味噌用麹菌の酸性ホスファターゼ遺伝子に関する研究

全文

(1)Title. 味噌用麹菌の酸性ホスファターゼ遺伝子に関する研究( 本文 (Fulltext) ). Author(s). 安田, 庄子. Report No.(Doctoral Degree). 博士(農学) 乙第144号. Issue Date. 2014-09-24. Type. 博士論文. Version. ETD. URL. http://hdl.handle.net/20.500.12099/50394. ※この資料の著作権は、各資料の著者・学協会・出版社等に帰属します。.

(2) ȽƘɒēà2ĨƄoYkI][ǙĬ 1À#DîÑ. ǹ. ÄȠư´ư´ɩĒǽ´îÑ ǚ Ť Ĭ.

(3) Ʉĸ. šɫ--------------------------------------------------------------------------------------------------------1 Ʊŧ ȽƘɒēà1DĐțǝǙĬƟǧƛ½ä2²ɜ Šõ--------------------------------------------------------------------------------------------8 ŀðĢɟA6ŀðȴȵ----------------------------------------------------------------11 ŀðéA6ċĥ----------------------------------------------------------------------15 ɓɇ-------------------------------------------------------------------------------------------21 Ʊŧ ȽƘɒēà1DĨƄoYkI][ A ǙĬ aphA. 2Ǿ¥. AphA 2ŠƄĿ2¤ɀ Šõ-------------------------------------------------------------------------------------------22 ŀðĢɟA6ŀðȴȵ----------------------------------------------------------------23 ŀðéA6ċĥ----------------------------------------------------------------------27 ɓɇ-------------------------------------------------------------------------------------------38 Ʊŧ ȽƘɒēà2Ňɓ0ĨƄoYkI][ C ǙĬ aphC. 2ǫǒ. AphC 2ŠƄĿ2¤ɀ Šõ-------------------------------------------------------------------------------------------40 ŀðĢɟA6ŀðȴȵ----------------------------------------------------------------40 ŀðéA6ċĥ----------------------------------------------------------------------48 ɓɇ-------------------------------------------------------------------------------------------64 Ơ¶A6éɫ----------------------------------------------------------------------------------------65 ńļ-------------------------------------------------------------------------------------------------------68 ɒȨí-------------------------------------------------------------------------------------------------69 ´ɫȨ2Âƙ.0D´řɫȨA6Åȅȗ´řɫȨ-------------------------------------76.

(4) šɫ. ȽƘ3ǵȺ2ǙǢǔ0ȅĎǉȽŵȚ-CũɊŌȽɭŽ.ǧɑ1ôġ? ǵȺ2ɟə1ç%0ǉȽɟ-DȽƘ3Ğá-3&2ŸǉȽƄ(-0 ìćGơź#DÆǼƄ1,?ǂɄ. EĆƂª-ɘɒ. E,DȽƘ1. 3Źƫ1Ȕż0ɔƅȦ-DȔżJsgĨ@®ŋj]s}ɣȷȟ1Á<E, D´ǔ13ȽƘŔ2Əň1ACH@Ƈ·Œ¼ș2yYSǑ#D.ɀ B1. E,CĲȸĨ@nm^bǠ&2Ňɓ0ÆǼƄƅȦ-D.ċBE,. D<'òȅĵû1A*,ōDìćȏ#0F)ȅH2ɕƃ2ĂǼ1+, ?ǂɄ. E,D B1ȽƘŔGƏň#DǵȺŵãƮ&2?2ɔƏň2¿ǘ. -əƝǔ0hx}YG?'B!Ż2ìć1Ãɐ#D.!,ïǋ *,ȽƘ2ťȐɡGăŪ. E,D!'. %D.3ǵȺł£&!,Ƃª1.*,ưȫÍŸ. .-D =&ȚĿȗĺÂś ȩƅ 23 ǹ 10 ê 31 ǵťȐŃǅĔĺƱ 11 đ 1AD.=& Ƚ Ƙ 2ǒÍ3 ưǤņ!3ưǤÏ6ȪȄǠ2ĖɣGŰŁ!'?21ȪȄ Ǡ2ĖɣGŰŁ!,"àGȁɔ!'?2G'?2Ȼ3ưǤGŰŁ!, "àGȁɔ!'?2ņ!3E1ȪȄǠ2ĖɣGŰŁ!'?2G'?21ŵ GĜĒ!EGȅĎ. %Ï6ŗƅ. %'?22)Ȉúãů2?21. ĝǡɣ ĝǡǡ=+Ï6ǡɣG ȢȽòɟ +7!Ł»ÕɣH7 Ǡ2ȧȼȻ3ǁŘǻŖȥÕũɊ'HȃſȦ¤ȥĎȲMQY&2ƩEB1ɣ #DŵȚGǧ". ǠG'?22)Ȉúãů2?2

(5) .. E,D. !. ǵȺ2ȽƘ2Ëó13ŠƒDøƯǀĕ2 ü

(6) G0ȅĎưǤŵȚ. 5!. D3 ũ

(7) ÕǴ2ǏnYaŵȚ 1óD.ǵȺ2øȨş13Ê E,Dǀĕ13 ȽƘ

(8) 1ɣĶ!'ÿ2Êɪ30<'ǰȨŻ2Ƈ·ƍ B3ǰȨķƯ1/HCGĩɒ!'ȽƘ2A0ŵȚ*'.F*,D. 1.

(9) $E1!,?ôġ2ǵȺ2ȽƘ3ǀĕ2&E.3ƊƣÌř.ȚĿãƮɒǜư 0*,D.BǵȺ-ǬĻ1ƣBE,'?2-D ǀɅƆĈ, 1982 ǀĕ@ǞǱJXJ2ĕ-3ĖȥȧGſ-ɨ*'?21SvgYOj Rhizopus Ʀ. GƇ. %' ?)ē

(10) Gɘɒ#D&E1Ƭ!ǵȺ2ȽƘ-3Ű!'Ȫ ȪȽƘ. @ưȄ ȄȽƘ A6ưǤ ǤȽƘ 1ēà Aspergillus ³!á2Ǝŋ!,ȁɔ !' 4Bē

(11) Gē.!,ĩɒ#D.Ğư2ǫǆ-D ǣȕ, 1987 ã¬ ". Aspergillus oryzae ÜÔ¦µµìÉ]¤Ü`~®+3#. Ü`~®Ü`~®9R?ME>H4?>µó l'00-!~ ®"ã¬!Ò~¥ îæf2_ƊēąǓ2ýȪȽƘ@ȄȽƘ2ūĒ3 ȪēȄē1Ű!'ưǤ.ſGǤȽƘ2ūĒ3ǤēY]]+Ȧ¤Â Ŀ-D'>ſ2=G,ƁêƁǹŗƅ#D.1AC ñȽƘ

(12) .ù4 EDƇ2ȽƘƊƣ. EDƊēǀA6ŗƅǀ13ȽƘɒēà A. oryzae !&. òɟ@Ɗƣôū2ǩâ1ɍɖ#DƭƄǶĨà Tetragenococcus halophila @ƭƄ ĎȲ Zygosaccharomyces rouxiiCandida versatilis 0/2ɑ0ȓƇȥȤĒǔ1ģ ɒ!űƣŞǫɌ2ȽƘ2Ƚ@ȢďȽȝɐ. EDȽƘ2ȚĿGȗ#Ņǝ13 Ƚ. ďCŴ

(13) ÔBEDűƣŞ1A*,3ȽƘ2 ȽďCŴ

(14) 2ăŪGɄǔ .!,ŋē2l{}b@ǫǒ2ǶĨà@ĎȲGǖ#D0/2Ɓ2ąȞG!, D ôġǵȺ13Ɓȕǹ?2øɥĪG?+ŋēuOȤƁƨġ!Ǌǹ1F' Cŏœ¦ɠ!,'ēàà¹V{SWw}2ǀBȽƘ2ŋɣ@űƣŞ2ƊƣŮ ë1ĒF%,ēàȶĬGl{}b!ŋē.!,×Ò!,DŋēuO2ɥĪG Ʒ*,.Å1ľǇķƯ1Ō@ȽƘũɊƊƣ1 ē

(15) GȌȂ#D ?@!

(16) .!,·ɉ!,' ǮɠòőŎ, 1987 ȍB3ɋɠ0ē1JzOyƄ2Ƀ© Ň1 ǐ GŶCD.-Ʃ26@ĦàGǿŢ! B1ēàȶĬ2ƭÎƄGăŪ %D.ǫŊÌřG·ɒ!,ɋɠ0ē2ŋGƇħ!,'.ċBED&2ý. 2.

(17) ɀĹķƯ1ºǕȁƾGɒ'ēà2ƸɚŜƀȁɔȵ¨¬BǨǷ. EŜƀȁɔ. !,ǪBE'ēàȶĬGl{}b!' ŋē

(18) Ō@ȽƘũɊ0/2űƣ1ĩɒ EDA10*'. ȽƘɒēà1ɓÐ. ED EDȋǔ0ƄĿ.!,3ɮȊųƥǝƥɯm|`J. [ÛɰÆ§Ɗē1DǎȢ2'>ǀɂƺɂ-Dɱē2ďCɠ0 /ÔBEDǙǢǔ0ȽƘ2Ɗȵ+<CɃGɒ'ǕƖűƣ-3ē2ȚĿ 3ĩɒ#Dēà2 AC?ąū1DƊēƞģŮë2 1ưÜ. E,. '!!áƯǔƐȒGǨǷ!'ąū-3ƊēŮë2ƃþDǓǝǼ.0* ''>ēà2¦ɠȽƘ2ȚĿăŪ1ȉ. E@#0*'&-űƣuO. -31960 ǹęBĻƖȫ@ŻǭƖȫ ɈĠUV Ǡ 1ACɋɠēà2ŋ ƔȆGƉɢǔ1Č*,'!!2A0ŋƔȆȴȵ13&2Ǒ²ɛ@ ƔȆ¹2Șȴȵ²ɜ0/Ěǲ0ǘƪ'>ēà2Ą<!ƄĿ2=G¦ɠȝ ɐ#D.3ĳǲ2Ý-D21 ƂÈ1ǷCƇȿ´2źȯ3ǃ!ēà A. oryzae 2 Ƈȿ2Ɛåž

(19) -DƗUgtȀɦ Aspergillus nidulansAspergillus fumigatus Ê. 2005 ǹ1èǒ. E' Machida et al., 2005 A. oryzae 2Ugt¤Ƌ13\. xruȦɚó-ɅƇ¹1á.. EDŌɣƠĒîÑŞ2Ȯƨ¹ RIB40 ¹ĩɒ. E'&2ý Aspergillus nigerAspergillus flavusAspergillus sojae Sato et al., 2011. Aspergillus Ʀİůà2ƗUgtȀɦèǒ. E'&2éE<-ɀ². -0*'ŕɓ0ƽïŷ'1ǪBE'ɤ4E<-ēà A. oryzae 2m|`J [1À!,3ɇ 20 ŋɣƽBE,'UgtŪ13 126 ?2m|`J[ɑ ǙĬƨġ!,' Machida et al., 2005 <' Tominaga B3 A. oryzae RIB40 g 2JkxaQW}ƇĒƅǙĬov|TSxY] 46 kb GS|d}T!&2ǀ 2ǙĬ2ȡçɗ!,D1ACJkxaQW}GƇħ-0. Ɨ- D. Gĺ!' UgtŪ1Ʃ2JS`Kl0JkxaQW}ƇĒƅǙĬo v|TSxY]Ȁɦ0

(20) .ĺ. 3. EƗƄ2ěÓACɀ²10*'.

(21) Tominaga et al., 2006 &!,:;ǧķ1ēà2ǙĬƞģÌř2)E<ȑŬ1Ăɛ2~*'ǙĬ]U_`K}TÌřưăŪ!' Ũğ3Ʊŧ -Ś9D. 2A1ǽ´´Ƈ´Ǡ1,ŕɓ0 Aspergillus Ʀİůà 2Ugtŭȳā«. Eēà2ǙĬƞģÌřăŪ!'.3ǵȺ2űƣħÝ. 1.*,Ɗƣm|ZY2³ŷ@ŷ'0ìćÆǼƄGȝɐ!'ŷƊȚ«ȅGČŪư0ƕź-D.ċBED ǵȺ-3áǹťȐŃ2ɬĄȫ@ɘȭƄǍÐ2ÜGōñȽƘ2ĭū3Ŗţ !±ĨäǉȽɟGǖ!'ǉȽȽƘ (!ǷCȽƘąȚ 2ĭū¯ư!,D (!ǷCȽƘąȚ13Ň.!, 5' ypeS{N^bcayLt#0F)¸Ƒ2 ĮȽŇƅȦ 5-IMP A6ǌƴ2ĮȽŇƅȦ 5-GMP 2cayLt Fig. I-1 ǖ E,CEBñȽƘ1Á<EDTz]s}Ĩ.Ùǉǔ1ǥ, Ƚ

(22) Gæǔ 1ăŪ. %D(!ǷCȽƘąȚ2Ɗƣ1,3ƇȽƘGǎŬ 85°C15 ȦƟ. ǟ2ǸŝəGČȽƘǀ1Á<ED(!ƅȦȦ¤ĎƚoYkI][GȜ· #DȔɓD Oike et al., 1984 2ĐǸąǓ13ưÉɁ0ǸƐȒǺɟȐ A6ŻëȐȔɓ-D'>(!ǷCȽƘąȚGƊƣ-DuO3ǒÉ ɁŪ2uO1öǒ. E,D!!ȽƘ3ôġ?ƾŴ2ÛǉȽɟ-. Cƾ2ţÉɁuO2ƊȚ1?ěÛŻÇD!'*,ĐǸŝə GȔɓ.!0ŷ'0ǉȽɟƊƣÌřG«ȅ#E4ƾ2űƣuO2ǫŴD ƾŵĢ«ȅ1čí-ťȐŃ2ƔƳı?Ć*,ȷ0ȱB!1čí-D< 'ôů2(!ǷCȽƘąȚ2ƊƣąǓ1,?Đ2ǸąǓŦɝ-E 4Ǜɢ@Ǻɟ2ĩɒɡɕƃ1čí-D.Ø1Ǹ1ADƊȚ2ɧȹį-D '>ȚĿăŪ1Ɇɜ+.ċBED ñȽƘɍɖ2ſȦ¤Ďƚ2ŋĨƄoYkI][ Acid phosphataseAph. 3 ȽƘ1ǖ. ED 5' ypeS{N^bGƶy}Ĩ!,ĮȽ20eS{N. WbGƇƅ#DŇɓ0Ȧ¤Ďƚ-D.ċBED Fig. I-2Oike et al., 1984 ȽƘ. 4.

