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Identification and characterization of starch and inulin modifying network of Aspergillus niger by functional genomics

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Identification and characterization of starch and inulin modifying network of Aspergillus niger by functional genomics

Yuan, X.L.

Citation

Yuan, X. L. (2008, January 23). Identification and characterization of starch and inulin modifying network of Aspergillus niger by functional genomics.

Institute of Biology Leiden (IBL), Group of Molecular Microbiology, Faculty of Science, Leiden University. Retrieved from

https://hdl.handle.net/1887/12572

Version: Corrected Publisher’s Version

License: Licence agreement concerning inclusion of doctoral thesis in the Institutional Repository of the University of Leiden

Downloaded from: https://hdl.handle.net/1887/12572

Note: To cite this publication please use the final published version (if applicable).

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ᨬ㽕









咥᳆䳝ᰃϔ⾡ᑓ⊯ᄬ⌏Ѣỡ⠽⅟ԧЁⱘϱ㦠ԧᓣⳳ㦠DŽ԰Ўϔ⾡㜤⫳㦠ˈᅗ㛑ѻ⫳໻䞣ϡৠ ⱘ∈㾷䝊ˈ⫼ᴹ䰡㾷⦃๗Ёỡ⠽ⱘ催ߚᄤ໮㊪៤ߚЎᇣߚᄤ࣪ড়⠽ˈҢ㗠Ў݊ᴀ䑿ᦤկ㛑⑤

੠⺇⑤DŽ䖭ѯ䝊໻䛑᳝ᕜ໮˄┰೼ⱘ˅⫼䗨ˈ↨བ˖೼䴶ࣙϮˈ⎔㉝Ϯˈ㒛⠽Ϯҹঞ亳ક੠

但᭭ᎹϮㄝDŽ䖭ѯ⫼䗨䖥ᴹ਌ᓩњ໻䞣ⱘⷨお݈䍷DŽ䗮䖛䘫Ӵǃ⫳࣪ǃ⫳⠽ֵᙃㄝ䗨ᕘᴹᓔ থ咥᳆䳝ᮼ೼᳈ࡴ⏙Ἦഄњ㾷݊ᇍ㊪㉏⠽䋼ⱘ䰡㾷ǃׂ佄㔥㒰ˈҢ㗠Ўᦤ催䝊ⱘѻ䞣੠ᑩ⠽

ⱘ߽⫼⥛ᦤկ⾥ᄺձ᥂DŽ঺໪ˈҢ咥᳆䳝Ёথ⦄ᮄ⌏ᗻ䝊гᰃϔϾЏ㽕಴㋴DŽ䖥ᑈᴹ⬅㥋݄

ⱘ⫳⠽ᡔᴃ݀ৌ DSM ੠㕢೑ⱘ DOE ෎಴㒘ⷨお᠔ᇍ咥᳆䳝ⱘ෎಴㒘⌟ᑣⱘᅠ៤ˈҹঞ咥᳆

䳝ⱘ෎಴㢃⠛ᡔᴃⱘ݋໛ˈՓᕫҢ咥᳆䳝Ёথ⦄ᮄ⌏ᗻ䝊ҹঞҢ෎಴㒘∈ᑇϞᴹߚᵤ݊ᴎԧ ݙⱘ⺇∈࣪ড়⠽˄㊪㉏˅䰡㾷ⱘ䕀ᔩ䇗᥻㔥㒰៤Ўৃ㛑DŽ

䖭㆛䆎᭛ˈ៥ӀⱘЏ㽕ⷨおⳂᷛᰃ᥶㋶咥᳆䳝䰡㾷ǃׂ佄⺇∈࣪ড়⠽ⱘߚᄤᴎࠊDŽ

↨བˈ咥᳆䳝ᰃབԩᛳফࠄ਼ೈ⦃๗Ёᄬ೼ⱘϡৠ⺇⑤ⱘ˗ᶤ⾡⡍ᅮ䇅ᇐ⺇⑤ⱘᄬ೼៪ѻ⫳

জᰃབԩ▔⌏䖭ѯ䰡㾷䝊෎಴ⱘ䕀ᔩ㸼䖒ⱘDŽ៥Ӏⱘⷨお䞡⚍೼Ѣᇏᡒ໮㊪㉏⠽䋼བ⎔㉝ǃ 㦞㊪ǃҹঞᅗӀⱘ㸡⫳⠽೼咥᳆䳝ԧݙⱘ䰡㾷䗨ᕘЁߎ⦄ⱘ䇅ᇐߚᄤ੠䕀ᔩ䇗㡖಴ᄤDŽ঺

