58 AAL-TA
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
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
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
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
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
64
a ɿɿ
b
26ɿ A. alternatả̎˙Ƒhʓʾʳˊ˥˙ƑhʰLaeAKOĸ (AaLAEAKOĸ ʓʾʳAsLAEAKOĸ) ʰHPLCȌİ
a; AaLAEAKOĸʰHPLCȌİ IFO8984; ̉̎˙ĖŵǭǮƑǬ H3ʓʾʳH5; AaLAEAKOĸ b; AsLAEAKOĸʰHPLCȌİ NAF8; ˊ˥˙ɾĖƑǬ
H16ʊH20ʓʾʳH25; AsLAEAKOĸ
65
27ɿ AsLAEAKOĸʰƑhèĽ«ʓʾʳŏDŽƇƈǝĽ«
Ǯʱínèˊ˥˙ƭƖÁ16r˅*Ɖʝʤʋ NAF8; ˊ˥˙ɾĖƑǬ
H16ʊH20ʓʾʳH25; AsLAEAKOĸ
; ƑhèĽ«
1ʀ1051/mlʯȞȂʝʤǜ¥ðŲŤ˅*Ɖʝʤʋ ; ŏDŽƇƈǝĽ«
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)ʋ ʰ¬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
67
ƕɇ ¥ʰŅǝȌİ˅˞́̐˟ʯǹʑʚʬʕqǝʫʏˁʋʸʤʊ˪˿˪Ƒhʫʏˁ ǫĵƑǬʱʊCDC ʰ?ɋMéʓʾʳ˫̈˶˪˗˯́ȌİʔʿÞʤʶʷ?ʪʰ˗˯
́éʕQƉqǝʫʏˁʋʢʚʫʊīƠưʫʱʊ°ŽƏƕ AAL ŏDŽƇtñɇ
¥ʫʏˁALT˔̈˞ˣ̐ɇ ¥ʮʭCDCʰɇ ¥ʬECʯÑʝʊA. alternata ʕAȹʝʪ.§ʟˁɇ ¥˅˗˯́é˅ʯĽDžʝʊʢ˂ʿɇ ¥ʰŅǝȌİ˅
ǹʧʤʋ
CDC@)6;$
ALT@
ɿ CDCʰ?ɋMéʔʿALT˔̈˞ˣ̐ʱCDCʯ2ˠ˦˪§ʝʪʐˁʚʬʕƣ
ʜ˂ʪʐˁʋALT˔̈˞ˣ̐ʰʑʦ8ɇ ¥ʰŅǝʱěʯĝʿʔʬʮʧʪʐˁʋīƠ ưʫʱʊŋˀʰ5ɇ ¥ALT5ʊ6ʊ10ʊ11ʓʾʳ12ʰŅǝȌİ˅ǹʧʤʋʚ˂ʿɇ ¥ ʱ ʢ ˂ ʣ ˂ ABC transporterʊshort chain dehydrogenase/reductaseʊfatty acyl-CoA synthetaseʊphytanoyl-CoA synthetaseʓʾʳpeptide synthetase˅˘̐˫ʝ ʪʐˁʬĆūʜ˂ʪʐˁʋALT5ʊALT11 ʓʾʳ ALT12 ʰÙȩȯĉʫʱʊʢ˂ʣ˂
500 ʰÙȩȯĉ&ʕÞʿ˂ʤʕʊ˯˦˔ˈˋ˪ (KO) ĸʱÞʿ˂ʮʔʧʤʋ ALT11ʓʾʳALT12ʱ˔̈˞ˣ̐Dʯ2˘˵̐§ʟˁʤʻʊ˗˯́Dʯ4˘˵̐§
ʟˁʋʝʤʕʧʪʊɊʰ˘˵̐˅ơʟˁʚʬʱqǝʫʏʧʤʕʊÐʰÙȩȯ ĉʫ?ʪʰ˘˵̐˅ơʟˁʚʬʱƢƁƕʯɢʫʏʧʤʰʔʼʝ˂ʮʐʋĥȵʊA.
