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

30

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΋૓

ᕉნΠ୻Ꭶफ़եǶ

31 3.2.2 ਥਥߏϩ݋

җ Wang ฻Γϐࣴزύࡰрߓ܎դ޺ϐਥߏڙډଯᡶ଍ნቹៜԶ׭ڋځғߏǴ ٠Ъऩ٬ҔԼ୏ೈқᆫӝᛙۓᏊ཮ࡠൺځਥ೽ғߏ༈ǴԶబуԼ୏ೈқᆫӝ׭ڋ Ꮚ߾׳уቃΑߓ܎դ޺ਥ೽ڙډϐଯᡶ଍ნॐ্ (Wang et al., 2008) ǶӢԜჹ 5 Ϻ εϷ 10 Ϻεϐߓ܎դ޺෌ਲ਼຾Չᡶҽ଍ნೀ౛ᢀჸځਥߏғߏϐᡂϯǶஒ୻Ꭶܭ

΋૓ᕉნ୻Ꭶ୷ϐഁғࠠᆶ 35S::OsADF2 ᙯ෗ਲ਼୻ᎦԿ 5 Ϻǵ10 Ϻࡕ౽෌Կ֖Ԗ 150 mM NaCl + 1/2 MS ୻Ꭶ୷ (௓ڋಔࣁ౽෌Կ 1/2 MS ୻Ꭶ୷) Ǵᢀჸ౽෌ࡕ 4 Ϻϐਥ೽ғߏᖿ༈аϷӸࢲ౗Ƕҗ่݀ᡉҢӧ҅தᕉნΠϐഁғࠠ Col-0 Ϸ 35S::OsADF2 ᙯ෗ਲ਼ਥߏคᡉ๱ৡ౦ (კΟ) Ǵௗ๱а҅தғߏᕉნΠϐਥߏբࣁ

኱ྗϯ୷ྗǴᢀჸܭ 150 mM NaCl ᡶ଍ნೀ౛Πϐਥ೽ғߏߏࡋৡ౦Ǵ่݀ᡉҢ 35S::OsADF2 ᙯ෗ਲ਼ϐਥߏᡉ๱ߏܭഁғࠠ Col-0 (კΟ) ǴЪ౽෌ 4 ϺࡕϐӸࢲ౗

Ψᡉ๱ၨଯ (კΟ) Ƕ

3.2.3 ԋਲ਼ᢀჸ

ࣁΑှНዿ OsADF2 ӧόӕ଍ნΠӧ෌ނᡏϣ܌ୖᆶޑቹៜаϷғ౛ϸᔈǴ Ԝаόӕϐߚғނ଍ნೀ౛ 21 Ϻεϐ 35S::OsADF2 ᙯ෗ਲ਼ߓ܎դ޺Ǵᢀჸځӧ଍

ნΠޑϸᔈаϷӣൺࡕޑғ౛߄౜Ƕ

3.2.3.1. ଳײ଍ნΠ 35S::OsADF2 ᙯ෗ਲ਼ϐϸᔈᢀჸ

ჹ 21 Ϻεϐഁғࠠߓ܎դ޺ (Col-0) аϷ 35S::OsADF2 ᙯ෗ਲ਼ߓ܎դ޺຾Չ ᘐНೀ౛ǴᘐНೀ౛ 7 Ϻࡕഁғࠠς໒ۈр౜๺঒аϷ޸ߙનಕᑈϐ౜ຝǴฅԶ ӧ 35S::OsADF2 ᙯ෗ਲ਼ߓ܎դ޺߾ࢂϝᆢ࡭҅தғߏ٠Ъค޸ߙનಕᑈǶௗ๱а ଳײೀ౛ 5 Ϻ (ಕᑈଶЗ๏Н 12 Ϻ) ࡕբࣁჴᡍ௓ڋಔϐ N3 ँᡂਲ਼ς࿶ֹӄԝΫǴ

