ˚˛̎Ȍİ˅ǈʹt˄ʡʤĚ ŘʫKOĸʔʭʑʔʰƢȘ˅ʝʤʋ

ɿ ALT511ʓʾʳ12ʫʱʊ500ʰÙȩȯĉ&ʕÞʿ˂ʤʕʊPCRʯʾˁƚuǈ ȴʹʰƢȘʫ3ȁĸʱÞʿ˂ʮʔʧʤʋALT6ʫʱ65ʰÙȩȯĉ&ʕÞʿ˂ʊʢʰ ʑʦʰ 12 Ǭĸ˅ PCRʫƢȘʝʤǊĳʊ2 ǬĸʫŃƕɊKʰ˱̎˫ (insert) ʕÞʿ

˂ʠʊ˄ˀʯƚuǈȴʹʯʾʧʪƇʞˁ˱̎˫ (homo) ʕĽJʜ˂ʤ ( 2a)ʋʚ

˂ʿ 2 Ǭĸʰ˚˛̎ȌİʫʱʊƚuǈȴʹʯʾʧʪǕĉʜ˂ˁɋM˅˸̌̐˷ʬʝ ʤtʊ˱̎˫ʱʹʿ˂ʮʔʧʤ (2b, c)ʋʚ˂ʿʰǊĳʔʿʊʚ˂ʿ2Ǭĸ˅KO ĸ (2 ˘˵̐ʬʼơʜ˂ʤĸ)ʊ6H1 ʓʾʳ6H8ʬʝʊɘʰ¬ɹʯƉʐʤʋʸʤ 1

˘˵̐KO ĸ (1 ˘˵̐ʱơʜ˂ʪʐˁʕŋˀʰ˘˵̐ʱŶ7ʮĸ) ʬƣʜ˂ʤ 6H10ĸ˅˘̎˪̌̐̊ĸʬʝʪɘʰ¬ɹʯƉʐʤʋALT10ʫʱʊ90ʰÙȩȯĉ&

ʕÞʿ˂ʊ8ǬĸʕKOĸʬƣʜ˂ʤʋʚ˂ʿ8Ǭĸʰʑʦ2Ǭĸ (10H36ʓʾʳ 10H39) ˅KOĸʬʝʊʸʤʊ1˘˵̐KOĸʬƣʜ˂ʤ10H44ʓʾʳ10H45˅˘̎

˪̌̐̊ĸʬʝʪɘʰ¬ɹʯƉʐʤʋ ɿ

1-2-1. ALT6KO'/ AAL*3-.6(

ɿ ALT6KO ĸʰƑhè˅Ľ«ʟˁʤʻʊĬėʬĚŘʫȶʵʤʾʑʯǜ¥˅ȞĔʝʊ

ínè˪˿˪ƭʊìƝ˶ˇ̐˞˪ʯąƭʝʤʋKOĸʊ6H1ʓʾʳ6H8ʬʼʯɐƇĸ ʫʹʿ˂ʤƑĖʱʹʿ˂ʮʔʧʤʋĚʊ1˘˵̐KOĸʫʏˁ6H10ʱɐƇĸʬu

(33)

32

a

b

c

2ɿ PCRʓʾʳ˚˛̎˷̌˦˨ˉ̎˕ʯʾˁALT6KOĸʰɆø a; PCRʯʾˁALT6KOĸʰɆø

homo; ƚuǈȴʹʯʾˀƇʞˁ˱̎˫

insert; ƚuǈȴʹʯʾˀhphːˠ˦˪ʬǕĉʜ˂ˁ˱̎˫

hph; ˰ˊ˕̌˿ˊ˜̎Bǚèɇ ¥ʰDɊɯ

exp; ɷ5ĜƘʰRNA˅úJʝcDNA˅tñÜʊALT6ʰƒƃ˅RT-PCRʫƢȘ ʝʤʋ

b; ALT6KOĸʰ˚˛̎˷̌˦˨ˉ̎˕

˸̌̐˷; ALT6 A

c; ALT6KOĸʰ˚˛̎˷̌˦˨ˉ̎˕

˸̌̐˷; ALT6 insert

As-27; ɐèĸʊ6H1, 6H8; ALT6KOĸʊ6H10; 1˘˵̐KOĸ

(34)

33

ƶʰƑĖʕʹʿ˂ʤ (3a)ʋĬėʬĚŘʰ15-1-1ʓʾʳ15-2ʯÝʐʊŏǄúJʓ ʾʳŏǄƇƈǝĽ«˅ǹʧʤʋʢʰǊĳʊɐƇĸʓʾʳ1˘˵̐KOĸʊ6H10ʫʱ

Ŋʕʹʿ˂ʤʋĚʊALT6KOĸʊ6H1ʓʾʳ6H8ʫʱʊ?ʗŊʱȇʿ˂ʮʔʧ ʤ (3b)ʋAALŏǄʰ«ɑʰʤʻHPLCȌİ˅ǹʧʤʋɐƇĸʓʾʳ1˘˵̐KO ĸʫʱʊAAL-TA˅ƣʟ˵̐˔ʕĽJʜ˂ʤʋĚʊKOĸʊ6H1ʓʾʳ6H8ʫʱʊ AAL-TA˅ƣʟ˵̐˔ʱĽJʜ˂ʮʔʧʤ (3c)ʋŏǄɑʰ«ɑʯʾˀʊɐƇĸʫ ʱ71.5 µg/mlʊKOĸʊ6H1ʓʾʳ6H8ʫʱʬʼʯ0 µg/mlʊ1˘˵̐KOĸʊ6H10 ʫʱ24 µg/mlʰŏǄʕĽJʜ˂ʤ (ǽ8)ʋ