(23) B. A. 5'-IMP2Na. Sodium Phytate. 5'-GMP2Na. Fig. I-1. Chemical structures of (A) sodium phytate and (B) disodium 5-nucleotides supplemented in miso products as umami flavor enhancers. The illustrations were quoted from Japan Chemical Substance Dictionary Web service.. Fig. I-2. A hypothetical primary pathway for degradation of 5-IMP (5-nucleotides) added to unheated miso.. 5.

(24) 2űƣ¡Ǔ13ɑ0ȓƇȥÀɐ!,D'>ȤƁ2ȓƇȥɍɖ2ƪŋɣ2Ďƚ Á<ED!!ŪŚ!'A1ñȽƘǀ1Á<EDſȦ¤Ďƚ2ƪ3ȽƘ ē1ɍɖ#D'>&2ưȡȦ3ȽƘɒēà A. oryzae 1ɍɖ#D.ċBED& -ȺîÑ-3ĨƄoYkI][·Ƅ2ǑñȽƘ2ƊƣGŀô#D'>1ȽƘ ē1,ĨƄoYkI][·Ƅ2Þ>,Ǒ A. oryzae GȦĬƇȥ´ǔŉȵ1A Cŋ#D.GɄȖ.!' y}3#9,2Ƈȥ2Ȕżñƚ-CƇƫǯ-3Ň1 DNA @ RNA 0/2±Ĩ ATPADP 0/2MfzRǄƢƫƌǌǦȥ2ė°ƅȦ-Dy}ĨOzWLt. ã-ƨġ!,D&!,y}Ĩ3MfzR2Ưń@ğȶWTcz2ŭȳǙ Ƶ0/ɑ0Ƈƫȉ1Àɐ!,D!'*,y}ĨGƫ¬BňCĘH-ɘ ɒ#DÆĊ3Ƈȿ1.*,Ȕż-DȽƘɒē1,3ēà A. oryzae 3ȪȄ ưǤ.*'ŲȥŋĬGɔó.!,D'>ŲȥŋĬ2Ňɓ0y}ĨǄƿãƮDkK^}Ĩ@&2Ʃ2y}ĨMY`zGȦ¤!,y}ĨGňCĘH-D.ċ BEDA. oryzae ȦȎ#DĨƄoYkI][A6ĨƄoYkI][2ŋ -DkK][1À!,3E<-1+2îÑȳĔ. E,D. Fujishima et al., 1964; Wang et al., 1980; Oike et al., 1984; Shimizu., 1993; Fujita et al., 2003a, 2003b kK][3kK^}ĨGĂɛǔ1ſȦ¤!y}ĨGɎɚ#D İůàɍɖ2kK][3ÂĿǫƄǑ·Ƅ2Ƥǒ1ɒBEDixda|y }Ĩ PNPP GÂĿ.!'ūĒ1?ƶy}Ĩ·ƄGĺ# Mitchell et al., 1997; Wyss et al., 1999b A. oryzae RIB40 ¹ɍɖ2kK][ǙĬħȥ3ƉƊ ȡɀB1. EƄĿ2. E,D Uchida et al., 2006 kK][¬2ĨƄoYkI][. 1+,3Ʃ2İůà Aspergillus niger MacRae et al., 1988 @ Penicillium chrysogenum Hass et al., 1992 ɍɖ2?21ƽïC¾Úǀ1y}Ĩȷȟ0ūĒ13&2 ĎƚƇħ ǙĬȅô ɕƃ. ED.F*,DŪ2A1ēà A.. oryzae ɍɖ2ĨƄoYkI][1À!,3ȡ¤ɀ. 6. E,DưȡȦ3Ȝɀ.

(25) 2<<-D<'¤ɀ. E'Ďƚ1À!,? 5' ypeS{N^b1Ƭ#Dƶy. }Ĩ·Ƅ1+,3:.H/ǉ9BE,0 A. oryzae 1,3ãĿǗ½ȵǙĬǾ¥ȵ@ǙĬĤŢȵA6{q] ɏĒǙĬ2ǨǷȵ.*'ȦĬƇȥ´ǔŉȵDǓǝ²ɜ!,C¢?E <-1ũɊēàŀɒ¹1,ĐțǝǙĬƟǧƛ½äG«ȅ!,DƱŧ3ȽƘɒ2ēàŀɒ¹1DĨƄoYkI][ǑƇħ¹2ȦĬŋGČ'> 1¹Ľ£łjN_SB×ɐGō'ȽƘɒēà A. oryzae KBN630 ¹Gɒ,Ă ɛǔ0Ɵǧƛ½ä2«ȅGĴ='#0F)ƜȴăƄǙĬrO-D pyrG ǙĬA6ȑƟǧȼƻéĒŐȣä2ĊƅĬ Ku70 ]}iSĿGVb#D ku70 ǙĬ2ǳŕǾ¥¹G A. oryzae KBN630 ¹BňǪ!'Ʊŧ-3Ʊŧ-«ȅ! 'ȽƘɒēà A. oryzae KBN630 ¹2ĐțǝƟǧƛ½äG·ɒ!,A. oryzae RIB40 ¹ɍɖ2kK][ǙĬG aphA ǙĬ.ȿȾ!,Ǿ¥!ǀÖƾǫħ2ǤȽƘ ɒ2ȽƘßē Ǥē GģƊ!ĨƄoYkI][·Ƅ2ȫGƤǒ!'<' A. oryzae 1, aphA ǙĬGĐȅô. %ƉƊ!' AphA ]}iSĿ1+,. 5' ypeS{N^b1Ƭ#DÂĿǫƄ@ pHǝ1À#DŠƄĿG¤Ƌ!'Ʊ ŧ-3Ǥēǀ2Ňɓ0 5-IMP ƶy}Ĩ·ƄGƹĨƄoYkI][GɀB 1#D'>17 ÷2ĀȰ2 aph ǙĬǾ¥¹GģŘ!Ǥēǀ2ĎƚƇħƄGĴ ð!'&2éaphC ǙĬ&ȽƘɒēà A. oryzae KBN630 ¹2Ǥēǀ2 ĨƄoYkI][·ƄA6 5-IMP ƶy}Ĩ·Ƅ2ɞ·Ƅ1,Ňɓ0Ɇµ G'#.ȇɀ!'&-A. oryzae 1, AphC ]}iSĿGȦȎƇħ %ƉƊĎƚ2ŠƄĿGɀB1!AphC 2 5' ypeS{N^bȦ¤82ÀɐGċ ĥ!'. 7.

(26) Ʊŧ ȽƘɒēà1DĐțǝǙĬƟǧƛ½ä2²ɜ Šõ ã¬§²

(27) q A. oryzae éï!~®_æf2`å/"ã¬§× !×y+ã¬§²!zÂbª æñn·0/ A. oryzae.

(28) "0'.

(29) !qg

(30) tÅiàYÇÐaà+YÇà,$SK? êYÇ!ËÎà

(31) mÃYÇ°àeñ0

(32) /Yu et al., 2004 A. oryzae 2ǙĬƞģ13<$ēàğȶǯ1¬ɖ DNA GǨǷ#D'>2ãĿǗ½ äG²ɜ#D.Ȕɓ-Dēà3ƪ±-D'>ɔɓÐƄȫ¹GȦɚ#D .ǲ!<'ēà3ĉƇȥĿ0/2ƪ2ɈĠ1ƭƄGĺ#'>ưǈà@ ĎȲ2A1ɈĠƭƄǙĬGɌƄƔƳrO.!,ɘɒ#D.Řɖ0& 2A0ǀ-?ēà2ɋƄrO.!,ɘɒǼ0ǙĬ+ȳĔ D Table 1-1 áǹ&2ɌɒƄ1ACƈH1ĩɒ. E,. E,D2 pyrG ǙĬ. rO-DpyrG ǙĬ3qX`KlZ{SWw}.fP`KlZ{SWw} 2ɞƜȴăƄ2Z{SWw}rO.!,ɘɒ-D'>ɩƧǔǙĬǾ¥#0 Table 1-1. Selective marker genes used for Aspergillus oryzae transformation Gene name. Encoded protein. Phenotype Complementation of arginine. argB. Ornithine carbamoyltransferase auxotrophy. pyrG. Orotidine-5'-phosphate decarboxylase. Complementation of uracil auxotrophy. niaD. Nitrate reductase. Assimilation of nitrate. amdS. Acetamidase. Assimilation of acetamide. Sulfate adenylyltransferase (ATP). Assimilation of sulfate. Thiazole biosynthetic enzyme. Resistance to pyrithiamine. Phosphoribosylaminoimidazole-. Complementation of adenine. succinocarboxamidesynthase. auxotrophy. sC prtA adeA. 8.

(33) F) YÇL6S=7Oà

(34) ¸0æf2_ A. oryzae 2ǙĬƞģ13ãĿǗ½ä2²ɜ2:1?ŕɓ0 Ʋ*'&E 3ǙĬ]U_`K}TĂɛ2Ǒ. -Dɤ4ũɊēà A. oryzae KBN616 g. ! pyrG ǙĬrO1ADãĿǗ½ä- amyR YÇÐa2(v_ǙĬ] U_`K}TĂɛ3 4.5% tÅig 200 g¾ 9 g Kitamoto et al., 2006 rA. oryzae RIB40 g+ KBN616 g

(35) YÇL6S=7Oà,$ ØÈ±Ê¯iueñ0 Takahashi et al., 2006 b; Kitamoto and Yasuda, 2008; Mizutani et al., 2008 £d¦Û!©¡¶"Í¨ºØÓ p/£d¦Û"h «©¡¶2X/) Í¨º2Ä¨ Ù/o{2

(36) /YÇ° !jÆ"!Í¨º!Ù cï DNA ©¡¶ ¯'0/|-0/ Í¨ºÙo{ "±Ê¯iÙuÕ±Êâ¹vÙu! õ´

(37) /Kanaar et al., 1998Fig.1-1~Ý Saccharomyces cerevisiae " ±Ê¯i Ùu ,.Í¨ºÙ}10/)cï DNA "©¡¶!±ÊôZ%¯('0 /ØÈ

(38) [ßq! q" Õ±Êâ¹vÙu ,.Í¨ ºÙ}10/)cï DNA "©¡¶!QU@NV¼ ¯'0/ØÈ YÇÐaxð"Õ Á

(39) Õ±Êâ¹Ùu" DNA W´sITB5U7E> ¢ÑPFAC(DNAPkc)KU70-KU80 JBT@5LDNA LIG4-XRCC4 Ú¶kë

(40) /Walker et al., 2001; Critchlow and Jackson et al., 19980-!?U G82;D/YÇ![2Ða/ ,.Õ±Êâ¹vÙu2º/ ^Ï .±Ê¯iÙu ,/YÇ?:AB4U9xð2¿/ ï/. Neurospora crassa " ku70 YÇÐa ,.YÇÐaxð. 100% Ninomiya et al., 2004A. fumigatus " ku80 YÇÐa ,.YÇÐ axð 80% Ferreina et al., 2004Þ0

(41) /'A. oryzae.

(42). * RIB40 g" ku70 YÇÐa ,.YÇÐaxðç 60% Takahashi et. 9.

(43) Fig. 1-1. Intergation machinery of exogenous DNA into chromosome.. al., 2006bèq KBN616 g" ku70 YÇÐa ,.YÇÐaxðç 80% Kitamoto and Yasuda, 2008 ' ligD YÇ!Ða , A. oryzae RIB40 gYÇÐaxð 100% Mizutani et al., 2008!, A. oryzae

(44) xð!ò

(45) YÇÐa<=BNeñ, +!ä»!YÇ!oÏ+ÌÀ2 Ö\/^Ïw/ã¬í! A. oryzae íqg

(46) " !, xðÃ±Ê¯iu2{½öÞ0

(47)

(48) &-Ʊŧ"ȽƘɒēà A. oryzae KBN630 ¹1DĐțǝǙĬƟǧƛ½ ä2«ȅGĴ='ƜȴăƔƳƄ2ǙĬrO-D pyrG ǙĬ2ãĿǗ½ ȵGɘɒ#D'>1ī>1 A. oryzae KBN630 ¹2ƓŴƫ DNA B pyrG ǙĬG ĤŢ!'ĸ1ǨǷ#D DNA ƼȬ2Ɵǧƛ½ĂɛGơư. %D'>1ȑƟǧ. ȼƻéĒŐȣä2ĊƅĬ Ku70 ]}iSĿGVb#D ku70 ǙĬGpyrG Ǚ ĬĤŢȫ¹2ƓŴƫ DNA BĤŢ!'. 10.

