水稻肌動蛋白去聚合因子OsADF2及OsADF11基因異位表達於阿拉伯芥中之功能性分析

୯ҥᆵ᡼εᏢғނၗྍᄤၭᏢଣၭ᛬Ꮲس ᅺγፕЎ

Department of Agronomy

College of Bio-Resources and Agriculture

National Taiwan University Master Thesis

НዿԼ୏ೈқѐᆫӝӢη OsADF2 Ϸ OsADF11 ୷Ӣ౦Տ ߄ၲܭߓ܎դ޺ύϐфૈ܄ϩ݋

Functional Analysis of Rice Actin Depolymerizing Factor

OsADF2 and OsADF11 by Heterologous Expression in

Arabidopsis

ଭऩ◚

Row-Yu Ma

ࡰᏤ௲௤Ǻ஭ۏ୷ റγ Men-Chi Chang, Ph.D.

ύ๮҇୯ 103 ԃ 7 Д July , 2014

ii

ᇞ ᇞᖴ

ӧࣴز܌೭ٿԃаϷૈ໩ճֹԋ೭ጇፕЎǴाགᖴޑΓߚதޑӭǴคፕࢂӧ ჴᡍ΢ޑᔅշ܈Γғ΢ޑЍ࡭ǴؒԖεৎࢂคݤֹԋޑǶ२ӃाགᖴࡰᏤ௲௤஭

ۏ୷Դৣ΋ၡ΢஥ሦךǵ௲ᏤךӚᅿᜢܭ෌ނӧ଍ნ΢ޑϸᔈϐ࣬ᜢޕ᛽Ǵ೭ٿ ԃ܌ளډޑޕ᛽Ϸ࿶ᡍǴࢂа߻ֹӄคݤགྷႽޑǶௗ๱ाགᖴ೚ӭԴৣӧჴᡍ׷

਑ϐЍ࡭ǵሺᏔ΢ޑගٮаϷБӛ΢ޑࡌ᝼Ƕགᖴࢫ໺ླྀԴৣගٮჴᡍ׷਑Ǵ٠ Ъ๏ϒᏢғόӕޑࡌ᝼٬ளჴᡍ׳уֹ᏾ǶགᖴύࣴଣᇳӀျԴৣаϷύࣴଣᎄ

࿤ᑫԴৣගٮჴᡍ׷਑ǵჴᡍמೌаϷሺᏔ٬೭ጇፕЎ׳уޑֹ๓Ƕགᖴჴᡍ࠻

ᖄӝ཮᝼ਔ݅ࡏᆺԴৣǵጰػᄆԴৣǵЦలࣔԴৣගٮᏢғ೚ӭόӕБӛޑᢀᗺǴ

٬ჴᡍޑҞ኱ϷࣴزБӛ׳уܴዴϷӄБय़Ƕགᖴ໳Ў౛Դৣǵࢫ໺ඦԴৣǵᖴ ԓߝԴৣǵᇳӀျԴৣϷ஭ۏ୷Դৣα၂ਔගрޑୢᚒϷ૸ፕǴӧፕЎ߃ዺޑঅ ׯ΢ڐշᏢғ٬ϐ׳уֹ᏾Ƕ

ӚՏჴᡍ࠻უՔॺޑЍ࡭ႴᓰϷཀـΨᔅշΑךёа໩ճֹԋ೭ጇፕЎǴག ᖴ໡ृᏢۆǵ⵻◖ᏢۆϷߓӓᏢߏӧჴᡍ΢ගٮࡐӭࣔ຦ޑ࿶ᡍǵಒЈࡰᏤаϷ ࡌ᝼Ǵᡣך׳уΑှჴᡍޑБӛǵচ౛ϷБݤǴ٬ளࣴز׳уֹ᏾Ƕགᖴℱ໦Ꮲ ߏࡰᏤךࡌҥᄬᑐᆶౢғᙯ෗ਲ਼ᡣךࡕុޑࣴز׳у໩ճǶགᖴჴᡍ࠻ޑᏢߏۆ ഓҏۆǵηሺǵ٩ᅩکӕᏆ੦։ǵ඲඲ǵ✎ϡکᏢ׌ۂ݊ᑣکᖰᖰӧჴᡍ΢๏ϒ

೚ӭڐշǵཀـǵᔅԆϷႴᓰǴаϷ੤΢ӧځдჴᡍ࠻ޑӕՔ྆Ӽǵ٫ᑉǵ䐁ЎǴ ᗋԖவεᏢ൩΋ޔഉՔךޑ׵ሑǴନΑӧჴᡍ΢ޑᔅշکၗ਑ϩ݋ޑ૸ፕϐѦǴ ВதғࢲύޑҬࢬಠϺΨ٬ளךࣴز܌ޑғఱύόࢂٗሶޑ࢏ᔿЮښǴкᅈΑ៿

኷Ƕ

നࡕ΋ۓाགᖴךޑৎΓǴӧךۺࣴز܌ޑ೭ࢤВηόଶޑЍ࡭ךǵႴᓰךǴ ӧךѨပեዊਔόᘐޑࣁךѺ਻Ǵ٬ךёа໩ճޑֹԋᏢ཰ǴؒԖգॺ൩ό཮Ԗ

౜ӧޑ೭ጇፕЎΑǴᖴᖴεৎǼ

iii

ύ

ύЎᄔा

Լ୏ೈқѐᆫӝӢη (Actin Depolymerizing Factor, ADF) ࣁ΋ᆶԼ୏ೈқ่

ӝϐೈқ፦Ǵځфૈࣁᙖҗᆶ actin ่ӝஒ F-actin ှᆫࣁ G-actinǴӧಒझύୖᆶ Αಒझମࢎϐ୏ᄊ܄ᡂϯǶҞ߻ςޕ ADFs ک෌ނಒझϐғߏวػǵ׎ᄊǵಒझϩ

຋ǵૻ৲໺ᏤϷғނ܄଍ნ࣬ᜢǴฅԶ ADFs ᆶ෌ނߚғނ଍ნऐڙ܄ϐᜢೱϷځ фૈϝԖࡑ௖૸ǶӢԜҁፕЎϐჴᡍа౦Տ߄ၲϐБԄǴճҔ 35S ௴୏ηၸໆ߄

౜Нዿ OsADF2 Ϸ OsADF11 ୷Ӣܭߓ܎դ޺ύǴ٠ᢀჸᙯ෗ਲ਼ᆶഁғࠠѦ߄ࠠϷ ځӧߚғނ଍ნΠޑϸᔈǶ่݀ᡉҢ T3ӕ่ࠠӝηϐ OsADF2 ک OsADF11 ᙯ෗ਲ਼ ӧ΋૓ғߏ௃ݩΠᙯ෗ਲ਼ယТελၨഁғࠠߓ܎դ޺ࣁεǴځܜऱਔ໔ᆶ໒޸ਔ ໔ΨၨഁғࠠࣁԐǶ΋૓ᕉნΠว޵ਔ໔ᆶഁғࠠคৡ౦ǶฅԶӧ 150 mM NaCl ޑᕉნΠǴ35S::OsADF2 Ϸ 35S::OsADF11 ᙯ෗ਲ਼ޑว޵ਔ໔ၨഁғࠠߓ܎դ޺ࣁ ԐǶќѦஒ 5 ϺεϷ 10 Ϻεϐߓ܎դ޺౽෌Կ֖Ԗ 150 mM NaCl ϐ୻Ꭶ୷ࡕว౜

OsADF2 Ϸ OsADF11 ᙯ෗ਲ਼ϐਥ೽ғߏϷӸࢲ౗ࣣεܭഁғࠠߓ܎դ޺ǶќѦа ଳײǵଯᡶǵեྕ଍ნೀ౛ 21 Ϻεϐ OsADF2 Ϸ OsADF11 ᙯ෗ਲ਼ԋਲ਼Ǵ่݀ᡉ Ң OsADF2 Ϸ OsADF11 ࣣၨഁғࠠऐײǴฅԶ OsADF11 ӧեྕೀ౛ࡕ෌ނಔᙃ Ԗڙ໾ޑ௃׎วғǴՠԜ౜ຝѝӧηယ೽ϩᢀჸډǴԶཥғယ߾ᆶഁғࠠคܴᡉ ৡ౦ǶճҔआѦጕ዗ቹႽᔠෳ෌ਲ਼ယྕϐᡂϯǴӧ΋૓చҹΠᙯ෗ਲ਼ᆶഁғࠠယ

ྕคৡ౦Ƕќ΋Бय़а 200 mM NaCl ೀ౛Οڬεϐ OsADF2 Ϸ OsADF11 ᙯ෗ਲ਼ Ϸഁғࠠߓ܎դ޺Ǵ߾ว౜ OsADF2 ᙯ෗ਲ਼ϐယТྕࡋᆶഁғࠠคᡉ๱ৡ౦ǴԶ OsADF11 ᙯ෗ਲ਼ϐယྕ߾ࢂၨഁғࠠࣁଯǶճҔҏԯ ubiquitin promoter ߄౜Нዿ OsADFs ୷Ӣৎ௼ϐ GFP ᑼӝೈқǴௗ๱аኩਔ܄߄ၲϩ݋Нዿ OsADFs ϐԛಒ झۓՏǴ่݀ᡉҢНዿ OsADF2ǵOsADF3 Ϸ OsADF11 ᆶ GFP ϐᑼӝೈқܭಒझ ਡϷಒझ፦ύࣣԖ߄౜Ǵځύ OsADF2 ᆶ GFP ϐᑼӝೈқӧယᆘᡏύΨԖ߄౜Ǵ Զ OsADF1ǵOsADF4ǵOsADF5ǵOsADF6 ᆶ GFP ϐᑼӝೈқ߾ࢂࣣ߄౜ܭယᆘ

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ᡏύǶҗа΢ϐ่݀௢ፕНዿ OsADF2 Ϸ OsADF11 ёૈᆶ෌ނय़ᖏଯᡶ଍ნаϷ ଳײ଍ნਔޑऐڙᐒڋԖᜢǶ

ᜢ

ᜢᗖӷǺНዿԼ୏ೈқѐᆫӝӢηǵಒझମࢎǵ౦Տ߄ၲǵ෌ނғߏวػǵߚғ ނ଍ნ

v

Abstract

Actin Depolymerizing Factors (ADFs) are small actin-binding proteins that involved in cytoskeleton remodeling in cells. Recently, ADFs were reported to play a role in plant growth, development, cell division, signal transduction and pathogen resistance. However the functions of rice ADFs (OsADFs) under abiotic stress still remained to explore. In this thesis, we took heterologous gene expression approach to overexpress rice OsADF2 and OsADF11 in Arabidopsis, and observed the phenotype difference between transgenic and wild type plants under normal growth and abiotic stress. The phenotype of OsADF2 and OsADF11 transgenic plants showed bigger leaves size than Col-0, moreover, the bolting time and flowering time was earlier. The

germination rate of transgenic plants had no difference compared to wild-type under normal condition while the germination rate of transgenic plants was faster after treatment with 150 mM NaCl. We also transferred the 5-day old and 10-day old seedlings to the 150 mM NaCl medium and found that the root growth and survival ratio of transgenic plants were both higher than wild-type. Next we grown 21-day old OsADF2, OsADF11 transgenic and wild-type plants under drought, salt and cold stresses, and OsADF2 and OsADF11 transgenic plants tended to be drought tolerance while injury tissue occurred in OsADF11 transgenic plant under cold treatment. Using non-destructive infrared thermography camera recorded the leaf temperature change under salt stress condition, the result showed that no obvious differences were found between transgenic and wild-type plants under normal condition. However OsADF11 transgenic plants showed higher leaf temperature compared to wild-type but not OsADF2 after treatment with 200 mM NaCl. In addition, to understand the subcellular localization of rice OsADFs, we used maize ubiquitin promoter to express rice

OsADFs-GFP fusion protein through rice protoplast mediated transformation. The

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result showed that OsADF3-GFP and OsADF11-GFP were located in the nucleus and cytosol, and OsADF2-GFP was located in the nucleus, cytosol and also in chloroplasts.

OsADF1, OsADF4, OsADF5 and OsADF6 were located in the chloroplasts. Taken together from above results, our study implied that rice OsADF2 and OsADF11 maybe involve in the plant response to drought and salt stress.

Keywords : Actin depolymerizing factor, Cytoskeleton, Heterologous expression, development and growth, abiotic stresses

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Ҟ Ҟᒵ

α၂ہ঩ቩۓਜ ... i

ठᖴ ... ii

ύЎᄔा ... iii

Abstract ... v

߄Ҟᒵ ... xi

კҞᒵ ... xi

ߕᒵҞᒵ ... xii

ᕭቪӷჹྣ ... xiii

ಃ΋കġ ߻ق ... 1

ಃΒകġ ߻Γࣴز ... 3

΋ǵġ෌ނӧߚғނ଍ნΠϐϸᔈ ... 3

ΒǵġಒझମࢎᆶԼ୏ೈқܭ෌ނϐғߏวػϷߚғނ଍ნऐڙ܄ ... 4

1. ෌ނಒझମࢎϐ୏ᄊѳᑽ ... 5

2. ಒझମࢎϐԼ୏ೈқགᔈѦࣚᕉნӢηϷϣғૻဦ ... 6

2. 1. ྕࡋ ... 6

2. 2. ᡶϩ ... 8

2. 3. ଳײ ... 9

2. 4. ख़ߎឦ ... 10

ΟǵġԼ୏ೈқѐᆫӝӢη(ADF) ... 10

1. Լ୏ೈқѐᆫӝӢη (ADF) ୖᆶ෌ނϐғߏวػ ... 11

2. Լ୏ೈқѐᆫӝӢη(ADF) ୖᆶ෌ނϐғނ଍ნऐڙ܄ ... 12

3. НዿԼ୏ೈқѐᆫӝӢηϐ࣬ᜢࣴز ... 12

ѤǵġࣴزҞޑϷჴᡍࢎᄬ ... 13

ಃΟകġ ׷਑Бݤ ... 15

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΋ǵġOsADF ୷Ӣৎ௼ϐғނၗૻϩ݋ ... 15

Βǵġ၂ᡍ׷਑ ... 15

1. ߓ܎դ޺ᅿη ... 15

2. Нዿᅿη ... 15

Οǵġߓ܎դ޺ғߏచҹϷόӕߚғނ଍ნೀ౛ ... 16

1. ߓ܎դ޺ғߏచҹ ... 16

2. ߓ܎դ޺ғ౛ϷѦ߄ࠠᢀჸϐߚғނ܄଍ნೀ౛చҹ ... 16

2.1 ว޵౗ϩ݋ϐ଍ნೀ౛చҹ ... 16

2.2 ਥߏϩ݋ϷӸࢲ౗ϐ଍ნೀ౛చҹ ... 17

2.3 ԋਲ਼ϩ݋ϐ଍ნೀ౛చҹ ... 17

2.4 ᇻआѦጕ዗གᔈቹႽϩ݋ᡶ଍ნೀ౛ᙯ෗ਲ਼చҹ ... 17

Ѥǵġߓ܎դ޺ँᡂਲ਼Ϸᙯ෗ਲ਼ϐϩη᠘ۓ ... 18

1. ෌ނ DNA ϐ๧ڗ ... 18

2. DNA ࢩ๼ᛏિጤᡏႝݚϩ݋ ... 19

ϖǵġߓ܎դ޺ँᡂਲ਼Ϸᙯ෗ਲ਼ϐ୷Ӣ߄౜ϩ݋ ... 19

1. RNA ϐܜڗϷᇙഢ ... 19

2. RNase Free DNase I ೀ౛ ... 19

3. cDNA ӝԋϐϸᙯᒵϸᔈ ... 20

4. ϸᙯᒵᆫӝ䁙ೱᙹϸᔈ (RT-PCR) ... 20

Ϥǵġߓ܎դ޺ᙯ෗ਲ਼ϐ፦ಈᄬᑐϷ଍ნऐڙ܄ෳ၂ ... 20

1. 35S::OsADF2 Ϸ 35S::OsADF11 ፦ಈᄬᑐࢬำ ... 20

2. ౦Տ߄౜(heterologous expression)Нዿ୷Ӣϐߓ܎դ޺ᙯ෗ ... 21

3. εဉఎ๵ᙯࠠբҔ ... 22

3.1 εဉఎ๵യҺಒझᇙഢ ... 22

3.2 ፦ಈεဉఎ๵ᙯࠠ ... 22

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3.3 ᔠෳᙯࠠࡕεဉఎ๵፦ಈ colony PCR ... 22

