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
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咥᳆䳝ᰃϔ⾡ᑓ⊯ᄬ⌏Ѣỡ⠽⅟ԧЁⱘϱ㦠ԧᓣⳳ㦠DŽЎϔ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ᴹᓩњ䞣ⱘⷨお݈䍷DŽ䗮䖛䘫Ӵǃ⫳࣪ǃ⫳⠽ֵᙃㄝ䗨ᕘᴹᓔ থ咥᳆䳝ᮼࡴ⏙Ἦഄњ㾷݊ᇍ㊪㉏⠽䋼ⱘ䰡㾷ǃׂ佄㔥㒰ˈҢ㗠Ўᦤ催䝊ⱘѻ䞣ᑩ⠽
ⱘ߽⫼⥛ᦤկ⾥ᄺձDŽˈҢ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⠛ᡔᴃⱘˈՓᕫҢ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⦃๗Ёᄬⱘϡৠ⺇⑤ⱘ˗ᶤ⾡⡍ᅮ䇅ᇐ⺇⑤ⱘᄬѻ⫳
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᭛ⱘϔ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ᔩᄤ˄བ CreAǃAreAǃPacC ㄝ˅ˈ⡍ᅮ㊪㉏䰡㾷䗨ᕘⱘ䕀 ᔩᄤ˄བ AmyR InuR˅ᴹࠊⱘDŽ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ⷕⱘ䇗㡖ᴎࠊҹঞ ϡৠ䕀ᔩᄤⱘϔѯ䞡㽕⡍ᕕDŽ
Ўњথ⦄䰡㾷⎔㉝ǃ㦞㊪᳝݇ⱘᮄ⌏ᗻ䝊ˈ៥Ӏ䞛⫼њϸ⾡ᮍ⊩DŽϔ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߿ᵘᓎҹ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ⱑDŽᴀ᭛ⱘϝ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 ᆊᮣˈℸϝ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ᑺⱘᑣ߫ⳌԐᗻˈԚ᳝݊ⱘ C ッֵো㚑ᑣ߫,े㊪࣪⻋㛖䝄㙠 䝛˄GPI ˅ⱘ䰘ⴔֵোᑣ߫ˈᑊϨ㔎Уᶤѯ ⎔㉝䝊ⱘ催ᑺֱᅜⱘ⇼ᑣ߫DŽ䆹䆎᭛ⱘಯ ゴᦣ䗄њ䖭ѯ㲟ⱑ䝊咥᳆䳝Ёⱘ䖛䞣㸼䖒ǃᦤপ㒃࣪ⱘࡳ㛑ᗻ䋼ˈҹঞ݊㓪ⷕⱘߴ 䰸䖛䞣㸼䖒᠔㸼⦄ߎⱘ⫳⧚㸼㾖⡍ᗻDŽ៥Ӏথ⦄ AgtAAgtB ഛ㸼⦄ߎ㊪㣋䕀⿏䝊ⱘ⡍ᕕ DŽ݊Ё AgtA ⱘߴ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㋏ⱘDŽ䰸њᏆⶹⱘϝ⾡㚲䝊
SucAˈInuE InuA ˈ៥Ӏ䖬থ⦄њϸ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ᡥࠊᄤ CreA ⱘࠊDŽ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ⱑ᳝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
ℷབѨ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ᕘЁᄬⴔϔϾ݅ৠⱘ䕀ᔩ䇗㡖ᄤˈ䇅ᇐ⠽
ߎ⦄ⱘᚙމϟˈ䆹䇗㡖ᄤ▔⌏݊㒧ᵘⱘ䕀ᔩ㸼䖒DŽᴀ᭛ⱘϗゴЁˈ៥Ӏ䆺㒚䯤䗄 њ䖭Ͼᮄⱘ㦞㊪䕀ᔩ▔⌏ᄤInuR ⱘথ⦄ঞ݊⡍ᕕDŽInuR ⱘথ⦄ᰃѢ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)DŽinuRⱘߴ䰸ᇐ㟈咥᳆䳝㦞㊪㫫㊪ЁⱘϹ䞡⫳䭓㔎䱋DŽ㦞㊪㫫㊪ᇍ 㚲㦞㊪䝊ҹঞ䖭㊪䖤䕧㲟ⱑⱘ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ⱑⱘԧࡳ㛑ˈ⡍߿ᰃ An15g00310 㓪 ⷕⱘ㊪䖤䕧㲟ⱑᰃϾᕜ᳝ᛣНⱘ䇒乬ᑊϨℷ⫼⫳࣪ߚᵤⱘ↉ⷨおЁDŽ⬅⬉㛥ᢳߚᵤ᠔
᳝ InuR 䇗ⱘⱘਃࡼᄤ៥Ӏথ⦄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
AmyR ⱘ⣀ゟ䇗ࡳ㛑DŽおコ InuR AmyR ᰃབԩ䕼䅸ᅗӀ㞾ⱘⳂᷛҹঞҔМ
㋴އᅮᅗӀⱘ㒧ড়⡍ᓖᗻᇚᰃϾ᳝ᛣНⱘ䇒乬ᑊ᳝ᕙѢ䖯ϔℹⷨおDŽ
䖭㒘㦞㊪䝊䈅Ёˈ៥Ӏথ⦄ inuE ᰃফࠄ㦞㊪㫫㊪䇅ᇐ᳔ᔎⱘDŽᴀ᭛
ⱘܿゴˈ៥Ӏ߽⫼inuE ⱘਃࡼᄤˈߚ߿㒧ড় racAG12V GFP ਞˈᡒࠄњϔ 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ᔩᄤDŽ䆹ᮍ⊩ৃҹ㹿ᑓ⊯䖤⫼ࠄᇏᡒ䕀ᔩᄤⱘさবԧDŽᓎ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ൟԧ䰜䗄ܿゴⱘሒDŽ
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