(49)

(50) ÏÁŘ60ÏŇň.

(51) ÇŐ£ ÍÑcEZI7Łè=Õ"ŊûŐ¹£ A. oryzae KBN630 = pyrG ~ğ È~ğÈÃß-Łŕ ku70 ~ğÈÃß-Łŕ60øéĊ DNA -ęô,Ő" Escherichia coli DH5 α =Ô- DNA Ēń-Ix_yJ,ÇŐ".

(52)

(53) ıē60ıŒåª A. oryzae KBN630 *. -Įó-¯ĊıŒ,.Czapek-DoxCD ıē3.0%. glucose0.3% NaNO30.1% K2HPO40.05% KCl0.005% MgSO4.7H2O0.001% FeSO4 7H2O , 1.5% Agar =Ĝ"5-=ÇŐ"đMx_,.7, 0.25% Triton-X100 60 1.0% Trace element0.014% CaCl20.0039% MnCl2.4H2O0.01% ZnSO40.0005% CuCl2. 2H2O0.00005% Na2MoO4.2H2O0.000015% CoCl2.6H2O0.01% FeCl3 0.0372% citric acid =Ĝ(ÇŐ"A. oryzae pyrG YÇ¨ć-lQ[ @fSwIPsy,.ãěıē, 0.1% 5-fluoroorotic acid5-FOA * 0.15% uridine 60 0.07% uracil =Ĝ"5-=ÇŐ"A. oryzae -ĊıŒ,.J vMRluig\yıēGP ıē 2% glucose1% polypeptone0.5% KH2PO4 0.5% KCl0.1% NaNO30.05% MgSO4.7H2O =ÇŐ"?ntTð-¬Ú, .CD ½àěıēĖ-JvMR=RWY,Ĕ"RWYěıē=ÇŐ "A. oryzae -ıŒ. 30°C ,(µĊıŒË.Ō 160 rpm -ĝÍë*= µ%" E.coli -ıŒ,. 2xYT ıē1.6% Bacto tryptone1.0% Bacto yeast extract0.5% NaCl =Ő ķő,(ĬĢ 50μg/mL - ampicillin sodium =Ĝ¯ĊıŒË,. 1.5% Agar =Ĝ"ıŒ. 37°C ,(µ%".

(54) A. oryzae -¦Îĝ. 11.

(55) A. oryzae = MP ıē2.0% malt extract0.1% Bacto peptone2.0% glucosepH6.0 Ė)pyrG YÇ¨ć. 0.25% uridine =" MPU ıēĖ) 30°C24 Ëı Œ°£Ċ=gtRYZIe@vW)Ø£"£ĊŌ 4 g = 40 mL -gx\gt R\ęô0.38% Novozyme2340.12% Cellulase “ONOZUKA” R-100.8 M NaCl 10 mM NaH2PO4 (pH 5.8) Ė) 30°CŌ 2 ËAyHrh\"-=gtR YZIe@vW);°;= êŁŕ(ĚĠ=Ö0.8 M NaCl ŏ) 2 Ģ÷æ 0.8 M NaCl-50 mM CaCl2 ŏ) 1 Ģ÷æ"Ħ7:"ĚĠ, 0.8 M NaCl-50 mM CaCl2 ŏ=Ō 100μl («Đ:=gx\gtR\*"gx\g tR\ 50 μl , DNA ĒńŌ 5μg * 12.5 μl - PEG ŏ25% PEG6000 in 50 mM CaCl2, 10 mM Tris-HClpH 7.5 =(ĸã) 20 ŁŅÊ7,ã- PEG ŏ= 500μl (ĸã) 5 ŁŅÊ"-, 1 mL - 0.8 M NaCl-50 mM CaCl2 ŏ =(º¸"°ŋ£gtRYZIPqw,Łė10 mL -¼óıē21.9 % Sorbitol, 1% Glucose, 0.6% NaNO3, 0.15% KH2PO4, 0.05% KCl, 0.05% MgSO47H2O, a trace of FeSO4H2O, 2% Bacto agarpH 6.5 =(¯30°C )Ō ×ıŒ (¦Îĝ=ÓĦ".

(56) A. oryzae øéĊ DNA -ęôPCR 60 DNA PIDyR A. oryzae -øéĊ DNA .GP ıē) 30°C3 ĩıŒ" A. oryzae -£Ċ= Ø£(îŁ=ß¡"°ģ©ā(ł¾eCbv-Ixxjvoň)ę ô"Sambrook and Russell, 2001; Raeder and Bronda, 1985 PCR Ő¶ü.TaKaRa Ex Taq DNA luptTTakara Bio, Otsu, Shiga 60 PfuUltra II Fusion HS DNA polymeraseStratagene, La Jolla, CA, USA =ÇŐ. :. !:-gx\Mv,Ü(ĳ=µ%"PCR ăĔ. GeneAmp9700 OmvOAI tApplied Biosystems, Foster City, CA, USA =ÇŐ"Ď â)ÇŐ"Eu N`IwEY]gtAm= Table 1-2 ,Ì". 12.

(57) Ò+Ix_yJR[Zg).model 4000LS DNA PKyOLI-COR, Lincoln, NE, USA 60 GenomeLab GeXPBeckman Coulter, Brea, CA, USA =Ő "PIDyPyJ,68óñŀ=ī".

(58) pyrG ~ğÈÃßŐhIW60 ku70 ~ğÈÃßŐhIW-³ĕ pyrG ~ğÈÃßŐhIWpDispyrG .z-6,³ĕ"pyrG ~ğÈ GenBank/EMBL/DDBJ accession no. AB017705, NITE DOGAN ID: AO090011000868 - 5’-Śöř 1.6-kb = A. oryzae øéĊ DNA *gtAmi? pyrG11/pyrG12 =Ő (Ąľï®¶ü SacI * XbaI )á"pyrG ~ğÈ- 3’-Śöř 1.4-kb = A. oryzae øéĊ DNA *gtAmi? pyrG13/pyrG14 =Ő(Ąľ ï®¶ü XbaI * HindIII )á"

(59) &-Ēń= pUC18 - SacI-HindIII ļ{,ĀĪpyrG ~ğ ÈÃßŐhIWpDispyrG =Âô" ku70 ~ğÈÃßŐhIWpDelku70-2 .z-6,³ĕ"ku70 ~ğÈ GenBank/EMBL/DDBJ Accession No. AB214649 - 5’-60 3’-Śöř. :!:. 1.0-kb = A. oryzae øéĊ DNA *gtAmi? ku70-1/ku70-6 60 ku70-72/ku70-82 =Ő(Ąľ" ku70 ~ğÈM]ř-ļ 1.0-kb Ēń= A. oryzae øéĊ DNA *gtAmi? ku70-9/ku70-10 =Ő(Ąľ":7- 3 &- PCR Åŀ=º ¸"5-=[ygw\*(gtAmi? ku70-1/ku70-10 =Ő(erQ sy PCR =µ%"erQsy PCR Åŀ= pUC18 - SmaI ļ{,OfIxy ( pDelku70-1 =Âô"A. oryzae øéĊ DNA *gtAmi? pyrGN2/pyrGC2 =Ő(Ąľ" pyrG ~ğÈĒń 1.8-kb = StuI )á( pDelku70-1 - Aor51HI ļ{,ĀĪku70 ~ğÈÃßŐhIWpDelku70-2 =Âô" A. oryzae -Lboäņ.ħŖµòňíôĺĹÛĴ³NITE http://www.bio.nite.go.jp/dogan/top 7ÓĦ". 13.

(60) Table 1-2. Oligonucleotide primers used in Chapter 1 Primer. Sequence. pyrG11. 5'-GCGAGCTCTACATTGGCAAAGGAAT-3'. pyrG12. 5'-GGTCTAGAATATTTAATCAGCTACC-3'. pyrG13. 5'-TGTCTAGACACTAGCTATACCGCCC-3'. pyrG14. 5'-CTAAGCTTATCAGCTGCATATCTCT-3'. pyrGN2. 5'-CAAGGCCTGCTGGAATTGACATTATTATGG-3'. pyrGC2. 5'-AAAGGCCTGATCAATACCGTACGGGAGATT-3'. ku70-1. 5'-TGGAATTCGGACCATTTTCGGATAGG-3'. ku70-6. 5'-AGAAGCTTTCAGGTGTGTTTGAAAG-3'. ku70-72. 5'-TCAAACACACCTGAAAGCTTCTGCCAACTTCCTGCAC-3'. ku70-82. 5'-CTGCGTCCCATATAATAGCGCTTTCGTCCGTTTGGATATA-3'. ku70-9. 5'-AAAGCGCTATTATATGGGACGCAGG-3'. ku70-10. 5'-CCGTCGACCATAGCCCCCAGAACAG-3'. ku70-11. 5'-AGCATGCATTTCTGGGATTAGACAGG-3'. amyRN1 5'-TTGAATTCGATCCCTGACTAGAGTC-3' amyRC1 5'-TTGGATCCAATCCTTCGGTTTACTA-3'. 14.

(61) Ï©60´Ä. pyrG ~ğÈÃß-ÂÚ ĉ- A. oryzae -¦ÎĝŇň.ś/ niaD ~ğÈKitamoto et al., 1995 sC ~ğÈYamada et al., 1997 pyrG ~ğÈKitamoto and Yoshino, 1999 argB ~ğÈ Gomi et al., 1987 60 adeA ~ğÈJin et al., 2004 -6+Œő ~ğÈm F-ÇŐ,'(9Table 1-1 . -Ė)ExYQy-5′-uyÆFvkH. PtT=M]9 pyrG ~ğÈ-ÇŐ.lQ[@fSwIPsy*aG[@f SwIPsy-ŗŇĭ)9"4č+œĞ9BuQyBtPvĶő ð.BuQyBtPvĶĜıēĖ)ùĲ:ŇBuQyBtPvő ð. 5-fluoroorotic acid5-FOA ĜıēĖ)ùĲ:95-FOA .BtPvú ¤ĊExYyÆ-?^xJŀÎ)8Éç£-ó=Ęþ9"%( BuQyBtPvĶő ð. 5-FOA ĆÀ).ó)+. O F. HN O. O. N H. HN. COOH. O. 5-FOA. O HN O. N H. O. PyrF COOH N H Orotic acid. O. O. PyrG. HN N. COOH. RP. CTP. HN O. N RP. UTP. Fig. 1-2. Uracil biosynthesis pathway 15. Uracil. UDP. UMP.

(62) A. B M. pDispyrG. pyrG11. 2. 3. (kb) 23.1 9.4 6.6 4.4. (5.7 kb). pyrG14. KBN630. 1. pyrG. 2.3 2.0. 4.8 kb Transformation 5-FOA selection KBN630-17 pyrG). pyrG11. 0.6. pyrG14. 3.0 kb. Fig. 1-3. Construction scheme and confirmation of the pyrG deletion mutant. A. Scheme of the pyrG deletion is shown. PCR-amplified DNA fragments from a pyrG deletion vector, pDisPyrG, were transformed to A. oryzae KBN630, and 5-FOA resistant transformants were selected. A pyrG deletion mutant, A. oryzae KBN630-17, was obtained. Black and gray boxes indicate the 5’- and 3’-flanking regions of the pyrG gene, respectively, and the open arrow indicates the pyrG gene region. The direction of the arrow indicates the orientation of the pyrG gene. Small arrows indicate the position of oligonucleotide primers used. B. Agarose gel electrophoresis of amplified DNA fragments in the pyrG gene region of the 5-FOA resistant transformants using primer pair pyrG11/pyrG14. Parental strain, A. oryzae KBN630 (Lane 1), the pyrG deletion mutant. A. oryzae KBN630-17 (Lane 2), and the pyrG undeleted mutant, A. oryzae KBN630-19 (Lane 3) are shown.. 16.

(63) ŊûŐq A. oryzae KBN630 - pyrG ~ğÈÃßŃ|=ÓĦ9"4, pDispyrG =[ygw\*(gtAmi? pyrG11/pyrG14 =Ő( PCR Ąľ= µĦ7:" DNA Ēń=Ő( A. oryzae KBN630 =¦Îĝ"Fig. 1-3 A Uvc\vBuQy60BtPv=3 CD ½àıēã,ó("¦Î ĝ-ã,5-FOA Čð¦Îĝ=ùĲ9"4,7, 5-FOABuQy 60BtPv=3 CD ½àěıē=Ùÿ"Ħ7:" 9 - 5-FOA Čð- $2 BuQyBtPvő ð=Ì"pyrG ~ğÈ-Ãß.gtAmi? pyrG11/pyrG14 =Ő" PCR ,6%(ī"ì A. oryzae KBN630 -øéĊ DNA 7. 4.8-kb ĒńĄľ: Fig. 1-3 B, Lane 1 pyrG ~ğÈÃß-øéĊ DNA 7. 3.0-kb ĒńĄľ:"2 -BuQyBtPvő ð-$ŇA. oryzae KBN630-17 . pyrG ~ğÈÃßŃ|)8Fig. 1-3 B, Lane 2 ĈŇA. oryzae KBN630-19 . pyrG ~ğÈÃßŃ|).+%"Fig. 1-3 B, Lane 3 A. oryzae KBN630-17 = pyrG ~ğÈKitamoto and Yoshino, 1999 =Ő(¦Îĝ 9*)BuQyBtPvĶő ðĽ":7-©7ŊûŐ ¹£ A. oryzae KBN630 ,(pyrG ~ğÈ=ùďmF*9¦Îĝ§ Ŗ"*Ì:".