3.4 εဉఎ๵፦ಈ DNA λໆપϯ ... 23

4. DNA Тࢤޑᇙഢᆶӣԏ ... 23

5. ߓ܎դ޺୷Ӣᙯ෗ ... 24

5.1 ෌ނ׷਑Ⴃഢ ... 24

5.2 ၭఎ๵ᙯࠠ ... 24

5. 2. 1 ၭఎ๵യҺಒझᇙഢ ... 24

5. 2. 2 ፦ಈၭఎ๵ᙯࠠ ... 24

5. 2. 3 ᔠෳᙯࠠࡕϐၭఎ๵፦ಈ colony PCR ... 25

5. 2. 4 ߓ܎դ޺ᙯ෗ ... 25

6. ߓ܎դ޺ᙯ෗ਲ਼ᑔᒧ ... 26

6.1 לғનᑔᒧᙯ෗ਲ਼ ... 26

ΎǵġOsADF ୷Ӣৎ௼ϐ GFP ᑼӝೈқϐԛಒझՏ࿼ϩ݋ ... 26

1. Ub::OsADFs-GFP ፦ಈᄬᑐࢬำ ... 26

2. Нዿ׷਑Ⴃഢ ... 27

3. ܜڗНዿচғ፦ᡏ ... 27

4. ᆫΌΒᎇ(PEG) ᙯ෗Нዿচғ፦ᡏ ... 27

5. Ӆ೫ขᡉ༾᜔ (confocal microscope) ᢀჸᑻӀೈқ߄౜ ... 28

ಃಃѤകġ ่݀ ... 29

΋ǵ OsADF2 Ϸ OsADF11 ୷Ӣܭߚғނ଍ნΠϐ୷Ӣфૈϩ݋ ... 29

1. ౦Տ߄౜Нዿ OsADF2 Ϸ OsADF11 ୷Ӣϐߓ܎դ޺ᙯ෗ਲ਼ϩη᠘ۓ ... 29

2. ౦Տ߄౜Нዿ OsADF2 Ϸ OsADF11 ୷Ӣϐߓ܎դ޺ᙯ෗ਲ਼Ѧ߄ࠠᢀ ჸ ... 29

2.1. Нዿ OsADF2 Ϸ OsADF11 ୷Ӣቹៜߓ܎դ޺෌ਲ਼ελ ... 29

x

2.2. Нዿ OsADF2 Ϸ OsADF11 ୷ӢගԐ෌ਲ਼ܜऱ໒޸ਔ໔ ... 30

3. Нዿ OsADF2 ୷Ӣϐ୷Ӣфૈ܄ϩ݋ ... 30

3.1. ߓ܎դ޺ 35S::OsADF2 ᙯ෗ਲ਼ϐว޵౗ෳۓ ... 30

3.2. ߓ܎դ޺ 35S::OsADF2 ᙯ෗ਲ਼ܭߚғނ଍ნΠϐᢀჸ ... 30

3.2.1. ว޵౗ϩ݋ ... 30

3.2.2. ਥߏϩ݋ ... 31

3.2.3. ԋਲ਼ᢀჸ ... 31

4. Нዿ OsADF11 ୷Ӣϐ୷Ӣфૈ܄ϩ݋ ... 33

4.1. ߓ܎դ޺ 35S::OsADF11 ᙯ෗ਲ਼ϐว޵౗ ... 33

4.2. ߓ܎դ޺ 35S::OsADF11 ᙯ෗ਲ਼ܭߚғނ଍ნΠϐᢀჸ ... 33

4.2.1 ว޵౗ϩ݋ ... 33

4.2.2 ਥߏϩ݋ ... 34

4.2.3 ԋਲ਼ᢀჸ ... 34

Βǵġ НዿԼ୏ೈқѐᆫӝӢηৎ௼ (Actin depolymerizing factor) ϐ GFP ᑼ ӝೈқܭНዿচғ፦ᡏϐԛಒझۓՏ ... 37

ಃಃϖകġ ૸ፕ ... 38

΋ǵġ௖૸Нዿ OsADF2 Ϸ OsADF11 ୷Ӣᆶ෌ނғߏวػϐᜢ߯ ... 38

1. Нዿ OsADF2 Ϸ OsADF11 ୷Ӣёૈୖᆶ෌ނϐғߏวػϷ໒޸ ... 38

Βǵġ௖૸Нዿ OsADF2 Ϸ OsADF11 ୷ӢܭߚғނΠϐфૈКၨ ... 40

1. OsADF2 Ϸ OsADF11 ୷Ӣୖᆶ෌ނय़ᖏଯᡶ଍ნਔޑऐڙ܄௖૸ 42 2. OsADF2 Ϸ OsADF11 ୷Ӣӧեྕ଍ნΠϐϸᔈ௖૸ ... 44

3. OsADF2 Ϸ OsADF11 ୷Ӣୖᆶ෌ނय़ᖏଳײ଍ნਔޑऐڙ܄௖૸ 45 Οǵġ Нዿ OsADF ୷Ӣৎ௼ϐ GFP ᑼӝೈқԛಒझۓՏ่݀௖૸ ... 47

Ѥǵġ ่ᇟ ... 48

ୖԵЎ᝘ ... 49

xi

߄

߄Ҟᒵ

߄΋ǵ НዿԼ୏ೈқѐᆫӝӢη)OsADFs*!ৎ௼ԋ঩୷Ӣׇဦ࣬ᜢၗૻ ... 58!

߄Βǵ НዿԼ୏ೈқѐᆫӝӢη)OsADFs*!ৎ௼ԋ঩ϐԛಒझۓՏ ... 58!

კҞᒵ კ΋ǵġ ߓ܎դ޺ၸࡋ߄౜Нዿ OsADF2 Ϸ OsADF11 ᙯ෗ਲ਼ϐϩη᠘ۓᆶѦ߄ࠠ ϩ݋ ... 59

კΒǵġ ߓ܎դ޺ၸࡋ߄౜Нዿ OsADF2 Ϸ OsADF11 ᙯ෗ਲ਼ϐܜऱϷ໒޸ਔ໔ᆶ ഁғࠠ Col-0 ϐϩ݋Кၨ ... 61

კΟǵġ ߓ܎դ޺ၸࡋ߄౜Нዿ OsADF2 ܭ଍ნΠਥߏϩ݋ᆶӸࢲ౗ϩ݋ ... 63

კѤǵġ ߓ܎դ޺ၸࡋ߄౜Нዿ OsADF11 ϐܭ଍ნΠਥߏϩ݋ᆶӸࢲ౗ϩ݋ ... 65

კϖǵġ 35S::OsADF2 ᙯ෗ਲ਼ԋਲ਼ϐଳײ଍ნᢀჸ ... 67

კϤǵġ 35S::OsADF2 ᙯ෗ਲ਼ԋਲ਼ϐଯᡶ଍ნᢀჸ ... 69

კΎǵġ 35S::OsADF2 ᙯ෗ਲ਼ԋਲ਼ϐեྕ଍ნᢀჸ ... 70

კΖǵġ 35S::OsADF11 ᙯ෗ਲ਼ԋਲ਼ϐଳײ଍ნᢀჸ ... 71

კΐǵġ 35S::OsADF11 ᙯ෗ਲ਼ԋਲ਼ଯᡶϐ଍ნᢀჸ ... 73

კΜǵġ 35S::OsADF11 ᙯ෗ਲ਼ԋਲ਼ϐեྕ଍ნᢀჸ ... 74

კΜ΋ǵġआѦጕ዗གᔈቹႽϩ݋ߓ܎դ޺ၸࡋ߄౜Нዿ OsADF2 Ϸ OsADF11 ᙯ෗ ਲ਼ܭ 200 mM NaCl ೀ౛ϐᡶҽ଍ნΠϸᔈ ... 75

კΜΒǵġНዿ OsADFs ୷Ӣৎ௼ϐ GFP ᑼӝೈқܭНዿচғ፦ᡏϐԛಒझۓՏ ... 77

xii

ߕ

ߕᒵҞᒵ

ߕᒵ΋ǵġ Нዿکߓ܎դ޺ϐԼ୏ೈқѐᆫӝӢηৎ௼ữ୷ለޑׇӈКჹϷᒃ

ጔᄽϯᐋϩ݋... 79

ߕᒵΒǵġ НዿԼ୏ೈқѐᆫӝӢηৎ௼୷Ӣܭόӕಔᙃᆶғߏයϐ߄౜კ᛼ ... 80

ߕᒵΟǵġ ୷Ӣᙯ෗߄౜ၩᡏᄬᑐϐ፦ಈკ... 81

ߕᒵѤǵġ ૻ৲໺ሀϩηᆶ෌ނԼ୏ೈқମࢎϐᜢᖄკ ... 82!

ߕᒵϖǵġ ҁࣴز܌٬ҔϐЇηჹ΋ំ ... 83!

ߕᒵϤǵġ ᆶԼ୏ೈқ࣬ᜢϐࣴز᏾౛ ... 84!

xiii

ᕭ

ᕭቪӷჹྣ

ADF Actin depolymerizing factor ABPs Actin binding proteins AF Actin filament

At Arabidopsis thaliana

CDPK Calcium-dependent protein kinase DNA Deoxyribonucleic acid

DREB Dehydration response element binding factor DW Dry weight

KD knock-down KO Knock-out

LEAs Late embryogenesis abundant protein Lat B Latrunculin B

M Molar

MDA Malondialdehyde MF Microfilament

MS Murashige and Skoog medium MT Microtubule

N3 atnced3 mutant Os Oryza sativa L.

OE2 35S::lfNCED overexpressing line PCR Polymerase chain reaction PEG Polyethylene glycol

PK Protein Kinase RNA Ribonucleic acid

xiv ROS Reactive oxygen species

RT-PCR Reverse transcription polymerase chain reaction SOD Superoxidase dismutase

TCN1 Taichung Native1 TNG67 Tainung 67

Ubi Ubiquitin WT Wild type

1

ಃ

ಃ΋കġ ߻ق

߈ԃٰӦౚ਻ং౦தϷᕉნԡࢉ฻ࣣቹៜΑբނғߏϷวػǴԜᕉნᡂϯࢂ

ज़ڋ٠ቹៜբނౢໆϷ፦ໆϐख़ाӢηǴ຾ԶፂᔐΑၭ཰ғౢǴᏤठբނғߏ౦ தϷౢໆΠफ़ǶቹៜբނౢໆϷ፦ໆޑ଍ნёϩࣁғނ܄଍ნᆶߚғނ܄଍ნǴ ғނ܄଍ნӵᎁڙੰচ๵གࢉǵܲᙝϷ୏ނࠈ໾ǹԶߚғނ଍ნ߾ӵଯྕǵեྕǵ ଳײǵଯᡶϷख़ߎឦԡࢉ฻ǹ߈ԃٰჹܭբނߚғނ଍ნऐڙ܄ϐϸᔈϷૻ৲໺

ᏤςԖ೚ӭᕕှǴՠۘ҂ว౜෌ނௗڙѦࣚᕉნׯᡂԶౢғޑڈᐟૻဦޑௗԏᏔ (stress sensor) ǴҞ߻Ԗ೚ӭࣴزࡰр྽෌ނय़ᖏ଍ნਔǴ཮ౢғ΋ೱՍፄᚇޑፓ

௓ᐒڋǴགᔈѦٰޑڈᐟ٠ஒૻဦ໺ሀډಒझϣ (Osakabe et al., 2013) Ǵ຾Զౢғ

፾྽ޑғ౛ϸᔈ܈׎ᄊ΢ޑ፾ᔈǴаගଯ଍ნΠޑғӸ౗ǴՠჹܭբނӵՖགᔈ

܈ჸ᝺Ѧӧߚғނ଍ნϐᡂϯҞ߻ϝឦ҂ޕǶ

Нዿ (Oryza sativa L.) ࢂШࣚΟεᙂ१բނȐλഝǵҏԯǵНዿȑϐ΋Ǵ१Ҕ ΓαኧനӭǴჹܭ٥ࢪԶق׳ࢂЬाޑғౢᆶ੃຤୔ୱǴฅԶҗܭΓαᗊቚϷ਻

ংᡂᎂᏤठౢໆΠफ़Ǵёૈ཮Ꮴठ҂ٰᙂ१ٮᔈόىǴӢԜ௖૸਻ংᡂᎂΠቚу НዿౢໆǴࢂ౜Ϟ΋εፐᚒǶᙖҗቚуНዿܢלߚғނ଍ნऐڙ܄аගଯځౢໆ Ϸࠔ፦ϐࣴز׳ࣁख़ाǶ౜ϞЬाᙖҗ໺಍ػᅿׯؼቚуНዿౢໆϷ଍ნऐڙ܄Ǵ ฅԶНዿύቹៜғނ଍ნऐڙ܄ޑ୷Ӣεӭឦܭኧໆ܄ރ୷ӢǴӢԜऩࢂа໺಍

ػᅿБԄ຾Չׯؼਏ݀Ԗज़ǶԶᒿ๱ϩηғނמೌޑว৖Ǵ୷Ӣᙯ෗מೌ೴ᅌԋ ዕǴ߈ԃٰΨ໒ۈճҔ୷ӢπำׯؼНዿჹܭ଍ნޑऐڙ܄Ƕ

྽෌ނڙډᕉნޑ଍ნਔǴ෌ނޑಒझጢϷಒझᏛന२྽ځፂԶ଺рϸᔈǶ ԶಒझମࢎࢂಒझጢϷಒझᏛޑख़ाಔԋԋϩǴҞ߻ςԖ೚ӭ᛾ᏵࡰрǴӢᔈಒ झጢࢬ୏܄ϐׯᡂǴ຾Զቹៜಒझମࢎख़᏾Ϸځ΢ϐᐒఓགᔈᏔ (mechanical sensor) Ǵᆶ෌ނགᔈѦࣚڈᐟࡕౢғޑૻ৲໺ሀ᜘Ԗᜢ (Orvar et al., 2000) ǶЪ ߈ԃٰޑࣴزΨࡰрಒझମࢎϐख़ಔǴӧࢲϯߚғނ଍ნϐૻ৲໺ሀϷΠෞ୷Ӣ ߄౜΢ёૈתᄽΑ࣬྽ख़ाޑགᔈفՅǴӢԜ௢ᘐಒझମࢎϐ୏ᄊѳᑽӧբނߚ

2

ғނ଍ნϐᡂϯགޕ΢࣬྽ख़ा (Blanchoin et al., 2010) ǶADF ࢂեϩηໆ (15ȉ 21 kDa) ϐλೈқ፦Ǵӧ܌Ԗޑ੿ਡғނύࣣёว౜Ǵ٠Ъӧữ୷ለׇӈ΢ڀԖଯ ࡋޑߥӺ܄ǶADF ёזೲޑஒ Filamentous form actin (F-actin) ѐᆫӝࣁ Globular form actin (G-actin) Ǵаፓ௓ಒझύԼ୏ೈқϐ୏ᄊѳᑽǴࢂቹៜ෌ނಒझମࢎᆫ ӝ୏ᄊ܄ᡂϯനख़ाӢηϐ΋Ƕ෌ނϐ ADFs ୷ӢࣴزଆۈܭԭӝᆶҏԯǴԶኳԄ

෌ނϐߓ܎դ޺ޑ AtADF ӭ୷Ӣৎ௼Ǵ߾ёϩࣁᔼᎦғߏکғ෗ғߏය߄౜ϐٿ εဂǴϩձ߄౜ܭόӕޑಔᙃᆶғߏวػයǴ٠Ъᆶ੝ۓޑፓ࿯фૈԖᜢ (Ruzicka et al., 2007) ǶନΑᆶ෌ނϐ҅தғߏวػǴӵ໒޸ǵ޸ણᆅϐғߏԖᜢѦǴAtADFs ӭ೏᛾ܴᆶ෌ނੰ๵ (plant-pathogen interaction) ϐ࣬ᜢऐ܄ǴڀԖ҅ӛፓ௓ϐф

ૈǶ

Ҟ߻ჹНዿ OsADFs ୷Ӣৎ௼ϐࣴز٠όӭǴЀځࢂԖᜢϐ୷ӢфૈϷᆶߚ ғނ଍ნϐ࣬ᜢ܄ǶӢԜҁፕЎԑӧϩ݋Нዿ OsADF ୷Ӣৎ௼ϐ୷ӢфૈǶ२Ӄ Ӄафૈ܄୷ӢᡏᏢБԄౢғᕇளфૈϐ gain of function (GOF) ౦Տ߄౜Нዿ୷ Ӣϐߓ܎դ޺ᙯ෗ਲ਼Ǵ຾Չғ౛ϷѦ߄ࠠϩ݋аϷӧߚғނ଍ნΠϐऐڙ܄ෳ၂Ƕ

ௗ๱ϩ݋ OsADFs-GFP ᑼӝೈқӧಒझύޑ߄౜ۓՏǶᙖҗ΢ॊНዿԼ୏ೈқѐ ᆫӝӢη୷Ӣৎ௼ϐғ౛ǵऐ܄ෳ၂Ϸೈқ፦ۓՏǴஒё٬ךॺ׳уΑှНዿ OsADF ୷Ӣৎ௼ӵՖቹៜНዿғߏวػǴ٠ёૈӧझጢࢬ୏܄Ϸಒझମࢎख़᏾΢Ǵ ӵՖբࣁ΋གޕϐૻ৲аࢲϯߚғނ܄଍ნΠෞϐ୷Ӣ߄౜Ƕ

3

ಃ

ಃΒകġ ߻Γࣴز

җܭ෌ނᆶ୏ނόӕǴؒԖၮ୏ᐒૈаϷઓ࿶س಍Ƕ෌ނ೯தғߏܭڰۓՏ

࿼Ǵ྽ᕉნวғᡂϯਔǴคݤЬ୏ᚆ໒೭٤଍ნǴӢԜჹܭᕉნૻဦϷϣғวػ

ૻဦϐགᔈǴӧ෌ނޑ׎ᄊϷऐ܄΢Ԗ܌ᡂϯǴ٬෌ނளаय़ჹ଍ნԶӸࢲࢂߚ தख़ाޑǶ෌ނճҔགᔈ೭٤Ѧࣚᡂϯૻ৲Ǵ٬෌ނౢғჹᔈڈᐟϐ୷Ӣ߄౜ᡂ ϯǵౢғ଍ნჹᔈೈқ፦ϷжᖴނǴаׯᡂԾيޑғߏวػǴ຾Զ٬෌ނᡏளа य़ჹᕉნᡂϯǴԶቚуӸࢲᐒ౗Ƕ܌аڈᐟૻ৲ޑௗԏаϷૻ৲໺ሀჹܭ෌ނޑ ғߏวػǵᕉნϸᔈԶقࢂΜϩख़ाޑǶ྽෌ނᎁၶډߚғނ଍ნȐӵଯᡶǵଯ