1-2-2. ALT6KO''1:D

ɿ ALT6KOĸʰƚȁʱʊĬėʬĚŘʯÝʐʊ˘˪̈̎˞˶ˎ̐̂̐˜̇̎ʯʾˀǹʧʤʋ

˝ˌˮ˨ˉ˜̎ǚèɇ ¥ʓʾʳ·>ʝʤɇ ¥ʰDɊɯ˅PCRʫĽJʟˁʚʬ ʫʊɇ ¥ʰ·>˅ƢȘʝʤʋKO ĸ 6H8 ĸʰƚȁʯʾˀʊƘƕɇ ¥ʕ·>ʜ˂

ʤĸʕ2ĸÞʿ˂ʤʋʚ˂ʿǬĸʰƑhèʓʾʳŏǄƇƈǝĽ«˅ǹʧʤʕʊƑh èʓʾʳŏǄƇƈǝʱàÈʝʮʔʧʤʋ

1-3. ALT10KO '1:D B+>/ AAL *3-.6

(

ɿ ALT10KOĸʰƚȁʱʊALT6ʬuŁʯǹʧʤʋPCRʓʾʳ˚˛̎Ȍİʯʾˀƚȁ ĸ10H36::G1ʓʾʳ10H36::G8ʕÞʿ˂ʤ (4)ʋALT10KOĸʓʾʳƚȁĸʰƑ hèĽ«ʰǊĳʊKO ĸʊ10H36 ʯɐƇĸʫʹʿ˂ʤƑĖʱʹʿ˂ʮʔʧʤʋĚʊ ƚȁĸ10H36::G1ʓʾʳ10H36::G8ʱɐƇĸʬuƶʰƑĖʕʹʿ˂ʤ (5a)ʋŏ ǄƇƈǝĽ«˅ǹʧʤǊĳʊɐƇĸʓʾʳƚȁĸʊ10H36::G1ʓʾʳ10H36::G8 ʫʱŊʕʹʿ˂ʤʋĚʊALT10KOĸʊ10H36ʫʱʊ?ʗŊʱʹʿ˂ʮʔʧʤ (5b)ʋHPLCȌİ˅ǹʧʤǊĳʊɐƇĸʓʾʳƚȁĸʫʱʊAALŏǄ˅ƣʟ˵̐

˔ʕĽJʜ˂ʤʋĚʊKOĸʊ10H36ʫʱʊAAL-TA˅ƣʟ˵̐˔ʱĽJʜ˂ʮʔ ʧʤʕĩƝʰ˵̐˔ʕĽJʜ˂ʤ (6a)ʋʚʰ˵̐˔ʱLC-MSȌİʰǊĳʊ˪̉ː

̊˽̎ɍʰʮʐpentolamineʬuʰȩɑʕƣʜ˂ʤ (˩̐ˣʮʝ)ʋŏǄɑʰ«ɑ ʯʾˀʊɐƇĸʫʱ26.0 µg/mlʊKOĸʊ10H36ʫʱ0 µg/mlʊƚȁĸʊ10H36::G1ʫ ʱ42.0 µg/mlʊ10H36::G8ʫʱ17.0 µg/mlʰŏǄʕĽJʜ˂ʤ (ǽ9)ʋǬƿñɔŐ ȱ˅ǹʧʤʬʚ˃ʊPDAʫʱÅʱʹʿ˂ʠʊ˰ˊ˕̌˿ˊ˜̎BxĦʫʱʊ ɐèĸʱñɔʯÅʱʹʿ˂ʮʔʧʤʋ˝ˌˮ˨ˉ˜̎xĦʫʱʊɐèĸʓ ʾʳKOĸʊ10H36ʯʓʐʪǬƿʰñɔʱʹʿ˂ʮʔʧʤʋƚȁĸʊ10H36::G1ʓ ʾʳ10H36::G8ʫʱǬƿʰñɔʯÅʱȇʿ˂ʮʔʧʤ (6b)ʋ

(35)

34

As-27 6H1 6H8 6H10

10

0 0 10 0 10 0 10

a

b

c

3ɿ ALT6KOĸʰƑhèĽ«ʊŏǄƇƈǝĽ«ʓʾʳHPLCȌİ a; ALT6KOĸʰƑhèĽ«

b; ALT6KOĸʰŏǄƇƈǝĽ«

c; ALT6KOĸʰHPLCȌİ

As-27; ɐèĸʊ6H1, 6H8; ALT6KOĸʊ6H10; 1˘˵̐KOĸ

(36)

35 ǽ8ɿ HPLCʯʾˁAALŏǄʰ«ɑ

AAL-TA (µg/ml)

As-27 71.5 µg/ml 6H1 0 µg/ml 6H8 0 µg/ml 6H10 24 µg/ml HPLCȌİʯʩʐʪAAL-TAʰ«ɑ˅ǹʧʤʋ

As-27; ɐèĸ

6H1, 6H8; ALT6KOĸ 6H10; 1˘˵̐KOĸ

(37)

36

a

b

c

4ɿ PCRʓʾʳ˚˛̎˷̌˦˨ˉ̎˕ʯʾˁALT10KOĸʬƚȁĸʰɆø a; PCRʯʾˁALT10KOĸʓʾʳƚȁĸʰɆø

npt; ˝ˌˮ˨ˉ˜̎ǚèɇ ¥ʰDɊɯ

b; ALT10KOĸʓʾʳƚȁĸʰ˚˛̎˷̌˦˨ˉ̎˕

˸̌̐˷; ALT10 insert

c; ALT10KOĸʓʾʳƚȁĸʰALT10ʰƒƃȌİ

Ǭ&˅4ĜɖɷÜʊRNA˅úJʝcDNAtñʝʤʋÞʿ˂ʤcDNA˅˨̎˸̋̐

˪ʯALT10ʰƒƃ˅RT-PCRʫƢȘʝʤʋ

As-27; ɐèĸʊ10H36; ALT10KOĸʊ10H36::G1, 10H36::G8; ALT10ƚȁĸ

(38)

37

a

b

5 ALT10KOĸʓʾʳƚȁĸʰƑhèʓʾʳŏǄƇƈǝĽ«

a; ALT10KOĸʬƚȁĸʰƑhèĽ«

b; ALT10KOĸʓʾʳƚȁĸʰŏǄƇƈǝĽ«

As-27; ɐèĸ 10H36; ALT10KOĸ

10H36::G1, 10H36::G8; ALT10ƚȁĸ

(39)