ഁғࠠ߾ࢂယТ௔Ԕำࡋуቃ๺঒௃׎׳уᝄख़ǴԶ 35S::OsADF2 ᙯ෗ਲ਼Ԝਔω ໒ۈр౜๺঒ϐ౜ຝǴฅԶځယТϐ๺঒ำࡋϝλܭഁғࠠǶௗ๱຾ՉൺНჴᡍǴ

32

ൺНࡕ΋Ϻ 35S::OsADF2 ᙯ෗ਲ਼ယТϐ௔ԔϷ޸ߙનಕᑈջԖั༾ӣൺϐ౜ຝǴ

ௗ๱࡭ុࡠൺ 5 Ϻࡕഁғࠠ Col-0 ߓ܎դ޺Ԗ٤༾ࡠൺϐ౜ຝȐယТ๺ᕭำࡋ෧ϿǴ ՠϝԖያᕭϐ௃׎ȑǴբࣁ҅ӛፓ௓ಔϐ OE2 ߾ࢂယТ๺঒ำࡋࡠൺ൯ࡋεǴନ࢏

໳ϐယТѦځдယТӣൺߙᆘՅϷ෧Ͽያᕭ௃׎ǴԶ 35S::OsADF2 ᙯ෗ਲ਼߾ࢂ൳ ЯֹӄӣൺǴယТև౜ߙᆘՅЪคያᕭϐ௃׎ (კϖ) Ƕ

3.2.3.2. ଯଯᡶ଍ნΠ 35S::OsADF2 ᙯ෗ਲ਼ϐϸᔈᢀჸ

а 200 mM NaCl ೀ౛ 21 Ϻεϐഁғࠠߓ܎դ޺ (Col-0) аϷ 35S::OsADF2 ᙯ෗ਲ਼ߓ܎դ޺Ǵ٠ೱុᢀჸ 12-15 ϺǴҗ่݀ᡉҢа 200 mM NaCl ೀ౛ 14 Ϻࡕ

ഁғࠠ Col-0 р౜ယТယጔ໳ϯϐယᆘનफ़ှ౜ຝǴբࣁ҅ӛፓ௓ಔϐ OE2 ߾ࢂ

ϝᆢ࡭ߙᆘՅǴॄӛፓ௓ಔϐ N3 ߾ࢂև౜׳ᝄख़ϐယᆘનफ़ှЪယТр౜ያᕭ౜

ຝǴԶ 35S::OsADF2 ᙯ෗ਲ਼߾ࢂԖ٤೚ယТр౜ယᆘᡏफ़ှϐ౜ຝǴЪӧಳϷယ ύԖ޸ߙનಕᑈǴԶယТ߄य़Ԗ٤༾ᛪ፦ރᄊǴҗᙯ෗ਲ਼ϐယᆘᡏफ़ှ౜ຝၨഁ

ғࠠࣁᇸ༾ǴЪӧࡕុᝩុаᡶҽೀ౛ᢀჸߏਔ໔ϐᡶҽ଍ნჹ෌ނϐቹៜǴᡉ ҢഁғࠠϷ௓ڋಔϐယТև౜೬ც౜ຝǴԶᙯ෗ਲ਼ϐယТ߾ڀԖၨখฯϐ᝻གǴ

௢ෳ OsADF2 ᔈӧ෌ނय़ᖏᡶҽ଍ნਔԖୖᆶځғ౛ϸᔈԶቚуځᡶҽ଍ნऐڙ

܄ (კϤ)Ƕ

3.2.3.3. եྕ଍ნΠ 35S::OsADF2 ᙯ෗ਲ਼ϐϸᔈᢀჸ

ஒ 21 Ϻεϐഁғࠠߓ܎դ޺ (Col-0) аϷ 35S::OsADF2 ᙯ෗ਲ਼ߓ܎դ޺ᙯ౽ Կ 4ʚեྕғߏጃǶեྕೀ౛ 7 ϺࡕᢀჸځѦ߄ࠠǴ่݀ύว౜คፕࢂഁғࠠ