(64) pyrG, ku70 ĨÙÃßŃ|-ÂÚ ku70 ~ğÈÃßFSZ\=¿"gtRn] pDelku70-2 = A. oryzae KBN630 pyrG, ku70 ĨÙÃßŃ|-³ĕ,ÇŐ"Fig. 1-4 A gtAmi? ku70-1/ku70-10 [ygw\ pDelku70-2 =ÇŐ" PCR ĄľĒń=Ő( A. oryzae KBN630-17 = ¦Îĝ"¦Îĝ7gtAmi? ku70-1/ku70-11 =ÇŐ( PCR =µ Ąľ" DNA Ēń=¬Ú9Ňň)¦Îĝ=ę2"ę2" 100 -¦Î ĝ-$ 8 ,&(6.3-kb ĒńFig. 1-4 B, Lane 2 ¬Ú:, Ňì) . 3.6-kb ĒńFig. 1-4 B, Lane 1 ¬Ú:"-*.PCR )Ąľ" ku70. 17.

(65) A. B pDelku70-2 (7.5 kb). (kb). pyrG. Ku70-1. 1. 2. 3. 23.1 9.4 6.6 4.4. Ku70-11. KBN630-17. M. ku70 3.6 kb ku70 gene disruption. KBN630-17K (ku70) Ku70-1. Ku70-11. pyrG. ku70. 2.3 2.0. 6.3 kb. 0.6. 5-FOA selection KBN630-17K3. Ku70-11. Ku70-1. 2.1 kb. Fig. 1-4. Construction scheme and confirmation of the ku70 deletion mutant. A. Scheme of the ku70 deletion is shown. PCR-amplified DNA fragments from a ku70 deletion vector, pDelku70-2, were transformed to A. oryzae KBN630-17, and ku70 disruption mutants were selected. A ku70 disruption mutant, A. oryzae KBN630-17K3, was obtained. Then, a ku70 deletion mutant was selected on 5-FOA treatment of A. oryzae KBN630-17K3. Black and gray boxes indicate the 5’- and 3’-flanking regions of the ku70 gene, respectively. The open and black arrows indicate the pyrG and ku70 genes, respectively. The direction of the arrow indicates the orientation of the ku70, pyrG genes. Small arrows indicate the position of oligonucleotide primers used. B. Agarose gel electrophoresis of amplified DNA fragments in the ku70 gene region of the ku70 disruption and deletion strains using primer pair ku70-1/ku70-11. Parental strain, A. oryzae KBN630-17 (Lane 1), the ku70 disruption mutant, A. oryzae KBN630-17K (Lane 2), and the ku70 deletion mutant, A. oryzae KBN630-17K3 (Lane 3) are shown. 18.

(66) ~ğÈÃßFSZ\ ku70 ~ğÈ»,ĥĪ:"*=ÌĦ7:" ku70 įŃ |-$-)9 A. oryzae KBN630-17K =z·-Ï,ÇŐ"ku70 ~ ğÈÃßFSZ\ ku70 ~ğÈ»1ĥĪ:9*,6%(ku70 ~ğÈ- 1.0-kb - 3′-Śöř pyrG ~ğÈ* ku70 ~ğÈ=¢>#ŗą,XAwI\ud\-¦ )ÙĿ9:7-ĤİŜ)ĂĤý9*,685-FOA )ÞŔ 9* pyrG ~ğÈ60 ku70 ~ğÈÃß:9pyrG ~ğÈ60 ku70 ~ğÈ= Ãß9"4, A. oryzae KBN630-17K -ŁóÈ= 5-FOABuQy60BtPv =Ĝ" CD ěıē,ġŉ5-FOA ČðMx_=ùĲ":7-Mx_ .Ō 1 10-2 -ĻĢ)Ħ7:"pyrG ~ğÈ60 ku70 ~ğÈ-¬Ú.gtAm i? ku70-1/ku70-11 =ÇŐ" PCR ,68µ%"Ąľ" DNA Ēń=¬Ú9 Ňň)¦Îĝ=ę2"A. oryzae KBN630-17K -øéĊ DNA 7. 6.3-kb Ē ńFig. 1-4 B, Lane 2 Ąľ:5-FOA Čð A. oryzae KBN630-17K3 -øé Ċ DNA 7Fig. 1-4 B, Lane 3 Ąľ:":7-©7KBN630-17 - ku70 ~ğÈÃß,ñ±"*Ì:". pyrG, ku70 ĨÙÃßŃ|,9~ğÈWLZ[@yJĻĢ ku70 ~ğÈÃß-~ğÈWLZ[@yJĻĢ,Ŏ9²=ę29"4,A. oryzae KBN630-17K3 = amyR ~ğÈįŐhIWpDisAmR100Kitamoto et al., 2006 -ï®¶ü EcoRI 60 SalI áŀ=Ő(¦Îĝ" ¼ó" pyrG+ ¦ Îĝ= CD ěıēã)ùĲ(ÝRWYěıē,}"AmyR . ?ntT~ğÈ¥-ōĥĝÐÈ)9"4Petersen et al., 1999 amyR+-¦Î ĝ.RWYěıēã)ó"°, KI-I2 øé=µ*Iu?Vy=¦ ñ9amyR ~ğÈį. Iu?Vy=¦ñ+50 -¦ÎĝĖ 46 .Iu?Vy=¦ñ+%"+<$92%-¦Îĝ amyR ~ğÈ į)8:7-,(ĂĤý%"*´7:"amyR ~ğ. 19.

(67) A. B pDisAmR100 (6.8 kb). M. 23.1 9.4 6.6 4.4. amyRC1. amyRN1. amyR 2.9 kb amyR gene disruption. 2.3 2.0 0.6. amyRC1. amyRN1. amyR. 2. (kb). pyrG. KBN630-17K3. 1. pyrG 3.6 kb. Fig. 1-5. Construction scheme and confirmation of the amyR disrupted strain. A. Scheme of the amyR disruption is shown. The amyR disruption vector pDisAmR100 digested with EcoRI and SalI was transformed to A. oryzae KBN630-17K3, and the transformants with no amylase activity were selected. Black and white arrows indicate the amyR and pyrG genes, respectively, and the direction of the arrows indicates the orientation of the respective genes. Small arrows indicate the position of oligonucleotide primers used. B. Agarose gel electrophoresis of amplified DNA fragments in the amyR gene region of the transformants with no amylase activity using primer pair amyRN1/amyRC1. Parental strain, A. oryzae KBN630-17K3 (Lane 1), the amylase-negative transformant (Lane 2) are shown.. ÈįhIWpDisAmR100 ,6%(ì- amyR ~ğÈĔ:"ĵ=g tAmi? amyRN1/amyRC1 =Ő" PCR õ,6%(4"Fig. 1-5 A ì A. oryzae KBN630-17K3 -øéĊ DNA ). 2.9-kb ĒńĄľ"-,ċFig. 1-5 B, Lane 1 ?ntTĶóÅð¦Îĝ-øéĊ DNA ). 3.6-kb ĒńĄľ "Fig. 1-5 B, Lane 2 A. oryzae KBN630-17K3 ,9~ğÈWLZ[@y. 20.

(68) RƖŻ0A. oryzae KBN616 §/ ku70 ŹûƉ §82.3%Kitamoto and Yasuda, 2008; A. oryzae RIB40 / ku70 ŹûƉ §63.4%Takahashi et al., 2006b/ "A=?ã'$A>/Å>ƒŌſĶƭşđƙ/ƖŻ0 A. oryzae §®*: ê/@+ĄéA$/Å0¹.ƥæA$È¸ÅTakahashi et al., 2006b+0-')$ A6*/ƥæ+ſƺ.Takahashi et al., 2006a; Takahashi et al., 2006bku70 Źû/Ɖ 0ĺőŻ;ƨû/Ũĺ=1ƌ+'$ƔÎ¿.0¼Dƹ- '$ku70 ŹûƉ §+ 5-FOA D$ pyrG Źû/ôĞ/ďƧDŊ7äC!@ +.=?vND~WIQ)ſ§ũ*ØǃŴ.ƛĴ/ŹûDôĞ. @. +ƈ*@Takahashi et al., 2008<.pyrG, ku70 ƂĕôĞƢ§*@ A. oryzae KBN630-17K3 §0ƮŇå½ĈƼ§ A. oryzae KBN630 /Ɓų/ĪŏƁĶ ƲDƳŴ+)ÜƎ/ŹûôĞĽDà.$?»ǉ-a+-@/ ØǃŴ-ŹûŤ¬XZbxDƼ@+.=?ƺ-Źû/Ɖ Dü$Ʈ Ň/ļŏ.ŵ$ƮŇå½ĈƼ§/óė³ŗ*@. ƽƴ ƮŇƼå½ Aspergillus oryzae KBN630 §.) pyrG ŹûM_Y- 5’ -~ øcNtOX}]ŹûôĞ§DƝǂpyrG ŹûDńŜvN+ @ÀćŸ¬ÂD¡ƌ$ŌſŊ¬.=@¢ǀŹû/łĬŕĤ3/Ŋç7ƖŻD ÚĤ!@$9.A. oryzae KBN630 §.ƶǀ. @ pyrG ŹûôĞ§/TjxĤ. > DNA ƂƫìĿŠđƙ´Þ/ (ƒŌſƭşÅäđƙÂ.¯C@ Ku70 ^lQ ćDUe. @ ku70 ŹûDĞ¹$Ďƀ$ pyrG, ku70 Źû ĕôĞ§.. ) amyR Źû/Ɖ Dā7$+B90% Ĥ/ƖŻ* amyR ŹûDƉ . @. +*$/Å=?ƮŇƼå½ A. oryzae KBN630 §.)ãƖŻŹû ŌſŊ¬ÂD£Ǆ. @+*$. 21.

(69) ś Ģ ƮŇƼå½.@øĶsZnF^] A ŹûaphA/Ɖ + AphA / ĝĶć/Ʋ ĝÏ ƮŇ/ƮDÚĤ!@$9. 5'IjXøƂfd~Jx5'IMP; 5'R GgøƂfd~Jx5'GMP/=- 5'-~thQM_eƂfd~Jxŷ A$ŬƮƮŇ%Ƅ?ƮŇÛƕļŏA)@ËƮŇũ.05'-~th QM_eƂfd~JxDŝ~ø)ýƮ/-~thQMXeDĺķ. @. âŉsZnF^]°6A)@"*ƮŇåũ/sZnF^]¦ĶŪ ű$ƮŇƼå½ A. oryzae ½§Dóė. @+.=')sZnF^]Ć¦. /$9/ãƆěǁDƑ$Įµ-ġLiP¿/%Ƅ?ŬƮƮŇļŏ¶ Ę/¡ƌƈ*@+ß>A@ A. oryzae ƝƐ. @sZnF^]+nH^].¯)A6*.(/. È¸ƥæA)@"A>/âŉ/ 5'~thQM_e.Ŗ. @Ɲ¦Ķ.. ()05+E,š>A)-Fujishima et al., 1964, Wang et al., 1980, Oike et al., 1984, Shimizu., 1993, Fujita et al., 2003a, 2003bnH^]0sZnF^]WoQ }Z/mZ_YøĶsZnF^]nFw~.Œ. @nH^]0īƜĐû. .@čƽ-~øūŧÀŘ*@nH_øDØǃŴ.ĳƝ~øDƷ ǂ. @ÿĨ½ƶǀ/nH^]0øĶsZnF^]Acid phosphataseAph. ¦Ķ/Őų.Ƽ>A@l}gd~øPNPPD±ć+$ĥä.:¦ĶDĄ +š>A)@Mitchell et al. 1997, Wyss et al. 1999bA. oryzae RIB40 §ƶǀ / n H ^ ] Ź û / cDNA Ɗ ǋ 0 c ^ q Z Ĥ . × ¡ A ) @ GenBank/EMBL/DDBJ accession no. AB042805"/Źû÷Ɯ0ĻļAĶć/ ƘƲ>.A)@Uchida et al., 20060"/Źû÷ƜnH_ ø=?: PNPP D±ć.$ĥä.=?ã¦ĶDĄ$+>"/ŹûDø ĶsZnF^] A ŹûaphA Źû+Ʊư$. 22.