alternata ʫʱˣ̎˝ˌ̉̎Ƒhʫ RNA ˚ˊ̋̎˜̎˕ŘʕƢƲʜ˂ʤʚʬʔʿ
(Miyamoto et al. 2008)ʊʚ˂ʿɇ ¥ʯʨʐʪʱʊ˚ˊ̋̎˜̎˕ŘʯʾˁŅǝȌİ ʕĦ\ʥʬç˄˂ˁʋALT5ʯʨʐʪʱʊKOĸʕÞʿ˂ʤtʊʢʰ?ʪʫCDCʕ ǞǭʝʪʐʤʋʚʰʤʻʊALT5 ʰʹʰŅǝ˅ȌİʟˁʚʬʕJįʮʔʧʤʋÜʊˣ
̐˗˦˨ˉ̎˕ʰ$Ǖʮʭ˅ʒʪÙȩȯĉ˅ǹʑäȅʕʏˁʋ
ɿ ALT6 ʰ KOĸʱʊAALŏDŽƇƈǝʓʾʳƑhèʕ¢˄˂ʪʐʤʋALT6 ʱ˶̃
˭˜̎Ƈtñɇ ¥ (FUMɇ ¥) ʯʓʘˁFUM13 ʬƚuèʕɺʗʊshort chain dehydrogenase/reductase˅˘̐˫ʝʪʐˁʬĆūʜ˂ʪʐˁʋFUM13ʰŅǝȌİʯʾ ˀʊFum13pʱ˶̃˭˜̎ʯʓʘˁC3ʰ˖˪˅œɍʯɈ:ʟˁʚʬʕƣʜ˂ʪʐ ˁ (Butchko et al. 2003, Yi et al. 2005)ʋ ʚ˂ʿʰʚʬʔʿʊALT6ʱ AALŏDŽʯʓ ʘˁC2ʰ˖˪˅œɍ_ʟˁ reductaseʫʏˁʬĆūʜ˂ˁʋ
ɿ ALT10ʰKOĸʱʊAALŏDŽʰ˵̐˔ʱ¢˄˂ʪʐʤʕʊęȈ˵̐˔ʕʹʿ˂ʤʋ ʚʰ˃Ť˅*Ɖʝʊ˱ˊˏˈ˦ˠˊ˅ǹʧʤʬʚ˃ʊŊʱʹʿ˂ʮʔʧʤʚʬʔʿę Ȉ˵̐˔ʱʊ°ʯ´ʝʪŏèʕʮʐʚʬʕƣʜ˂ʤʋƑhèĽ«ʯʓʐʪʼKO ĸʱƑhè˅¢ʧʪʐʤʋʸʤʊʚʰęȈ˵̐˔ʰȩɑȌİʯʾˀʊAAL ŏDŽʰ˪̉
68
ː̊˽̎ɍʕ¢˄˂ʤpentolamineʬuʰȩɑ˅ƣʝʤʚʬʊʓʾʳ FUMɇ ¥
ʯʓʘˁALT10˼̃̌˕ʊFUM10ʰŅǝȌİʯʓʐʪʼuŁʰNJijʕÞʿ˂ʤʚʬ
ʔʿ (Butchko et al. 2003)ʊīɇ ¥ʰ˘̐˫ʟˁˣ̎˲˔ȩʰŅǝʱʊ˪̉ː̊˽̎
ɍʴʰCoAʰZʬǙʒʿ˂ʤʋ
AaTRF1-4
ɿ ȯFȞƹ¥˅˘̐˫ʟˁʬĆūʜ˂ˁTRF1-4ʱCDCʫȇJʜ˂ʤɇ ¥ʫ ʏˀʊTRF1ʱALT˔̈˞ˣ̐Dʯ§ʟˁʋʚ˂ʿ4ɇ ¥˅ʢ˂ʣ˂KOʝʊAAL ŏDŽƇƈǝʓʾʳƑhè˅Ľ«ʝʤʕʊɐƇĸʬŐʵĦëʮÅʱʹʿ˂ʮʔʧʤʋ TRF1ʰKOĸʯʓʐʪALTɇ ¥ʰƒƃ˅ɐƇĸʬŐȱʝʤʬʚ˃ʊALT1ʰ˱̎
˫ʕ_ʝʪʐʤʋʸʤʊʶʬˆʭʰALTɇ ¥ʕɐƇĸʯŐʵʊ˜˕ˬ̊ʕÖʗʮ ʧʪʐʤʋʝʔʝʮʕʿʊ ʰ¬ɹʫʱʊ̉ˈ̊ˣˊ́PCRʰʾʑʮ«ɑ˅ǹʧʪʐ ʮʐʤʻʊÜʊʼnƢʯƒƃɑ˅ȞʵˁäȅʕʏˁʋTRF1 ʱʊ̉̎˙Ƒhʓʾʳ ˊ˥˙Ƒhʫ˼̃̌˕ʕȇJʜ˂ʪʓˀʊ̉̎˙Ƒhʫʱ AM ŏDŽʰƇƈ˅ȥ ʯRßʝʪʐˁʚʬʕƣʜ˂ʪʐˁʋĚʊˊ˥˙ƑhʫʱʊAF ŏDŽʰƇƈʯ Úɪ˅jʷʜʮʐʚʬʕƣʜ˂ʪʐˁ (ċīƦ/)ʋʚ˂ʿʰNJijʔʿʊTRF1 ʱŏ DŽɑʰ_ʯʱǤʿʮʐƬÐʰ˄ʠʔʮƒƃɑʰRß˅ûʧʪʐˁqǝèʼǙʒ ʿ˂ʤʋʸʤʊ ʱɷĮʕŘ̉˥̄̐˟ʰʹʫʏʧʤʤʻʊĮ˅ʒ ˁʚʬʫʊʚ˂ʿTRFɇ ¥ʰŅǝʕĝʿʔʯʮˁqǝèʼʏˁʋ