ྕǵեྕǵଳײǵख़ߎឦǾ฻ȑਔǴځғߏวػ཮ڙډቹៜǴӢԶ೷ԋӧѦ߄׎

ᄊǵғ౛аϷжᖴ΢ޑׯᡂǴ܈ࢂЇวୖᆶૻ৲໺ሀ܈ऐڙ܄ϸᔈ୷Ӣޑ߄౜Ǵ ߈ԃٰޑࣴزࡰрǴಒझମࢎޑ׎ᄊ΢ϐ୏ᄊᡂϯǴаϷୖᆶ෌ނᡏϣϐૻ৲໺

ሀԶቹៜΠෞғ౛ϸᔈ (Marylin et al., 2002) ǴӢԜಒझମࢎϐख़ಔ (remodeling) ܭऐ଍ნϐғ౛ғϯфૈǴӧ෌ނᡏύёૈתᄽख़ाޑفՅǶ

΋ǵġ ෌ނӧߚғނ଍ნΠϐϸᔈ

྽ᕉნวғᡂϯǴӵߚғނ଍ნ฻Ǵ෌ނᡏགᔈډԜᡂϯࡕ཮ౢғ΋ೱՍޑ ғ౛жᖴϸᔈǶ߃යϸᔈࣁፓ௓࣬ᜢ୷Ӣ߄౜ໆǴ຾ԶׯᡂжᖴౢނғԋǴ٬෌

ނᡏளаय़ჹᕉნᡂϯޑׯᡂԶӸࢲǶ෌ނӧय़ᖏᕉნ଍ნਔԖ೚ӭόӕӢᔈᐒ ڋǴӵᖿᗉ܄ (escape)ǵٛᑇ܄ (avoidance) Ϸऐڙ܄ (tolerance) (Chaves et al., 2003)Ǵᖿᗉ܄εӭр౜ܭғߏܭ؅ᅃϐอғڮຼයϐ෌ނǴځࣁᗉ໒ѳਔଳײ଍

ნԶѝӧอኩफ़ߘύ๴޵٠ِೲֹԋғڮຼයǶٛᑇ܄߾ࢂ෌ނᙖҗቚуҁيϐ

଍ნٛᑇૈΚǴӵ؅ᅃύ෌ނਥسวၲǴ܈ࢂᄽϯрଞރယǵࠆف፦ቫ฻а෧Ͽ НϩණѨԶளаܭଳײᕉნύӸࢲǶ෌ނౢғ଍ნऐڙ܄Ьा೸җٿБय़Ǻ΋ࣁ

෌ނᡏӧௗԏډѦࣚϐ଍ნࡕᇨᏤ੝ۓ୷Ӣ߄౜Ǵ຾Զౢғჹᔈϐжᖴౢނ܈ೈ

қ፦ԶளаԾՉ੃ࡎ܈অൺ଍ნჹ෌ނᡏ܌೷ԋޑཞ্Ǵ٬෌ނߥ࡭҅தғ౛ф

4

ૈԶӸࢲǴΒ߾ࣁѦٰӢનϷΓπᒧ᏷୻ػǴӵ೯ၸᚇҬػᅿǵ୷Ӣπำǵᇨᡂ ᒧܘ฻Ƕ୷ܭၲᅟЎޑނᝡϺ᏷ǵ፾ޣғӸޑ౛ፕǴғނᡏӧԾฅ௃ݩΠᎁၶ଍

ნਔڀഢԖճᡂ౦చҹޣӸࢲǴԶ೷ԋԖճޑ୷Ӣࠠӧ௼ဂύКٯ೴ᅌ΢ϲǴߏ ਔ໔ࡕ೴ᅌಕᑈᡂ౦Ǵനࡕ׎ԋԾฅΠคݤᆶচᅿ௼ဂѫػΠ΋жϐཥ௼ဂ (Darwin, 1859) ǶّϞࣁЗӧ෌ނܢל଍ნޑࣴزБय़Ǵคፕࢂӧжᖴᐒڋǵϩη ቫԛ୷ӢۓՏ܈ࢂᒪ໺ࣴزБय़ςԖࡐεϐ຾৖ǴҞ߻ӧቚу෌ނܢל଍ნऐڙ

܄ϐ೼৩ԖаΠٿᅿǺ΋ࣁճҔᒪ໺Ꮲ໺಍ϐػᅿǴჹ෌ނ׎ᄊ่ᄬϷғߏวػ

຾Չፓ௓ᑔᒧǹΒࣁ೸ၸғނמೌჹ෌ނϐжᖴ೼৩аϷ୷Ӣ߄ၲ຾Չፓ௓Ƕฅ Զ౜Ϟϐᕉნᡂϯቃਗ਼ǴԜౢғڀԖऐڙ܄ࠔᅿϐೲࡋᇻόϷᕉნϐᡂϯׯᡂǴ ӢԜғނᙯ෗מೌࣁќ΋ᅿቚуऐ܄ࠔᅿϐᒧ᏷ǶճҔᙯ෗מೌёஒҞ኱܄ރ࣬

ᜢ୷ӢǴකΕటׯؼϐҞ኱ғނᡏύǴ٠Ъ௓ڋځ߄౜ໆǵ߄౜೽Տ฻ǴԜמೌ

ёँઇόӕނᅿ໔ޑᕷ෗႖ሖǴёᒧ᏷ϐ୷ӢёٰԾܭόӕނᅿࣗԿࢂΓࣁ୷ӢǴ ԶёቚуҞ኱܄ރޑ߄౜Ƕӧᙯ෗բނ΢ϐᔈҔӵቚуբނౢໆǵౢғ੝ۓೈқ аϷڀԖࢌ٤଍ნऐڙ܄฻Ǵ຾Զ୻ػрёܢל଍ნϐբނǶّϞࣁЗςԖ೚ӭ

ࣴزஒߚғނ଍ნԖᜢϐѦྍфૈ܄ೈқ୷ӢȐӵН೯ၰೈқǵᅖ೸ᓸፓ࿯ೈқǵ לএೈқϷ዗Ҷլೈқ฻ȑᏤΕ෌ނಒझ٠εໆ߄౜ϐǴ่݀ᡉҢёቚу෌ނჹ ߚғނ଍ნϐऐڙ܄ (review by Arbona et al., 2013) Ƕ

Β

Βǵġ ಒझମࢎᆶԼ୏ೈқܭ෌ނϐфૈ

ಒझମࢎЬाҗಒझ༾ᆅ (microtubules) ǵಒझ༾ํ (microfilament) Ϸύ໔

ํ (intermediate filament) ܌ಔԋǴځύ߻ΒޣЬाୖᆶΑ෌ނಒझύϐಒझମࢎ

ϐ୏ᄊ܄ѳᑽ (dynamic rearrangement of cytoskeleton) ǴԜ୏ᄊѳᑽӧ෌ނϐғߏ วػǵ಻ᅟᆾբҔࣗԿѦࣚૻ৲ϐགޕϷ໺ሀ΢ࣣתᄽख़ाϐفՅ (Drøbak, Franklin-Tong et al., 2004; Hussey et al. 2006) Ƕ෌ނಒझମࢎ΋૓ࣁೈқᠼᆢಔԋ ϐΟᆢᆛރ่ᄬǴӧ෌ނಒझϐύୖᆶନΑᆢ࡭ಒझ୷ҁޑ׎ރᛙۓѦǴӧ෌ނ

5

ଯࡋ੝౦ϯޑಒझ Ȑӵ޸ણᆅǵਥЛǵ૙Л (trichome) Ϸ਻Ͼߥፁಒझ฻ȑ׎ᄊ วғϷфૈԖख़ाޑբҔǴ٠ЪΨୖᆶΑಒझ೚ӭғ౛ࢲ୏ӵǺझϣၮᒡǵझᏔ ၮ౽ǵᚆη೯ၰϐ໒௴/ᜢഈǵૻ৲໺ᏤǵಒझғߏϷϩ຋ǵಒझ׎ރᡂϯǵಒझ ӛ܄ғߏаϷಒझ፦ࢬ฻ (Henty-Ridilla et al., 2013; Deeks and Hussey 2009; Ye et al.,2013) Ƕಒझମࢎᙖҗୖᆶа΢ғ౛ࢲ୏Զᆢ࡭෌ނಒझϐ҅தфૈǴ٬෌ނ ᡏளа҅தғߏวػǹ߈ԃٰΨԖ೚ӭࣴزࡰрಒझମࢎୖᆶΑߚғނ଍ნϷғ ނ଍ნΠǴ෌ނགᔈԶౢғϐૻဦ໺ሀၡ৩ (review by Nick, 2008; review by Schmelzer, 2002) Ƕ

1. ෌෌ނಒझମࢎϐ୏ᄊѳᑽ

෌ނಒझମࢎޑ୏ᄊѳᑽჹܭᆢ࡭΋ঁಒझӧᛙۓޑރᄊԶقΜϩख़ाޑǴ ԶԜ୏ᄊѳᑽЬाࢂࡰಒझ༾ᆅϷԼ୏ೈқόଶӦҗౚ׎ൂᡏԼ୏ೈқ (Globular form actin, G-actin) ᆫӝࣁᆫӝᄊํރԼ୏ೈқ (Filamentous form actin, F-actin) Ǵ аϷ F-actin ѐᆫӝࣁ G-actin ܌ᄬԋǶᙖҗᆶԼ୏ೈқ่ӝೈқ፦ဂ (Actin Binding Proteins, ABPs) Ǵӵ Arp2/3 ፄӝᡏ (Actin related protein 2/3 complex) , Լ୏ೈқ

׭ڋೈқ (profiling) ǵԼ୏ೈқѐᆫӝӢη (Actin depolymerizing factor,

ADF/cofilin) ǵԼౚೈқ (myosin) ǵᠼᆢೈқ (fibrin) Ϸ Geosolin/villin ฻܌Ӆӕ ፓ࿯ (Wasteneys et al., 2004) Ƕ೸җа΢ ABPsǴᆶԼ୏ೈқํϐ C ᆄ܈ N ᆄϐ G-actin ่ӝǴஒํރԼ୏ೈқှᆫ܈ᆫӝԶ׎ԋಒझϣಒझମࢎϐ୏ᄊѳᑽǶӧ ғ౛ᡶᐚࡋΠϷӸӧ ATP Ϸ Mg²⁺ޑᕉნΠǴౚރԼ୏ೈқ཮ԾวᆫӝԋํރԼ୏

ೈқǴԜᆫӝբҔԖаΠѤ؁ᡯǺ(1) ࢲϯԼ୏ೈқ (activation) ǴԼ୏ೈқൂᡏ ᆶᡶᜪ่ӝǴ٠Ъׯᡂᄬࠠǹ (2) ԋਡϸᔈ (nucleation) Ǵҗൂᡏ่ӝϐեᆫᡏ

೴ᅌ่ӝԋํރԼ୏ೈқӭᆫᡏǹ (3) Լ୏ೈқ༾ํۯߏ (elongation) Ǵҗӭᆫ ᡏٿᆄ೴ᅌۯߏǹ (4) Լ୏ೈқ༾ํᆫӝ (annealing) Ǵٿచς׎ԋϐํރԼ୏ೈ

қٿᆄ࣬ϕௗӝ (Pollard et al., 1986) Ƕӧ΋૓௃ݩΠǴᛙۓޑಒझϣϐಒझମࢎ

ೀܭ΋୏ᄊ܄ѳᑽǴෞᚆϐ G-actin ᐚࡋ཮ೀܭᖏࣚᐚࡋǴԜਔ่ӝډٿᆄϐԼ୏

6

ೈқᆶவٿᆄှᚆϐԼ୏ೈқ΋ኬӭǴཀջಒझମࢎϐѐᆫӝೲ౗ᆶᆫӝೲ౗࣬

฻ၲډѳᑽ (Ono et al., 2007) Ƕ

2. ಒಒझମࢎϐԼ୏ೈқགᔈѦࣚᕉნӢηϷϣғૻဦ

Ҟ߻ςԖ೚ӭࣴزϷ᛾Ᏽ᛾ܴ෌ނಒझମࢎୖᆶ೚ӭόӕޑૻ৲໺ሀ᜘Ǵхࡴ

෌ނགᔈѦࣚߚғނૻဦӵག᝺ǵᅖ೸ᓸǵྕࡋ (Nick, 2008) аϷғނૻဦ (Schemelzer, 2002)Ƕ෌ނགᔈૻဦёϩࣁΟঁЬा٣ҹǺགޕڈᐟǵౢғૻ৲ǵૻ

ဦ໺ሀǴ຾ԶቹៜډࡕុΠෞғϯϸᔈ೼৩ (Yamaguchi et al., 2000) ǶԶ Eun Ϸ Lee ܭ 1997 ԃว౜Լ୏ೈқӧ਻ϾᜢഈਔǴ཮җಔᙃֹ᏾ޑރᄊ (well-organized) ᙯࣁှᡏ᚞ණ (disorganized) ޑࠠᄊ (Eun and Lee, 1997) ǴЪ Hwang Ϸ Lee ܭ 2014 ԃΨว౜Լ୏ೈқୖᆶፓ௓ߥፁಒझᡏᑈǴԶ௢ፕԼ୏ೈқୖᆶፓ௓ಒझጢ

΢ᚆη೯ၰࢲ܄ (Hwang and Lee, 2014) Ƕӧϣғғ౛ૻဦ΢ǴҞ߻ςԖ೚ӭࣴز

ࡰр෌ނಒझମࢎΨୖᆶΑ෌ނғߏวػфૈǴӵғߏન(Auxin) ӧ෌ނಒझύޑ ၮ౽ΨکಒझମࢎᄬࠠԖஏϪᜢ߯Ǵᙖҗಒझମࢎᆶ PIN ೈқ໔ޑҬϕբҔǴԶ ቹៜғߏનӧಒझϣޑၮ౽Ǵ຾Զቹៜ෌ނϐғߏ (Nick, 2013; Paque et al., 2014)Ƕ

྽෌ނᎁڙډமӀਔǴယᆘᡏޑࠟޔ౽୏Ψڙډಒझϣϐಒझମࢎ܌ፓ௓ǴԶ෌

ނགᔈӦЈЇΚԶڀԖӛӦ܄ǴΨᆶಒझମࢎᜢ߯ஏϪǴӢԜ෌ނಒझମࢎޑᛙ ۓ܄ᆶᆢ࡭΋ঁಒझޑ҅தфૈ৲৲࣬ᜢǶ

2. 1. ྕࡋ

෌ނϐלհ܄΋ޔ೿ࢂࣴز෌ނ଍ნऐڙ܄ޑख़ाҞ኱ϐ΋Ǵࣴزว౜ཱུᆄ

ྕࡋޑׯᡂȐଯྕ܈եྕȑǴ཮೷ԋԼ୏ೈқಒझମࢎޑ่ᄬᡂϯǴեྕ଍ნ཮೷

ԋ෌ނԼ୏ೈқѐᆫӝϯǴڈᐟ້ᚆη຾ΕಒझǴЪ཮٬෌ނᡏϣಕᑈεໆԾҗ

୷ԶᏤठጢڙډཞ্ (Mazars et al. 1997; Orvar et al. 2000; Pokorna et al. 2004;

D’Angeli and Altamura 2007) Ƕаեྕೀ౛оݨ๼ᝌੌ୻ᎦಒझޑᠼᆢࠠԼ୏ೈқ (Fibrous actin, F-actin) Ǵ཮٬ள F-actin ှᚆࣁౚࠠԼ୏ೈқ (Globular actin, G-actin)

7

(Egierszdorff and Kacperska, 2001) ǶНዿ LTRPK1 (low-temperature response protein kinase 1) ࣁ΋ឦႀೈқᐟ䁙 I (Casein kinase I, CKI) ৎ௼ϐೈқ፦Ǵӧ੿ਡғނ ύբࣁૻ৲໺ሀၡ৩ϐፓ௓ӢηǴ٠ЪᆶԼ୏ೈқ࣬ᜢೈқڀԖҬϕբҔǴӧ Liu

฻Γ (2012) ޑࣴزύࡰрǴԜೈқёаᛙۓНዿϐಒझጢᛙۓ܄Ǵ٠ЪቚуНዿ ޑեྕऐڙ܄ (Liu et al., 2012) ǶAbdrakhamanora ฻Γ (2003) ᛾ჴλഝаեྕϷ ABA ೀ౛ࡕว౜ಒझମࢎԖख़᏾ (re-organized) ౜ຝǴа 4ʚೀ౛ڀऐհ܄ϐλ ഝਥ೽ 7 ϺǴว౜ಒझମࢎ཮Ӣեྕೀ౛Զอኩ஝ှǴՠᒿ๱೴ᅌӣྕࡠൺғߏ చҹǴऐհ܄λഝϐಒझମࢎёख़ཥࡠൺᆫ໣Ǵՠόऐհޣ߾คݤ׎ԋᆫ໣ǴԜ