(70) ś Ģ*0ś Ģ*¡ƌ$ƮŇƼå½ A. oryzae KBN630 §/ãƖŻŹûŌſ Ŋ¬ÂD¦Ƽ) aphA ŹûDƉ ŽƮŇƼ/å.@øĶsZnF^ ]¦Ķ/ƢDö$ă.å½.@»ǉ-pz^*@ TEF1 Ź ûpz^Kitamoto et al., 1998D¦Ƽ) aphA ŹûDãƌÎ!AphA DƝƐĺ÷!@+º.AphA DĻļ)âŉ¥ŴĝĶćDĽ$. ĈÊòǆ=1ĈÊƦƧ úƼ½§ ƮŇƼå½§ A. oryzae KBN630 §D DNA Ŭļ.úƼ$ś 1 Ģ*óė$ A. oryzae KBN630 §ƶǀ/ pyrG, ku70 ŹûƂĕôĞƢ§*@ A. oryzae KBN630-17K3 §=1ģƵƼå½ KBN616 §ƶǀ/ alp, pyrG ŹûƂĕƉ Ƣ §*@ A. oryzae PDE1 DÀćŸ¬ƼĖč+)úƼ$A. oryzae PDE1 §0 Ŋ¬^lQćDØǃ=ĺ÷!@$9.GN~pbG]Źûalp Źû Murakami et al., 1991DƉ $§*@ƫ§/óļƦƧ.()0, ơ/´.ƌƔ. @Ƹų*@. E. coli DH5 α DĐ/ DNA Šƣ/QgR.úƼ$. ƋŢ=1ƋƾħÆ A. oryzae /ŕƋƾ.0Ơ*E2EƋŢRS ƋŢ 3% rice starch1% polypeptone 1% NaNO30.2% KCl0.1% KH2PO40.05% MgSO4.7H2ODúƼ) 30°Cƴ 160 rpm /Ÿąĭ+ƋƾDà'$ ŽåƋƾ0ĩĊŚŽr`dDƋƾ±+) 30°C43 Ă®/ƋƾDà'$ĩ ĊŚŽr`d0ŚŽDĳ. 2 Ă®įŰÒ35 ƝĩĊ)*$ĩĊŚŽDw_ ´*ŮÁƴ 10 mmŭ 15 ~ 20 mm .ķÀ)úƼ$ E.coli /Ƌƾ.0 2xYT ƋŢ 1.6% Bacto tryptone1.0% Bacto yeast extract0.5% NaCl. 23.

(71) DƼ Ɠƽ.)ƇŻ 50μg/mL / ampicillin sodium DŷÑŕƋƾĂ.0 1.5% Agar Dŷ$Ƌƾ0 37°C .)à'$.

(72) A. oryzae /ÀćŸ¬ A. oryzaepyrGD MPU ƋŢ2.0% malt extract0.1% Bacto peptone2.0% glucose 0.25% uridinepH6.5ũ* 30 24 Ă®ƋƾÒ

(73) .Ą$ƦƧ+ſƺ. Novozyme234 =1 Cellulase “ONOZUKA” R-10 DƼ)pdp}Zd$ƀ >A$pdp}Zd. PEG œñ* DNA Šƣ/Ď?ç7DàC!)ÀćŸ¬Dà íĺ©ŶƋŢũ* 30°Cƴ Ē®Ƌƾ)ÀćŸ¬§DĎƀ$. A. oryzae łĬŕ DNA /ŬļPCR =1 DNA XQLZ A. oryzae /łĬŕ DNA 0GP ƋŢ* 30°C3 ƃ®Ƌƾ$ A. oryzae /½ŕD ē½)ĳƝDĞ¹$ÒżÅ¨ŋ)ƞïnKj-QsxƧ*Ŭ ļ$Sambrook and Russell, 2001; Raeder and Bronda, 1985 PCR Ƽâŉ0TaKaRa Ex Taq DNA u~y}]Takara Bio, Otsu, Shiga=1 PfuUltra II Fusion HS DNA polymeraseStratagene, La Jolla, CA, USADúƼ"A #A/pdU.Ě)ƍDà'$PCR ōŤ0 GeneAmp9700 WvWIQ }Applied Biosystems, Foster City, CA, USADúƼ$ś Ģ*úƼ$M~ VhQM_ep}IvD Table 2-1 .Ą$ č-QgRZb`p*0model 4000LS DNA XSWLI-COR, Lincoln, NE, USA=1 GenomeLab GeXPBeckman Coulter, Brea, CA, USADƼ$X QLXR.=?ĺķƜD£ƅ$. aphA ŹûƉ ƼqQ^/ÞŦ aphA ŹûƉ ƼqQ^pDisAphA D/=.ÞŦ$aphA Źû/. 24.

(74) Table 2-1. Oligonucleotide primers used in this study Primer. Sequence. phyA1. 5'-TCGAGCTCGGTACCCCTGTTCATTTTGGTTGAGAA-3'. phyA2. 5'-CAGCGGCTTGATCAACTCGTACAAGGCGAC-3'. phyA3. 5'-TTGATCAAGCCGCTGCTGGAATTGACATTA-3'. phyA4. 5'-GTGACGGGAGATTGTACGAACAGATGGCCC-3'. phyA5. 5'-ACAATCTCCCGTCACGGTGCACGGTATCCA-3'. phyA6. 5'-CTCTAGAGGATCCCCAGTTTCCACCCGATGTAACG-3'. fupyrGN 5'-CGGTACCCGGGGATCCAAGCCGCTGCTGGAATTGACA-3' pyrGC2. 5'-AAAGGCCTGATCAATACCGTACGGGAGATT-3'. pyrGtef. 5'-ATTGATCAGGCCTTTCACTGTGGACCAGACAGGC-3'. tefPrev. 5'-CATTTTGAAGGTGGTGCGAACTTTG-3'. tefaphA. 5'-ACCACCTTCAAAATGGCGGTCCTTAGCGTGCTCCTTC-3'. aphASal. 5'-ATGCCTGCAGGTCGACTGAGGAGAGGAAGGATGGG-3'. 5’-Ǌŀǈ 1.0-kb + 3’-Ǌŀǈ 1.1-kb D"A#A A. oryzae łĬŕ DNA +p}I vrG phyA1/phyA2 =1 phyA5/phyA6 DƼ) PCR Ŏƚ$pyrG ŹûŠ ƣ 1.8-kb D A. oryzae łĬŕ DNA +p}IvrG phyA3/phyA4 DƼ) PCR Ŏ ƚ$ƀ>A$ 3 (/ PCR ŎƚŠƣ+ SmaI *Ġ$qQ^pUC18 /äÃ 4 ŠƣDèäIn-Fusion Advantage PCR Cloning KitTakara BioDƼ) In-Fusion ƍDà'$/=.)ƀ>A$aphA Źû/ 5’-Ǌŀǈ+ 3’-Ǌŀǈ/ ®. pyrG Źû Ť. @p}ZweD pDisAphA +$. aphA ŹûƌÎƼqQ^/ÞŦ aphA ŹûD A. oryzae TEF1 Źû/pz^ĵÓ*ƌÎ!@$9. 25.

(75) ƌÎƼp}Zwe pTFAphA D/=.ÞŦ$pyrG ŹûŠƣ 1.8-kb D A. oryzae łĬŕ DNA +p}IvrG fupyrGN/pyrGC2 DƼ) PCR Ŏƚ$A. oryzae TEF1 Źûpz^/ 0.8-kb ŠƣD A. oryzae łĬŕ DNA +p}Iv rG pyrGtef/tefPrev DƼ)Ŏƚ$aphA Źû 2.0-kb ŠƣD A. oryzae łĬŕ DNA +p}IvrG tefaphA/aphASal DƼ)Ŏƚ$ƀ>A$ 3 (/ PCR Ŏ ƚŠƣ+ BamHI/SalI *Ġ$qQ^pUC18 /äÃ 4 ŠƣDèäIn-Fusion Advantage PCR Cloning Kit DƼ) In-Fusion ƍDà'$/=.)ƀ>A$ TEF1 Źû/pz^ aphA Źû/Ueǈ/Ǌ.ĸ£. Ť. @p}Z. weD pDisAphA +$. A. oryzae ãĺ÷§>/ AphA ^lQć/ĻļGwjøƊǋÄų =1ŝR~UX A. oryzae ÀćŸ¬§ APA4 DƠ*E2EƋŢRS ƋŢ* 5 ƃ®ĭ+Ƌƾ $½ŕDB)Ğ¹$ÒƋƾĤĹ 150 ml .°6A@^lQćD 80% ƪǌ /ǅ*ůź10 mM Tris-HCl bufferpH 7.0.ƻſk`nF.Ŗ)ž Ľ$ňĻļâŉDſk`nF*Ɵá$ HR16/20 Fast Flow Q-Sepharose IMÔ¬N}xGE Healthcare, Buckinghamshire, UK./!0.0 M > 0.3 M NaCl /ŮŃÙƊ.=?ƻė$AphA D°8ƝDſk`nF.Ŗ)žĽ íŻ HR16/20 Fast Flow Q-Sepharose IMÔ¬N}x./!$")·Ũ$^ lQćD 0.0 M > 0.3 M NaCl /ŮŃÙƊ.=?ƻė$ N ƭşGwjøƊǋDĽ. @$9.ĻļâŉD ProSorbApplied Biosystems. ōŤDƼ) PVDF Ƭ.ŸĉApplied Biosystems Procise 491 XQLWDƼ )ŁƲĜApplied Biosystems.Ĕ')ƊǋDÄų$Ļļâŉ/ŝR~UX 0 endoglycosidase H Glyko, Novato, CA, USA DƼ)yNŁƲĜ.Ĕ') à'$endoglycosidase H Dŷ. @Ņ.^lQć/ƢĶDà'$. 26.

(76) âŉ¦Ķ/Őų øĶsZnF^]¦Ķ0Śţ>Oike et al., 1984/ƦƧDČ«ƢÝ)à'$ ųǇ/âŉD 100 mM ıøk`nFpH 4.0.ƻ$ 1 mM p-gdnK g~øPNPPƻũ* 40°C10 ƝIO{qd$âŉƍD 10% d ~QıøƻD)Ųþ$ 2 M Şøfd~JxƻD$ÒƷǂ $ p-gdnKjPNPǇD 405 nm /·ÖŻ.=?Őų$âŉ |g` d0ƫħÆ* 1 Ɲ®. 1 mol / PNP DƷǂ. @âŉǇ+$ŽåƋƾ/ĥä0. øĶsZnF^]¦ĶŐų/ƍħÆ0 40°C10 Ɲ.ř)37°C20 Ɲ+ $âŉ/Āŵ pH 0ƺ- pH3.0 to 7.0/ 100 mM ıøfd~JxpH 4.0 ƻũ*âŉD 40°C10 ƝIO{qd)Őų$ âŉ/ĀŵŻ0 100 mM ıøfd~Jxƻũ*âŉDƺ-Ż25°C to 65°C* 10 ƝIO{qd )Őų$ŻųĶ+ pH ųĶ0ƺ-Ż25°C to 65°C* 30 ƝIO {qd$Ò+ƺ- pH3.0 to 7.0* 30°C1 Ă®IO{qd$Ò/ âŉ¦ĶD"A#AŐų$±ćƁĶ0/=.Ŭ4$100 mM ıøk` nFpH 4.0.ƻ$ 2 mM /±ćƻũ*âŉD 40°C30 ƝIO{qd $Ʒǂ$Ư´~øǇD Phosphor C Test KitWako Pure Chemical, Osaka, Japan DƼ)Őų$ GnFGw}]¦Ķ0×ųƧNishiya 1993.Ĕ')Őų$ũĶpb G]¦Ķ0×ųƧNishiya 1993DČ«ƢÝ)Őų$k`nF0 McIlvaine bufferpH 3.0.ř) McIlvaine bufferpH 6.0DúƼ$.

(77) ĈÊÅ=1ßö

(78) aphA Źû/ in silico QgR+ŹûƉ A. oryzae 0ƛĴ/øĶsZnF^]Aph=1nH^]Dĺ÷. @". /&(0ĻļAN ƭşGwjøƊǋÄųA)@Fujita et al., 2003a,. 27.

(79) A. B pyrG. 1. 2. 3. 4. 5. 6. 7. 8. (kb) phyA1. phyA6. 23.0 9.4 6.6 4.4. aphA KBN630-17K3 2.6 kb. 2.3 2.0. aphA gene disruption phyA1. phyA6. pyrG. aphA. 0.56. aphA 3.9 kb. Fig. 2-1. Construction scheme and confirmation of the aphA disrupted strain. A. Scheme of the aphA disruption is shown. PCR-amplified DNA fragments from a aphA disruption vector, pDisAphA, were transformed into A. oryzae KBN630-17K3. Gray and black boxes indicate the 5’-flanking region and a part of the coding region of the aphA gene, respectively, and the closed and open arrows indicate the aphA gene and pyrG gene regions, respectively. The direction of the arrow indicates the orientation of the aphA and pyrG genes. Small arrows indicate the position of oligonucleotide primers used. B. Agarose gel electrophoresis of amplified DNA fragments in the aphA gene region of the transformants using primer pair phyA1/phyA6.. 2003b"A>/GwjøƊǋ.Ŗ ZĤ.Éė%. @ŹûD A. oryzae Tjxc^q. +ėǀ-'$"/$90 A. oryzae Tjxc^q. Z> BLAST Çõ.=?ĲųøĶsZnF^]ŹûDŬë$"/Å5 Ð/ĲųøĶsZnF^]Źû+ 8 Ð/ĲųnH^]ŹûÉ('$Ĳ ųøĶsZnF^]Źû/ƌÎ0ŚŽ.°6A@nH_øƶǀ/ƩƗ-~. 28.