38

a

b

6 ALT10KOĸʓʾʳƚȁĸʰHPLCȌİʓʾʳǬƿñɔŐȱ

a; ALT10KOĸʬƚȁĸʰHPLCȌİ

ɥƜfʫƣʝʤ˵̐˔ʱALT10KOĸʰʹȇʿ˂ʤęȈʰ˵̐˔

b; ALT10KOĸʬƚȁĸʰǬƿñɔŐȱ

÷ƇżȩʰųÐʱ100 ppm As-27; ɐèĸ

10H36; ALT10KOĸ

10H36::G1, 10H36::G8; ALT10ƚȁĸ

(40)

39 ǽ9ɿ HPLCʯʾˁAAL-TAʰ«ɑ

AAL-TA (µg/ml)

As-27 26 µg/ml 10H36 0 µg/ml 10H3::G1 42 µg/ml 10H3::G8 17 µg/ml HPLCȌİʯʼʬʩʐʪAAL-TAʰ«ɑ˅ǹʧʤʋ

As-27; ɐèĸ 10H36; ALT10KOĸ

10H36::G1, 10H36::G8; ALT10ƚȁĸ

(41)

40

1-4-1. TRF1-4@@<=" KO'?!

ɿ CDCʯ§ʟˁ4ʨʰȯFȞƹɇ ¥ʊTRF1- TRF4ʰŅǝȌİ˅ǹʧʤʋʚ˂

ʿɇ ¥ʱʢ˂ʣ˂Zn(II)2Cys6ʰȯF¥ (TRF1ʓʾʳTRF2)ʊC2H2 finger domain protein (TRF3) ʓʾʳC6 zinc finger domain containing protein (TRF4) ˅˘

̐˫ʝʪʐˁʬêʜ˂ʪʐˁʋPCR ʓʾʳ˚˛̎ȌİʰǊĳʔʿʊTRF1 ʫʱʊ TRF1H5 ˅ KO ĸʬʝʊɘʰ¬ɹʯƉʐʤ ( 7)ʋuŁʯ TRF2 ʫʱʊTRF2H9 ʓʾʳ TRF2H45 ˅ KO ĸʬʝʊɘʰ¬ɹʯƉʐʤ ( 8)ʋuŁʯ TRF3 ʫʱʊ TRF3H2 ˅ KO ĸʬʝʊɘʰ¬ɹʯƉʐʤ ( 9)ʋʸʤʊuŁʯ TRF4 ʫʱʊ TRF4B4ʓʾʳTRF4D2˅KOĸʊTRF4A16˅˘˵̐KOĸʬʝʊɘʰ¬ɹʯ Ɖʐʤ (10)ʋ

1-4-2. TRF1-4@92 B+>HPLC;$ 0,;$

ɿ sǬĸ˅PDAʓʾʳV8ʯļǬʝʊ1ȽɖÜʰǬʰÙî˅Ȋ³ʝʤʕʊɐè ĸʬ KOĸʯÅʱʹʿ˂ʮʔʧʤʋAALŏǄʰ«ɑʰʤʻ HPLCȌİ˅ǹʧʤǊ ĳʊTRF1-4ʰ?ʪʰǬĸʯʓʐʪAAL-TA˅ƣʟ˵̐˔ʕĽJʜ˂ʤ (7d) ( 8d) (9d) (10c)ʋTRF1H5ʯʓʐʪALT˔̈˞ˣ̐ɇ ¥ʰƒƃȌİ˅ǹʧʤ ǊĳʊɐèĸʾˀʼKO ĸʰĚʕʽʽƒƃɑʕZʝʪʐʤʋʸʤʊALT1 ʯʓʐʪ KOĸʫʱĥ¸ʰ˱̎˫ʕĥʼƒƃɑʕʔʧʤ (11)ʋ

1-5-1. AVE1@<=" KO'?!

ɿ CDCʯ§ʟˁavenacinase˅˘̐˫ʝʪʐˁʬêʜ˂ʪʐˁAVE1ɇ ¥ʰŅ ǝȌİ˅ǹʧʤʋPCRʓʾʳ˚˛̎ȌİʰǊĳʔʿAVH6˅KOĸʬʝʊɘʰ¬

ɹʯƉʐʤ (12a)ʋ

1-5-2. AVE1KO'/( 4(

ɿ ƑhèĽ«˅ǹʧʤʬʚ˃ʊɐèĸʬKOĸʯɲǯʮÅʱʹʿ˂ʮʔʧʤ (12b)ʋ ʸʤʊ˪˿˥̎ǚèĽ«˅ǹʧʤǊĳʊɐèĸʓʾʳKO3ȁĸʰǜ¥ƒǪʯɲǯ ʮÅʱʹʿ˂ʮʔʧʤ (˩̐ˣŶʝ)ʋ

2. &7@)6;$

2-1-1. AaGPA1@<=" KO'?!

ɿ ÉĶǩ&ʯ§ʟˁ G protein 〈−subunit ˅˘̐˫ʟˁʬĆ«ʜ˂ˁɇ ¥ʊ

AaGPA1ʰŅǝȌİ˅ǹʧʤʋPCRʓʾʳ˚˛̎ȌİʰǊĳʔʿ GPA1H1ʓʾʳ

GPA1H2˅KOĸʬʝʊɘʰ¬ɹʯƉʐʤ (13a)ʋ

(42)

41

TRF1H5 As-27

a b

c

d

7 PCRʬ˚˛̎˷̌˦˨ˉ̎˕ʯʾˁTRF1KOĸʰɆøʓʾʳHPLCȌİ a; PCRʯʾˁTRF1KOĸʰɆø

exp; RNA˅úJʝcDNA˅tñÜʊTRF1ʰƒƃ˅RT-PCRʫƢȘʝʤʋ b; TRF1KOĸʰ˚˛̎˷̌˦˨ˉ̎˕ʊ˸̌̐˷; TRF1 A

c; TRF1KOĸʰ˚˛̎˷̌˦˨ˉ̎˕ʊ˸̌̐˷; TRF1 insert d; TRF1KOĸʰHPLCȌİ

As-27; ɐèĸʊ1H5; TRF1KOĸ

(43)