AaAVE1
ɿ avenasinaseʰ˼̃̌˕ʬĆ«ʜ˂ʤAVE1ʰŅǝȌİʰNJijʊƑhèʓʾʳAAL ŏDŽƇƈʯÚɪʱʹʿ˂ʮʔʧʤʋʸʤ˪˿˥̎Iƅʝʤǜ¥ʼɐƇĸʬuŁʯƒ ǪʝʤʋʸʤʊŘ̉˥̄̐˟ʫʰƒƃʕɨÉʯ%ʗʊuĮʫʼƒƃʕʹʿ
˂ʮʐʚʬʕʏʧʤʋʚ˂ʿʰNJijʔʿʊAVE1 ʱʊƑhèʯɗʝʪʐʮʐʊʏˁʐ ʱŅǝ˼̃̌˕ʕ˗˯́ʯ§ʟˁqǝèʕƣʜ˂ʤʋ¬ɞʊǫĵƑǬ˫̈˶˪
˗˯́˩̐ˣ˅*ʧʤ˼̃̌˕ʰĽDžʰNJijʊȃēʰ˼̃̌˕ʕȇJʜ˂ʪʐˁʋ ɿ ʊCDCʫALTɇ ¥ʰƑhèɗȼɇ ¥˅ȇJʟʚʬʱJįʮʔʧ ʤʋʝʔʝʮʕʿʊ ʰ¬ɹʱ AAL ŏDŽƇƈʯĦQʮĮʰʹʫƒƃȌİ˅ǹ ʧʪʐˁʚʬʔʿʊ°ǮʮʭʰĮʫEЬɹ˅ǹʑʚʬʫʊǽƃʰ_˅
ȇJʡˁqǝèʼʏˁʋ
EC@)6;$
AaGPA1
69
ɿ G ˣ̎˲˔ȩȃt&ʱʊʁʊʂʓʾʳʃ˚˷̆˭˦˪ʔʿŀñʜ˂ʪʓˀʊɊʔʿ ʰSű˅˜˕ˬ̊ʬʝʪǡȦȹn¯&ʔʿdžǜǡDɊʰn¯&ʴʬ Ƀʝʪʐˁ (Neer et al. 1995, Idnurm and Howlett 2001)ʋʚ˂ʸʫʯʊA. alternataʰˣ̎ˡ̉̎Ƒ hǬɿ ʓʾʳ̉̎˙ƑhʯʓʘˁGˣ̎˲˔ȩ〈˚˷̆˭˦˪˼̃̌˕ʰŅǝȌİ ʫʱʊīɇ ¥ʕǜ¥Ùñʯɗʟˁʚʬʕƣʜ˂ʪʐˁ (Wang et al. 2010, Yamagishi et al. 2006)ʋG ˣ̎˲˔ȩʁ˚˷̆˭˦˪˅˘̐˫ʟˁʬĆ«ʜ˂ˁ
AaGPA1ʰKOĸʱʊǜ¥ƇƈʕɐƇĸʯŐʵʊŨ¹ʝʪʐʤʋʸʤʊǬʢʑʕɐè
ĸʬʱƏʮʧʪʐʤʋʚ˂ʿʰNJijʔʿʊAaGPA1 ʱˣ̎ˡ̉̎Ƒhʓʾʳ̉̎˙Ƒ hǬʫzʜ˂ʤNJijʬuŁʊǜ¥ʰƇƈʯɗʟˁʚʬʕĝʿʔʯʮʧʤʋʸ ʤƑhèĽ«ʰNJijʊɐèĸʯŐʵʊAaGPA1KO ĸʫʱƑhèʕŨ¹ʝʪʐʤʚ ʬʔʿ (˩̐ˣŶʝ)ʊAaGPA1ɇ ¥ʰƑhèʴʰɗʕƣʜ˂ʤʋ
AaGPR1-3
ɿ G ˣ̎˲˔ȩAÛn¯& (GPCR) ʬĆ«ʜ˂ʤ AaGPR1-3ʰʑʦʊAaGPR3 ʰ KO ĸʰʹǽƃʯ_ʕʹʿ˂ʊʶʬˆʭʰǜ¥˚ˊ˟ʕƞ_ʝʊɝÙñʕĩ Źʫʏˁʼʰʕʗʹʿ˂ʤʋuğʯƑhX%ʕȘʻʿ˂ʤʋƚȁ¬ɹʯʾˀʚʰ ǽƃʱɐƇĸʬuƶʯàÈʝʤʚʬʔʿʊīɇ ¥ʱʊǜ¥ʰñŹʯɗʝʪʐ ˁqǝèʕƣʜ˂ʤʋAaGPR3ʼxʻʊʚ˂ʿ3 ɇ ¥ʱ GPCRʰKɳcyclic AMPˣˊ˸ʯKɳʜ˂ˁ (Lafon et al. 2006)ʋʚʰˣˊ˸ʰŅǝʱǬɳʫʱʶʬˆʭ