(Col-0) ܈ࢂ 35S::OsADF2 ᙯ෗ਲ਼ߓ܎դ޺ࣣԖр౜ڙډեྕԶ׭ڋғߏǴ٠Ъว ғۯ጗ځܜऱǵ໒޸ϐਔ໔ϐ౜ຝǴӕਔΨԖӕኬр౜ၨวၲޑ૙Л (trichome) Ǵ

࡭ុեྕೀ౛ 14 ϺࡕǴ၂ᡍಔϐߓ܎դ޺໔คܴᡉৡ౦Ǵᆶჹྣಔ (22ʚ/18ʚ) ߓ

܎դ޺࣬КࣣԖۯ጗วػ౜ຝǴ5 ຼࡕϝۘ҂຾Εғ෗ғߏය (კΎ) Ƕ

33

3.2.4 35S::OsADF2 ၸၸࡋ߄౜ᙯ෗ਲ਼ܭ଍ნΠϐᇻआѦጕ዗གᔈቹႽϩ݋

྽෌ނय़ᖏଯᡶ଍ნਔǴ཮ᙖҗፓ௓਻Ͼϐ໒ᚂа௓ڋᡏϣϐНϩ֖ໆаᆢ

࡭෌ނᡏϐᅖ೸ᓸǴаϷࡕុϐғ౛ϸᔈǴ྽෌ނ਻Ͼᜢഈਔယྕ཮ϲଯǴԶ਻

ϾѺ໒ਔ߾ࢂယྕ཮Πफ़ǴӢԜճҔߚઇᚯ܄ᇻआѦጕ዗གᔈቹႽϩ݋ٰᢀჸယ

ྕаΑှ 35S::OsADF2 ᙯ෗ਲ਼ӧଯᡶ଍ნΠϐϸᔈǴځύᚑՅୃӛᙔՅж߄ྕࡋ

ၨեǴԶୃӛआՅ߾ࢂྕࡋၨଯǶࣁΑ௖૸ᡶҽ଍ნჹܭ෌ނᇃණբҔаϷ਻Ͼ ໒ӝޑቹៜǴၱа 200 mM NaCl ೀ౛ 3 ຼεϐߓ܎դ޺ഁғࠠ (Col-0) ǵ

35S::LfNCED3 (OE2) ǵatnced3 (N3) ǵ35S::OsADF2 ᙯ෗ਲ਼ 24 λਔǴ௓ڋಔ߾ࢂ

዆а΋૓ԾٰНǴ႖Ϻӧӕ໔ғߏ࠻ϣ຾ՉܡឪǴаᇻआѦጕ዗གᔈሺᢀჸယྕǶ җ่݀ᡉҢӧ΋૓ᕉნΠ 35S::OsADF2 ᙯ෗ਲ਼ᆶഁғࠠϐယྕคܴᡉৡຯ (კΜ

΋ (A) ) ǴԶӧа 200 mM NaCl ೀ౛ 24 λਔϐ 35S::OsADF2 ᙯ෗ਲ਼ယྕ߾ࢂၨ

௓ڋಔࣁଯǴՠᆶഁғࠠคܴᡉৡ౦ (კΜ΋ (B) ) Ƕ

4. OsADF11 ୷Ӣϐ୷Ӣфૈ܄ϩ݋

4.1 ߓ܎դ޺ϐ 35S::OsADF11 ၸࡋ߄౜ᙯ෗ਲ਼ϐว޵౗

ࣁΑᕕှ౦Տ܄߄ၲНዿ OsADF11 ܭߓ܎դ޺ύࢂց཮ቹៜډځว޵౗Ǵၱ

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

sucrose, 8 g/L Agar 1/2 MSȑύ٠Ъ಍ीځว޵౗Ϸว޵༈Ǵҗ่݀ύᡉҢ

35S::OsADF11 ϐߓ܎դ޺ᙯ෗ਲ਼ϐว޵༈ӧ΋૓ᕉნΠᆶഁғࠠ Col-0 ค౦ (კ Ѥ) Ƕ

4.2 35S::OsADF11 ᙯ෗ਲ਼ܭߚғނ଍ნΠϐᢀჸ

4.2.1 ว޵౗ϩ݋

ࣁϩ݋ 35S::OsADF11 ᙯ෗ਲ਼ࢂցቹៜډଯᡶ଍ნΠޑว޵౗Ϸว޵༈Ǵ٬Ҕ όӕᐚࡋϐ NaCl (100 mMǵ150 mMǵ200 mM) ຾Չෳ၂Ǵځύа 150 mM NaCl