42

AAL-TA

As-27 2H9 2H45

a b

c

d

8ɿ PCRʬ˚˛̎˷̌˦˨ˉ̎˕ʯʾˁTRF2KOĸʰɆøʓʾʳHPLCȌİ a; PCRʯʾˁTRF2KOĸʰɆø

exp; RNA˅úJʝcDNA˅tñÜʊTRF2ʰƒƃ˅RT-PCRʫƢȘʝʤʋ b; TRF2KOĸʰ˚˛̎˷̌˦˨ˉ̎˕

˸̌̐˷; TRF2 B

c; TRF2KOĸʰ˚˛̎˷̌˦˨ˉ̎˕

˸̌̐˷; TRF2 insert d; TRF2KOĸʰHPLCȌİ ƜfʱAALŏǄ˅ƣʟʋ

As-27; ɐèĸʊ2H9, 2H45; TRF2KOĸ

(44)

43

AAL-TA

As-27

3H2

5.8 3.5 5.8 (kb) As-27 3H2

As-27 3H2

a b

c d

exp; RNA˅úJʝcDNA˅tñÜʊTRF3ʰƒƃ˅RT-PCRʫƢȘʝʤʋ b; TRF3KOĸʰ˚˛̎˷̌˦˨ˉ̎˕ʊ˸̌̐˷; TRF3 B

c; TRF3KOĸʰ˚˛̎˷̌˦˨ˉ̎˕ʊ˸̌̐˷; TRF3 insert d; TRF3KOĸʰHPLCȌİ

ƜfʱAALŏǄ˅ƣʟʋ

As-27; ɐèĸʊ3H2; TRF3KOĸ

(45)

44

AAL-TA

A16 B4 D2

As-27

a

b c

exp; RNA˅úJʝcDNA˅tñÜʊTRF4ʰƒƃ˅RT-PCRʫƢȘʝʤʋ b; TRF4KOĸʰ˚˛̎˷̌˦˨ˉ̎˕ʊ˸̌̐˷; TRF4 A

c; TRF4KOĸʰHPLCȌİ ƜfʱAALŏǄ˅ƣʟʋ

As-27; ɐèĸʊB4, D2; TRF4KOĸʊA16; ˘˵̐KOĸ

(46)

45

As-27

TRF1H5

1 2 3 4 5 6 7 8 9 10 11 12 13 TF1H

(bp) 700 280 700 280

11ɿ TRF1KOĸʯʓʘˁALT˔̈˞ˣ̐ɇ ¥ʰƒƃȌİ

RNAʱ̉˥̄̐˟Ť&ʫ5ĜɖāʬʑɷʝʤǬ&ʔʿúJʝʊcDNAtñÜʊ ALT˔̈˞ˣ̐ɇ ¥ʰƒƃ˅RT-PCRʫƢȘʝʤʋ

As-27; ɐƇĸ TRF1H5; KOĸ

(47)

46

As-27 AVH6

a

b

12ɿ PCRʯʾˁAaAVE1KOĸʰɆøʓʾʳƑhèĽ«

a; PCRʯʾˁAaAVE1KOĸʰɆø homo; ƚuǈȴʹʯʾˀƇʞˁ˱̎˫

exp; RNA˅úJʝcDNA˅tñÜʊAaAVE1ʰƒƃ˅RT-PCRʫƢȘʝʤʋ b; ƑhèĽ«

1ʀ10⁵1/mlʯȞȂʝʤǜ¥ðŲŤ˅*Ɖʝʤʋ As-27; ɐèĸ

AVH6; AaAVE1KOĸ

(48)

47 (bp) homo

hph

insert

exp

839

356 409

356 As-27 GPA1H1GPA1H2

a

b

c

13ɿ PCRʯʾˁAaGPA1KOĸʰɆøʓʾʳƑhèĽ«

a; PCRʯʾˁAaGPA1KOĸʰɆø

homo; ƚuǈȴʹʯʾˀƇʞˁ˱̎˫ʊinsert; ƚuǈȴʹʯʾˀ hph ːˠ˦˪ʬǕĉ ʜ˂ˁ˱̎˫ʊhph; ˰ˊ˕̌˿ˊ˜̎Bǚèɇ ¥ʰDɊɯ

exp; RNA˅úJʝcDNA˅tñÜʊAaGPA1ʰƒƃ˅RT-PCRʫƢȘʝʤʋ b; ǬƿñɔŐȱ

c; ¬&ɲáɓʯʾˁǬʢʑʰŐȱ

As-27; ɐèĸʊGPAH1, GPAH2; AaGPA1KOĸ

(49)

48 2-1-2. AaGPA1KO''92 B+>

ɿ ǬƿñɔŐȱ˅ǹʧʤǊĳʊɐƇĸʬŐȱʝʪ?ʪʰKOĸʫŒ ǬƿʕŨ¹ʝʊ Ǭʢʑʕ_ʝʪʐʤ ( 13b)ʋʸʤʊǬƿʰȊ³˅ǹʧʤǊĳʊɐèĸʫǜ¥ʰ Ƈƈʕȇʿ˂ˁĚʊKO ĸʊGPA1H1 ʓʾʳGPA1H2 ʫʱʶʬˆʭȇʿ˂ʮʔʧ ʤ (13c)ʋ

2-1-3. AaGPA1KO'/ AAL*3-.6(

ɿ ƑhèĽ«ʱǜ¥ʰlʕɢʝʔʧʤʤʻʊ˞˾˦˪ąƭʯʾˀǹʧʤʋɐèĸʫ ƑĖʕē1ȇʿ˂ˁĚʊKOĸGPA1H1ʓʾʳGPA1H2ʫʱƑĖʕűŨʝʪʐ ʤʋAAL ŏǄƇƈǝĽ«˅ǹʧʤǊĳʊɐƇĸʫʹʿ˂ˁŊʕKO ĸGPA1H1 ʓʾʳGPA1H2ʫʱZʝʪʐʤ (˩̐ˣŶʝ)ʋ

2-2-1. 8%/9 GPCRC

2-18*3+04 GPCR "()7% "

6GPCR&7-8NCBI12/$5 AaGPR1cAMP.GPCRAaGPCR2AaGPCR3carbon/amino!