่݀ᡉҢ෌ނಒझମࢎϐѐᆫ໣ᆶλഝեྕϐགޕԖᜢ (Abdrakjamanora, 2003)Ƕ Örvar ฻Γ(2000) а DMSO (membrane rigidifier) ܈ cytochalasin D (actin

microfilament destabilizer) ೀ౛रጴϐᝌੌ୻ᎦಒझǴ᛾ჴёӧ 25ʚኳүհႯϯϐ చҹΠǴᇨ٬ CAS30 (cold acclimation specific gene) ߄౜Ƕ࣬ϸޑջ٬ӧ 4ʚΠǴ ऩа Benzyl alcohol (membrane fluidizer) Ϸ Jaspakinolide (actin microfilament stabilizer)ೀ౛रጴಒझǴ߾཮׭ڋ້ᚆηϐࢬΕЪ٬ CAS30 ୷Ӣคݤ߄౜ǶќѦ Sangwan ฻Γ (2001) аݨ๼ (Brassica napus) ᙯ෗ਲ਼ϐ༝ယዬ (leaf disc) ࣁ׷਑Ǵ ճҔ BN115 (cold-induced gene) ௴୏η០୏ GUS ൔᏤ୷ӢǴऩӧ 25ʚа DMSO Ϸ Latrunculin B (micro filament destabilize) ܈ oryzalin/colchicine (microtubule destabilize) ёᇨᏤ GUS ϐ߄౜Ǵϸϐջ٬ӧ 0ʚΠǴа Benzyl alocoholǵ

Jaspakinolideǵtaxol (microtubule stabilizer) Ϸ Gd³⁺(calcium channel blocker)߾཮׭

ڋ GUS ϐ߄ၲǶԜ٤᛾Ᏽࡰрಒझମࢎϐख़᏾ӧեྕϐૻ৲໺Ꮴǵ້ᚆηࢬΕϷ

୷Ӣ߄౜ύࢂѸሡޑǶOullet ฻Γ (2001) ϩᚆ٠᠘ۓΑ΋཮೏եྕᇨᏤϐλഝԼ

୏ೈқѐᆫӝӢη (TaADF)ǴӧհႯϯऐհޑλഝࠔᅿύǴว౜Ԝ TaADF ޑεໆ ಕᑈǴЪԜಕᑈ੝܄ѝჹեྕᇨᏤ߄౜ڀԖ஑΋܄ǴӢԜ௢ፕԜλഝ TaADF ёૈ

ᆶեྕႯϯǴԶߚଆۈϐեྕགޕԖᜢ (Oullet et al., 2001) ǶԜѦଯྕೀ౛Ψ཮ቹ ៜ෌ނϐಒझମࢎԼ୏ೈқޑ่ᄬǶMýller ฻Γ (2007) аߓ܎դ޺ਥ೽ࢲޑ߄Ҝ

8

ಒझࣁ׷਑Ǵว౜а 40~41ʚϐଯྕอኩೀ౛཮٬ಒझମࢎวғѐᆫ໣౜ຝǹԶ྽

ӣൺډ 20ʚࡕ 1-3 λਔϣǴջёख़ཥࡠൺচӃϐ่ᄬǴЪԜၸำᆶಒझᅿᜪϷว ػਔයԖᜢ (Mýller et al., 2007; Malerba et al., 2010)Ƕ

ᆕӝ΢ॊ߻Γᜢܭྕࡋᆶ෌ނಒझମࢎǵԼ୏ೈқ໔ޑࣴزൔ֋ύǴёаΑ

ှѦࣚϐྕࡋᡂϯǴคፕࢂଯྕ܈ࢂեྕǴࣣ཮ׯᡂԼ୏ೈқಒझମࢎϐ่ᄬǴ ٠Ъӧ೭٤ڈᐟ่״ࡕڀԖёӣൺϐૈΚǴӕਔΨளޕಒझମࢎᔈୖᆶ෌ނಒझ གᔈѦࣚྕࡋׯᡂϐૻ৲໺ሀၡ৩ǴᗨҞ߻ჹԜૻ৲໺ሀၡ৩ϝۘࡑࣴزǶ

2. 2. ᡶᡶϩ

βᝆޑࠔ፦ჹܭ෌ނғߏวػࣗԿࢂౢໆԶقቹៜࣗ႐Ƕ߈ԃٰШࣚ΢ςԖ೚

ӭӦБϐβᝆςᎁڙډᡶᡵϯǴӢԜפ൨٬෌ނჹܭᡶҽ׳уڀԖऐڙ܄ޑ୷Ӣ

܈ࢂᐒڋǴࢂҞ߻ғނמೌ΢ှ،ԜୢᚒޑБݤϐ΋Ƕᡶҽ଍ნอයϣ཮೷ԋᅖ

೸ᓸϐׯᡂǴߏය߾໊ᚆηϐಕᑈǶLu ฻Γ (2007) ϐࣴزᡉҢǴऩа microtuble destabilizer ܈ stabilizer ೀ౛ҏԯਥӾǴёගଯҏԯਥӾಒझჹᅖ೸ᓸ଍ნϐ௵ག

܄Ǵ٠ߦ຾ᚆቫለ (ABA) ϐ֖ໆቚуǶԜࣴز߄ҢǴᙖҗಒझମࢎϐख़᏾ᆶᅖ೸

ᓸ଍ნ໔۶ԜϐҬϕբҔǴёᏤठಒझϣ ABA ғӝԋϐቚуԶගϲऐڙ܄ǴฅԶ ځύዴϪޑᐒڋϝόϼమཱ (Lu et al., 2007) Ƕӧࣴزᡶҽ଍ნჹܭಒझମࢎޑࣴ

ز΢ǴWang ฻Γа paclitaxel ೀ౛ߓ܎դ޺ѴभǴว౜Ꮴठ׳ӭѴभܭᡶ଍ნΠ ԝΫǴ࣬ϸޑऩа oryzalin ܈ࢂ propyzamide ೀ౛ǴϸԶ෧Ͽߓ܎դ޺Ѵभӧᡶ଍

ნΠϐԝΫ౗ (Wang et al., 2011a) Ƕ྽уΕ້ᚆηܭ୻Ꭶ୷ύǴ߾ёӣൺಒझ༾

ํϐख़ཥಔᙃ٠ගଯӸࢲ౗ǴฅԶऩࢂуΕΑ້ᚆη೯ၰೈқ׭ڋᏊ (ӵ LaCl3) Ǵ

཮फ़եߓ܎դ޺Ѵभܭᡶҽ଍ნΠޑӸࢲ౗Ǵа΢่ࣣ݀߄Ңಒझ༾ํόᛙۓ཮

೷ԋಒझϣ້ᚆηᐚࡋϐᡂϯǴ຾Զ೷ԋ෌ނಒझჹᡶ଍ნϐགޕᡂ౦ (Wang et al., 2007) ǶԶଯᡶೀ౛ନΑჹ෌ނ೷ԋଯᡶ଍ნѦǴΨ཮೷ԋᚆη଍ნϐᅖ೸ᓸ ׯᡂаϷࡕុޑຬ਼ϯނಕᑈ଍ნӢԜ೷ԋ෌ނ਼ϯ଍ნ (Mittler et al. 2004) Ƕ

9

Ԝၸ਼ϯނಕᑈޑ౜ຝӕਔӧаԼ୏ೈқᛙۓᏊаϷԼ୏ೈқᆫӝ׭ڋᏊೀ౛ࡕ ܭ෌ނਥ೽ύว౜ǴӢԜઇᚯԼ୏ೈқচғϐ୏ᄊѳᑽ཮Ꮴठၸ਼ϯނಕᑈ຾Զ

٬෌ނڙډӒ্ ( Liu et al., 2012) ǶќѦӧᅖ೸ᓸჹܭԼ୏ೈқᛙۓࡋޑࣴز΢Ǵ Komis ฻Γว౜ଯᅖ೸ᓸೀ౛Ӗើ (Chlorophytum) ϐယ೽ಒझǴ཮೷ԋಒझ༾ํ

ϐख़ಔ (Komis, 2002)ǶLiu ฻ΓࡰрճҔׯᡂ෌ނԼ୏ೈқ୏ᄊѳᑽϐᛰᏊ (stabilizer/ destabilize) Ǵ཮ቹៜၸ਼ϯނӧߓ܎դ޺ਥ೽ޑಕᑈǴЪӧᡶҽ଍ნΠ ऩࢂӆаԼ୏ೈқᆫӝ׭ڋᏊ (ӵ Latrunculin B) ೀ౛Ǵ཮٬෌ਲ਼ჹᡶ଍ნ׳у௵

གǴฅԶऩࢂаԼ୏ೈқᛙۓᏊ (ӵ phalloidin) ೀ౛෌ਲ਼Ǵ߾཮ቚу෌ਲ਼ޑӸࢲ

౗ (Liu et al., 2012) ǶќѦ Actin-related protein 2/3 complex ࢂ΋ᆶԼ୏ೈқ่ӝϐ

ೈқ፦ǴZhao ฻Γܭ 2013 ԃճҔϩ݋ Arp2/3 ँᡂਲ਼ܭᡶҽ଍ნΠޑғ౛ϸᔈǴ җ่݀ᡉҢǴԜԼ୏ೈқ่ӝೈқϐँᡂਲ਼ჹᡶҽ଍ნၨ௵གǴЪ཮೷ԋಒझϣ

້ᚆηᐚࡋଯࡋ΢ϲǴ٠ቹៜډࡕុϐૻ৲໺ሀ೼৩ (Zhao et al., 2013) Ƕа΢ࣴ

زࣣᡉҢ෌ނಒझମࢎϷԼ୏ೈқ཮ڙډѦࣚᕉნׯᡂԶቹៜځᄬࠠϷᛙۓ܄Ǵ

຾Զቹៜډ෌ނჹᡶϐғ౛ϸᔈаϷऐڙ܄Ƕ

2. 3. ଳଳײ

ଳײࢂቹៜբނౢໆϐख़ाज़ڋӢηϐ΋Ǵࣁࣴزߚғނ଍ნϐख़ाࣴزҞ

኱Ƕ2005 ԃ Ghulam ฻ΓᙖҗჹٿຼεϐНዿ຾Չଳײෳ၂Ǵܜڗځယᓋϐೈқ

፦຾Չϩ݋Ǵ่݀ᡉҢڀԖ 10 ᅿೈқ፦ܴᡉεໆ߄౜ǴԶԖٿᅿೈқ፦߾ࢂ֖ໆ ෧ϿǶஒ೭٤ೈқ፦຾Չϩ݋ࡕว౜Ǵ೭٤ೈқ፦ᆶ෌ނܢᑇ܄ǵૈໆౢғǵж ᖴբҔǵಒझ่ᄬаϷૻ৲໺ᏤԖᜢǶԶࣁΑᕕှᆶଳײ଍ნϐᜢ߯ǴGhulam ฻ ΓΞаόӕϐ଍ნೀ౛ϩ݋Нዿϐೈқ፦Ǵว౜΋Нዿ OsADF ཮ڙډଳײϷᅖ೸

ᓸ଍ნᇨᏤԶεໆಕᑈǴԶό཮Ӣեྕೀ౛܈ລᅟᆾೀ౛Զׯᡂځ֖ໆǴЪӧऐ ײϐНዿࠔύύջ٬ࢂ҂࿶ଳײೀ౛ջڀԖεໆϐ OsADF ϐ߄౜ǴᡉҢНዿ OsADF ೈқ፦ࣁଳײ଍ნΠϐᇨᏤ߄౜Ҟ኱ϐ΋ (Ghulam et al., 2005) ǶԶ Liu

10

฻Γаೈқ፦ᡏᏢϩ݋όӕଳײऐڙ܄ϐНዿǴаଳײೀ౛ࡕ่݀ว౜ OsADF5 ӧଳײ଍ნΠϐᔼᎦวػਔය཮εໆಕᑈǴՠऩख़ཥឲН߾཮੃ѨԶؒԖಕᑈǶ

྽຾΋؁а IR64 ܜᕞ߻ 3 Ϻϐ෌ਲ਼຾Չଳײೀ౛Ϸ ADF ߄౜ϩ݋Ǵёޕ ADF ҭӸӧܭቆယǵᕞ࢓Ϸ໢ᑮύǴ߄ҢԜ ADF ᆶНዿଳײ଍ნऐڙ܄Ԗᜢ (Liu, 2005)Ƕа΢ࣴزࣣࡰрǴଳײ଍ნೀ౛཮ቹៜډ ADF ϐ߄౜ǴฅԶࡕុϐ෌ނϸ ᔈ߾ϝۘࡑࣴزǶ

2. 4. ख़ख़ߎឦ

ख़ߎឦԡࢉࢂࣴزߚғނ଍ნϐख़ाࣴزҞ኱ǴΨࢂҞ߻Ӓ্Ѡ᡼ၭ཰ख़ा

Ӣηϐ΋ǶЬाࢂޔௗჹ෌ނ֎ԏނ፦Ꭶϩϐਥ೽೷ԋ໾্Ǵ຾ԶᏤठ෌ਲ਼ԝΫǶ ӵᙿ (Cd) Ϸ᎑ (Al) ޑࢥ܄཮זೲӦჹ෌ނౢғࢥ܄Ǵ٠Ъ٬ਥ೽ଶЗғߏǶ 2012 ԃ Krtková ฻ΓޑࣴزύࡰрǴа᎑ೀ౛ߓ܎դ޺ਥ೽ว౜߃යջр౜ಒझ ጢسᛙۓࡋڙډཞ্Ǵ೷ԋ෌ނਥ೽ଶЗғߏǴӢԜբޣ௢ፕǴ෌ނӧᎁၶډ᎑

ࢥ্Ǵ܌೷ԋޑ෌ނཞ໾کಒझጢޑᛙۓࡋԖᜢ (Krtková et al., 2012) ǶԶӧᙿჹ ܭ෌ނϐࢥ্ࣴزǴ྽෌ނௗڙډ Cd ᚆη཮Ꮴठ෌ނಒझϣޑ້ᚆηᐚࡋΠफ़Ǵ ٠Ъ཮೷ԋϣ፦ᆛϷಈဏᡏޑᄬ೷ઇᚯǴௗ๱җܭ້ᚆηޑᐚࡋׯᡂΨ཮ቹៜډ Լ୏ೈқ༾ํޑှᆫǴ຾ԶᏤठ෌ނਥ೽ଶЗғߏ (Wan et al., 2012) Ƕ

Οǵġ Լ୏ೈқѐᆫӝӢη(ADF)

ჹܭ୏ނޑ ADF Ҟ߻ςԖεໆޑࣴزǴԶჹܭ෌ނޑ ADF ߾ࢂࣴزၨϿǶ ߈ԃٰҗҏԯǵԭӝ໒ۈᅌᅌԖ܌ࣴزǴௗ๱ӧߓ܎դ޺ǵ๨૛аϷා޸฻෌ނ ύΨ೿Ԗ࣬ᜢޑࣴزൔᏤ (Hussey et al., 1998; Jiang et al., 1997a; Jiang et al., 1997b;

Gungabissoon et al., 1998 ; Li et al., 2010) ǴԶԜ٤Ԑයᜢܭ෌ނ ADF ε೿ࢂ᛾ܴ

ځᆶ෌ނϐࠠᄊวғаϷࠠᄊᄬԋԖᜢǶ

Լ୏ೈқѐᆫӝӢηࣁ 16-20 kDa ޑλϩηೈқǴࢂፓ௓ಒझମࢎϐԼ୏ೈқ ϐᆫӝᆶѐᆫӝϐ୏ᄊѳᑽϐख़ाӢη (Maciver et al., 2002) ǶԼ୏ೈқᙖҗ

11

Mg²⁺ǵK⁺ک ATP ޑբҔΠǴҗൂᡏౚࠠϐ G-actin (globular) ᆫӝ׎ԋڀԖཱུ܄ޑ ᚈᖥ௽ํރᆫӝᡏጕ׎ϐ F-actin (filamentous) ǴԶԼ୏ೈқѐᆫӝӢηᙖҗᆶ G-actin ่ӝǴஒ F-actin ѐᆫӝࣁൂᡏౚࠠϐ G-actinǴADF ᆶԼ୏ೈқϐ໔ޑҬ ϕբҔڙ pH ॶǵё଍܄ᕗለϯϷ੝ۓᕗለԼᎇ܌ፓ௓ (Allwood et al., 2002;

Smertenko, 2010; Dong et al., 2013) Ƕ

ߓ܎դ޺ AtADF ୷Ӣৎ௼ڀԖ 11 ঁԋ঩ǴRuzicka ฻Γ (2007) ճҔᙯ෗ߓ

܎դ޺ AtADF ௴୏η AtADFpro::GUS ϩ݋ߓ܎դ޺ AtADF ϐ߄౜Տ࿼аϷಔᙃ஑

΋܄Ƕว౜೭٤୷Ӣ཮ڙډόӕวػਔයϐፓ௓Ƕаᄽϯ΢ٰϩ݋ߓ܎դ޺ AtADF ԋ঩ёϩࣁѤঁԛဂ (subgroup) Ǵӧ୷Ӣޑቫભ΢Ǵಃ΋ঁԛဂЬाࢂ߄౜ܭᔼ ᎦғߏਔයϷନΑ޸ણᆅϐѦޑғ෗ಔᙃύ࡭ុ܄߄ၲǹಃΒঁϩဂࢂ஑΋܄ޑ ଯࡋ߄౜ܭԋዕޑ޸ણаϷ޸ણᆅύǴӕਔΨԖӧਥ೽߄Ҝޑ૙Л (trichome) Ϸ ਥЛύว౜ځ߄౜ǴԜ౜ຝӧൂηယޑНዿύΨԖว౜ǹಃΟঁԛဂޑ AtADF ߾