zʜ˂ʪʐʮʐʤʻʊ ʰNJijʔʿʊʚʰˣˊ˸ʰęʤʮŅǝʕƣʜ˂ʤʋ
AaSTE2
ɿ GPCR ʰʑʦ STE2 ˣˊ˸ʯ¾ʟˁɇ ¥ʱʊǬɳʫèK_ʯɗʝʪʐˁʚʬʕ
zʜ˂ʪʐˁ (Burkholder and Hartwell 1985, Kim and Borkovich 2004)ʋīɇ
¥˼̃̌˕ʰKOĸʱʊǽƃʯÚɪʕʹʿ˂ʮʔʧʤʋA. alternataʱɋʕǢŷ ƍʫʼ¬ɹ̋˺̊ʫʼȇJʜ˂ʪʐʮʐʤʻʊʯ¥ʰʑōÙñʕqǝʮĮʕ ȇJʜ˂ʤtʊīɇ ¥ʰŅǝʕȇJʜ˂ˁqǝèʕʏˁʋ
AtLAEAAsLAEA AaLAEA
ɿ A. nidulansʫȇJʜ˂ʤLaeAʱmethyltransferase˅˘̐˫ʝʊȃēʰŇȡƈ żƇtñɇ ¥˔̈˞ˣ̐˅Rßʝʪʐˁʚʬʕzʜ˂ʪʐˁʋAspergillus ¾ʫʱ
˿ˊ˘˪˒˜̎ʫʏˁˈ˶̈˪˒˜̎ʽʢʰTɸ&ʰ˞˨̉˕˿˪˜˞˥̎ʮʭ˅ʼnʯ Rßʝʪʐˁʚʬʕzʜ˂ʪʐˁʋʸʤʊLaeA ʱ velvetˣ̎˲˔ʬȃt&˅Ùñʟ ˁʚʬʕƝʿ˂ʪʐˁʋʸʥʱʧʖˀʬʱKʔʧʪʐʮʐʕʊʚ˂ʿȃt&ʏˁʐʱʢ
70
˂ʣ˂ʕ˳˞˪̎˅0ɶʟˁʚʬʫʊɇ ¥˔̈˞ˣ̐˅ʼnʸʤʱȥʯRßʝʪʐˁʬ Ǚʒʿ˂ʪʐˁɿ (Palmer and Keller 2010)ʋ
ɿ ī¬ɹʫʱʊ˫̈˶˪˗˯́˜̐˔ˍ̎˞˩̐ˣʔʿ˪˿˪Ƒh (AtLAEA)ʊˊ˥˙
Ƒh (AsLAEA) ʓʾʳ̉̎˙Ƒh (AaLAEA) ʰ 3 Ƒhʔʿʢ˂ʣ˂ LaeA
˼̃̌˕˅ȇJʝʊŅǝȌİ˅ǹʧʤʋ˪˿˪ƑhʰLaeA˼̃̌˕ʊAtLAEAʰKO ĸʱʊAAL ŏDŽƇƈǝʊƑhèʊǬƿñɔʊǜ¥Ƈƈǝʓʾʳ̂̈˭̎_ʮʭȃē ʰǽƃʯÚɪʕʹʿ˂ʤʋʸʤʊALT ɇ ¥˅xʺ CDC ʰɇ ¥ʰƒƃ˅̉
ˈ̊ˣˊ́PCRʫĽȏʝʤNJijʊKOĸʱʊ?ʪʰALTɇ ¥ʰƒƃʕŨ¹ʝʤʋ ʸʤʊALT ɇ ¥ʰ˔̈˞ˣ̐Dɇ ¥ʼƒƃʕŨ¹ʝʪʐʤʋĚʫʊ˔̈˞
ˣ̐Dʯ§ʟˁ metallo protease ʱƒƃɑʯ_ʕʹʿ˂ʮʔʧʤʋmetallo proteaseʱCDCʰPʰòʯʼ§ʝʊ̋˺̊ʫʼʐʯƚuèʕɺʐʚʬ ʔʿʊ˔̈˞ˣ̐ʰmetallo protease˅ĽJʝʪʐˁqǝèʕʏˀʊ2ɇ ¥ɖʫƢ
¬ʯƏʮˁɯ˅˸̈ˊ˿̐ʬʝʪ*ƉʝʊEÐĽȏʟˁäȅʕʏˁʋ ʰƒƃ ȌİʫʊAtLAEAʱALT˔̈˞ˣ̐˅ʼnʯRßʝʪʐˁʚʬʕƣʜ˂ʤʋALT˔̈˞
ˣ̐ʱCDCʰ˜̐˔ˍ̎˞Ȍİʔʿʊ˚˷˨̌̂ˈɯʯ$Ǖʝʪʐˁʚʬʕƣʜ˂