(80) ø.=')ƿĵA@+ß>A$"/ǁƶ0"A>/Źû Aspergillus niger MacRae et al., 1988; Penicillium chrysogenumHass et al., 1992ƶǀ/~øƿ ĵĶøĶsZnF^]+ŌſĶ@$9*@ù?/ÕƤŹû/ũ*Ķć =ƲA)@ A. niger phyA Źûvan Hartingsveldt et al., 1993+î:Ō ſĶ/ãŹûNITE DOGAN ID: AO090023000692DńŜ$/Źû0 A6*.ƥæA)@ A. oryzae RIB40 /nH^]ŹûGenBank/EMBL/ DDBJ accession no. AB042805Uchida et al., 2006+ſ*?aphA Źû+Ʊ ư$")0 AphA 5'-~thQM_e.ŖĳƝŝ~ø ¦ĶDĄ. ,DŬ4$. aphA ŹûƉ §DĎƀ. @$9.p}Zwe pDisAphA Dbpd+. p}IvrG phyA1/phyA6 DƼ$ PCR Ŏƚ.=?ƀ>A$ DNA ŠƣDúƼ )\nQgR.=? A. oryzae KBN630-17K3 DÀćŸ¬$Fig. 2-1 A ÀćŸ¬§.()p}IvrG phyA1/phyA6 DƼ$ PCR DàŎƚ$ DNA Šƣ/WI[.=?ÀćŸ¬§DŬ4$ Ŭ4$ 8 §/& 6 §.() 3.9-kb ŠƣÇėA$Fig. 2-1 B, lane 2 > 7Ʀù?/ 2 §.()0 3.9-kb Š ƣ.) 2.6-kb ŠƣÇėA$Fig. 2-1 B, lanes 1 =1 8A>/Å= ?Ņċ/ 6 §.) aphA ŹûƉ A$+ĄéA$ƀ>A$ aphA ŹûƉ §/& 3 §Dă/āÊ.úƼ. @+.$. aphA Źû/Ɖ Žåũ/øĶsZnF^]¦Ķ.ƹ@¼DŬ4@$9 .aphA ŹûƉ §/âŉĺ÷ĶDİ§+Ə¤$3 §/ aphA ŹûƉ §0 º.ĩĊŚŽr`d.ĸĦ.ĺ$$')aphA Źû/Ɖ 0ĩĊ ŚŽDƾÍ+$ĥä/ĺ.¼Dƹ-+C'$Table 2-2 .Ą. =. .aphA ŹûƉ §/øĶsZnF^]/ĺ÷0İ§.Ə4)ƴ 20% Ìğ $ƍŖ.Gw}]+ũĶpbG]/ĺ÷0İ§.Ə4)ƴ 10% Ŏ$ A>/Å>aphA Źû/Ɖ Gw}]/=-Ŕ/âŉĺ÷D>. 29.

(81) Table 2-2. Effect of aphA gene disruption on enzyme production Strain. -Amylase. Neutral protease. Acid phosphatase. (% of control). (% of control). (% of control). aphA-1. 778 ± 54.4 (122). 31,883 ± 1460.2 (110). 324 ± 30.5 ( 84). aphA-2. 721 ± 43.1 (113). 30,061 ± 1803.8 (104). 304 ± 32.7 ( 79). aphA-3. 695 ± 68.6 (109). 30,547 ± 601.0 (105). 309 ± 13.1 ( 80). A. oryzae KBN630. 636 ± 14.8 (100). 29,028 ± 338.0 (100). 387 ± 22.3 (100). The enzyme activity is shown as U/g koji. The activity of two independent experiments is presented as the average ± standard deviation.. Ŏ!@ƈĶĄéA$<.aphA ŹûƉ §*0Ŕ/øĶsZn F^]/ĺ÷DŎ$ƈĶ?aphA ŹûƉ /Ø0Ĉð=?űÉ ľ:>A)@:A-aphA Źû÷Ɯ/ņøĶsZnF^]¦Ķ. @²ƹǃDŬ4@$9.0Ŕ/øĶsZnF^]Źû¾/Ɖ §DĎƀ) Ľ. @Ɠƽ@.

(82) AphA /ãƌÎ+Ļļ aphA Źû÷Ɯ/âŉ¥ŴĶćDƲ>.. @$9.aphA ŹûD A. oryzae. TEF1 Źûpz^/ĵÓ*ãƌÎ!$TEF1 Źûpz^0 A. oryzae .)î:»pz^/(*@A. oryzae TEF1 Źû/pz ^ĵÓ. aphA ŹûDŊçE%ƌÎqQ^pTFAphA D *ę4 $=.ÞŦ$A. oryzae KBN630 §ƶǀ/ aphA Źû0Fig. 2-268 bp /I d 1 ÐD°8 1,469 bp >ķ?"/±Ɗǋ0 A. oryzae RIB40 /nH^ ]ŹûGenBank/EMBL/DDBJ accession no. AB042805+ªņ.ť$ aphA 30.

(83) ATGGCGGTCCTTAGCGTGCTCCTTCCCATTACCTTCCTTCTCTCGAGgtaagctcacccatagatgctgccctatagtggatgccctaat M A V L S V L L P I T F L L S S. 90 16. ctaacagcggctgatcttcattcagTGTTACCGGCACTCCGGTGACCAGCCCGAGACAACAGTCGTGCAATACCGTTGACGAAGGCTACC V T G T P V T S P R Q Q S C N T V D E G Y Q. 180 38. AGTGCTTCTCCGGGGTCTCTCACTTGTGGGGCCAGTATTCGCCTTACTTCTCGGTCGACGACGAGTCTTCCTTGTCCGAAGACGTTCCGG C F S G V S H L W G Q Y S P Y F S V D D E S S L S E D V P D. 270 68. ACCACTGCCAGGTTACCTTTGCCCAAGTGCTCTCCCGTCACGGTGCACGGTATCCAACGAAGAGCAAGTCTGAGAAGTACGCCAAGCTCA H C Q V T F A Q V L S R H G A R Y P T K S K S E K Y A K L I. 360 98. TCAAGGCCGTCCAGCATAATGCTACCTCGTTCTCCGGGAAGTATGCGTTCCTGAAATCTTACAACTACTCCCTCGGCGCCGATGACCTTA K A V Q H N A T S F S G K Y A F L K S Y N Y S L G A D D L T. 450 128. CGCCTTTTGGAGAGAACCAGTTGGTGGATTCGGGGATCAAGTTCTACCAGCGCTATGAGGAGCTCGCCAAGAACGTCGTTCCTTTCATTA P F G E N Q L V D S G I K F Y Q R Y E E L A K N V V P F I R. 540 158. GGGCATCGGGTTCGGATCGGGTAATCGCATCCGGCGAGAAATTCATCGAGGGCTTCCAGAAGGCAAAGCTTGGTGACTCTAAGTCTAAGC A S G S D R V I A S G E K F I E G F Q K A K L G D S K S K R. 630 188. GGGGCCAGCCTGCTCCTATTGTCAACGTAGTTATTACTGAGACCGAGGGTTTCAACAACACGTTGGACCACAGTCTCTGCACGGCCTTTG G Q P A P I V N V V I T E T E G F N N T L D H S L C T A F E. 720 218. AGAACAGCACAACAGGGGATGACGCAGAGGACAAGTTCACCGCTGTTTTTACGCCCTCGATTGTTGAGCGTCTGGAGAAGGACCTCCCAG N S T T G D D A E D K F T A V F T P S I V E R L E K D L P G. 810 248. GAACCACGCTCTCCAGCAAAGAGGTGGTTTATCTGATGGACATGTGCTCATTCGACACCATCGCCTTGACCCGTGACGGCAGTCGGCTAT T T L S S K E V V Y L M D M C S F D T I A L T R D G S R L S. 900 278. CCCCCTTCTGCGCTTTGTTCACCCAGGAAGAATGGGCACAATATGACTACCTGCAGTCAGTCTCTAAGTACTACGGCTACGGTGGAGGAA P F C A L F T Q E E W A Q Y D Y L Q S V S K Y Y G Y G G G N. 990 308. ACCCTCTCGGACCTGCGCAGGGCATCGGCTTCGCTAACGAGCTGATCGCTCGCCTGACCAAGTCTCCGGTTAAGGATCACACCACCACCA 1080 P L G P A Q G I G F A N E L I A R L T K S P V K D H T T T N 338 ATACCACGCTGGACTCAAATCCCGCCACCTTCCCGCTGAATGCTACGCTCTATGCGGACTTCTCGCACGATAACACGATGACCTCCGTTT 1170 T T L D S N P A T F P L N A T L Y A D F S H D N T M T S V F 368 TCTTCGCGCTTGGTCTGTATAATACGACCGAGCCCCTCTCTCAGACTTCGGTGCAGTCCACTGAGGAGACGAACGGATATTCATCCGCCC 1260 F A L G L Y N T T E P L S Q T S V Q S T E E T N G Y S S A R 398 GGACCGTTCCATTCGGGGCCAGAGCCTACGTCGAGATGATGCAGTGCACGGATGAGAAGGAGCCTCTCGTCCGCGTACTGGTCAACGACC 1350 T V P F G A R A Y V E M M Q C T D E K E P L V R V L V N D R 428 GGGTCATTCCGCTGCAAGGCTGTGATGCTGATGAGTATGGCCGGTGTAAACGGGACGATTTCGTCGAAGGACTGAGCTTCGTTACATCGG 1440 V I P L Q G C D A D E Y G R C K R D D F V E G L S F V T S G 458 GTGGAAACTGGGGAGAGTGCTTTGCTTAA 1470 G N W G E C F A * 466. Fig. 2-2. Nucleotide and deduced amino acid sequences of the aphA gene from A. oryzae KBN630. Numbers on the right refer to nucleotide sequence and amino acid sequence. Intron sequence is in lower-case letters. An asterisk (*) marks the translation stop codon. The N-terminal amino acid sequence analyzed chemically is thick-underlined. Potential N-glycosylation sites are fine-underlined.. 31.

(84) Table 2-3. Summary of purification of AphA from A. oryzae APA4 Specific Total. Total. Purification step. Recovery. Purification. (%). (fold). Activity activity (U) protein (mg) (U/mg). Culture filtrate. 1,414. 56.4. 25.1. 100.0. 1.0. 485. 18.4. 26.4. 34.3. 1.1. Q-Sepharose HP (1st). 361. 3.9. 92.5. 25.5. 3.7. Q-Sepharose HP (2nd). 303. 2.8. 108.4. 21.4. 4.3. Ammonium sulfate precipitation. ~ĻÖ½Ŏ¯hMWpTFAphA B A. oryzae PDE1 0Łņ RS ōĨ,ōŹ Đŏ. åł. &§áĺB. &ōŹĬ1 Aph ĆBĚĴ">-0;=AphA ½ĉÐB. &30 1§áĺ1jTeDWVĆ2 2.3 ~ 12.1 U/ml21 ~ 112 mg/l. ,=$?0Ġ. +Ġ÷"/A' pyrG ~ĻÖBŁņ. & A. oryzae PDE1 2. đĆBÞ/(& Ç8 AphA Ć1½(& APA4 ōŹ<1GKwµMvm\Nr eF0;(+ AphA BĤ1WwcMá05,Ċċ. &Table 2-3 AphA 1Ċċ. ŌŽ2 4.3 Ō,çŽ2 21.4%,(&Ċċ AphA 1WwcMááƀľ&=1Ŕ Ć2 108.4 U/mg ,(& AphA 2 SDS-PAGE ú, 58.0 < 65.0 kDa Fig. 2-3, lane 2 1fv]/bw],(&1ŝÖƀ2ćìWwcMá1EnaÒŋƄ0 *+ªÑ. &ŝÖƀ 47,581 Da ;=8ů 10.0 ~ 17.0 kDa ġħ,(&. AphA 1ŝÖƀ1ªÑħ-âÊ1ħ-1Ä2WwcMá1NsPSt0;>8 1,>-º<?>1-2AphA 1ăĴEnaÒŋƄ0 Asn-X-Ser / Thr W Gg1 N ¬¾¦NsPStŚz 7 òFig. 2-2 1Éď1EnaÒAsn-104,. 32.

(85) Fig. 2-3. SDS-polyacrylamide gel electrophoresis of AphA purified from an A. oryzae transformant, APA4. AphA was purified as described in the Materials and Methods. The gel was stained with Coomassie Brilliant Blue. The protein band corresponding to the endoglycosidase H is indicated by the arrow. Lane 1, molecular-mass markers: [rabbit muscle phosphorylase b (97.4 kDa), bovine serum albumin (66.3 kDa), rabbit muscle aldolase (42.4 kDa), bovine carbonic anhydrase (30.0 kDa), soybean trypsin inhibitor (20.1 kDa), and egg white lysozyme (14.4 kDa)]; lane 2, purified AphA; lane 3, endogylcosidase H treated AphA.. 33.