໪ˈ⬅䖭ѯ㒧ᵘ෎಴㓪ⷕⱘ䝊ᰃབԩڀ࣪ϡৠⱘ⺇⑤ⱘˈҹঞ䖭ѯ䝊ⱘ⫳⧚ࡳ㛑ᰃҔМˈг ᰃ៥Ӏⷨおⱘ䞡㽕ᮍ䴶DŽ

ᴀ䆎᭛ⱘ㄀㄀ϔゴὖᣀњ咥᳆䳝԰Ў䝊ѻ⫳㦠ⱘ䞡㽕ᗻҹঞᔧ᳝ࠡ݇⎔㉝੠㦞㊪䰡㾷ׂ佄䝊ⱘ ⶹ䆚DŽ᳝݇⺇∈࣪ড়⠽䰡㾷ⱘ䝊ⱘ䕀ᔩ㸼䖒੠݊㲟ⱑѻ䞣೼᳝ᴎԧݙᰃ催ᑺ䇗㡖ⱘDŽ䖭⾡䇗 㡖Џ㽕⬅৘⾡ϡৠⱘᑓ⊯䕀ᔩ಴ᄤ˄བ CreAǃAreAǃPacC ㄝ˅ˈ੠⡍ᅮ㊪㉏䰡㾷䗨ᕘⱘ䕀 ᔩ಴ᄤ˄བ AmyR ੠ InuR˅ᴹ᥻ࠊⱘDŽ䖭ϔゴЏ㽕㒭䇏㗙њ㾷᳝݇䇗㡖෎಴㸼䖒ⱘ䕀ᔩ಴

ᄤⱘϔѯ㚠᱃ֵᙃˈৠᯊг䆺㒚䯤䗄њϢ⺇∈࣪ড়⠽䰡㾷᳝݇ⱘ䝊㓪ⷕ෎಴ⱘ䇗㡖ᴎࠊҹঞ ϡৠ䕀ᔩ಴ᄤⱘϔѯ䞡㽕⡍ᕕDŽ

Ўњথ⦄䰡㾷⎔㉝ǃ㦞㊪᳝݇ⱘᮄ⌏ᗻ䝊ˈ៥Ӏ䞛⫼њϸ⾡ᮍ⊩DŽ㄀ϔ⾡ᮍ⊩೼㄀㄀ Ѡゴ䞠䯤䗄DŽ䆹ᮍ⊩ᰃ߽⫼ܟ䱚ᡔᴃᴹᵘᓎ⎔㉝੠㦞㊪⡍ᅮⱘ咥᳆䳝෎಴㸼䖒᭛ᑧDŽ䖭ѯ෎

಴㸼䖒᭛ᑧߚ߿ᵘᓎ೼ҹ㒚㦠੠䝉↡Ў䕀࣪ԧⱘ䕑ԧϞDŽߚᄤߚᵤ䆕ᯢ䖭ѯ෎಴㸼䖒᭛ᑧ䋼 䞣ᇮདᑊ㹿䕀࣪ࠄ㒚㦠੠䝉↡ԧݙDŽ⬅Ѣ cDNA 㗏䆥ⱘ㲟ⱑ㸼䖒䞣ҹঞ㲟ⱑ㸼䖒䕀࣪ԧⱘ᭄

䞣ⱘϡ䎇ˈⳂࠡাㄯ䗝ߎᇥ䞣ⱘ䕀࣪ԧDŽ䆹ゴ᳿ሒᦤߎњ޴⾡ᬍ୘ cDNA 㸼䖒᭛ᑧⱘᵘᓎঞ ㄯ䗝ᮍ⊩ⱘᓎ䆂੠᥾ᮑDŽ

㄀Ѡ⾡ᮍ⊩ᰃ߽⫼Ꮖⶹ⌏ᗻ䝊ⱘᑣ߫ᴹ䇗ᶹ咥᳆䳝ⱘ෎಴㒘ˈҢ㗠㦋ᕫⳌԐᑣ߫㓪 ⷕⱘ㲟ⱑDŽ೼ᴀ᭛ⱘ㄀㄀ϝゴˈ៥Ӏҟ㒡њỡ⠽⎔㉝ৃ㹿 ⎔㉝䝊ˈ㨵㧘㊪࣪䝊ঞ 㨵㧘㊪㣋 䝊䰡㾷៤ᇣߚᄤⱘ㨵㧘㊪DŽ䖭ѯ䝊ߚ߿ሲѢ㊪㣋∈㾷䝊㋏ 13ǃ15 ੠ 31 ᆊᮣˈ಴ℸ೼㄀ϝゴ