ʪʐˁʋA. nidulansʰLaeAKOĸʰȌİʫʼuŁʯ˚˷˨̌̂ˈɯʰ˔̈˞ˣ̐˅
Rßʝʪʐˁʚʬʕzʜ˂ʪʓˀ (Shwab et al. 2007)ʊƏʮˁǬʯʓʐʪʼRßʰ ʅ$Ǖ\ijʆʕAȹʝʪʐˁʚʬʕƣʜ˂ʤʋ
ɿ Ť&ʯʾˀǬƿ˅ŐȱʝʤɞʊKO ĸʱɐƇĸʬʱƏʮˀʊʶʬˆʭ̂̈˭̎_
ʝʪʐʮʔʧʤ (˩̐ˣʮʝ)ʋʸʤʊ̂̈˭̎Ƈtñɇ ¥ʱ5.7 MbʰĶǩ&
ʯÑʝʪʐˁʚʬʕzʜ˂ʪʐˁ (Akamatsu et al. 1999, Akagi et al. 2009a, b)ʋ ʝʤʕʧʪʊAtLAEAʰRßʱCDCʰʹʮʿʠʊʰĶǩ&ʯʼÚɪ˅ʒˁʚʬʕ ƣʜ˂ʤʋ
ɿ AsLAEA ʰ KO ĸʫʱʊAF ŏDŽƇƈǝʓʾʳƑhèʕŨ¹ʝʤʋʸʤʊAaLAEA ʯʓʐʪʼAMŏDŽƇƈǝʕŨ¹ʝʤʋ ʊƑhèĽ«ʱJįʮʔʧʤʕʊŏDŽ ɑʰŨ¹ʯʾˀƑhèʕ%ʟˁʚʬʔʿ (Harimoto et al. 2007)ʊKOĸʼƑhèʯ ÚɪʕJˁʚʬʕǙʒʿ˂ʤʋ
ʰNJijʔʿʊLaeAʰ˼̃̌˕ɇ ¥ʱʊA. alternataʯʓʘˁƑhʯɗ-ʮ ʗHSTƇtñ˅ʼnʯRßʝʪʐˁʚʬʕƣʜ˂ʤʋÜʊȖdžʮƒƃȌİ˅ǹʑ
«ʫʏˁʋ
ɿ ī¬ɹʫʱʊ˫̈˶˪˗˯́é˅ʯěƝʰƑhèɗȼɇ ¥ʰ˼̃̌˕˅ĄDž ʝʊŅǝȌİ˅ǹʧʤʋʢʰNJijʊ˗˯́éʕʮʐžîʫʱʊ˔̌̐˭̎˕ʕɢ ʥʧʤGPCRɇ ¥ (AaGPR1-3ʓʾʳAaSTE2) ʮʭʗʰɇ ¥˅˔̌̐˭̎˕
ʟˁʚʬʕqǝʬʮʧʤʋ QƉʝʤ˗˯́éʱʊª?ʮ˗˯́ȌİʔʿÞʿ˂
71
ʤ˩̐ˣʫʱʮʔʧʤʕʊŅǝȌİ˅ǹʑʰʯʱ`KʫʏʧʤʋPKS ʽ NRPS ʮʭ ɨÉʯɔʐɋMʰtʊª?ɔʕÞʿ˂ʮʐʚʬʕʏˁʕʊɊKɋM˅QƉʝʪʼ KO ¬ɹʱqǝʫʏˁʤʻʊŅǝʕɏȅʫʏˁʔʭʑʔʰOĘʯʱ˫̈˶˪Ȍİʰ˩
̐ˣʫ`Kʫʏʧʤʋ
ɿ ʊǫĵƑǬʯʓʘˁ 16 ɇ ¥ʬ̉̎˙ʓʾʳˊ˥˙Ƒhʯʓʘˁ AtLAEA ʰ˼̃̌˕ʰ2ɇ ¥ʰȎ18ɇ ¥ʰKO¬ɹ˅Ȕʹʤʋ3ɇ ¥ʯʨʐʪʱKO ʟˁʚʬʕJįʮʔʧʤʕʊ15ɇ ¥ʰKOʯñYʝʤʋʸʤʊʚ˂ʿ15ɇ ¥ʰʑ ʦʊ6 ɇ ¥ʕƑhèʯɗʟˁʚʬʕƣʜ˂ʤʋī¬ɹʯʾˀȇJʜ˂ʤʊLaeA
˼̃̌˕ʫʏˁ3ɇ ¥ʱʊƏʮˁƑhɖʯʼɗ˄ʿʠʊʢ˂ʣ˂ʰ HSTƇƈ˅
ʼnʯRßʝʪʐˁʚʬʕƣʜ˂ʤʋʚ˂ʿɇ ¥ʱɨÉʯƚuèʕɺʐʚʬʔʿʊī ɇ ¥˅ŃƕʯʝʤDzUʮʭ˅QƉʟˁʚʬʫʊȃēʰƑŒʯ´IJįˁĜʕįˁ ʔʼʝ˂ʮʐʋ
72 Ƶ
Ƶ 4 Ƴ ɿ ° Ž Ə ƕ AAL ŏ DŽ Ƈ t ñ ɇ ¥ ˔ ̈ ˞ ˣ ̐ ʰ ɨ Ƒ h è A.