(86) Asn-119, Asn-206, Asn-219, Asn-338, Asn-351, Asn-375 ĜË">-5&Jw] NsPSXV H ,Ċċ¼ēBðŻ. & AphA 2Fig. 2-3, lane 3 ŝÖƀ 48.0 kDa. 0õ/(&-08×Û?>Ċċ AphA 1ŝÖƀ2?5,0ŤÀ?& Š1 A. oryzae ¤,½Ŏ¯?&~ĻÖÐŜ Uchida et al., 2006 -2|/(+& $1}2NsPSt1ĵļ1},2/-º<?1-2A. niger 0 A. fumigatus 1eFWVBŎ¯#>ST[o0+NsPSt1ĵļ2b ZYŨ0|/>-Ï?+>-Wyss et al., 1999b <8×Û?> Ċċ AphA 1 N ũEnaÒŋƄ2Gln-Ser-X-Asn-Thr-Val-Asp-Glu-Gly-Tyr-Gln X 2«Ĵřŉ ,(&Fig. 2-2Ğď1EnaÒ 1ŋƄ2Ķ0«Ĵ ,/(& Cys-30 Bô Gln-28 < Gln-38 1ăĴEnaÒŋƄ0+đ 0Ī. &SignalP 3.0 gvNroBŷ&Č0;=AphA 1 N ũĥ< 19 1E. naÒŋƄ2SN^tSMJwT,>-Ŵĕ?&:0AphA 1 20 < 27 œŭ1EnaÒŋƄ2ĝ1Øþ¤eFWV Wyss et al., 1999a, Lassen et al. 2001 -ŀŶ0gvigY]-ăĴ?&AphA 1 N ũĥEnaÒŋƄ2A. oryzae RIB128 1 2 )1 Aph ;3 2 )1eFWV-2ġ|/(+&1<AphA 2 A. oryzae RIB128 0+2ŝŕ?+/81-º<?&. AphA 1¼ēĶĆá Ċċ AphA 0)+1¼ēĶĆáBÚ® xĴĆ;3 ļxĴĆBÚ®. &Õ70Ùķ pHÙķ ļpH. &Fig. 2-4Fig. 2-5 AphA 1Ùķ pH 2 4.0Ù. ķ ļ2 40°C ,(&AphA 2 pH3.0 < pH 7.0 1¸ pH Œ{,xĴ,(& AphA 2 ļ 35°C 5,2xĴ,35°C Bį> ļ,¡ě0à. 34. &.

(87) 100.0. 100.0. 80.0. 80.0. 60.0. 60.0. 40.0. 40.0. 20.0. 20.0. 0.0. 0.0 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 pH. Residual activity (%). 120.0. Relative activity (%). 120.0. Fig. 2-4. Effects of pH on enzyme activity and stability of AphA. The effects of pH on enzyme activity were investigated by measurement at 40°C in various pH acetate buffers. Symbols: , activity;

(88) , stability.. 35.

(89) 120. 100. 100. 80. 80. 60. 60. 40. 40. 20. 20. 0. 0. Residual activity (%). Relative activity (%). 120. 25 30 35 40 45 50 55 60 65 Temperature Fig. 2-5. Effects of temperature on enzyme activity and stability of AphA. The effects of temperature on enzyme activity were investigated by measurement in pH4.0 at various temperatures. For the temperature stability, residual activity was measured at 40°C after incubation in acetate buffer (pH4.0) at various tempereatures. Symbols: , activity;

(90) , stability.. 36.

(91) Table 2-4. Comparison of the properties of AphA with those of other acid phosphatases from A. oryzae AphA. ACP-Ia. ACP-IIa. ACP-IIIa. AphK1b. 58.0 to 65.0. 110. 58. 56. 70. pH optimum. 4.0. 4.5. 5.0. 5.5. 5.5. pH stability. 3.0 to 7.0. 4.3 to 5.5. 4.8 to 6.5. 5.0 to 5.5. 2.0 to 7.0. Optimum temperature (°C). 40. 60. 40. 45. 60. Thermal stability (°C). <35. <45. <35. <40. NDc. Molecular mass (kDa). a. The results of ACP-I, ACP-II and ACP-III were taken from the report by Fujita et al. (Fujita. et al. 2003a). b c. The results of AphK1 were taken from the report by Shimizu (Shimizu 1993).. ND, not determined.. Ý0 AphA 1áŃ|ĆBÚ®. &Table 2-5 á2Ý181BÔŷ. &p-. _\veI_tswÒPNPP eFYwÒphytate α-NsUvswÒ glycerophosphate dvswÒpyrophosphate NtPT 6 swÒ D-glucose-6-phosphate 5' NE_tÒ5’-GMP ;3 5' GaSwÒ5’-IMP ,>AphA 2 PNPP BÇ8ġőěļ,Ąŝ. & . eFYwÒ. phytate α-NsUvswÒglycerophosphate ;3NtPT 6 swÒ D-glucose-6-phosphate 0Ġ. +8 2 ŝ1 1 ĵļ1ĆBÞ. AphA ¸áŃ. |ĆBű">¼ē,>-Ŭ<0/(&AphA 2 5’-GMP - 5’-IMP 0Ġ +87-8%1ģswÒĆBÞ. &1¬;=AphA 2ŪĒ0Ĺ?> 5'-. sk`MuKY]^\sHo1ģswÒ0>ĵļA(+>ŉĆÞÃ ?&AphA 2ĝ1 A. oryzae ŲźÒĆjTeDWV-2|/>áŃ|ĆBÛ(+ &1;0 N ũĥEnaÒŋƄ¼ēĶĆá;3áŃ|ĆBŔ">- 37.

(92) Table 2-5. Comparison of AphA substrate specificity with those of other acid phosphatases from A. oryzae Relative activitya (%) Substrate AphA. ACP-Ib. ACP-IIb. ACP-IIIb. AphK1c. Sodium p-nitrophenylphosphate. 100. 100. 100. 100. 100. Sodium phytate. 54.0. 14. 255. 23. 3.3. Sodium glycerophosphate. 49.2. 10. 0. 81. 93.3. Sodium pyrophosphate. 33.3. NDd. ND. ND. 183.3. D-Glucose-6-phosphate. 43.4. 52. 0. 45. ND. 5’-GMP. 8.1. ND. ND. ND. ND. 5’-IMP. 6.9. ND. ND. ND. ND. a. Hydrolysis rate of p-nitrophenylphosphate was taken as 100%.. b. The results of ACP-I, ACP-II and ACP-III were taken from the report by Fujita et al. (Fujita. et al. 2003a). c. The results of AphK1 were taken from the report by Shimizu (Shimizu 1993).. d. ND, not determined.. AphA 2 Fujita et al Fujita et al., 2003a, 2003b 9 ShimizuShimizu, 1993 ŤÀ &ÒĆjTeDWV9eFWV-2Ŭ0|/(+&5&?2 A. oryzae Ųź1ÒĆjTeDWV 5’-GMP - 5’-IMP <swÒBųż">-BıčĶ0 Þ. &ñ7+1ŤÀ-/(&. Ÿů ŪĒŷ¿¤ Aspergillus oryzae KBN630 1½ŘļėŀĔ©Bŷ oryzae KBN630 1ÒĆjTeDWV A ~ĻÖaphA ~ĻÖ BŊ 38. +A. &åł.

(93) & aphA ~ĻÖŊ2Ŀ¿,2Ĉü0ĉ Ć2 A. oryzae KBN630 -Ŕ WBŷ. &. +ů 20%ĳ. . ÒĆjTeDWV1. +&¿¤ TEF1 ~ĻÖgvq. + aphA ~ĻÖB½Ŏ¯#&-@AphA WwcMáBōĨĬ0. ŝŕ#>-,&AphA WwcMá1ŝÖƀ2 58.065.0 kDa ,Ùķ pH 2 4.0Ùķ ļ2 40°C ,(&AphA WwcMá2 5’-GMP 3 5’-IMP Bŝ +ūswÒBųż#&. 39.

(94) Ģø ŪĒŷ¿¤1äŸ/ÒĆjTeDWV C ~ĻÖaphC 1ŃĴ- AphC 1óĆá1Ŭ ó° Ģø,2A. niger phyA ~ĻÖ-Ç8ėŀĆ1½ A. oryzae KBN630 Ųź aphA ~ĻÖGenBank accession number AB042805 / AP007157 0)+~ĻÖŊ-~ ĻÖ½Ŏ¯0;>ČB»(&$1¬aphA ~ĻÖ1P]">ÒĆjTeD WV AphA 25’-IMP ;3 5’-GMP 1ģswÒĆB>ĵļű">- Ŭ<0/(&. . ;(+ 20% ĳ. Ŀ¿Ĭ1ÒĆjTeDWVĆ2aphA ~ĻÖŊ0 /(&?<1¬<Š1 A. oryzae 1ÒĆjTeD. WVĿ¿Ĭ1ÒĆjTeDWVĆ0ġŵ. +>-º<?&. $,Ģø,25’-IMP ģswÒĆBű">äŸ/ÒĆjTeDWVBŬ <0">&70A. niger phyA ~ĻÖ-ėŀĆ1>Ó= 7 ±1¶ţ1 aph ~ĻÖ ŊBÍí. Ŀ¿Ĭ1¼ēĉÐĆBÚ®. &$1¬aphC ~ĻÖ$. Ŀ¿Ĭ1ÒĆjTeDWVĆ;3 5’-IMP ģswÒĆ1žĆ0+ä Ÿ/ŮB&"-ŐŬ. &$,A. oryzae 0+Ç8£Ƃ/gvqW. 1),>WLEnrV~ĻÖtaaG2 ~ĻÖ 1gvqWBŷ+ aphC ~ĻÖB½Ŏ¯#AphC WwcMáBĊċ <0. +$1óĆá;3áŃ|ĆBŬ. &. â®Ìſ;3â®ťŦ Ôŷ¤ ŪĒŷ¿¤ A. oryzae KBN630 B DNA Įċ0Ôŷ. &Ģø,Íí. & A.. oryzae KBN630 Ųź1 pyrG, ku70 ~ĻÖńêÎôš|,> A. oryzae KBN63017K3 ;3ùŰŷ¿¤ KBN616 Ųź1 alp, pyrG ~ĻÖńêŊš|, > A. oryzae PDE1 B§áĺŷëä-. +Ôŷ. 40. &A. oryzae PDE1 2ĔW.

(95) wcMáB·Ž;ĉÐ#>&70EtLsgv[EV~ĻÖ alp ~ĻÖ Murakami et al., 1991 BŊ. &,>ŧ1ÍċťŦ0)+2.Š1. 0Ŏŗ">ŴĴ,>5&Ģø,Íí ÖŊ-. +Ôŷ. & A. oryzae aphA B aphA ~Ļ. &. E. coli DH5 α Bæ1 DNA ĦŢ1Mv_wN0Ôŷ. &. ōĨ;3ōŹý A. oryzae 1ğōŹ02ş,C4CōĨRS ōĨ 3% rice starch1% polypeptone 1% NaNO30.2% KCl0.1% KH2PO40.05% MgSO4.7H2O BÔŷ. + 30°Ců 160 rpm. 1ĺßā-ōŹB»(& Ŀ¿ōŹ2ÿãġĿiuZ\BōŹ-. + 30°C43 Ü1ōŹB»(&ÿ. ãġĿiuZ\2ġĿBĄ0 2 ÜĂĲ³35 ŝÿã ,ı¨ů 10 mmİ 15 ~ 20 mm 0ć§. +Ôŷ. +,&ÿãġĿBnwY. &. E.coli 1ōŹ02 2xYT ōĨ1.6% Bacto tryptone1.0% Bacto yeast extract0.5% NaCl BŷŖŸ0!+ňļ 50μg/mL 1 ampicillin sodium BĹ Agar BĹ. ²ğōŹÜ02 1.5%. &ōŹ2 37°C 0+»(&. A. oryzae 1§áĺ A. oryzaepyrG B MPU ōĨ2.0% malt extract0.1% Bacto peptone2.0% glucose 0.25% uridinepH6.5 Ĭ, 30 24 ÜōŹ³ 0Þ. &ťŦ-ŀŶ0. Novozyme234 ;3 Cellulase “ONOZUKA” R-10 Bŷ+gv\grT\. &ł. <?&gv\grT\0 PEG ĜË, DNA ĦŢ1å=Á6B»A#+§áĺB» ÆĉĸōĨĬ, 30°CůèōŹ. +§áĺBåł. &. A. oryzae ĎĀğ DNA 1ĮċPCR ;3 DNA SMJwT. 41.