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থ⦄њ 17 Ͼᮄ㲟ⱑ䝊DŽ䗮䖛෎಴㒘ᑣ߫ҹঞᅗӀⱘ䕀ᔩ㸼䖒∈ᑇ䇗㡖ⱘ㓐ড়ߚᵤˈ㒧ᵰᰒ⼎

໻䚼ߚ෎಴㸼⦄ߎϡৠᇏᐌⱘ䕀ᔩ䇗㡖῵ᓣDŽгህᰃ䇈ˈᅗӀ᮶ϡফ⎔㉝䰡㾷䗨ᕘⱘ䇅ᇐ⠽

呺㢑㊪ⱘ䇅ᇐˈгϡফ⎔㉝䰡㾷䇗㡖಴ᄤAmyR ⱘ䇗᥻DŽা᳝ϸ⾡䝊ˈ㊪㣋䝊 AgdB ੠

⎔㉝䝊 AmyC, ৃ㛑݋໛䰡㾷⎔㉝ⱘ⌏ᗻDŽ݊ᅗⱘ㲟ⱑ䝊ࣙᣀ㚲ݙ䝊ǃ㚲ຕⳌ݇ⱘ䝊ˈᕜ᳝

ৃ㛑⍝ঞࠄ㒚㚲ຕⱘ㒘៤៤ߚ 㨵㘮㊪ⱘׂ佄䖛⿟ˈ㗠ϡ⍝ঞ⎔㉝ⱘ䰡㾷䖛⿟DŽ䖭ѯ؛䆒೼

㄀ϝゴЁᕫࠄ䆺㒚䅼䆎DŽ

೼ࠡϔゴᦤࠄⱘ GH13 䈅㋏ⱘ㲟ⱑЁˈ᳝ϝϾ㲟ⱑ䝊˄AgtAǃAgtBǃAgtC˅ሑㅵ Ϣⳳ㦠⎔㉝䝊㉏᳝催ᑺⱘᑣ߫ⳌԐᗻˈԚ݊݋᳝঺໪ⱘ C ᳿ッֵো㚑ᑣ߫,े㊪෎࣪⻋㛖䝄㙠 䝛˄GPI ˅ⱘ䰘ⴔֵোᑣ߫ˈᑊϨ㔎Уᶤѯ  ⎔㉝䝊ⱘ催ᑺֱᅜⱘ⇼෎ᑣ߫DŽ䆹䆎᭛ⱘ㄀㄀ಯ ゴᦣ䗄њ䖭ѯ㲟ⱑ䝊೼咥᳆䳝Ёⱘ䖛䞣㸼䖒ǃᦤপ੠㒃࣪ⱘࡳ㛑ᗻ䋼ˈҹঞ݊㓪ⷕ෎಴ⱘߴ 䰸੠䖛䞣㸼䖒᠔㸼⦄ߎⱘ⫳⧚㸼㾖⡍ᗻDŽ៥Ӏথ⦄ AgtA੠AgtB ഛ㸼⦄ߎ㊪㣋䕀⿏䝊ⱘ⡍ᕕ DŽ݊Ё AgtA ⱘ෎಴ߴ䰸㸼⦄ߎᇍ㒚㚲ຕᑆᡄ⠽ Calcofluor White (CFW)ᔎ⚜ⱘᬣᛳᗻˈ㸼 ᯢ䆹෎಴ⱘ㔎༅㟈Փ㒚㚲ຕݙ䚼㒧ᵘⱘᅠᭈᗻফࠄ⸈ണDŽ㲟ⱑᑣ߫ߚᵤᰒ⼎ AgtA ੠ AgtB ⱘ ৠ⑤㲟ⱑгߎ⦄೼݊ᅗ᳝ 㨵㘮㊪៤ӑⱘ㒚㚲ຕⱘⳳ㦠Ёˈ㗠ϡ೼㔎У 㨵㘮㊪ⱘ㒚㚲ຕⱘ 䝉↡ЁDŽ䖭ϔ⦄䈵䖯ϔℹ㸼ᯢ AgtA 㲟ⱑ䝊⌏ᗻϢ㒚㚲ຕ㒧ᵘ᳝݇DŽ