#STE2Ste2!#'9

2-2-1. AaGPR1-3@<=" KO'?!

ɿ ÉĶǩ&ʯ§ʟˁG protein coupled receptor˅˘̐˫ʟˁʬĆ«ʜ˂ˁɇ ¥ʊ

AaGPR1-3ʰŅǝȌİ˅ǹʧʤʋAaGPR1ʫʱʊPCRʓʾʳ˚˛̎ȌİʰǊĳʔʿ

GPR1N1 ʓʾʳ GPR1N2 ˅ KO ĸʬʝʊɘʰ¬ɹʯƉʐʤ ( 14)ʋuŁʯ AaGPR2ʫʱʊGPR2H1ʊGPR2H5ʓʾʳGPR2H8˅ KOĸʬʝʊɘʰ¬ɹʯƉ ʐʤ (15)ʋAaGPR3ʫʱʊGPR3H17˅KOĸʬʝʊɘʰ¬ɹʯƉʐʤ (16)ʋ

2-2-2. AaGPR1-3KO'92 B+> /(

ɿ sǬĸʰǬƿñɔ˅ŐȱʝʤʕʊɲǯʮÅʱʹʿ˂ʮʔʧʤ ( 17)ʋʸʤʊƑh èĽ«˅ǹʧʤǊĳʊɐèĸʓʾʳKOĸʯɲǯʮÅʱʹʿ˂ʮʔʧʤ (18)ʋ

2-2-3. AaGPR3KO'1:D

ɿ AaGPR3KOĸʰƚȁʱʊALT6ʬuŁʯʝʪǹʧʤʋPCRʓʾʳ˚˛̎Ȍİʯʾˀ ƚȁĸGPR3H17::G1ʓʾʳGPR3H17::G16ʕÞʿ˂ʤʚʬʔʿɘʰ¬ɹʯƉʐ ʤ (16b, c)ʋ

(50)

49

a

b

14ɿ PCRʬ˚˛̎˷̌˦˨ˉ̎˕ʯʾˁAaGPR1KOĸʰɆø a; PCRʯʾˁAaGPR1KOĸʰɆø

insert; ƚuǈȴʹʯʾˀnptːˠ˦˪ʬǕĉʜ˂ˁ˱̎˫

npt; ˝ˌˮ˨ˉ˜̎ǚèɇ ¥ʰDɊɯ

exp; RNA˅úJʝcDNA˅tñÜʊAaGPR1ʰƒƃ˅RT-PCRʫƢȘʝʤʋ b; AaGPR1KOĸʰ˚˛̎˷̌˦˨ˉ̎˕

˸̌̐˷; AaGPR1 insert As-27; ɐèĸ

GPR1N1ʓʾʳGPR1N2; AaGPR1KOĸ

(51)

50

(bp)

409 223 As-27

GPR2H1

GPR2H8

homo

hph insert

exp

223 1206 GPR2H5

a

b

c

15ɿ PCRʬ˚˛̎˷̌˦˨ˉ̎˕ʯʾˁAaGPR2KOĸʰɆø a; PCRʯʾˁAaGPR2KOĸʰɆø

exp; RNA˅úJʝcDNA˅tñÜʊAaGPR2ʰƒƃ˅RT-PCRʫƢȘʝʤʋ b; AaGPR2KOĸʰ˚˛̎˷̌˦˨ˉ̎˕

˸̌̐˷; AaGPR2 B

c; AaGPR2KOĸʰ˚˛̎˷̌˦˨ˉ̎˕

˸̌̐˷; AaGPR2 insert

As-27; ɐèĸʊGPR2H1ʊGPR2H5ʓʾʳ GPR2H8; AaGPR2KOĸ

(52)

51

a

b

c

16 PCRʯʾˁAaGPR3KOĸʰɆøʊƚȁĸʰ˛̎˷̌˦˨ˉ̎˕ʓʾʳƒƃȌ İ

a; PCRʯʾˁAaGPR3KOĸʰɆø homo; ƚuǈȴʹʯʾˀƇʞˁ˱̎˫

exp; RNA˅úJʝcDNA˅tñÜʊAaGPR3ʰƒƃ˅RT-PCRʫƢȘʝʤʋ b; AaGPR3ƚȁĸʰ˚˛̎˷̌˦˨ˉ̎˕

˸̌̐˷; AaGPR3 insert

As-27; ɐèĸʊGPR3H17; AaGPR3KOĸ 3H17::G1, 3H17::G16; AaGPR3ƚȁĸ c; AaGPR3ƚȁĸʰƒƃȌİ

(53)

52

N1

N2 WT

WT H1

H5 H8

H17 WT

PDA V8

GPR1

GPR2

GPR3

17ɿ AaGPR1-3KOĸʯʓʘˁPDAʊV8ʓʾʳ̉˥̄̐˟ʫʰÙîŐȱ a; PDAʓʾʳV8ʫʰAaGPR1-3KOĸʰÙîŐȱ

sǬĸ˅ļǬ4ĜÜʯČÚʝʤʋ WT; As-27 (ɐƇĸ)

N1ʓʾʳN2; AaGPR1KOĸ H1ʊH5ʓʾʳH8; AaGPR2KOĸ H17; AaGPR3KOĸ

(54)

53

GPR1N1 GPR1N2

AAL-TA

GPR2H5 GPR2H8

GPR2H1 GPR3H17

18ɿ AaGPR1-3KOĸʰƑhèĽ«ʓʾʳHPLCȌİ

GPR1N1, GPR1N2; AaGPR1KOĸʊGPR2H1, GPR2H5, GPR2H8; AaGPR2KOĸ GPR3H17; AaGPR3KOĸ

(55)

54

2-2-4. AaGPR311:'5+> /(

ɿ ǜ¥Őȱ˅ǹʧʤǊĳʊǜ¥ʰʖʜʱɐƇĸʫʱÌ 18 μmʊKO ĸʫʱ 15 μmʊƚȁĸʫʱ20 μmʥʧʤ (19a)ʋƑhèĽ«˅ǹʧʤǊĳʊAaGPR3 KO ĸʯʓʐʪƑhXʰ%ʕȘʻʿ˂ʤ̒18̓ʋ

2-3. AaSTE2@<="92 B+> /(

ɿ ÉĶǩ&ʯ§ʟˁG protein coupled receptor˅˘̐˫ʟˁʬĆ«ʜ˂ˁɇ ¥ʊ

AaSTE2ʰŅǝȌİ˅ǹʧʤʋPCRʓʾʳ˚˛̎ȌİʰǊĳʔʿ2ǬĸʰKOĸʕ

Þʿ˂ʤʋǬƿñɔŐȱʓʾʳƑhèĽ«˅ǹʧʤʬʚ˃ɐèĸʬ KO ĸʫɲǯʮ Åʱʹʿ˂ʮʔʧʤ (˩̐ˣŶʝ)ʋ

2-4-1. LAEA@<=" KO'?!