ࢂӧᔼᎦғߏය߄౜ՠ߄౜ໆၨեǴѝԖӧזೲғߏ܈ϩϯޑಒझύȐӵᘰӝಒ झ callusǵ߃ғယǵϩϯಔᙃȑၨଯǹനࡕಃѤঁԛဂޑ AtADF ࢂ࡭ុ܄ӧ܌Ԗಔ ᙃύ߄ၲǶԶӧೈқ፦ޑ߄౜΢Ǵ߾ࢂϩࣁٿঁԛဂǴಃ΋ԛဂՏܭߓ܎դ޺ယ Ժಒझϐಒझ፦аϷਡύ߄౜ǴԶಃΒԛဂ߾ࢂЬाӧਥЛаϷ޸ણᆅޑӾᆄϷ ۯߏ೽Տϐಒझ፦ύ߄౜ (Ruzicka et al., 2007)Ƕ

1. ԼԼ୏ೈқѐᆫӝӢη (ADF) ୖᆶ෌ނϐғߏวػ

Լ୏ೈқᠼᆢᙖҗᆶಒझ፦ǵಒझጢ܈ࢂಒझϣځдझᏔ࣬ೱௗǴаᆢ࡭ಒ झϐ҅தфૈǴӵਥЛޑۯ՜ǵ޸ણᆅϐ๴วǵယη้Лϐ՜ߏ฻౜ຝԼ୏ೈқ

೿ୖᆶځύǶDong ฻Γܭ 2001 ԃޑࣴزύࡰрǴߓ܎դ޺ AtADF1 ௓ڋߓ܎դ޺

ಒझۯߏаϷғߏǴ٠Ъၸࡋ߄౜ AtADF1 ёаගԐߓ܎դ޺໒޸ਔ໔Ǵϸϐ෧Ͽ AtADF1 ޑ߄౜཮ۯᒨߓ܎դ޺ޑ໒޸ਔ໔ (Dong et al., 2001) ǶԶߓ܎դ޺

AtADF7 Ϸ AtADF10 Ψӧࣴزύ೏ว౜ԜΒޣᆶߓ܎դ޺޸ણᆅϩϯਔޑ੝ۓಒ

12

झۯߏфૈԖᜢ (Daher et al., 2012; Zheng et al., 2013) Ƕ

ӧ ADF ޑࣴزᗋԖճҔ౦Տ߄ၲѦٰ୷Ӣ຾Չᙯ෗෌ނϐѦ߄ࠠϩ݋Ǵӵ 2013 ԃ Chi ฻ΓճҔ౦Տ܄߄ၲා޸ GbADF1 ᙯ෗ܭ๨૛ύ຾ՉࣴزǶ२ӃӃճ Ҕữ୷ለׇӈ aligment Кჹٿঁނᅿϐ ADF ࣬՟܄Ǵ٠ЪճҔࠄБϷчБᏀᗺᙯ ᅄמೌ (Southern and Northern blot) ᛾ܴ٠ዴᇡԜᙯ෗ GbADF1 ϐ๨૛ύёа҅

தޑౢғр GbADF1 ೈқ፦Ǵௗ๱ᢀჸډ T1ᙯ෗ਲ਼߄౜ၨอޑΠखືаϷڀԖၨ

ϿޑਥЛǴЪᙯ෗ਲ਼ϐ਻ϾԖ೏ᇨᏤᜢഈޑ໼ӛǴԶӧ໒޸ਔ໔ޑБय़߾ࢂว౜

ᙯ෗ਲ਼ၨഁғࠠࣁۯᒨϖϺǹௗ๱ᢀჸ GbADF1- GFP ᑼӝೈқϐᑻӀวғՏ࿼Α

ှځ߄౜Տ࿼ว౜Ԝᑼӝೈқ߄౜ӧࢩጳϐಒझਡаϷಒझମࢎϣǴа΢่݀ᡉ ҢԜ GbADF1 ӧා޸ޑಒझғߏϩϯύתᄽख़ाޑفՅǶ (Chi et al., 2013) Ƕ

2. ԼԼ୏ೈқѐᆫӝӢη(ADF) ୖᆶ෌ނϐғނ଍ნऐڙ܄

ߓ܎դ޺ϐ AtADF όѝୖᆶΑ෌ނϐғߏวػǴӕਔΨୖᆶΑ෌ނჹלғނ

଍ნΠޑऐڙ܄Ƕ2009 ԃ Tian ฻ΓճҔϸӛᒪ໺Ꮲ (Reverse genetics approach ) ϩ݋ߓ܎դ޺ AtADF4 ӧ Pseudomonas syringae pv tomato ΢ޑלੰ೼৩Ǵҗ่݀ύ ᡉҢک΋૓לੰ܄ϐಒझମࢎፓ௓ᐒڋόӕǶAtADF4 ࢂᙖҗፓ௓ಒझମࢎϐܢל

ૻ৲೼৩ٰቚу෌ނϐऐੰ܄ǴᡉҢ ADF ٬෌ނڀԖऐڙ܄ϐᐒڋόѝࢂᙖҗׯ ᡂಒझମࢎϐ୏ᄊѳᑽǴࢌᅿำࡋ΢ΨୖᆶΑ෌ނऐڙᐒڋϐϸᔈૻ৲໺ሀၡጕ (Tian et al., 2009; Katie et al., 2012) Ƕ

3. НዿԼ୏ೈқѐᆫӝӢηϐ࣬ᜢࣴز

ӵ߻य़ᇥܴଳײ଍ნᆶԼ୏ೈқ໔ϐᜢ߯ύගډ 2005 ԃ Liu ฻Γว౜Нዿύ ڀԖ΋ ADF ᆶНዿଳײ଍ნऐڙ܄Ԗᜢ (Liu, 2005)ǶԶ Feng ฻Γܭ 2006 ԃағ ނၗૻБݤ຾Չߓ܎դ޺ᆶНዿ୷Ӣၗ਑৤ޑ ADFs ၗ਑᏾ӝǴࣴزว౜ёૈԿϿ ӚԖ 11 ঁ ADF ୷ӢӸӧܭߓ܎դ޺ᆶНዿ୷Ӣಔύ (Feng et al. 2006) Ƕךॺჴ ᡍ࠻ޑ໳໡ृᏢှϩ݋ΑНዿ OsADFs ୷Ӣৎ௼ӧόӕਔයǵ೽ՏǵລᅟᆾаϷ

13

όӕߚғނ଍ნޑᙯᒵ߄౜Ǵ่݀ࡰр܌ԖНዿ OsADF ୷Ӣԋ঩ࣣܭНዿᑉ޸೽

Տ߄౜Ǵځύ OsADF2, 4, 5, 11 ୷Ӣ߄౜ܭ܌ԖಔᙃǴԶ OsADF2, 4, 5, 11 ୷Ӣܭ όӕޑғߏ໔߄౜ܭ܌ԖಔᙃύǴӧ୷Ӣ߄౜Бय़ OsADF1, 3, 9 11 ཮ڙډհᇨᏤ ߄౜ (ߕᒵΒ) ǴԶа ABA ೀ౛ࡕ߾ࢂ཮ᇨᏤ OsADF1, 3, 5, 9, 11 ߄౜Ǵᡶೀ౛߾

ࢂѝԖ OsADF3 ԖܴᡉεໆڙډᇨᏤ߄౜Ǵଳײೀ౛ޑ୷Ӣ߄౜่݀߾ࢂ OsADF3, 10 Ԗεໆ߄౜ޑ౜ຝวғ (ߕᒵΒ) Ƕௗ๱ճҔ 35S ௴୏ηၸࡋ߄౜Нዿ OsADF3

୷Ӣܭߓ܎դ޺ύϩ݋ OsADF3 ᙯ෗ਲ਼ӧ଍ნΠޑ߄౜Ǵว౜౦Տၸࡋ߄౜Нዿ OsADF3 ୷Ӣёቚуߓ܎դ޺ᙯ෗ਲ਼ჹᅖ೸ᓸϷଳײ଍ნޑऐڙ܄ (Huang et al.

2012) Ƕ Ѥ

Ѥǵġ ࣴزҞޑϷჴᡍࢎᄬ

җ΢ॊࣴزύёளޕҞ߻ჹܭߓ܎դ޺฻ނᅿޑ ADF ςԖ࣬྽ำࡋޑΑှǴ คፕࢂӧ෌ނ׎ᄊ΢܈ࢂߚғނ଍ნȐӵᡶ্ǵଯ/եྕǵᅖ೸ᓸ଍ნ฻ȑΠޑф

ૈǴςԖ೚ӭࣴزว߄ǴฅԶНዿޑ ADF ޑфૈҞ߻ۘࡑ௖૸ǶӆޣǴҗܭҞ߻

ςԖ೚ӭࣴزࡰр෌ނಒझମࢎǵԼ୏ೈқޑᛙۓ܄܈ࢂ୏ᄊѳᑽӧ෌ނޑғߏ วػаϷय़ᖏߚғނ଍ნȐӵեྕǵᡶ্฻ȑਔޑૻ৲໺ሀ೼৩ϷϸᔈתᄽΑΜ ϩख़ाޑفՅǴӢԜ௢ፕ ADF ёૈک෌ނय़ᖏߚғނ଍ნޑऐڙ܄ԖᜢǶҁጇࣴ

ز२ӃҗНዿၗ਑৤ύפр OsADFs ୷Ӣৎ௼ǴӃафૈ܄୷ӢᡏᏢБԄࣴز OsADF2 Ϸ OsADF11 Ȑӕឦܭ OsADF ୷Ӣৎ௼ϐ A ဂȑ౦Տ߄౜Нዿ୷Ӣܭߓ

܎դ޺ύǴ຾Չߚғނ଍ნΠϐ୷Ӣ߄౜ǵғ౛аϷѦ߄ࠠϩ݋ǴаΑှНዿ ADF ӧ෌ނय़ᖏߚғނ଍ნਔޑғ౛ϸᔈǶௗ๱ճҔ PEG transfectin ኩਔ܄߄ၲНዿ OsADF ୷Ӣৎ௼ᆶ GFP ϐᑼӝೈқܭНዿচғ፦ᡏύǴճҔӅ೫ขᡉ༾᜔ᢀჸځ ߄౜Տ࿼Ǵ٠௖૸ځёૈୖᆶϐಒझфૈǶჴᡍࢎᄬӵΠ।܌ҢǶ

14 კ

კ΋ǵჴᡍࢎᄬკ

15

ಃ

ಃΟകġ ׷਑Бݤ

΋ǵġ OsADF ୷Ӣৎ௼ϐғނၗૻϩ݋

ਥᏵҁჴᡍ࠻റγ੤໳໡ृᏢۆޑፕЎଞჹςှׇϐНዿ୷Ӣಔ຾ՉԼ୏ೈ

қѐᆫӝӢηৎ௼୷Ӣޑਡ㧿ለᆶೈқ፦ׇӈཛྷ൨Ǵ٠ୖԵҞ߻ςว߄Ў᝘᏾౛

ᇙ߄Ǵ٬Ҕ Vector NTI ೬ᡏϐ AlignX фૈ (Vector NTIǴv9.0ǴInformax) ຾Չׇ

ӈКჹǴаНዿ KOME ၗ਑৤Ϸ୷Ӣբ܌ᕇளޑữ୷ለׇӈ٬Ҕ ClustalX (vl.83) ᆶ Treeview (vl6.6) ຾Չᒃጔᄽϯᐋޑࡌҥ (ߕᒵ΋) Ƕཛྷ൨୷Ӣׇӈ࣬ᜢᆛ֟Ǻ NCBI database (http://www.ncbi.nlm.nih.gov) ǵKOME database

( http://cdna01.dna.affrc.go.hp/cDNA/)Ƕ

Βǵġ ၂ᡍ׷਑

1. ߓ܎դ޺ᅿη

ഁғࠠߓ܎դ޺ (Arabidopsis thaliana ecotype Columbia, Col-0) ᅿηϷ Col-0 ङඳϐ T-DNA ँᡂਲ਼ᅿηǴϩձҗᆵ᡼εᏢၭ᛬سЦలࣔ௲௤ჴᡍ࠻ගٮǴаϷ ऍ୯߮Ү߮ԀԀҥεᏢ Arabidopsis Biological Resource Center (ABRC, OH, USA) ᐒᄬ (http://abrc.osu.edu) ܌ҙፎǶќѦҗჴᡍ࠻റγࡕࣴز঩ഋᗶӓᏢߏගٮ (Chen et al., 2011) ޑၸࡋ߄౜៓ઍԭӝ (Lilium formosanum ) ABA ғӝԋ୷Ӣ NCED3 (9-cis-epoxycarotenoid dioxygenase 3) ϐ OE2 ࠔسȐ35S::LfNCED3ȑǴϷߓ

܎դ޺ NCED3 ँᡂࠔس N3 ᅿηȐatnced3ȑǴբࣁӧᡶҽ଍ნऐڙ܄΢ፏӵᅿη

๴޵౗ǵғߏ༈฻ޑ҅ӛፓ௓аϷॄӛፓ௓௓ڋಔǴаዴۓ଍ნೀ౛ࢂցԖਏǶ

2. Нዿᅿη

ఒዿВҁන (Oryza sativa L. cv. Nipponbare)ǵѠၭ 67 ဦ (Oryza sativa L. cv.

Tainung 67) ϷޛዿѠύӧٰ 1 ဦ (Oryza sativa L. cv. Taichung Native 1, TCN1)Ƕ TCN1 ࣁ 99 ԃಃΒයբᅿ෌ܭᆵ᡼εᏢၭ൑ǹTNG 67 ࣁ 99 ԃಃ΋යբǶ

16 Ο

Οǵġ ߓ܎դ޺ғߏచҹϷόӕߚғނ଍ნೀ౛

1. ߓ܎դ޺ғߏచҹ

ߓ܎դ޺ܭϟ፦βᝆϐ୻ᎦచҹǺڗ፾ໆߓ܎դ޺ᅿη࿼ܭ 1.5 mL ༾ໆᚆЈ ᆅύӇܭ 4ʚӇጃύٿϺǴ຾ՉࡾϯբҔаѺઇځҶ઀Զ٬ϐว޵౗΋ठǶࡾϯբ Ҕ่״ࡕ֡Ϭ᠀ኞӧςྐ๵ϐ୻Ꭶβ΢ (୻ᎦβǺ຃ҡǺࣔ੧ҡ = 2:1:1) Ǵௗ๱

౽Կ 22/18ʚ (Вྕ/ڹྕ) ǴߏВྣ 16/8 λਔӀྣ (Ӏྣமࡋ 120-150 μmol/m²/sec) Ϸ໵སڬයϐғߏጃύǴᔸࡋࣁ 60% (60% RW) Ǵ؂ຼ዆ 2 ԛНǴࡼ᠀ޥ਑Ȑ޸

ᝊ 3 ဦȑа΋ຼ΋ԛࣁज़Ƕߓ܎դ޺ܭ୻Ꭶҝϐค๵୻ᎦచҹǺஒ፾ໆߓ܎դ޺

ᅿη࿼ܭ༾ໆᚆЈᆅύǴܭค๵ᏹբѠύуΕ 1 mL 70% ଚᆒ᎜ᕏ 7 ϩដǴอኩ ᚆЈࡕѐନଚᆒǴуΕ 1 mL 1-2% NaOCl (֖ 0.05% Tween-20) ᎜ᕏ 10 ϩដࡕǴ ᚆЈѐନ NaOClǴаค๵Н᎜ᕏమࢱᅿηԿϿ 4-5 ԛǴޔډόӆр౜ݰݣǴуΕ 1 mL ค๵НܫΕ 4ʚ Ӈጃ຾ՉࡾϯբҔǴٿϺࡕѐନค๵Н٠уΕཥޑ 1 mL ค

๵Нஒᅿη֡Ϭ᠀ኞӧڰᄊ୻Ꭶ୷΢Ǵௗ๱а೸਻ጤ஥࠾Ր୻Ꭶҝ౽࿼ 22/18ʚ (В/ڹྕ) ǵ16/8 λਔӀྣϷ໵སڬයϐғߏጃύǶ

2. ߓ܎դ޺ғ౛ϷѦ߄ࠠᢀჸϐߚғނ܄଍ნೀ౛చҹ 2.1 ว޵౗ፓࢗϐ଍ნೀ౛చҹ

၂ᡍ׷਑ࣁߓ܎դ޺ഁғࠠ (Col-0) ǵ35S::LfNCED3 ᙯ෗ਲ਼ (OE2) ǵatnced3

ँᡂਲ਼ (N3) ᆶၸࡋ߄౜Нዿ OsADF2, OsADF11 ୷Ӣᙯ෗ਲ਼ᅿη (T3) Ӛऊ 100 ᗭǴ੃ࢥϷࡾϯೀ౛ࡕ֡Ϭ᠀ܭόӕ଍ნϐ 1% sucrose, 8 g/L Agar 1/2 MS (SIGMA)