34

ϐೀ౛ڀԖനܴᡉޑ߄౜ৡ౦ǴၱճҔԜᐚࡋ຾Չว޵౗Ϸว޵༈ϩ݋Ƕ

җ่݀ύᡉҢ୻Ꭶܭ 150 mM NaCl 1/2 MS ୻Ꭶ୷ύϐ 35S::OsADF11 ᙯ෗ਲ਼ ϐว޵ೲ౗ၨഁғࠠ Col-0 ࣁז (კѤ) ǴฅԶനಖว޵౗ᙯ෗ਲ਼ᆶഁғࣣࠠၨ΋

૓ᕉნΠ୻Ꭶफ़եǶ

4.2.2 ਥਥߏϩ݋

җ Wang ฻Γϐࣴزύࡰрߓ܎դ޺ϐਥߏڙډଯᡶ଍ნቹៜԶ׭ڋځғߏǴ ٠Ъऩ٬ҔԼ୏ೈқᆫӝᛙۓᏊ཮ࡠൺځਥ೽ғߏ༈ǴԶబуԼ୏ೈқᆫӝ׭ڋ Ꮚ߾׳уቃΑߓ܎դ޺ਥ೽ڙډϐଯᡶ଍ნॐ্ (Wang et al., 2008) ǶӢԜჹ 5 Ϻ εϷ 10 Ϻεϐߓ܎դ޺෌ਲ਼຾Չᡶҽ଍ნೀ౛ᢀჸځਥதғߏϐᡂϯǶஒ୻Ꭶܭ

΋૓ᕉნ୻Ꭶ୷ϐഁғࠠᆶ 35S::OsADF11 ᙯ෗ਲ਼୻ᎦԿ 5 Ϻǵ10 Ϻࡕ౽෌Կ֖Ԗ 150 mM NaCl + 1/2 MS ୻Ꭶ୷ (௓ڋಔࣁ౽෌Կ 1/2 MS ୻Ꭶ୷) Ǵᢀჸ౽෌ࡕ 4 Ϻϐਥ೽ғߏᖿ༈аϷӸࢲ౗Ƕҗ่݀ᡉҢӧ҅தᕉნΠϐഁғࠠ Col-0 Ϸ 35S::OsADF11 ᙯ෗ਲ਼ਥߏคᡉ๱ৡ౦ (კѤ) Ǵௗ๱а҅தғߏᕉნΠϐਥߏբ ࣁ኱ྗϯ୷ྗǴᢀჸܭ 150 mM NaCl ᡶ଍ნೀ౛Πϐਥ೽ғߏߏࡋৡ౦Ǵ่݀ᡉ Ң 35S::OsADF11 ᙯ෗ਲ਼ϐਥߏᡉ๱ߏܭഁғࠠ Col-0 (კѤ) ǴЪ౽෌ 4 ϺࡕϐӸ ࢲ౗Ψᡉ๱ၨଯ (კѤ) Ƕ

4.2.3 ԋਲ਼ᢀჸ

ࣁΑှНዿ OsADF11 ӧόӕ଍ნΠӧ෌ނᡏϣ܌ୖᆶޑቹៜаϷғ౛ϸᔈǴ Ԝаόӕϐߚғނ଍ნೀ౛ 21 Ϻεϐ 35S::OsADF11 ᙯ෗ਲ਼ߓ܎դ޺Ǵᢀჸځӧ