(96) A. oryzae 1ĎĀğ DNA 2GP ōĨ, 30°C3 ŅōŹ é¤. +ĄŝBô¢. &³Ľ¬Ė. +ŞÈ. & A. oryzae 1¤ğB. eIat-MvvjtoŦ,Į. &Sambrook and Russell, 2001; Raeder and Bronda, 1985 . ċ. PCR ŷ¼ē2TaKaRa Ex Taq DNA lsprVTakara Bio, Otsu, Shiga ;3 PfuUltra II Fusion HS DNA polymeraseStratagene, La Jolla, CA, USA BÔŷ. $?. %?1gv\Pt0ï!+őB»(&PCR Ęĩ2 GeneAmp9700 RmtRGM rApplied Biosystems, Foster City, CA, USA BÔŷ Q`MuKY]grGmB Table 3-1 0Þ. &Ģø,Ôŷ. &Ks. &. ä/Mv_wNT[Zg,2model 4000LS DNA SOwRLI-COR, Lincoln, NE, USA ;3 GenomeLab GeXPBeckman Coulter, Brea, CA, USA Bŷ&S MJwSwN0;=ĉćŜBŇ. &. aph ~ĻÖ¥1ŊŷhMW1¹ī aphB ~ĻÖŊŷhMWpDisAphB By1;0¹ī. &aphB ~ĻÖ1. 5’-ƃčƁ 1.0-kb - 3’-ƃčƁ 1.0-kb B$?%? A. oryzae ĎĀğ DNA -grG miE phyB1/phyB2 ;3 phyB3/phyB4 Bŷ+ PCR ęś. &pyrG ~ĻÖĦŢ. 1.8-kb B A. oryzae ĎĀğ DNA -grGmiE pyrGN2/ pyrGC2Table 1-1 Bŷ + PCR ęś. &ł<?& 3 )1 PCR ęśĦŢ- SmaI ,ö. 1¾ª 4 ĦŢBÂ¾. In-Fusion Advantage PCR Cloning KitTakara Bio Bŷ+. In-Fusion őB»(&1;0. +ł<?&aphB ~ĻÖ1 5’-ƃčƁ- 3’-. ƃčƁ10 pyrG ~ĻÖzĩ">grTn]B pDisAphB pDisAphB B[wgu\-. &grTn]. grGmiE phyB1/phyB4 Bŷ& PCR 0;=. aphB ~ĻÖŊŷ DNA ĦŢBęś. A. oryzae KBN630-17K3 B§áĺ. aphB ~ĻÖŊ1ŇÚ®02§áĺğ<ĭí \-. &hMWpUC18. &. &ĎĀğ DNA B[wgu. +ŷgrGmiE phyB1/phyB4 Bŷ& PCR B»(&Table 1 1gr. 42.

(97) Table 3-1. Oligonucleotide primers used in Chapter 3 Name. Sequence (5’ to 3’). Direction. Primers for gene disruption The aphB gene phyB1. CGGTACCCGGGGATCCCAACCGTACTCCTAGGAGTGG. Forward. phyB2. TTCCAGCAGGCCTTGAAAGCATGGCGCACTATCGCGG. Reverse. phyB3. ATTGATCAGGCCTTTCGACCAGCTAACCAAGACTAGT. Forward. phyB4. ATGCCTGCAGGTCGACATGATCAACGAATTCCTTCAACG. Reverse. The aphC gene phyC1. CGGTACCCGGGGATCCTATGCCGGGATACTAACACAAT. Forward. phyC2. TTCCAGCAGGCCTTGCTATTAGTGGGTAATGCATAGTG. Reverse. phyC3. ATTGATCAGGCCTTTGAATGAGTGGGAGTATGTGCTC. Forward. phyC4. ATGCCTGCAGGTCGACAAGTCCAGATCTCGGAAATCAG. Reverse. The aphD gene phyD1. CGGTACCCGGGGATCCATCCTTAACAGTAGACTACTTGG. Forward. phyD2. TTCCAGCAGGCCTTGCAGACTAACACTTCTAGACTAAG. Reverse. phyD3. ATTGATCAGGCCTTTAACTTCCGAACCCGCTATGCTAG. Forward. phyD4. ATGCCTGCAGGTCGACAACTTGACTCTCGTTCTTCGGTG. Reverse. The aphE gene phyE1. CGGTACCCGGGGATCCCTGTGATCACTTTGAATAACACC. Forward. phyE2. TTCCAGCAGGCCTTGCAACATAACTTACTTGAAGACCAG. Reverse. phyE3. ATTGATCAGGCCTTTGGTCGTGATATTTTGCTTGCCAC. Forward. phyE4. ATGCCTGCAGGTCGACAGGTCCAGATTTCGGAGATTAG. Reverse. 43.

(98) The aphF gene phyF1. CGGTACCCGGGGATCCAGAATACAACATGCTGACTATGG. Forward. phyF2. TTCCAGCAGGCCTTGATGCGATACTAAGCAAAGCAATC. Reverse. phyF3. ATTGATCAGGCCTTTGCTCAAAGGGTCACTTGATCGCC. Forward. phyF4. ATGCCTGCAGGTCGACATACTAGATGGAACCGGAAAGG. Reverse. The aphG gene phyG1. CGGTACCCGGGGATCCTTTAGACAATTTCAGTCCCGCTC. Forward. phyG2. TTCCAGCAGGCCTTGAAATGCAGCAGGTACGTTCTC. Reverse. phyG3. ATTGATCAGGCCTTTGGATCAAATACGGCAGGCTG. Forward. phyG4. ATGCCTGCAGGTCGACATAGAGGATCGGATCCAAAGAG. Reverse. The aphH gene phyH1. CGGTACCCGGGGATCCACACAGAACAAACAAAAGATCTG. Forward. phyH2. TTCCAGCAGGCCTTGGTGAAGTTCTCTAGACTCTAGG. Reverse. phyH3. ATTGATCAGGCCTTTCCTTAATGACTGGAGCTATGGGC. Forward. phyH4. ATGCCTGCAGGTCGACCTTCATGAACGTGTCAAGCTCAC. Reverse. Primers for aphC expression vector fupyrGN CGGTACCCGGGGATCCAAGCCGCTGCTGGAATTGACA. Forward. pyrGC3 TCAGAAGAAAAGGATGATCAATACC. Reverse. pyrGtaa ATCCTTTTCTTCTGAATTCATGGTGTTTTGATCATTTT. Forward. taaPrev CATAAATGCCTTCTGTGGGGTTTATTGTT. Reverse. taaaphC CAGAAGGCATTTATGCAGCAATTATTGCAATCAACGG. Forward. aphCSal ATGCCTGCAGGTCGACGGGTTGATAGAGCTTGTTCTGGTGATC Reverse. 44.

(99) GmiEBÔŷ. +$1ĝ1 6 ±1 aph ~ĻÖŊhMWpDisAphC <. pDisAphH 5,B pDisAphB -ŀŶ1ťŦ,¹ī. &6 ±1 aph ~ĻÖŊŷ DNA. ĦŢ0)+8 aphB ~ĻÖ1û¾-ŀŶ0ęś. A. oryzae KBN630-17K3 0Łņ. &~ĻÖŊ1ŇÚ®02§áĺğ<ĭí \-. &ĎĀğ DNA B[wgu. +ŷ aph ~ĻÖ0Ń|Ķ/grGmiEaphC ~ĻÖ0)+2. phyC1/phyC4aphD ~ĻÖ0)+2 phyD1/phyD4aphE ~ĻÖ0)+2 phyE1/phyE4aphF ~ĻÖ0)+2 phyF1/phyF4aphG ~ĻÖ0)+2 phyG1/phyG4aphH ~ĻÖ0)+2 phyH1/phyH BÔŷ. + PCR B»(&. A. oryzae taaG2 ~ĻÖgvqW1ą´0> aphC ~ĻÖ1Ŏ ¯ aphC ~ĻÖB A. oryzae taaG2 ~ĻÖgvqWTsukagoshi et al., 1989 1ą´ ,Ŏ¯#>&70aphC ~ĻÖ½Ŏ¯ŷhMWpTAAphC By1;0¹ ī. &pyrG ~ĻÖĦŢ 1.8-kb B A. oryzae KBN630 1ĎĀğ DNA -grGm. iE fupyrGN/pyrGC3 Bŷ+ PCR ęś &A. oryzae taaG2 ~ĻÖ1gvqW 0.6-kb ĦŢB A. oryzae ĎĀğ DNA -grGmiE pyrGtaa/taaPrev Bŷ+ęś &aphC ~ĻÖ 2.0-kb ĦŢB A. oryzae ĎĀğ DNA -grGmiE taaaphC/aphCSal Bŷ+ęś. &ł<?& 3 )1 PCR ęśĦŢ-BamHI/SalI ,ö. &hMWpUC18 1¾ª 4 ĦŢBÂ¾. In-Fusion Advantage PCR Cloning Kit B. ŷ+ In-Fusion őB»(&1;0. +ł<?&taaG2 ~ĻÖ1gvqW. . aphC ~ĻÖ1P]Ɓ1ƃ0Ĉ0zĩ">grTn]B pTAAphC $. +grTn] pTAAphC B A. oryzae PDE1 0Łņ. &. &. aphC ~ĻÖ1Gw\vwBŇ">&70High Fidelity RNA PCR KitTakara Bio -³î1 AphC ½ĉÐ<ł<?&\Wt RNA Bŷ+ RT-PCR B»(&ń ŧÅ cDNA ¾ć2 Adaptor Primer FB - taaaphC 1grGmiEBŷ+»(&. 45.

(100) 0' HincII $Éx. èěõĠ9ID?iJ5. pUC118 'DhUiE. .

(101) A. oryzae ªÛ³ 3( AphC LiXD»(ÜÝ;`V´đĺā 2*ðEfGIgx »ą ( A. oryzae APC15 9 RS Ēö$ 5 ĎĒı . ë97|#È. ĒıÌÚ 50 ml 9 10 mM Tris-HCl bufferpH 7.0$ĉÞ. (ä©å. HR16/20 Fast Flow Q-Sepharose q<AiBea. t9ĊWOZ:$Ğ¨x. '(0.0 M 3 0.25 M NaCl (þá£đ'24ĮÃ. AphC 9/ĝ~9ĊW. OZ:'ì#ĉÞ¯ć HR16/20 Fast Flow Q-Sepharose q<AiBea' (ĊÏ$¯ćĮÃ. AphC 9/ĝ~9ĊWOZ:'ì#ĉÞ. 40% ģĻ%&52'Ķj9z $Ğ¨x. 1(940% ģĻĶj9z. ĊWOZ:. HiLoad 26/10 Phenyl Sepharose HPGE HealthcareBea'. B. ea9Ğ¨xWOZ:$=@OIcúLiXD»9 40% 3 0% ģĻĶj (þá£đ'24ĮÃ. AphC 9/ĝ~9 10 mM Tris-HCl bufferpH 7.0$ĉ. ÞSDS-PAGE $ñpWiS'&5%9ď 5. N Ħô;`V´đĺ9Þ. 0'ÜÝ©å9 ProSorbApplied Biosystemsçø9į# PVDF ĥ'ą½. Applied Biosystems Procise 491 ID?iH9į#ßĨÇApplied Biosystems 'Âđĺ9ā. ÜÝ©å(ðEfGIgx) endoglycosidase HGlyko, Novato,. CA, USA9į#bBßĨÇ'Â!#§! 'LiXD»(ğ×9§!. endoglycosidase H 9Ąz5ã. .

(102) ©å×(éā ´×^JZ:LK×)î÷3Oike et al., 1984(ġĢ9¾ğ¤#§! pāķ(©åt9 100 mM Ô´WOZ:pH 4.0'Į Ugfi´PNPPĮtû$ 40°C 10 ĝ<iCc\R. 46. . 1 mM p-URhZ>. ©åēu9 10% Rf.

(103) DhhÔ´Įt9z#Ā·. 2 M ó´TRf=aĮt9z. p-URhZ>VgPNPķ9 405 nm ( ć'24éā ĤÏ$ 1 ĝ' 1 mol ( PNP 9ĪĲ5©åķ%. ĪĲ. ©å dUOR). Ĉ¬Ēı(Í«)´×. ^JZ:LK×éā(ēuÏ) 40°C10 ĝ'í#37°C20 ĝ%. . ©å(¸ă pH )ĭ& pH3.0 to 7.0( 100 mM Ô´TRf=apH 4.0Į tû$©å9 40°C10 ĝ<iCc\R#éā. ©å(¸ăvć)100 mM. Ô´TRf=aĮtû$©å9ĭ&vć25°C to 65°C$ 10 ĝ<iCc\R #éā c\R. vćjā×% pH jā×)ĭ&vć25°C to 65°C$ 30 ĝ<iC %ĭ& pH3.0 to 7.0$ 30°C1 ¹<iCc\R. (. 100 mM Ô´WO. ©å×966éā. »ċm×)kw(2'ý+. Z:pH 4.0'Į. 2 mM (»Įtû$©å9 40°C30 ĝ<iCc\. R. ĪĲ. 9į#éā. ħfi´ķ9 Phosphor C Test KitWako Pure Chemical, Osaka, Japan . ;gZ:;`eK×)¡āĢNishiya, 1993'Â!#éā ;K×)¡āĢNishiya 19939¾ğ¤#éā. WOZ:) McIlvaine. bufferpH 3.0'í# McIlvaine bufferpH 6.09µį 5’-IMP (ðfi´×)kw(2'§!. . 1 mL ( 20 mM IMP Įt9 0.2 mL. ( 200 mM Ô´WOZ:pH 4.0%«0.1 mL (ĉÞ 37°C $ 20 ĝ<iCc\R. û×[hP. Ēıt9z#. ēu9 0.1 mL ( 1 N NaOH Įt9z#Ā·. 0.1 mL ( 1 N HCl Įt9z#ûĻ. ĪĲ. fi´9 Pi ColorLock ALS. Innova Biosciences Ltd., Cambridge, UK9į#éā. ©å×(ā)Ĥ. éāÏw$ 1 ĝ' 1 μmol (fi´9ĪĲ5©åķ9 1 dUOR%. .

(104) GiYcL'25đĺÞ aph nĆ¶(æĊ×±)ČĴ§ØĢÓÝĘėyÄĔ¥NITE( the. 47.