ÉĶǩ&ʯ§ʟˁ˳˞˪̎ʰmethyltransferase˅˘̐˫ʟˁʬĆ«ʜ˂ˁɇ ¥ʊ LaeAʰ˼̃̌˕ʕA. alternataʰsƑhʯġɀƕʯ§ʟˁʔʭʑʔ˅PCRʯʾ ʧʪƢȘʝʤʋʢʰǊĳʊ?ʪʰƑhǬʫLaeA ʰ˼̃̌˕˅ƣʟ˱̎˫ʕĽJʜ

˂ʤʋǫĵƑǬʰ LaeA ˼̃̌˕ʫʏˁ AtLAEA ʰÙȩȯĉ˅ǹʧʤǊĳʊ LAEAH40ʊLAEAH41 ʓʾʳ LAEAH43 ʰ KO ĸʕÞʿ˂ʤʋ̉̎˙ƑhǬʰ LaeA˼̃̌˕ʫʏˁAaLAEAʰÙȩȯĉ˅ǹʧʤǊĳʊKOĸH13ʓʾʳH15ʕ Þʿ˂ʤʋˊ˥˙ƑhǬʰLaeA˼̃̌˕ʫʏˁAsLAEAʰÙȩȯĉ˅ǹʧʤǊĳʊ KOĸH16ʊH20ʊH25ʕÞʿ˂ʤʋ

2-4-2. AtLAEAKO '92 B+>5-.6+>9A+>/

AAL*3-.6(

ɿ Ǭƿñɔ˅ŐȱʝʤǊĳʊKOĸʊLAEAH40ʊLAEAH41ʓʾʳLAEAH43ʫʱ ɐèĸʫʹʿ˂ʤŒ ǬƿʕűŨʝʪʐʤ (20)ʋǜ¥ƇƈǝŐȱʓʾʳƑh èĽ«˅ǹʧʤǊĳʊKOĸʫʱʊɐƇĸʫʹʿ˂ʤǜ¥ʓʾʳƑĖʕűŨʝʪʐ ʤ (21)ʋʸʤʊŏǄƇƈǝĽ«˅ǹʧʤǊĳʊKOĸʫʱɐèĸʫʹʿ˂ʤŊ ĖʕűŨʝʪʐʤ (22a)ʋHPLCȌİ˅ǹʧʤǊĳʊ?ʪʰǬĸʯʓʐʪAAL ŏǄ˅ƣʟ˵̐˔ʕĽJʜ˂ʤ (22b)ʋŏǄɑʰ«ɑʯʾˀʊɐƇĸʫʱ16.1 µg/mlʊKOĸʊLAEAH40ʊLAEAH41ʓʾʳLAEAH43ʫʱʢ˂ʣ˂1 µg/mlʊ1.1 µg/mlʓʾʳ1.2 µg/mlʰŏǄʕĽJʜ˂ʤ (ǽ10)ʋǬ&ɑ˅ŐȱʝʤǊĳʊKO ĸʱɐèĸǬ&ɑʰǁ2/3ƬÐʯŨ¹ʝʪʐʤ (22c)ʋ

(56)

55

a

b

19ɿ AaGPR3KOĸʓʾʳƚȁĸʰǜ¥ñɔ a; AaGPR3KOĸʓʾʳƚȁĸʯʓʘˁǜ¥

V8ʫ2Ƚɖɷʝʊǜ¥˅lʝʤʋ b; AaGPR3KOĸʓʾʳƚȁĸʯʓʘˁǜ¥ɔ a, b, cʯʱʢ˂ʣ˂ĦëÅʕȘʻʿ˂ʤʋ As-27; ɐèĸ

3H17; AaGPR3KOĸ

3H17::G1ʓʾʳ3H17::G17; AaGPR3ƚȁĸ

(57)

56

20ɿ AtLAEAKOĸʰǬƿñɔ As-27; ɐƇĸ

LAEAH40ʊLAEAH41ʓʾʳ LAEAH43; AaLAEAKOĸ PDAʫɷÜ5ĜƘʯČÚʝʤʋɿ

(58)

57

5#

a

b

c

21ɿ AtLAEAKOĸʰÙîʊǜ¥ƇƈǝʓʾʳƑhèĽ«

a; AtLAEAKOĸʰ¬&ɲáɓʯʾˀȊ³ʝʤKOĸʰǬʢʑ b; AtLAEAKOĸʰ˸̋̐˪1Ĳʏʤˀʰǜ¥ēŐȱ

c; AtLAEAKOĸʰƑhèĽ«

As-27; ɐƇĸʊLAEAH40, LAEAH41, LAEAH43; AaLAEAKOĸ

(59)

58 AAL-TA

LaeAH40 LaeAH41 LaeAH43

AAL-TA

As-27

AAL-TA

10

0 0 10 0 10 0 10

a

b

c

22ɿ AtLAEAKOĸʰŏǄƇƈǝĽ«ʊHPLCȌİʓʾʳǬ&ɑ a; AtLAEAKOĸʰŏǄƇƈǝĽ«

b; AtLAEAKOĸʰHPLCȌİ c; AtLAEAKOĸʰǬ&ɑ

̉˥̄̐˟Ť&ʫ2ȽɖɷÜʊźʜʡɏɑ˅ū«ʝʤʋ

As-27; ɐƇĸʊLAEAH40ʊLAEAH41ʓʾʳ LAEAH43; AaLAEAKOĸ

(60)