଍ნΠޑϸᔈаϷӣൺࡕޑғ౛߄౜Ƕ

35

4.2.3.1. ଳଳײ଍ნΠ 35S::OsADF11 ᙯ෗ਲ਼ϐϸᔈᢀჸ

ჹ 21 Ϻεϐഁғࠠߓ܎դ޺ (Col-0) аϷ 35S::OsADF11 ᙯ෗ਲ਼ߓ܎դ޺຾

ՉᘐНೀ౛ǴᘐНೀ౛ 7 Ϻࡕഁғࠠς໒ۈр౜๺঒аϷ޸ߙનಕᑈϐ౜ຝǴฅ Զӧ 35S::OsADF11 ᙯ෗ਲ਼ߓ܎դ޺߾ࢂϝᆢ࡭҅தғߏ٠Ъค޸ߙનಕᑈǴᝩុ

аଳײೀ౛ 5 Ϻࡕբࣁჴᡍ௓ڋಔϐ N3 ँᡂਲ਼ς࿶ֹӄԝΫǴഁғࠠ߾ࢂယТ௔

Ԕำࡋуቃ๺঒௃׎׳уᝄख़ǴԶ 35S::OsADF11 ᙯ෗ਲ਼ϝᆢ࡭҅தғߏǴ໻ယТ р౜٤༾௔ԔϷϿໆ޸ߙનಕᑈǴௗ๱຾ՉൺНჴᡍࡕ΋Ϻ 35S::OsADF11 ᙯ෗ਲ਼ ယТϐ௔ԔϷယТ޸ߙનಕᑈջԖӣൺϐ౜ຝǴௗ๱࡭ុൺ 5 Ϻࡕഁғࠠ Col-0 ߓ

܎դ޺Ԗ٤༾ࡠൺϐ౜ຝȐယТ๺ᕭำࡋ෧ϿǴՠϝԖያᕭϐ௃׎ȑǴբࣁ҅ӛፓ

௓ಔϐ OE2 ߾ࢂယТ๺঒ำࡋࡠൺ൯ࡋεǴନ࢏໳ϐယТѦځдယТӣൺߙᆘՅ Ϸ෧Ͽያᕭ௃׎ǴԶ 35S::OsADF11 ᙯ෗ਲ਼߾ࢂ൳ЯֹӄӣൺǴယТև౜ߙᆘՅЪ

ֹӄ๤৖คያᕭ౜ຝǶҗᙯ෗ਲ਼ϐယᆘᡏफ़ှ౜ຝၨഁғࠠࣁᇸ༾ǴЪӧࡕុᝩ

ុаᡶҽೀ౛ᢀჸߏਔ໔ϐᡶҽ଍ნჹ෌ނϐቹៜǴᡉҢഁғࠠϷ௓ڋಔϐယТ և౜೬ც౜ຝǴԶᙯ෗ਲ਼ϐယТ߾ڀԖၨখฯϐ᝻གǴ௢ෳ OsADF11 ᔈӧ෌ނय़ ᖏᡶҽ଍ნਔԖୖᆶځғ౛ϸᔈԶቚуځᡶҽ଍ნऐڙ܄ (კΖ) Ƕ

4.2.3.2. ଯᡶ଍ნΠ 35S::OsADF11 ᙯ෗ਲ਼ϐϸᔈᢀჸ

а 200 mM NaCl ೀ౛ 21 Ϻεϐഁғࠠߓ܎դ޺ (Col-0) аϷ 35S::OsADF11 ᙯ෗ਲ਼ߓ܎դ޺Ǵ٠ೱុᢀჸ 12-15 ϺǴҗ่݀ᡉҢа 200 mM NaCl ೀ౛ 14 Ϻࡕ