㊪㣋∈㾷䝊㋏ 32 ᆊᮣ㲟ⱑ᳝ৃ㛑⍝ঞࠄ㦞㊪੠ᅗⱘড়៤ᑩ⠽㫫㊪ⱘ䰡㾷䖛⿟DŽ ೼

㄀Ѩゴ䞠ˈ៥Ӏ䇗ᶹњ咥᳆䳝෎಴㒘ᴹᇏᡒሲѢ GH32 䈅㋏ⱘ෎಴DŽ䰸њᏆⶹⱘϝ⾡㚲໪䝊

SucAˈInuE ੠ InuA ໪ˈ៥Ӏ䖬থ⦄њ঺໪ϸ⾡㚲ݙ䝊ˈSucB ੠ SucCDŽ䕀ᔩߚᵤ㸼ᯢ䖭ѯ 㚲໪䝊ᰃणৠ䇗㡖ⱘᑊফ㦞㊪੠㫫㊪䇅ᇐⱘDŽᅗӀⱘ䕀ᔩ∈ᑇгফࠄ⺇ڀ࣪ҷ䇶ᡥࠊ಴ᄤ CreA ⱘ᥻ࠊDŽ䖯ϔℹⱘᅲ偠ߚᵤ㸼ᯢ೼咥᳆䳝ԧݙˈᰃ㫫㊪៪ᅗⱘ㸡⫳⠽ˈ㗠ϡᰃᵰ㊪ˈ԰

Ў䇅ᇐ⠽ᴹ䇅ᇐ㦞㊪ߚ㾷䝊෎಴ⱘ䕀ᔩ㸼䖒ⱘDŽ

೼㄀㄀݁ゴ䞠ˈ៥Ӏ䇗ᶹњ݊ЁϔϾ GH32 䈅㋏ⱘ㚲ݙ䰡㾷䝊SucBˈẔᶹ݊ᰃ৺೼㦞

㊪៪㫫㊪ⱘ䰡㾷Ё䍋ⴔѻ⫳䇅ᇐ⠽ⱘ԰⫼DŽ៥Ӏᇍ SucB㲟ⱑⱘ㋏㒳䖯࣪ǃߚᄤ੠⫳࣪⡍ᗻ䖯 㸠њⷨおDŽSucB ⱘ䖛䞣㸼䖒ᑊ㒃࣪ⱘ㲟ⱑ݋᳝㚲ݙ䰡㾷㫫㊪䝊ⱘ⡍ᗻᑊݐ᳝䕀࣪ᵰ㊪ⱘ⌏

ᗻDŽ䆹෎಴ⱘߴ䰸ᑊ≵᳝ᬍব咥᳆䳝೼㦞㊪៪㫫㊪Ё⫳䭓⡍ᕕˈ㸼ᯢ SucB㲟ⱑᑊϡ೼㦞㊪៪

㫫㊪ⱘ䰡㾷Ё䍋ⴔѻ⫳䇅ᇐ⠽ⱘ԰⫼DŽԚᰃ SucB ᳝ৃ㛑೼㒚㚲ݙⱘ㫫㊪䕀࣪៤ᵰ㊪ǃ㨵㧘㊪

៪݊ᅗᇣߚᄤⱘᆵ㊪Ё㸠Փϔᅮⱘࡳ㛑DŽ

ℷབ೼㄀ѨゴЁᦤࠄⱘˈ㚲໪㦞㊪ߚ㾷䝊㉏೼䕀ᔩ∈ᑇϞᰃणৠ䇗㡖ⱘᑊফ㫫㊪੠

㦞㊪ⱘ䇅ᇐˈ䖭Ͼ㒧ᵰᦤ⼎೼㦞㊪ⱘ䰡㾷䗨ᕘЁᄬ೼ⴔϔϾ݅ৠⱘ䕀ᔩ䇗㡖಴ᄤˈ೼䇅ᇐ⠽

ߎ⦄ⱘᚙމϟˈ䆹䇗㡖಴ᄤ▔⌏݊㒧ᵘ෎಴ⱘ䕀ᔩ㸼䖒DŽ೼ᴀ᭛ⱘ㄀㄀ϗゴЁˈ៥Ӏ䆺㒚䯤䗄 њ䖭Ͼᮄⱘ㦞㊪䕀ᔩ▔⌏಴ᄤInuR ⱘথ⦄ঞ݊⡍ᕕDŽInuR ⱘথ⦄ᰃ෎Ѣ咥᳆䳝෎಴㒘Ё㦞