59 ǽ10ɿ HPLCʯʾˁAALŏǄʰ«ɑ

4, AAL-TA (µg/ml)

As-27 16.2 µg/ml

LAEAH40 1 µg/ml

LAEAH41 1.1 µg/ml

LAEAH43 1.2 µg/ml

AALŏǄʰ«ɑʱHPLCʯʩʐʪǹʧʤʋ As-27; ɐƇĸ

LAEAH40ʊLAEAH41ʓʾʳ LAEAH43; AaLAEAKOĸ

(61)

60

2-4-3. AtLAEAKO''@0,;$

ɿ RNA˅úJʝʊɇ ¥ʰƒƃȌİ˅ǹʧʤʬʚ˃ʊKOĸʫʱALT˔̈˞ˣ̐ɇ

¥ ʰ ƒ ƃ ɑ ʕ ɐ è ĸ ʾ ˀ ʼ Ũ ¹ ʝ ʪ ʐ ʤ ʋ Ě ʊ ˔ ̈ ˞ ˣ ̐ D ʯ § ʟ ˁ metalloproteaseɇ ¥ʱɐèĸʬKOĸʫƒƃɑʯ_ʱʮʔʧʤ (23a)ʋʸʤʊ ƒƃɑʰǌ´ɑ˅Őȱʝʤʬʚ˃ʊɐèĸʫɺʔʧʤ˔̈˞ˣ̐ɇ ¥ʰƒƃʕKO ĸʫŨ¹ʝʪʐʤʋALT˔̈˞ˣ̐ʰɇ ¥ʱɐèĸʓʾʳKOĸʫƒƃɑʕ

¹ʮʔʧʤ (23b)ʋÉĶǩ&ʯ§ʟˁɇ ¥ʰƒƃȌİ˅ǹʧʤǊĳʊKOĸ ʰƒƃɑʱɐèĸʰƒƃɑʬŐȱʝʪAKSʫʱ72ʊVKSʱǁ32ʯZʝʪʐ ʤʋMAT1 ʓʾʳ ALMʫʱƒƃɑʯɲǯʮÅʱʹʿ˂ʮʔʧʤʋɐèĸʫʱ 4 ɇ ¥ʰƒƃɑ?ʪʕβ-tublinʬŐȱʝʪ¹ʮʔʧʤ (24)ʋ

2-4-4. AaLAEA AsLAEAKO'92 B+>/ AAL

*3-.6(

ɿ ǬƿñɔŐȱ˅ǹʧʤǊĳʊAaLAEAʓʾʳAsLAEAKOĸʬʼʯʊɐèĸʫʱʹ ʿ˂ʤŒ Ǭƿʕʶʬˆʭʹʿ˂ʮʔʧʤ ( 25)ʋHPLC Ȍİ˅ǹʧʤǊĳʊ?ʪ ʰǬĸʯʓʐʪŏǄ˅ƣʟ˵̐˔ʕĽJʜ˂ʤ ( 26)ʋƑhèĽ«ʓʾʳŏǄƇ ƈǝĽ«˅ǹʧʤǊĳAsLAEAKOĸʫʱʊɐèĸʫʹʿ˂ˁƑĖʓʾʳŊĖʱ ʶʬˆʭʹʿ˂ʮʔʧʤ (27)ʋ

(62)

61

a

b

23ɿ AtLAEAKOĸʓʾʳɐƇĸʯʓʘˁCDCɇ ¥ʰŐȱƒƃȌİ

Ȫ; ALT˔̈˞ˣ̐ɇ ¥ ɽǩ; ȯFȞƹ¥

ɥ; ȑʰɇ ¥ a; ɐƇĸʬʰƚ´4

sɇ ¥ƒƃʱʂ-tublinɇ ¥˅1ʬʝʪʸʠē4_ʝʤʋ

ɐƇĸʫÞʿ˂ʤē4˅1ʬʝʪKOĸʰƒƃɑ˅˕̈˶ʯƣʝʪʐˁʋ b; ƒƃɑʰǌ´4

ʂ-tublinɇ ¥˅1ʬʝʤğʰƒƃɑ

(63)

62

a

b

24ɿ AtLAEAKOĸʰÉĶǩ&ɇ ¥ʰƒƃȌİ

̉˥̄̐˟ʫāʬʑɷ8ĜÜʰǬ&ʔʿRNA˅úJʝʊcDNA ˅tñÜʊ̉ˈ

̊ˣˊ́PCRʯʾʧʪƒƃɑ˅«ɑʝʤʋ

a; ʂ-tublinɇ ¥ʓʾʳɐèĸʰsɇ ¥ƒƃ˅1ʬʝʤtʰAtLAEAKOĸɇ ¥ʰɐƇĸʬʰƚ´4

b; ʂ-tublinɇ ¥˅1ʬʝʤtʰsɇ ¥ʰƒƃɑʰǌ´4 ɽ: As-27; ɐèĸ

ɥ: H40; AtLAEAKOĸ

(64)

63

a

b

25ɿ A. alternatả̎˙Ƒhʓʾʳˊ˥˙ƑhʰLaeAKOĸ (AaLAEAKOĸ ʓʾʳAsLAEAKOĸ) ʰǬƿñɔ

a; AaLAEAKOĸʰǬƿñɔ

PDAʯļǬÜʊ5ĜƘʯČÚʝʤʋ IFO8984; ̉̎˙ĖŵǭǮƑǬ H3ʓʾʳH5; AaLAEAKOĸ b; AsLAEAKOĸʰǬƿñɔ PDAʯļǬÜʊ5ĜƘʯČÚʝʤʋ NAF8; ˊ˥˙ɾĖƑǬ

H16ʊH20ʓʾʳH25; AsLAEAKOĸ

(65)

64

a ɿɿ

b

26ɿ A. alternatả̎˙Ƒhʓʾʳˊ˥˙ƑhʰLaeAKOĸ (AaLAEAKOĸ ʓʾʳAsLAEAKOĸ) ʰHPLCȌİ

a; AaLAEAKOĸʰHPLCȌİ IFO8984; ̉̎˙ĖŵǭǮƑǬ H3ʓʾʳH5; AaLAEAKOĸ b; AsLAEAKOĸʰHPLCȌİ NAF8; ˊ˥˙ɾĖƑǬ