alternataǬʴʰ·>
Ƶ1ƹɿ Ïȟ
ɿ A. alternataʰƑhǬʱ HST˅ƇƈʝʊŽ«ʰ°ʯƑŒ˅Ôʖȫʚʟʋǫĵ ƑǬʊ̉̎˙ĖŵǭǮƑǬʓʾʳˊ˥˙ɾĖƑǬʱ HST ʫʏˁ AALʊAM ʓʾʳ AF ŏDŽ˅ʢ˂ʣ˂ƇƈʝʊŽ«ʰƭʯƑŒ˅Ôʖȫʚʟ (Hrimoto et al. 2007, Johnson et al. 2001, Johnson et al. 2000a, Johnson et al. 2000b, Hatta et al. 2002)ʋʚ˂
ʿŏDŽʰƇƈʯʱē`1ʔʿʮˁƇtñɇ ¥ʕɗʝʪʐˁʚʬʊ˔̈˞ˣ̐_ʝ ʪʐˁʚʬʕzʜ˂ʪʐˁʋȵÍʰƠưʔʿʚ˂ʿA. alternataʰHSTƇtñ˔̈
˞ˣ̐ʱ1.8 MbʰĶǩ&ʯÑʝʪʐˁʚʬʕĝʿʔʬʮʧʪʐˁʋ
ɿ 3ƳʫʼȶʵʤʾʑʯʊǫĵƑǬʰ13ʔʿʮˁAALŏDŽƇtñɇ ¥˔̈˞ˣ̐
(ALT˔̈˞ˣ̐) ʱ1 MbʰCDĶǩ&ʯÑʝʪʐˁʋǫĵƑǬʰCDĶǩ&ʱ
˜̐˔ˍ̎˞ȌİʔʿuǠĶǩ&ʫʏˁʚʬʊALT ˔̈˞ˣ̐ʱīĶǩ&ʰĻʯ 1
˘˵̐ʠʨʰȎ 2 ˘˵̐Ñʝʪʐˁʚʬʕĝʿʔʬʮʧʪʐˁʋʚ˂ʸʫʰƠ ưʔʿʚ˂ʿ 13 ʰɇ ¥ 8 ɇ ¥ʕŏDŽƇƈʓʾʳƑhèʯɗʝʪʐˁʚʬ ʕƣʜ˂ʪʐˁʋʜʿʯʊ̉̎˙ƑhǬʫ AM ŏDŽƇtñɇ ¥ (AMT) ʕ 1.1-1.7 Mb ʰ (Johnson et al. 2000, Johnson et al. 2001)ʊˊ˥˙ƑhǬʫʱAFŏ DŽƇtñɇ ¥ʕ 1.05 Mb ʰ CD Ķǩ&ʯÑʝʪʐˁʕĝʿʔʬʮʧʪʐˁ (Hatta et al. 2002)ʋ
ɿ ØƠưʫʱTʊǫĵƑǬʰCDĶǩ&ʕɨƑhèA. alternataǬʯœÌƫ]
ʟˁʚʬʯʾˀʊęʤʮǫĵƑǬʕșƇʝʤʬʟˁʊCD Ķǩ&ʰœÌƫ]Ȝ˅ȓĝ ʝʪʐˁ (Akagi et al. 2009a, b)ʋĚʫʊALT˔̈˞ˣ̐ɇ ¥ʰʹ˅ɨƑhèA.