୻Ꭶ୷ύǺჹྣಔࣁ 1/2 MS ୻Ꭶ୷ǹଯᡶ଍ნࣁ֖Ԗ 150 mM (J.TBaker)ϐ 1/2 MS

୻Ꭶ୷Ƕа೸਻ጤ஥࠾Ր୻ᎦҝǴ࿼ܭ 22/18ʚ (В/ڹྕ) Ǵ16/8 λਔӀྣϷ໵ས ڬයϐғߏጃύ 1~7 ϺǴीᆉ؂Ϻว޵౗ǴԶว޵౗ϐ኱ྗࣁаखਥँઇᅿҜࡕ

ीᆉǶ

17 2.2 ਥਥߏෳໆϷӸࢲ౗ϐ଍ნೀ౛చҹ

၂ᡍ׷਑ࣁߓ܎դ޺ഁғࠠ (Col-0) ǵ35S::LfNCED3 ᙯ෗ਲ਼ (OE2) ǵatnced3

ँᡂਲ਼ (N3) ᆶၸࡋ߄౜Нዿ OsADF2, OsADF11 ୷Ӣᙯ෗ਲ਼ᅿη (T₃) Ӛऊ 100 ᗭǴ੃ࢥϷࡾϯೀ౛ࡕ֡Ϭ᠀ܭ 1% sucrose, 8 g/L Agar, 1/2 MS (SIGMA) ୻Ꭶ୷ύǴ

࿼ܭ 22/18ʚ (В/ڹྕ) Ǵ16/8 λਔӀྣϷ໵སڬයϐғߏጃύғߏԿ 5 ϺεϷ 10 ϺεǴ౽෌Կόӕ଍ნϐ 1/2 MS (SIGMA)୻Ꭶ୷ύ୻Ꭶ 4 ϺӟෳໆਥߏаϷӸࢲ

౗Ǻჹྣಔࣁ 1/2 MS ୻Ꭶ୷ǹଯᡶ଍ნࣁ֖Ԗ 150 mMNaCl (J.T Baker) ϐ 1/2 MS

୻Ꭶ୷ǴӚೀ౛ 3 ख़ፄǴа೸਻ጤ஥࠾Ր୻ᎦҝǴࠟޔ࿼ܭ 22/18ʚ (В/ڹྕ) Ǵ 16/8 λਔӀྣϷ໵སڬයϐғߏጃύǴғߏ 4 Ϻࡕྣ࣬इᒵǴۓကယТқϯޣࣁ ԝΫǴԶᆢ࡭ᆘՅယТޣ߾ࣁӸࢲǶ

2.3 ԋਲ਼ᢀჸϐ଍ნೀ౛చҹ

၂ᡍ׷਑ࣁߓ܎դ޺ഁғࠠ (Col-0) ǵ35S::LfNCED3 ᙯ෗ਲ਼ (OE2) ǵatnced3

ँᡂਲ਼ (N3) ᆶၸࡋ߄౜Нዿ OsADF2, OsADF11 ୷Ӣᙯ෗ਲ਼Ǵ࿶ၸࡾϯբҔࡕ֡

Ϭ᠀ܭ֖Ԗϟ፦ϐ 3 әࣧ΢Ǵ࿼ܭ 22/18ʚ (В/ڹྕ) Ǵ16/8 λਔӀྣϷ໵སڬය ϐғߏጃύǴғߏԿ 10 Ϻࡕϩձ౧भǴௗ๱؂ڬа޸ᝊΟဦ຾ՉࡼޥǶғߏԿ 21 Ϻεࡕ຾Չ଍ნೀ౛Ǻჹྣಔࣁό଺ೀ౛҅த዆НǴ4 ख़ፄǹଳײ଍ნೀ౛ࣁ຾Չ ᘐНೀ౛ᢀჸ 12-15 ϺǴԶࡕӆࡠൺٮНǴ4 ख़ፄǹଯᡶ଍ნೀ౛ࣁа 200 mM NaCl

ೀ౛ᢀჸ 12-15 ϺǴࡠൺ҅தٮН 7 ϺǴӚ 4 ख़ፄǹեྕ଍ნࣁ୻Ꭶܭ 4ʚೀ౛ᢀ ჸ 12-15 ϺǴࡠൺ҅தྕࡋ 7 ϺǶྣ࣬इᒵϷीᆉӸࢲ౗Ƕ

2.4 ᇻआѦጕ዗གᔈቹႽϩ݋ᡶ଍ნೀ౛ᙯ෗ਲ਼చҹ

ճҔߚઇᚯ܄ᇻआѦጕ዗ቹႽמೌȐNon-destructive infrared thermographyȑᔠ

ෳ෌ނௗڙଯᡶ଍ნೀ౛Πϐғ౛ϸᔈǴԜচ౛߯ճҔ෌ނယТᇃණೲ౗ᆶယТ

ྕࡋ໔ϐ࣬ᜢ܄ǴᙖҗᔠෳယТྕࡋаΑှᆶځଯࡋ࣬ᜢϐ௓ϐယТᇃණೲ౗ޑ

18

਻ϾᏤࡋ (Stomata conductance) (Costa et al., 2013) Ƕ

၂ᡍ׷਑ࣁߓ܎դ޺ഁғࠠ (Col-0) ǵ35S::LfNCED3 ᙯ෗ਲ਼ (OE2) ǵatnced3

ँᡂਲ਼ (N3) ᆶၸࡋ߄౜Нዿ OsADF2, OsADF11 ୷Ӣᙯ෗ਲ਼Ǵ࿶ၸ 4ʚࡾϯբҔ ࡕ֡Ϭ᠀ܭ֖Ԗϟ፦ϐ 3 әࣧ΢Ǵ࿼ܭ 22/18ʚ (В/ڹྕ) Ǵ16/8 λਔӀྣϷ໵ས ڬයϐғߏጃύǴғߏԿ 10 Ϻࡕϩձ౧भԿ 4 ස/ࣧǴғߏԿ 21 ϺεࡕӃ౽࿼ట

຾ՉᇻआѦጕቹႽϩ݋ϐύѧࣴزଣ෌༾܌ғߏ࠻Ȑ29ʚǴ55% RWȑႯϯ΋Ϻࡕ

຾Չଯᡶ଍ნೀ౛Ǻа 200 mM NaCl ೀ౛΋ϺࡕǴܭࡡྕғߏ࠻ύаᇻआѦጕ዗

གᔈቹႽϩ݋ሺ (IRM-R300SR) ܡឪቹႽइᒵϷϩ݋ယТྕࡋǶ

Ѥ

Ѥǵġ ߓ܎դ޺ँᡂਲ਼Ϸᙯ෗ਲ਼ϐϩη᠘ۓ 1. ෌ނ୷Ӣᡏಔ DNA ๧ڗ

ڗ 50-100 mg ယТܫΕ 2 mL ༾ໆᚆЈᆅ (QSP) ύ׫Εనᄊේϣ࡚ೲեྕհ ࠅǴௗ๱аς੃ࢥϐᙻηܫΕ΋ᗭ 5 mm ᒳ੧ǴճҔ֡፦ᐒа 1,200 rpm ᎜ᕏ 10

ࣾஒယТᑃԋણރǴऩ҂ֹӄᑃ࿗ёӆख़ཥܫΕనᄊේࡕख़ཥ֡፦΋ԛǶௗ๱у Ε 1 mL Urea extraction buffer ِೲ᎜ᕏ٢ϯǴܭ࠻ྕΠᓉ࿼ 5 ϩដǶа 12,000 rpm ᚆЈ 10 ϩដǴڗ΢మన 800 μL Կཥޑ 2 mL ༾ໆᚆЈᆅύǴуΕ 400 μL phenol (pH=8.0, Amresco)Ϸ 400 μL Chloroform ِೲ᎜ᕏ٢ϯǹа 12,000 rpm ᚆЈ 10 ϩ ដǴڗ΢మన500 μL Կཥޑ 1.5 mL ༾ໆᚆЈᆅύǴуΕ฻ᡏᑈϐ Isopropanol (Merck) షϬǴа 12,000 rpm ᚆЈ 10 ϩដǴѐନ΢మన٠੮Π DNA ؈ᐘނ (pellet) ǹ а 70% ଚᆒమࢱ DNA ؈ᐘނǴа 12,000 rpm ᚆЈ 7 ϩដǴख़ፄԜ؁ᡯ΋ԛǹѐ ନଚᆒࡕஒ DNA ؈ᐘނ࿼ܭ࠻ྕΠ॥ଳǴ྽ DNA ؈ᐘނև౜ъ೸ܴጋރਔǴу Ε20 μl ѐᚆηНྋှ DNA ؈ᐘނࡕᇸᄟǴܭ࠻ྕΠᓉ࿼ 10 ϩដࡑ DNA ӣྋܭ ѐᚆηНύǴڗ2 μl DNA ྋనаӀࡋी NanoVue ۓໆǶ

19

2. DNA ࢩࢩ๼ᛏિጤᡏႝݚϩ݋

ଛ࿼ 1%ᐚࡋϐࢩ๼ᛏિጤ (Agarose gel, 1 X TAE buffer) Ǵу዗ஒ Agarose ӄ ኧྋှࡕуΕ ethidium bromide ٠ॹΕ᠗ጤ৒ᏔǶӧኬࠔύуΕ 6 x loading dye ష ϬǴࡑጤᡏᏉڰࡕ࿼ܭးԖ 1 X TAE ጗ፂనϐႝݚኲϣǴஒኬࠔݙΕጤТύǴа 100V ႝᓸႝݚԿՅ஥ډጤᡏ 2/3 Տ࿼ࡕǴջёܭ UV ጃϣྣ࣬Ƕ

ϖǵġ ߓ܎դ޺ँᡂਲ਼Ϸᙯ෗ਲ਼ϐ୷Ӣ߄౜ϩ݋

1. RNA ϐܜڗϷᇙഢ

ڗ 50-100 mg ኬࠔ࿼Ε 2 mL ༾ໆᚆЈᆅύǴаనᄊේհࠅኬࠔǴӆܫΕႣ հၸϐ 5 mm ᒳ੧Ǵӕਔஒ֡፦ᐒϐ༟ጤࢎΨаనᄊේύհࠅǴа 1,200 rpm ᎜ᕏ 15 ࣾஒኬࠔᑃ࿗ԋણރǴௗ๱уΕ 1 mL TRIZoL (Invitrogen)٠ِೲషӝ֡ϬǴᓉ

࿼ܭӇ΢ 5 ϩដǶܭ 4ʚΠ 13,000 rpm ᚆЈ 10 ϩដǴ֎ڗ 900 μl ΢మనуΕཥޑ 1.5 mL ༾ໆᚆЈᆅǴௗ๱уΕ 240 μl Chloroform (Merck)᎜ᕏషϬǴᓉ࿼ܭӇ΢ 10 ϩដࡕܭ 4ʚΠ 13,000 rpm ᚆЈ 10 ϩដǶ֎ڗ 500 μl ΢మనԿཥޑ 1.5 mL ༾ໆ ᚆЈᆅǴуΕӕᡏᑈ IsopropanolǴ΢Πའਗ֡ϬǴᓉ࿼ܭӇ΢ 10 ϩដࡕܭ 4ʚΠ 13,000 rpm ᚆЈ 10 ϩដǶݮ๱ᆅᏛ֎ڗ΢మనҧకǴуΕ 75% DEPC ೀ౛ϐ EtOH ᝌੌᆅۭ RNA ؈ᐘނǴа 8000 rpm ᚆЈϖϩដѐ΢మన٠ख़ፄ΢ॊ؁ᡯٿԛǴ ѐନ΢మనࡕஒ RNA ؈ᐘނܭ࠻ྕΠ॥ଳ 5-10 ϩដǶуΕ 30 μl nuclear-free water ӣྋ RNAǴߥӸܭ-80ʚǶڗ 2 μl RNA ྋనаӀࡋी NanoVue ۓໆǶ

2. RNase Free DNase I ೀ౛

ࣁٛЗ RNA ኬҁڙډූ੮ genomic DNA ԡࢉǴа RNase Free DNase I ೀ౛

RNA ኬҁǴჴᡍ؁ᡯ٩ྣ Ambition TURBO DNase Free kit ٬ҔЋн຾ՉᏹբǶڗ 10 μg RNA уΕ 40 μl TURBO mix షӝ֡ϬǴܭ 37ʚ incubator ϣᓉ࿼ 30 ϩដǴ уΕ 5 ul ςӣྕϐ Inactivation Reagent ࠻ྕΠȐ25ʚȑϸᔈ 5 ϩដǴ΋ѿϩቫջ

20

ҥڅషӝǶа 13,500 rpm ᚆЈ 5 ϩដǴλЈڗ΢మనډཥޑ༾ໆᚆЈᆅϣǶаӀ ࡋी NanoVue ۓໆǴܭӇ-80ʚߥӸǶ

3. cDNA ӝӝԋϐϸᙯᒵϸᔈ

ჴᡍ؁ᡯ٩ྣ RNA ϸᙯᒵሇન Invitrogen^TMSuperScrupt^TMIII First-Stranf Synthesis System for RT-PCR kit ٬ҔᏹբЋнǴஒ RNA ϸᙯᒵԋಃ΋ިϕံ DNA (cDNA)а຾Չࡕុ୷Ӣ߄౜ϩ݋Ƕڗ 1μg RNA ኬҁǴуΕ 1 μl Oligo dTǵ2.5 μl 10 mM dNTPǴ٠а DEPC ೀ౛ϐΒԛНஒᡏᑈံԿ 28 ulǴа 65ʚೀ౛ 5 ϩដǴᙯ౽ ԿӇ΢ 1 ϩដǴௗ๱уΕ 22 μl cDNA synthesis mix (5 μl 10X RT bufferǵ10 μl 25 mM MgCl2ǵ5 μl 0.1M DTTǵ1 μl RNaseOUT^TMRecombinant RNase Inhibitorǵ1 μl SuperScript^®III RT ) ܫΕ PCR ᐒǴำԄ೛ۓ 50ʚ 50 ϩដǵ85ʚ 5 ϩដǴௗ๱࿼

ܭӇ΢уΕ1 μl RNase H ܭ 37ʚ ೀ౛ 20 ϩដѐନഭᎩ RNA ࡕǴߥӸܭ -20ʚӇ ጃǶ

4. ϸᙯᒵᆫӝ䁙ೱᙹϸᔈ (RT-PCR)

ڗ1 μl cDNAǵ2.5 μl 10X PCR bufferǵ2 μl 10mM dNTP mixǵ0.5 μl ੝ۓ୷Ӣ ϐ Forward (Fw)Ϸ Reverse (Rv)Їηჹǵ0.5 μl DNA Taq polymeraseǴаѐᚆηНံ

ىᡏᑈԿ25 μlǶPCR ำԄǺ94ʚ ೀ౛ϖϩដǴ94ʚ denature 30 ࣾ/55ʚ annealing 30 ࣾ/72ʚ extension 30 ࣾǴख़ፄ 25 ൻᕉǴӆа 72ʚೀ౛ 7 ϩដǴϸᔈ่״ࡕߥ࡭

ܭ 4ʚǴߥӸܭ-20ʚǶ

Ϥǵġ ߓ܎դ޺ᙯ෗ਲ਼ϐ፦ಈᄬᑐϷ଍ნऐڙ܄ෳ၂ 1. 35S::OsADF2 Ϸ 35S::OsADF11 ፦ಈᄬᑐࢬำ

ҁፕЎၩᡏᄬᑐၩᡏٰྍԾܭᆵ᡼εᏢၭ཰ϯᏢسࢫ໺ඦԴৣჴᡍ࠻ගٮϐ pCYH8 Ϸ pCAMBIA1320ǶаНዿ TNG67 ޑ cDNA library ࣁኳ݈Ǵа PCR ຾Չ

21

ᘉቚڗள OsADF2 Ϸ OsADF11ϐ Open Reading FrameǴ٬Ҕ PCR ຾ՉᘉቚڗளǴ

ঁձ܌٬ҔޑЇηჹӵߕᒵϖǶࡌҥၩᡏᄬᑐ؁ᡯӵΠǺ(1) ஒ΢ॊ܌ளϐ PCR ТᆄࣣӃௗΕ pGEM-T easy vector (Promega, USA) ᙯΕεဉఎ๵ύǴ࿶ᒧ෗ᆶۓ

ׇዴۓคᇤǶ (2) ٬Ҕज़ڋ䁙䁙Ϫ຾Չ DNA ТࢤᗹӝϸᔈǶ35S::OsADF2 Ϸ፦ಈ ᄬᑐϐࡌҥǴࢂҗКჹ TIGR ׇဦ LOC_Os03g56790 ϐ coding sequence ࣁ 438 bpǴ ܭځ5’ᆄϷ 3’ᆄ೛ी KpnI ϪՏЇη OsADF2_KpnI_FwǺ