㊪䝊㉏෎಴㕸ⱘ䇗ᶹDŽinuR ෎಴䴴䖥 SucB ෎಴ˈѠ㗙П䯈䱨ⴔϔϾ㊪㉏䖤䕧㲟ⱑ෎಴

(An15g00310)DŽinuR෎಴ⱘߴ䰸ᇐ㟈咥᳆䳝೼㦞㊪੠㫫㊪ЁⱘϹ䞡⫳䭓㔎䱋DŽ㦞㊪੠㫫㊪ᇍ 㚲໪㦞㊪䝊෎಴ҹঞ䖭㊪䖤䕧㲟ⱑ෎಴ⱘ䇅ᇐᰃձ䌪Ѣ InuR 䇗㡖಴ᄤⱘDŽ෎಴㒘ⱘ䕀ᔩ㸼䖒 ߚᵤথ⦄঺໪ϝϾ㊪䖤䕧㲟ⱑ෎಴(An15g04060,An15g03940andAn17g01710)ˈᅗӀгফࠄ 㫫㊪ⱘᔎ⚜䇅ᇐᑊϨձ䌪Ѣ InuR DŽ䖭ѯ㊪䖤䕧㲟ⱑ෎಴ⱘ݋ԧࡳ㛑ˈ⡍߿ᰃ An15g00310 㓪 ⷕⱘ㊪䖤䕧㲟ⱑᰃϾᕜ᳝ᛣНⱘ䇒乬ᑊϨℷ೼⫼⫳࣪ߚᵤⱘ᠟↉ⷨおЁDŽ⬅⬉㛥῵ᢳߚᵤ᠔

᳝ InuR 䇗᥻ⱘ෎಴ⱘਃࡼᄤ៥Ӏথ⦄ϔϾ┰೼ⱘ InuR 㒧ড়ԡ⚍CGGN8CGGˈ䆹ᑣ߫ᕜ㉏ ԐѢ⎔㉝䰡㾷䇗㡖಴ᄤAmyR 㒧ড়ԡ⚍CGGN8C/AGGDŽঠ䞡෎಴ⱘߴ䰸ߚᵤᧁ⼎њ InuR

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੠ AmyR ⱘ⣀ゟ䇗᥻ࡳ㛑DŽおコ InuR ੠ AmyR ᰃབԩ䕼䅸ᅗӀ৘㞾ⱘⳂᷛ෎಴ҹঞҔМ಴

㋴އᅮᅗӀⱘ㒧ড়⡍ᓖᗻᇚᰃϾ᳝ᛣНⱘ䇒乬ᑊ᳝ᕙѢ䖯ϔℹⷨおDŽ

೼䖭㒘㦞㊪䝊෎಴䈅Ёˈ៥Ӏথ⦄ inuE ᰃফࠄ㦞㊪੠㫫㊪䇅ᇐ᳔ᔎⱘ෎಴DŽ೼ᴀ᭛

ⱘ㄀㄀ܿゴˈ៥Ӏ߽⫼inuE ෎಴ⱘਃࡼᄤˈߚ߿㒧ড় racAG12V ੠ GFP ᡹ਞ෎಴ˈᡒࠄњϔ Ͼᮄⱘㄯ䗝ᮍ⊩ᴹߚ⾏ߎ㦞㊪៪㫫㊪ֵো䗨ᕘЁⱘさবԧDŽ䗮䖛䖭䘫Ӵㄯ䗝ᮍ⊩㒧ড়さবԧ ⱘ䕼߿៥Ӏ㛑໳ᡒࠄ䙷ѯѻ⫳䇅ᇐߚᄤˈ䖤䕧៪ᛳᑨ䇅ᇐߚᄤⱘ㲟ⱑˈҹঞ䙷ѯ▔⌏㦞㊪䰡 㾷䗨ᕘⱘ䝊෎಴䕀ᔩ಴ᄤDŽ䆹ᮍ⊩ৃҹ㹿ᑓ⊯䖤⫼ࠄᇏᡒ䕀ᔩ಴ᄤⱘさবԧDŽ೼ᓎゟ䖭Ͼৃ

㛑ᗻⱘㄯ䗝ᮍ⊩ⱘ䖛⿟Ёˈ៥Ӏথ⦄ẝ㉑㊪г᳝䇅ᇐ㦞㊪䝊෎಴ⱘ⡍ᗻDŽ

෎Ѣᴀ䆎᭛᳔ৢಯϾゴ㡖ⱘ㒧ᵰˈ៥ӀᦤߎњϔϾ咥᳆䳝ԧݙ㦞㊪䰡㾷ֵো䗨ᕘⱘ

؛䇈῵ൟDŽ䆹؛؛䇈῵ൟ݋ԧ䰜䗄೼㄀ܿゴⱘ᳿ሒDŽ

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ᨬ㽕



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