alternataǬʯ·>ʝʤtʊʢʰ·>Ǭʱ AAL ŏDŽƇƈǝʕÞʿ˂ˁʔwʔʊʸ ʤʊ·>Ǭʕ AAL ŏDŽ˅ƇƈʫʖʤtʊƑhèʼȨʜ˂ˁʭʑʔʱĝʿʔʬʮ ʧʪʐʮʐʋĚʊǫĵƑǬʰŏDŽƇƈǝʓʾʳƑhèʰ.ÿʯʱALT˔̈˞ˣ̐
ɇ ¥ʬʱPʯÉĶǩ&ʯÑʟˁɇ ¥ʕɗʝʪʐˁqǝèʼǙʒʿ˂ˁʋ ɿ TʾˀALT˔̈˞ˣ̐˅·>ʟˁĚŘʬʝʪʊBAC˔̌̐̎˅˺˔ˣ̐ʯƉʐˁĚ ŘʕǙʒʿ˂ʪʐʤʋ100 kbƬʰALT˔̈˞ˣ̐ʰ?ɔʕ·>ʜ˂ʪʐˁBAC˅ɨ ƑhèǬʯ·>ʟ˂ʲʊALT ˔̈˞ˣ̐·>Ùȩȯĉ&ʕ)Jʫʖˁʚʬʕêʜ
˂ʤʋĚʊBAC ʱ˚ˊ˟ʕʖʐʚʬʔʿʊƽȂʰ)ľ ʯLĘʜ˂ˁʚʬʕʐ ʚʬʊʸʤ˘˵̐ēʕ¹ʮʐʚʬʔʿɑʰDNA˅Þˁʚʬʕɢʫʏˁʚʬʮʭʔʿʊ Ùȩȯĉ&˅ÞˁʚʬʱJįʮʔʧʤʋ
73
ɿ ʢʚʫīƠưʫʱʊƼĞʯɑʰDNAʕÊqǝʮlong PCR˅*ƉʝʊALT˔
̈˞ˣ̐˅4ĘŻʯKʘʪÊʝʤʋʚ˂ʿʰPCRƈż˅ɨƑhèA. alternataǬ ĸʯ·>ʟˁʚʬʫʊʚ˂ʸʫʯȇJʜ˂ʪʐˁAALŏDŽƇtñɇ ¥ʫAALŏ DŽʕƇƈqǝʫʏˁʔʭʑʔ˅Ľȏʝʤʋ
74 Ƶ
Ƶ2ƹɿ ĬėʬĚŘ
1: +ȔǬ
ɿ AALŏDŽ˅ƇƈʟˁƑhèA. alternataǬʊAs-27ʓʾʳɨƑhèA. alternata ǬʊO-94Ǭĸ˅*Ɖʝʤʋ
2: DNAĎ)
ɿ DNAʽRNAʰúJʊƼcʮĎ)ʱƵ2ƳʯÝʐǹʧʤʋ
3: ALT˔̈˞ˣ̐ɇ ¥·>ĸʰ)ñ
ɿ As-27ʰDNA˅˨̎˸̋̐˪ʯALT˔̈˞ˣ̐˅4ĘŻʯKʘʊPCRʯʾˀÊʝ ʤ (28)ʋÊʯʱKOD FX (TOYOBO) ˅*ƉʝʤʋuŁʯ˰ˊ˕̌˿ˊ˜̎ǚ èɇ ¥ (hph) ʯʨʐʪʼ˸̈˞̀˫DNAʰpAN7-1˅˨̎˸̋̐˪ʯÊʝʤʋʚ
˂ʿ5ƭɳʰPCRƈż˅ʊO-94Ǭĸʯ2-10ʯÝʐco-transformationʝʤʋʤʥʝʊ ʱ˸̌˪˸̈˞˪80 μl (1.25ʀ1081/ml) ʯ´ʝʊ5ƭʰPCRƈż˅ǁ50 μlƉ ʐʤʋÙȩȯĉʯʾˀ 94H2 ǬĸʕÞʿ˂ʤʕʊʚʰǬĸʰ ALT10 ʯņ¢ʕȇʿ˂
ʤʋʢʰʤʻʊALT10 ʰ ORF (Alt10AF/Alt10-ComR) ʓʾʳ˝ˌˮ˨ˉ˜̎ǚèɇ ¥ (npt) ˅PCRʯʾˀÊʝʊȑʬuŁʮĚŘʫ94H2ʯco-transformationʝ ʤʋ
4: ALT˔̈˞ˣ̐ɇ ¥·>ĸʰ˞˔̉̐˭̎˕
ɿ 2-10 ʯÝʧʪɆøʜ˂ʤ˰ˊ˕̌˿ˊ˜̎ǚèǬĸ˅ɣ¥̋̎˝Řʯʾˀ DNAú JÜʊQuick Taq (TaKaRa) ʯʾˀʢ˂ʣ˂ALT˔̈˞ˣ̐4ĘŻʰɊʫʏˁALT1, ALT2, ALT3ʓʾʳALT10ʰDɊɯ˅PCRʯʾˀÊʝʤʋɣŒś]ʫ˱̎˫˅
ƢȘÜʊ3ī˱̎˫ʕĽJʜ˂ˁǬĸʯʨʐʪʰʹʊEÐʊŤ&ƱDŽŘʫDNA
˅úJʝʊPCRʯʾˀALT1-13 ʰDɊɯ˅ÊÜʊɣŒś]ʫ˱̎˫ʰĦŶ˅Ƣ Șʝʤʋ