5’-GGTACCATGGCGTTCATGCGTTCCCA-3’ǴOsADF2_KpnI_RvǺ

5’-ACTAGTTTAATGAGCCCGCTCCCGGA-3’Ǵа PCR ᘉቚځТࢤǴճҔ PCR-clean up kit ӣԏځ DNA ТࢤǴ4ʚΠ຾Չᗹௗϸᔈܭ pGEM-T easy vectorǴ٠ճҔᙔқ ᑔᑔᒧܜڗ፦ಈࡕۓׇǴஒ pTRA151 а KpnI Ϫѐ 1.1 kb ϐ HPT ୷ӢǴ٠а 16ʚ ᗹௗϸᔈௗ΢֖Ԗ KpnI ϪՏϐ OsADF2 ୷ӢǴҗ OsADF2_KpnI_Fw ЇηϷ tml-Rv Їηቚ൯рТࢤዴۓௗ΢БՏǶ35S::OsADF11 Ϸ፦ಈᄬᑐϐࡌҥǺКჹ TIGR ׇ ဦ LOC_Os12g43340 ϐ coding sequence ࣁ 438 bpǴܭځ 5’ᆄϷ 3’ᆄ೛ी BamHI ϪՏЇη OsADF11_BamHI_FwǺ5’-CGGGATCCATGGCATTCGTCAGATCACG-3’Ǵ OsADF11_BamHI_RvǺ5’-CGGGATCCTCAATGAGCACGCTCTCTAA-3’Ǵа PCR ᘉቚځТࢤǴճҔ PCR-clean up kit ӣԏځ DNA ТࢤǴ4ʚΠ຾Չᗹௗϸᔈܭ pGEM-T easy vectorǴ٠ճҔᙔқᑔᑔᒧܜڗ፦ಈࡕۓׇǴஒ pTRA151 а BamHI Ϫѐ 1.1 kb ϐ HPT ୷ӢǴ٠а 16ʚᗹௗϸᔈௗ΢֖Ԗ BamHI ϪՏϐ OsADF11 ୷ ӢǴҗ OsADF11_BamHI_Fw ЇηϷ tml-Rv Їηቚ൯рТࢤዴۓௗ΢БՏǶ

2. ౦౦Տ߄౜(heterologous expression)Нዿ୷Ӣϐߓ܎դ޺ᙯ෗

ஒ 35S::OsADF2 Ϸ 35S::OsADF11 ፦ಈϩձаၭఎ๵޸ׇݠᗹݤ(Floral dipping) (Clough et al., 1998) ᙯΕഁғࠠߓ܎դ޺ύ (Col-0) Ǵԏڗ T1 жᙯ෗ਲ਼Ǵа Hygromycin (25 mg/L) לғનᑔᒧԿ T₂жीᆉϩᚆКࣁ 3:1 ࠔسዴᇡࣁൂ΋ copy ϐᙯ෗ਲ਼ǴஒӸࢲ෌ਲ਼ϐࡕж෌ਲ਼ᝩុਭᅿԿԋዕǴԏڗᅿη٠ᑔᒧԿ T3жբࡕ

22

ុჴᡍ٬ҔǶຟӸచҹࣁߥӸܭ 25ʚࡡྕࡡᔸᕉნǴЪ໪؂ъԃख़ཥᅿ෌٠ԏڗ ᅿηǴаᆢ࡭ᅿηϐཥᗲࡋϷࢲΚǶ

3. εεဉఎ๵ᙯࠠբҔ

3. 1 εဉఎ๵യҺಒझᇙഢ

ҁጇፕЎ٬ҔϐയҺಒझ๵ਲ਼سࣁǺE. coli XL1-BlueǴа੃ࢥၸϐ࣒෇ஒയ Һಒझ֡Ϭ༡ܓܭ LB ڰᄊ୻Ꭶ୷Ȑ֖ tetracycline 10 μl/mLȑǴӧ 37ʚΠ୻Ꭶ 12-16 λਔǴࡷڗൂ΋๵ပа 5 mL LB నᄊ୻Ꭶ୷Ȑ֖ tetracycline 10 μl/mLȑܭ 37ʚའ ਗ୻Ꭶ 12-16 λਔǴуܭ 500 mL LB నᄊ୻Ꭶ୷Ȑ֖ tetracycline 10 μl/mLȑǴܭ 37ʚ

୻ᎦԿ OD₆₀₀ॶऊ 0.5-1.0Ƕ4ʚΠа 8,000 rpm ᚆЈ๵న 15 ϩដǴѐନ΢మనܭӇ

੎ύуΕ 200 mL ค๵Нᝌੌ๵ᡏǴख़ፄٿԛࡕуΕ 40 mL ςႣհޑ 10% glycerol ᝌੌ๵ᡏǴ4ʚΠа 8,000 rpm ᚆЈ๵న 5 ϩដࡕѐନ΢మనǴуΕᆶ๵ᡏ฻ໆϐ 10% glycerol ᝌੌ๵ᡏǴа 40 μl ϩးܭ 1.5 ༾ໆᚆЈᆅύǴҥջаనᄊේհࠅߥӸ ܭ-80ʚύǶ

3. 2 ፦ಈεဉఎ๵ᙯࠠ

ҁጇፕЎ٬Ҕϐεဉఎ๵ᙯࠠჴᡍБݤࣁ Heat Shock E. coli TransformationǴ ڗ2 μl ፦ಈ DNA уΕεဉఎ๵യҺಒझషϬǴ࿼ܭӇ΢ 15-20 ϩដǴ42ʚН੎

Heat shock ೀ౛ 1 ϩ 30 ࣾǴ࿼ܭӇ΢ 2 ϩដǴௗ๱ӧค๵ᏹբѠύуΕ 200 μl LB నᄊ୻Ꭶ୷షϬǴܭ 37ʚ୻Ꭶ 15-20 ϩដǴௗ๱ 4ʚΠа 12,000 rpm ᚆЈ๵న 1 ϩដѐନ೽ϩ΢మనǴஒഭᎩϐ΢మనᆶ๵ᡏషϬǴ༡ܭ֖Ԗלғનϐ LB ڰᄊ୻

Ꭶ୷΢Ǵܭ 37ʚ୻Ꭶ 12-16 λਔǶ

3. 3 ᔠෳᙯࠠࡕεဉఎ๵፦ಈ colony PCR

Ӄܭ༾ໆᚆЈᆅύଛ࿼ PCR ϸᔈྋనȐ2.5 μl 10X PCR bufferǵ2 μl 10M NTPǵ

23

0.5 μl Ӛ୷ӢჹᔈЇηჹ Forward ᆶ Reverseǵ0.5 μl DNA taq polymeraseȑǴௗ๱ܭ ᙯࠠࡕϐεဉఎ๵ϐ LB ڰᄊ୻Ꭶ୷΢ᒧڗൂ΋๵ပǴуΕ PCR ϸᔈྋనǴуΕ ѐᚆηНஒᡏᑈံԿ25 μlǶ຾Չ PCR ϸᔈǺ95ʚ ೀ౛ϖϩដǴ95ʚ denature 30

ࣾ/56ʚ annealing 30 ࣾ/72ʚ extension 30 ࣾǴख़ፄ 25 ൻᕉǴӆа 72ʚೀ౛ 5 ϩដǴ ϸᔈ่״ࡕߥ࡭ܭ 4ʚǴ຾Չࢩ๼ጤᡏႝݚϩ݋Ǵ٠ߥӸܭ-20ʚǶ

3. 4 εεဉఎ๵፦ಈ DNA λໆપϯ

ҁጇࣴز٩ྣ GeneMark® Mini plasmid kit ᏹբЋн؁ᡯǺࡷڗൂ΋๵ပа 5 mL ֖Ԗלғનϐ LB నᄊ୻Ꭶ୷ܭ 37ʚ୻Ꭶ 12-16 λਔǶ֎ڗ 2 mL ๵నа 14,000 rpm ᚆЈ 1 ϩដǴక΢మన٠ख़ፄԜ؁ᡯ΋ԛǹௗ๱уΕ 200 μl Solution I ᆶ๵న

֡ϬషϬǴуΕ200 μl Solution II ҥջᙌᙯ๵న٬ϐᙯᡂࣁዂమǴௗ๱ӆуΕ 200 μl Solution III ҥջᙌᙯ๵నǴа 14,000 rpm ᚆЈ 5 ϩដǶஒ spin column ᆅࢊܫΕ 2 mL collection tube ύǴஒ΢మనᙯ౽ԿځύǴа 14,000 rpm ᚆЈ 1 ϩដǴख़ፄԜ؁ᡯ

΋ԛѐନᘠనǶуΕ700 μl Wash SolutionǴа 14,000 rpm ᚆЈ 1 ϩដǴѐନᘠనǴ ख़ፄԜ؁ᡯ΋ԛǶа 14,000 rpm ᚆЈ 5 ϩដѐନഭᎩଚᆒǶஒ spin column ᙯ౽Կ ཥޑ 1.5 mL ༾ໆᚆЈᆅǴуΕ 30 mL ค๵НǴ࠻ྕΠᓉ࿼ 5 ϩដࡑ DNA ӣྋǴ а 14,000 rpm ᚆЈ 1 ϩដǴஒᚆЈࡕޑ DNA ТࢤྋనߥӸܭ-20ʚύǶ

4. DNA Тࢤޑᇙഢᆶӣԏ

ճҔჹᔈϐज़ڋሇનܭၩᡏ΢Ϫڗ܌؃Ҟ኱Тࢤ܈а PCR ᘉቚр੝ۓТࢤǴ

຾Չႝݚዴᇡచ஥Ǵ٠ܭ UV ᐩΠϪڗ֖Ԗ܌ሡϐ DNA Тࢤϐᛏિጤᡏ࿼ܭ 1.5 mL ༾ໆᚆЈǶҁጇࣴز٩Ᏽ GeneMark® Gel Elution kit ᏹբЋн຾Չჴᡍ؁ᡯǺ ஒးԖҞ኱ DNA ጤᡏϐ 1.5 mL ༾ໆᚆЈᆅગख़ǴуΕ฻ᡏᑈϐ Binding SolutionǴ а 60ʚН੎у዗ 10 ϩដǴϸᔈၸำሡ΢ΠᙌᙯԿጤᡏֹӄྋှǹௗ๱ஒ spin column ᆅࢊܫΕ 2 mL collection tube ύǴஒϸᔈనᙯ౽ԿځύǴа 13,000 rpm ᚆЈ 1

24

ϩដǴѐନ collection tube ύᘠనǴуΕ 500 μl Binding Solution а 13,000 rpm ᚆ Ј 1 ϩដǴѐନ collection tube ύᘠనǹуΕ 700 μl Washing SolutionǴа 13,000 rpm ᚆЈ 1 ϩដǴѐନ collection tube ύᘠనǴख़ፄԜ؁ᡯ΋ԛǹӆа 13,000 rpm ᚆЈ 7 ϩដѐନූᎩଚᆒǹஒ spin column ᙯ౽Կཥޑ 1.5 mL ༾ໆᚆЈᆅύǴуΕ 20 μl ѐᚆηНǴᓉ࿼ 5 ϩដࡕа 13,000 rpm ᚆЈ 1 ϩដǴஒᚆЈࡕޑ DNA Тࢤྋన ߥӸܭ-20ʚύǶ

5. ߓߓ܎դ޺୷Ӣᙯ෗

5. 1 ෌ނ׷਑Ⴃഢ

ഁғࠠߓ܎դ޺ᅿη(Col-0)ܭ 4ʚೀ౛ٿϺ຾ՉࡾϯբҔǴኞᅿܭးԖԖ୷୻

ᎦβϐΟκࣧǴᅿܭ 22/18ʚ (В/ڹྕ) Ǵ16/8 (Ӏྣ/໵ས) ғߏጃύғߏǴ10 Ϻ ࡕ຾Չ౧भǴ؂ࣧ੮ 4-6 ਲ਼ߥ࡭෌ਲ਼໔໔ຯǴࡑ 3-4 ڬ໒ۈܜऱਔᄔЈǴ1 ڬࡕ຾

Չߓ܎դ޺ၭఎ๵ᙯ෗Ǵ຾Չߓ܎դ޺ၭఎ๵ᙯ෗߻βᝆѸ໪٬ϐ֎ᅈНϩаխ βᝆ֎ԏӭܭ๵నԶᏤठ෌ਲ਼གࢉԝΫǶ

5. 2 ၭఎ๵ᙯࠠ

5. 2. 1 ၭఎ๵യҺಒझᇙഢ

ҁጇࣴز٬Ҕϐၭఎ๵๵سǺGV3101Ǵܭ LB నᄊ୻Ꭶ୷ (֖ Gentamycin 50 μl/mL) ӧ 28ʚ୻ᎦٿϺǴڗൂ΋๵ပ୻Ꭶܭ 5mL LB నᄊ୻Ꭶ୷ (֖ Gentamycin 50 μl/mL) 28ʚ᎜ᕏ୻ᎦٿϺǴᙯ౽Կ 50 mL LB నᄊ୻Ꭶ୷ (֖ Gentamycin 50 μl/mL) ܭ 28ʚ୻ᎦԿ OD600ॶऊ 0.5-1.0Ƕ4ʚΠа 4,500 rpm ᚆЈ๵న 5 ϩដǴѐ ନ΢మనуΕ 10 mL ςႣհޑ 10% glycerol ᝌੌ๵ᡏǴ4ʚΠа 4,500 rpm ᚆЈ๵

న 5 ϩដࡕѐନ΢మనǴуΕᆶ๵ᡏ฻ໆϐ 10% glycerol ᝌੌ๵ᡏǴа 40 μl ϩး

ܭ 1.5 ༾ໆᚆЈᆅύǴҥջаనᄊේհࠅߥӸܭ-80ʚύǶ

25 5. 2. 2 ፦፦ಈၭఎ๵ᙯࠠ

ڗ1 μl ፦ಈ DNA уΕၭఎ๵യҺಒझషϬǴ࿼ܭӇ΢ 5 ϩដǴуΕႣհϐႝ

ऀϾႝᏤᆅ (cuvette, Bio-Rad^®) ǴܫΕႝऀϾሺᏔ (Bio-Rad^®micropluser) ϐႝኲ ύǴᒧ᏷ AGO1 ࡕࡪ໒ۈ຾ՉႝᏤǴႝᏤࡕஒႝᏤᆅ࿼ܭӇ੎ύǴௗ๱ӧค๵ᏹ բѠύуΕ 1 mL LB నᄊ୻Ꭶ୷షϬǴᙯ౽Կ 1.5 mL ༾ໆᚆЈᆅύǴܭ 28ʚ୻Ꭶ 30 ϩដǴௗ๱ 4ʚΠа 8,000 rpm ᚆЈ๵న 1 ϩដѐନ೽ϩ΢మనǴஒഭᎩϐ΢మ నᆶ๵ᡏషϬǴ༡ܭ֖Ԗלғનϐ LB ڰᄊ୻Ꭶ୷΢Ǵܭ 28ʚ୻ᎦٿϺǶ

5. 2. 3 ᔠෳᙯࠠࡕϐၭఎ๵፦ಈ colony PCR

Ӄܭ༾ໆᚆЈᆅύଛ࿼ PCR ϸᔈྋనȐ2.5 μl 10 X PCR bufferǵ2 μl 10M NTPǵ 0.5 μl Ӛ୷ӢჹᔈЇηჹ Forward ᆶ Reverseǵ0.5 μl DNA taq polymeraseȑǴௗ๱ܭ ᙯࠠࡕϐၭఎ๵ϐ LB ڰᄊ୻Ꭶ୷΢ᒧڗൂ΋๵ပǴуΕ PCR ϸᔈྋనǴуΕѐ ᚆηНஒᡏᑈံԿ25 μlǶ຾Չ PCR ϸᔈǺ95ʚ ೀ౛ϖϩដǴ95ʚ denature 30 ࣾ /56ʚ annealing 30 ࣾ/72ʚ extension 30 ࣾǴख़ፄ 25 ൻᕉǴӆа 72ʚೀ౛ 5 ϩដǴ ϸᔈ่״ࡕߥ࡭ܭ 4ʚǴ຾Չࢩ๼ጤᡏႝݚϩ݋Ǵ٠ߥӸܭ-20ʚǶ

5. 2. 4 ߓ܎դ޺ᙯ෗

ଛᇙၭఎ๵ᙯ෗੆ዎన (infiltration medium) (100 mL/construct)Ǵஒ֖Ԗᙯ෗

፦ಈϐ GV3101 ၭఎ๵ௗᅿԿ 5 mL LB నᄊ୻Ꭶ୷ (֖ Gentamycin 50 μl/mL) Ǵܭ 28ʚ୻Ꭶ 2 ϺǴஒ๵న౽Կ 100 mL LB నᄊ୻Ꭶ୷ (֖ Gentamycin 50 μl/mLȑ ܭ 28ʚ ୻ᎦԿ OD₆₀₀ॶऊ 0.5-1.0Ǵܭ 4ʚΠа 4,500 rpm ᚆЈ๵న 10 ϩដǴѐ΢మ నуΕ፾ໆᡏᑈϐ Infiltration mediumǴஒ๵ᡏᝌੌԿ OD600ၲ 0.5Ƕటᙯ෗ϐߓ܎

դ޺ഁғࠠӧ຾Չ޸ׇݠᗹݤ (Floral dipping) ߻Ǵᔈ٬ϐਭᅿϟ፦֎ᅈНϩǴ٠ Ъ౽ନς่ϐ݀ಲϷς໒ϐ޸ׇǴஒߓ܎դ޺޸ׇॹԌܭ֖Ԗᙯ෗ၭఎ๵ϐ੆ዎ నύ 5 ϩដǴௗ๱ஒ᏾ਲ਼а༟ጤ೓хᙟ٠౽Կғߏጃύ໪ݙཀ೯॥Ǵ12-24 λਔࡕ