(66)

65

27ɿ AsLAEAKOĸʰƑhèĽ«ʓʾʳŏǄƇƈǝĽ«

Ǯʱínèˊ˥˙ƭƖÁ16r˅*Ɖʝʤʋ NAF8; ˊ˥˙ɾĖƑǬ

; ƑhèĽ«

1ʀ10⁵1/mlʯȞȂʝʤǜ¥ðŲŤ˅*Ɖʝʤʋ ; ŏǄƇƈǝĽ«

(67)

66 Ƶ

Ƶ4ƹɿ Ǚ³

ɿ ŇʌʯƓʟˁę˜̐˔ˍ̎˚̐ʰƒ½ʯʾˀʊɑʰ˗˯́éʕǱƮʜ˂

ʨʨʏˁʋǬɳʯʓʐʪʼAspergillus¾ʽNeurospora crassaʓʾʳˊˮʐʼʦƑǬ Magnaporthe griseaʮʭʗʰǬʯʨʐʪ˗˯́ȌİʕǇʝʤ (Dean et al. 2005, Galagan et al. 2003, Galagan et al. 2005, Machida et al. 2005)ʋĥȵʫʱʊʚ˂ʿ˗˯

́ʰŐȱȌİʕǹ˄˂ʊǬɳʯʓʘˁȾ_ʽK_ʮʭ˅ǏǖƕʯȌİʟˁʚʬʕ qǝʬʮˀʨʨʏˁ (Gao et al. 2011, Ma et al. 2010, Amselem et al. 2011)ʋ˗˯́?

&˅QƉʝʤŐȱȌİʱʊǬĸɖʫʰƚuɯʊʏˁʐʱȯÑʽʖʮ˩̉̐˜̇

̎ʮʭ˅ȇJʟtʯɨÉʯĦ\ʫʏˁʋʢʰʤʻʊƑhèǬʕÿʨɇ ƕŽâ ʮʭ˅ȇJʟʚʬʕqǝʫʏˁʋĚʊʗʰtʊɨÉʯȵǔʰǬĸɖʫʜʒʊʢ ʰ˗˯́ŀñʱƏʮˁʤʻʊ1ʌʰɇ ¥ʕʭʑʐʧʤŅǝ˅ʝʪʐˁʔʱʊʢ˂ʣ

˂ʰɇ ¥ʯʨʐʪɇ ¥ơʽ˚ˊ̋̎˜̎˕ʮʭ˅ʟˁäȅʕʏˁʋ

ɿ A. alternataʱʊʚ˂ʸʫʯ¹ʮʗʬʼ7ʨʰƑhʕzʜ˂ʪʓˀʊʢ˂ʣ˂Ə ʮˁ°ŽƏƕŏǄ (HST) ˅ƇƈʟˁʚʬʕƝʿ˂ʪʐˁʋʸʤʊ7Ƒhʰʑʦˣ

˱˘˅ʰʣʗƑhʫHSTƇƈʯɗʟˁɇ ¥ʕcɡʊu«ʜ˂ʪʐˁ (Aakagi et al. 2009, Akamatsu et al. 2003, Miyamoto et al. 2009, Tanaka et al. 1999, 2000, Hatta et al. 2002, Ruswandi et al. 2005, Johnson et al. 2000, 2001, Harimoto et al.

2007)ʋ˪˿˪ʊ̉̎˙ʓʾʳˊ˥˙ʰ 3 Ƒhʯʓʐʪʱʊʚ˂ʿɇ ¥ʕ˔̈˞ˣ̐

_ʝʪʓˀʊʔʨƇ§ʯʱäɮʫʱʮʐĶǩ&ʫʏˁ conditionally dispensable chromosome (CDC) ʯÑʝʪʐˁʚʬʕzʜ˂ʪʐˁ (Akagi et al. 2009, Harimoto et al 2007, Hatta et al. 2002)ʋ

ɿ sƑhʕ.ĦʟˁHSTƇtñɇ ¥ʱʊʶʬˆʭʰtʊʰHSTƇtñɇ ¥ʬƚuèʕʮʗʊǫĵƑǬʫȇJʜ˂ʤ 13 ʰ AAL ŏǄƇtñɇ ¥ (ALT ɇ ¥) ʰ˼̃̌˕ʱʊʰ A. alternata ʯʓʐʪʱȇJʜ˂ʪʐʮʐʋĚʫʊ essential chromosome (EC) ʰɇ ¥ʱƏʮˁƑhɖʫʼɨÉʯɺʐƚuèʕ Șʻʿ˂ʪʐˁ (Kusaba et al. 1995. Akagi et al. 2009)ʋʰ¬ɹʫʊǫĵƑǬʔ ʿÞʤLaeAʰ˼̃̌˕ʊAtLAEAʰɋM˅ʯ)Ȃʝʤ˸̈ˊ˿̐˅*ƉʝʤɞʊĽ

«ʝʤ?ʪʰƑhʫƘƕʰ˱̎˫ʕĽJʜ˂ʤʚʬʔʿʼʊCDC ʰɇ ¥ʱ ɨÉʯƚuèʕɺʐʚʬʕʑʔʕʒˁʋʚʰʾʑʯʊƑhèA. alternataʱʊɺÐʯ.

§ʜ˂ʤ EC ʬʊʬʪʼ̆˭̐˔ʮɋMʊʟʮ˄ʦ HST Ƈtñɇ ¥ʕÑʟˁ CDC ˅.ÿʝʤĿʻʪŽâƕʮ˗˯́ŀȻ˅ĦʝʪʐˁʬʰǙʒʕʜ˂£ʻʪʐ ˁʋ

ɿ A. alternata ʱʚ˂ʸʫʰƠưʫʊɇ ¥ˣ̐˗˦˨ˉ̎˕Řʕŀƻʜ˂ʪʓˀʊŃ

Alternaria alternata ļ ļżƑhǬʯʓʘˁ