5: ƒƃȌİ
ɿ ALT˔̈˞ˣ̐ʕ·>ʜ˂ʤʬǙʒʿ˂ˁǬĸʰƒƃȌİ˅ǹʧʤʋ2-4-1ʯÝʐʊ RNA ˅úJÜʊȸȯFkæ˅ǹʐʊALT ˔̈˞ˣ̐ɇ ¥ʢ˂ʣ˂ʰDɊɯ˅
Êʟˁ˸̈ˊ˿̐ʫPCR˅ǹʧʤʋ
75 6: DNA˜˜̐˔ˍ̎˞
ɿ sǬĸʰ MAT1-1-1 ɇ ¥ʰɋMʰȌİʱĸÓƥ˶ˇ˞˿˦˔ʰnȐ DNA ˜̐˖̎˞Ȍİ˅QƉʝʤʋÞʿ˂ʤɋMʱ GENETYX ˅*Ɖʝʊˈ̈ˊ̎̂
̎˪˅ǹʧʤʋƌ8ʰZÄʯʱAdobe Photoshop Elements 8˅*Ɖʝʤʋ
76 Ƶ
Ƶ3ƹɿ NJij
1: ˘̎˞˪̈˔˪ʰ)ñ
ɿ ?ALT˔̈˞ˣ̐ʰɇ ¥˅·>ʟˁʤʻʰ˘̎˞˪̈˔˪ʰŀƻ˅ȔʹʤʋPCRʫ ALT˔̈˞ˣ̐ʰ?ɔʓʾʳALT3-11ʰÊ˅ǹʧʤʕʊPCRVƈżʱÞʿ˂ʮ ʔʧʤ (˩̐ˣʮʝ)ʋʢʚʫʊALT˔̈˞ˣ̐ʰALTɇ ¥ɊKʰʹ˅PCRʫÊ ʝʊʔʨ·>ʟˁĘŻʕ¹ēʯʮˁʾʑʯ˘̎˞˪̈˔˪˅ŀƻʝʤ (28)ʋ
2: ·>ĸʰ)J
ɿ 5-2-3ʯÝʐʊAs-27ʰDNA˅˨̎˸̋̐˪ʯʝlong PCR˅ǹʧʤʋLong PCR Êƈżʊ4ĘŻ˅ɣŒś]ʯʾˀ˚ˊ˟˅ƢȘʝʤNJijʊ13.6 kb, 12.9 kb, 15.8 kbʓ ʾʳ17.7 kbʥʧʤʋǎʐʪ5-2-3ʯÝʐhphʰÊʼǹʧʤʋʚ˂ʿ5ʨʰPCR Êƈż˅2-10ʓʾʳ2-16ʯÝʐʊO-94Ǭĸʯco-transformationʝʤʋ
ɿ 5-2-4ʯÝʐʊALT1-13ɇ ¥ʰDɊɯ˅PCRʫÊʝʊ˰ˊ˕̌˿ˊ˜̎ǚè Ùȩȯĉ&136 Ǭĸʰ˞˔̉̐˭̎˕˅ǹʧʤʬʚ˃ʊ2 Ǭĸʫ?ʪʰ˱̎˫ʕĽJ ʜ˂ʤʋʚ˂ʿ2Ǭĸ˅94H2ʓʾʳ94H36ʬʝʊɘʰ¬ɹʯƉʐʤ (29a)ʋ
3: ƒƃȌİ
ɿ 2-4ʓʾʳ5ʯÝʐʊAs-27ʓʾʳ94H2Ǭ˅Ř̉˥̄̐˟ʫ7ĜɖɧǕɷʝʊ RNA˅úJÜʊcDNA˅ǹʐʊʚ˂˅˨̎˸̋̐˪ʬʝʪALT1-13ʰDɊɯ˅Ê ʟˁ˸̈ˊ˿̐ʫPCR˅ǹʧʤʋêȹˀAs-27ʰ˱̎˫ʬu˚ˊ˟ʰ˱̎˫ʕ94H2 ǬĸʫĽJʜ˂ʤʋĚʊȉĸʬʝʤO-94Ǭĸʕ.ÿʝʪʐʮʐMSASɇ ¥ʰD Ɋɯ˅Êʝʤ˱̎˫ʱǬĸʫĽJʜ˂ʮʔʧʤ (29b)ʋ
4: 94H2Ǭĸʰ HPLCȌİʓʾʳ LC-MSȌİ
ɿ 2-15-1 ʓʾʳ 2-15-3 ʯÝʐʊɷ˃Ťʔʿ AAL ŏDŽʰúJ˅ǹʧʤʋʢ˂ʿʰ HPLCȌİ˅ǹʧʤNJijʊ94H36ǬĸƊįʰɷ˃ŤʔʿʱƘƲʧʤ˵̐˔ʕĽJ ʜ˂ʮʔʧʤĚʊ94H2 Ǭĸʰɷ˃Ťʔʿʱ AAL ŏDŽʬʱƏʮˁ̉˨̎˜̇̎ˣ ˊ́ʯ˵̐˔ʕȇʿ˂ʤʋʚ˂ʱTʊALT10 ʰ KOĸʫÞʿ˂ʤ˵̐˔ʬ̉˨̎˜̇
̎ˣˊ́ʕuʥʧʤ ( 30a)ʋLC-MS Ȍİ˅ǹʧʤNJijʊʚ˂ʿʰ˵̐˔ʱ AAL-pentolamineʬuʥʧʤ (30b)ʋ
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