౽ନ༟ጤ೓ࡑ෌ਲ਼ԋዕࡕԏڗᅿηǶ

26 6. ߓߓ܎դ޺ᙯ෗ਲ਼ᑔᒧ

6. 1 לғનᑔᒧᙯ෗ਲ਼

ஒᙯ෗ࡕ T₁жߓ܎դ޺ᅿη੃ࢥࡕኜኞܭ֖Ԗלғન Hygromycin B (25 mg/L) ޑ 1/2 MS medium ΢Ǵ࿼ܭ 22/18ʚ (В/ڹྕ) Ǵ16/8 λਔӀྣ (Ӏྣமࡋ 120-150 μmol/m²/sec) Ϸ໵སڬයϐғߏጃύǴऊ΋ڬࡕջёᑔᒧډڀԖܢללғનૈΚ ϐ෌ਲ਼Ǵջࣁᙯ෗ਲ਼ǹࡑᙯ෗ਲ਼ߏрٿТ੿ယࡕ౽෌Կਭ୻ϟ፦Ǵܭ 22/18ʚ (В /ڹྕ) Ǵ16/8 λਔӀྣ (Ӏྣமࡋ 120-150 μmol/m²/sec) Ϸ໵སڬයϐғߏጃύ

୻ᎦǴ෌ਲ਼ܜऱࡕёᄔନಃ΋ঁ޸ׇǴаճࡕុԏڗၨӭᅿηǶ

Ύǵġ OsADF ୷Ӣৎ௼ϐ GFP ᑼӝೈқϐԛಒझՏ࿼ϩ݋

ҁፕЎၩᡏᄬᑐٰྍԾܭᆵ᡼εᏢၭ཰ϯᏢسࢫ໺ඦԴৣჴᡍ࠻ගٮϐ pCYH8 Ϸ pCAMBIA1320ǴаϷҗറγࡕࣴز঩ഋᗶӓᏢߏගٮϐ

pHCC1300::GFP ࣁ GFP ᘉቚϐጢ݈Ƕ 1. Ub::OsADFs-GFP ፦ಈᄬᑐࢬำ

ҁጇࣴزаНዿ TNG67 ޑ cDNA library ࣁጢ݈Ǵа PCR ຾Չᘉቚڗள OsADF1, 2, 3, 4, 5, 6, 7, 10, 11 ϐ Open Reading FrameǴӕਔ GFP open reading frame ߾а pHCC1300::GFP ፦ಈࣁጢ݈Ǵ٬Ҕ PCR ຾ՉᘉቚڗளǴӚձ܌٬ҔޑЇηჹӵ߄ ΟǶ຾Չၩᡏᄬᑐϐ؁ᡯӵΠǺ(1) ஒ΢ॊ܌ள PCR ТࢤӃௗΕ pGEM-T easy vector (Promega, USA) ᙯΕεဉఎ๵ύǴ࿶ᒧ෗ᆶۓׇዴۓคᇤǶ (2) ٬Ҕज़ڋ䁙䁙Ϫ

຾Չ DNA ТࢤᗹӝϸᔈǶӃஒНዿ OsADFs Ϸ GFP ճҔ SmaIǵHindIII Ϸ AvrII ϪՏ຾Չ䁙ϪǴճҔύ໔ၩᡏ pHCC1300 ᗹӝ OsADFs Ϸ GFPǴӆԛۓׇዴᇡׇ

ӈ҅ዴǶpCYH8 ፦ಈ੝܄ёٮҞ኱୷ӢբᏯਔ܄߄౜ϐϩ݋ǴڀԖҏԯ Ubiquitin

௴୏ηǴϩձஒНዿ OsADF1, 2, 3, 4, 5, 6, 7, 10, 11 ᆶ GFP ୷ӢТࢤа SmaI ک SpeI ϪՏௗӝΕ pCYH8 ϐ SmaI Ϸ SpeI ज़ڋ䁙ϪՏǴௗ๱ӆа Heat Shock ޑБԄᏤΕ εဉఎ๵Ǵεໆ୻Ꭶࡕܜڗ፦ᡏ DNAǶ

27 2. ННዿ׷਑Ⴃഢ

ܜڗচғ፦ᡏޑኬࠔࢂٰԾΜϺεϐ TNG67 НዿϐယᓋǶஒ TNG67 Нዿᅿ ηѐෘǴܭค๵ᏹբѠύуΕ 70% ଚᆒ᎜ᕏ 7 ϩដǴѐନଚᆒࡕуΕ 1-2% NaOCl (֖ 0.05% Tween-20) ᎜ᕏ 10 ϩដǴѐନ NaOClǴаค๵Н᎜ᕏమࢱᅿηԿϿ 4-5 ԛǴޔډόӆр౜ݰݣǶаᙻηஒНዿᅿη᏾ሸ௨ӈܭ 8% Agar ϐ 1/2 MS (SIGMA)

୻Ꭶ୷Ǵӧܜڗচғ፦ᡏޑ߻ٿϺᗉӀೀ౛ǴаᗉխНዿယᆘᡏኧໆϼӭᏤठό

ܰᢀჸǶ

3. ܜڗНዿচғ፦ᡏ

ྗഢ Enzyme solution (1.5% Onazuka RS, 0.75% Macerozyme R10, 0.8 M

mannitol, 0.1 M MES pH=5.7, 1 M CaCl2, 0.1% BSA)ǴஒΜϺεޑНዿϐယᓋܭ䁙Ϫ ྋనύϪ࿗፦ 0.5-1 mmǴܜ੿ޜ 15 ډ 30 ϩដ٬ሇનనܺຠܭယᓋ࿗৕Ǵܭ໵ས ύ 28ʚа 40 rpm ᎜ᕏ 4 λਔǴനࡕ΋ϩដа 80 rpm ᎜ᕏǶа W5 solution (3 M NaCl, 1 M CaCl₂, 1 M KCl, 2 mM MES pH=5.7 ) ዎࢱ nylon meshǴஒ֖ԖНዿယᓋ࿗৕ϐ 䁙ϪྋనၸᘠԿ 50 mL falcon tubeǴܭ 4ʚΠа 250 xg ᚆЈ 7 ϩដǶѐନ΢మనǴ а W5 ᝌੌচғ፦ᡏǴӆܭ 4ʚΠа 250 xg ᚆЈ 7 ϩដǴѐନ΢మనуΕ 2-3 mL W5 ᝌੌচғ፦ᡏǴճҔՈౚीኧᏔܭᡉ༾᜔Πीᆉচғ፦ᡏᐚࡋǴܭ 4ʚΠа 250 rpm ᚆЈ 7 ϩដǴѐନ΢మనǴа MMg buffer (0.8 M mannitol, 15 mM CaCl₂, 4 mM MES pH=5.7 )ፓ᏾ᐚࡋԿ 1 x 10⁶protoplast/mLǶ

4. ᆫΌΒᎇ(PEG) ᙯ෗Нዿচғ፦ᡏ

ӧཥޑ༾ໆᚆЈᆅύуΕ 10 μl DNA (֖Ԗ 10-20 μg DNA)ǴуΕ 200 μl চғ

፦ᡏྋనషϬǴуΕ฻ᡏᑈ (210 μl) PEG solution (PEG 4000) (40% w/v) ᇸᄟᆅᏛ షϬǴܭ໵ས࠻ྕΠᓉ࿼ 30 ϩដǴуΕ 1 mL W5 షϬǴܭ 4ʚΠа 250 xg ᚆЈ 7

28

ϩដǴѐନ΢మనуΕ 1mL Incubation solution (0.8M mannitol, 4mM MES pH=5.7, 4mM KCl)షϬǴӧ୻Ꭶ৒Ꮤ΢֡Ϭ༡ܓ 0.1% BSAǴܭ 26ʚ໵སύ୻Ꭶ 16 λਔǶ

5. ӅӅ೫ขᡉ༾᜔ (confocal microscope) ᢀჸᑻӀೈқ߄౜

ӧ຾ՉᢀჸᑻӀೈқ߄౜߻ሡ֎ڗϿໆ (1-2μl) ςᙯ෗ϐচғ፦ᡏྋనܭၩ

࣒Т΢Ǵᇂ΢ᇂݢТࡕа೸ܴࡰҘݨ࠾Րᇂ࣒ТϷၩ࣒Тϐ໔ሜǴλЈϮᇤᓸډ চғ፦ᡏྋనаխচғ፦ᡏઇ຋Ƕҁԛ຾ՉӅ೫ขᡉ༾᜔ᢀჸᑻӀೈқ߄౜٬Ҕ ϐሺᏔࣁ٬Ҕύࣴଣၭ཰ғނࣽמࣴزύЈϐӅ೫ขᡉ༾᜔࠻ϐॹҥӅ೫ขᡉ༾

᜔ (Zeiss LSM 510 META NLO DuoScan) аϷѠεғނၗྍᄤၭᏢଣӅӕሺᏔύ ЈϐӅ೫ขᡉ༾᜔ (Leica TCS SP5 II) ǶӅ೫ขᡉ༾᜔ϐ໒ᐒ؁ᡯǺ٩ׇѺ໒ᕴႝ

ྍǵհࠅس಍ǵႜ৔ϐ໒ᜢǴௗ๱Ѻ໒ᑻӀႝྍϷႝတЬᐒᆶᑻჿǶӧᇂ࣒Т΢

༡ܓϿໆଳݨаߡݨ᜔ᢀჸǶᢀჸኬࠔϐ؁ᡯࣁܭёـӀΠӧຎഁϣפډኬࠔ٠ ፓ᏾ขຯǴճҔ configuration ϪඤӚᑻӀᘠТߔᆶऀ೸Ӏ೛ۓǴᒧڗჹᔈჴᡍϐ ᑻӀႜ৔ݢߏ (ҁჴᡍϐᑻӀೈқࣁ GFPǴၱፓ᏾ݢߏࣁ 500 nm) ǴዴᇡዴჴԖ ᑻӀೈқ߄౜ϐኬࠔՏ࿼ࡕǴ຾ΕቹႽᘏڗኳԄ (ύࣴଣǺLSMǴѠεӅሺǺLeica)Ǵ ܭႣំቹႽΠፓ᏾௟ඔୖኧॶ (zoom)ϷຎഁΠϐ௟ඔ୔ୱǴ຾Չ҅Ԅ௟კǴջё ᕇளటᢀჸϐᑻӀೈқ߄౜Տ࿼კǶӄ೽ኬࠔᢀჸ่״ࡕᒡрკТᔞǴሡճҔޜ қӀᅷТ຾ՉᐨᒵǶ

29

ಃ

ಃѤകġ ่݀

΋ǵġOsADF2 Ϸ OsADF11 ୷Ӣܭߚғނ଍ნΠϐ୷Ӣфૈϩ݋

1. ౦Տ߄౜Нዿ OsADF2 Ϸ OsADF11 ୷Ӣϐߓ܎դ޺ᙯ෗ਲ਼ϩη᠘ۓ ஒӚԾа 35S ௴୏η០୏Нዿ OsADF2ǵOsADF11 ୷Ӣϐ፦ಈȐߕკΟȑǴ ᙖҗၭఎ๵ϐ޸ׇݠᗹݤȐAgrobacterium-mediated floral dippingȑǴஒ߄౜ၩᡏӚ ԾᙯΕഁғࠠߓ܎դ޺ (Col-0) ύǴ٠ஒᅿη࡭ុа Hygromysin B לғન (25 mg/L) ᑔᒧԿ T3жӕ่ࠠӝᅿηࡕǴ຾Չᙯ෗ਲ਼ϐϩη᠘ۓǶࣁዴۓНዿ OsADF2 Ϸ OsADF11 ୷ӢӚԾዴჴᙯΕߓ܎դ޺ύǴа PCR מೌୀෳ T3ж 35S::OsADF2ǵ 35S::OsADF11 ߓ܎դ޺ᙯ෗ਲ਼ϐ HPT ל܄୷Ӣ (٬ҔϐЇηׇӈӵߕᒵϖ) Ǵҗ ࢩ๼ᛏિጤᡏႝݚ่݀ᡉҢᙯ෗ਲ਼ύࣣԖ HPT ל܄୷Ӣϐచ஥Ǵዴᇡځࣁᙯ෗ਲ਼ (data not shown) Ƕௗ๱ࣁΑዴۓНዿ OsADF2 Ϸ OsADF11 ዴჴӧӚߓ܎դ޺ᙯ෗

ਲ਼ύεໆ߄౜Ǵа RT-PCR ( Reverse Transcriptase Polymerase Chain Reaction) מೌ

຾Չ୷Ӣ߄౜ϩ݋Ǵ่݀ᡉҢ 35S::OsADF2 ᙯ෗ਲ਼ύዴჴԖНዿ OsADF2 εໆ߄

౜ǵ35S::OsADF11 ᙯ෗ਲ਼ύዴჴԖ OsADF11 εໆ߄౜Ǵഁғࠠߓ܎դ޺߾ؒԖ OsADF2 ܈ OsADF11 ϐ୷Ӣ߄౜Ȑკ΋ȑǶ

2. ౦Տ߄౜Нዿ OsADF2 Ϸ OsADF11 ୷Ӣϐߓ܎դ޺ᙯ෗ਲ਼Ѧ߄ࠠᢀჸ 2.1 Нዿ OsADF2 Ϸ OsADF11 ୷Ӣቹៜߓ܎դ޺෌ਲ਼ελ

ӧ҅தਭᅿޑ௃ݩΠ 35S::OsADF2 ᙯ෗ߓ܎դ޺ᙯ෗ਲ਼Ϸ 35S::OsADF11 ᙯ෗

ਲ਼ߓ܎դ޺ᆶഁғࠠ (Col-0) ߓ܎դ޺ӧғߏวػ׎ᄊ΢ؒԖᡉ๱܄ޑৡ౦ǴԶ а Image J ำԄෳໆӚ෌ਲ਼܌ԖယТय़ᑈᕴک຾Չ಍ीϩ݋КၨǴҗ่݀ᡉҢ 35S::OsADF2 ᙯ෗ਲ਼Ϸ 35S::OsADF11 ᙯ෗ਲ਼ޑယय़ᑈελکഁғࠠߓ܎դ޺࣬К

ၨεȐკ΋ȑǶ

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2.2 ННዿ OsADF2 Ϸ OsADF11 ୷ӢගԐ෌ਲ਼໒޸ਔ໔

ഁғࠠޑߓ܎դ޺ว޵ࡕऊ΋ঁД཮ϩϯр޸޵٠Ъܜऱ (bolting) Ǵௗ๱຾

Εғ෗ғߏයǴԶᢀჸ 35S::OsADF2 ᙯ෗ਲ਼Ϸ 35S::OsADF11 ᙯ෗ਲ਼ϐܜऱਔ໔Ψ

ၨഁғࠠ Col-0 ࣁԐǴځύа OsADF2 ϐᙯ෗ਲ਼ගԐ 3-4 ϺܜऱǴԶ OsADF11 ߾

ࢂԜᖿ༈ၨόܴᡉǴՠϝԖගԐ 2-3 ϺၨഁғࠠࣁԐܜऱޑ౜ຝ (კΒ (A) (B) )Ǵ Զӧ໒޸ਔ໔ᢀჸ΢ჸ 35S::OsADF2 ᙯ෗ਲ਼Ϸ 35S::OsADF11 ᙯ෗ਲ਼ᡉҢගԐ 2-3 ΟϺǴฅځϐ rosette leaf number (RLN)΢คܴᡉৡ౦ (data not shown)Ƕ

3. Нዿ OsADF2 ୷Ӣϐ୷Ӣфૈ܄ϩ݋

3.1 ߓ܎դ޺ϐ 35S::OsADF2 ᙯ෗ਲ਼ϐว޵౗ෳۓ

ࣁΑᕕှ౦Տ܄߄ၲНዿ OsADF2 ܭߓ܎դ޺ύࢂց཮ቹៜډځว޵౗Ǵၱ

ஒӕਔ௦ԏϐᙯ෗ਲ਼ᅿηϷഁғࠠ Col-0 ᅿηӕਔᅿ෌ܭӕ΋ዬ୻Ꭶ୷Ȑ1%

sucrose, 8 g/L Agar 1/2 MSȑύǴ٠Ъ಍ीځว޵౗Ϸว޵༈Ǵҗ่݀ύᡉҢ 35S::OsADF2 ϐߓ܎դ޺ᙯ෗ਲ਼ϐว޵༈ӧ΋૓ᕉნΠᆶഁғࠠ Col-0 ค౦ (კ Ο ) Ƕ

3.2 35S::OsADF2 ᙯ෗ਲ਼ܭߚғނ଍ნΠϐᢀჸ

3.2.1 ว޵౗ᢀჸ

ࣁϩ݋ 35S::OsADF2 ᙯ෗ਲ਼ࢂցቹៜډଯᡶ଍ნΠޑว޵౗Ϸว޵༈Ǵ٬Ҕ όӕᐚࡋϐ NaCl (100 mMǵ150 mMǵ200mM) ຾Չෳ၂Ǵځύа 150 mM NaCl ϐೀ౛ڀԖനܴᡉޑ߄౜ৡ౦ǴၱճҔԜᐚࡋ຾Չว޵౗ᢀჸǶ

җ่݀ύᡉҢ୻Ꭶܭ 150 mM NaCl 1/2 MS ୻Ꭶ୷ύϐ 35S::OsADF2 ᙯ෗ਲ਼ϐ ว޵ೲ౗ၨഁғࠠ Col-0 ࣁז (კΟ) ǴฅԶനಖว޵౗ᙯ෗ਲ਼ᆶഁғࣣࠠၨ΋૓

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