ഁғࠠ Col-0 р౜ယТယጔ໳ϯϐယᆘનफ़ှ౜ຝǴբࣁ҅ӛፓ௓ಔϐ OE2 ߾ࢂ

ϝᆢ࡭ߙᆘՅǴॄӛፓ௓ಔϐ N3 ߾ࢂև౜׳ᝄख़ϐယᆘનफ़ှЪယТр౜ያᕭ౜

ຝǴԶ 35S::OsADF11 ᙯ෗ਲ਼߾ࢂܭԴယϷηယԖ٤೚ယТวғယᆘᡏफ़ှϐယТ

໳ϯǴЪӧಳϷယύԖ޸ߙનಕᑈ౜ຝǴԶယТ߄य़Ԗ٤༾ᛪ፦ރᄊ (კΐ) Ƕ

36

4.2.3.3. եեྕ଍ნΠ 35S::OsADF11 ᙯ෗ਲ਼ϐϸᔈᢀჸ

ஒ 21 Ϻεϐഁғࠠߓ܎դ޺ (Col-0) аϷ 35S::OsADF11 ᙯ෗ਲ਼ߓ܎դ޺ᙯ

౽Կ 4ʚեྕғߏጃǶեྕೀ౛ 7 ϺࡕᢀჸځѦ߄ࠠǴ่݀ύว౜คፕࢂഁғࠠ

(Col-0) ܈ࢂ 35S::OsADF11 ᙯ෗ਲ਼ߓ܎դ޺ࣣԖр౜ڙډեྕԶ׭ڋғߏǴځύ 35S::OsADF11 ᙯ෗ਲ਼ߓ܎դ޺ϐڙډ׭ڋғߏၨܴᡉǴ٠Ъࣣวғۯ጗ځܜऱǵ ໒޸ਔ໔ϐ౜ຝǴӕਔΨԖӕኬр౜ၨวၲޑ૙Л (trichome)Ǵ࡭ុեྕೀ౛ 14 ϺࡕǴ၂ᡍಔϐߓ܎դ޺໔คܴᡉৡ౦Ǵ໻ 35S::OsADF11 ᙯ෗ਲ਼ܭೀ౛ 7 Ϻࡕη ယ೽ϩр౜ڙཞ௃׎ǴฅԶӧځдယТϝᆢ࡭҅த߄౜คځдܴᡉϸᔈǴ௢ᘐё

ૈηယϐಔᙃၨૄ১Զр౜ڙ໾௃׎Ƕ࡭ុೀ౛ 14 Ϻࡕηယև౜ယጔ໳ϯЪያᕭǴ ᆶჹྣಔ (22ʚ/18ʚ) ߓ܎դ޺࣬КࣣԖۯ጗วػ౜ຝǴ5 ຼࡕϝۘ҂຾Εғ෗ғ ߏය (კΜ) Ƕ

4.2.4 35S::OsADF11 ၸࡋ߄౜ᙯ෗ਲ਼ܭ଍ნΠϐᇻआѦጕ዗གᔈቹႽϩ݋

྽෌ނय़ᖏଯᡶ଍ნਔǴ཮ᙖҗፓ௓਻Ͼϐ໒ᚂа௓ڋᡏϣϐНϩ֖ໆаᆢ࡭

෌ނᡏϐᅖ೸ᓸǴаϷࡕុϐғ౛ϸᔈǴ྽෌ނ਻Ͼᜢഈਔယྕ཮ϲଯǴԶ਻Ͼ Ѻ໒ਔ߾ࢂယྕ཮Πफ़ǴӢԜճҔߚઇᚯ܄ᇻआѦጕ዗གᔈቹႽϩ݋ٰᢀჸယྕ

аΑှ 35S::OsADF11 ᙯ෗ਲ਼ӧଯᡶ଍ნΠϐϸᔈǶࣁΑ௖૸ᡶҽ଍ნჹܭ෌ނᇃ ණբҔаϷ਻Ͼ໒ӝޑቹៜǴၱа 200 mM NaCl ೀ౛ 3 ڬεϐߓ܎դ޺ഁғࠠ

(Col-0) ǵ35S::LfNCED3 (OE2) ǵatnced3 (N3) ǵ35S::OsADF11 ᙯ෗ਲ਼ 24 λਔǴ

௓ڋಔ߾ࢂ዆а΋૓ԾٰНǴ႖Ϻӧӕ໔ғߏ࠻ϣ຾ՉܡឪǴаᇻआѦጕ዗གᔈ ሺᢀჸယྕǴځύᚑՅୃӛᙔՅж߄ྕࡋၨեǴԶୃӛआՅ߾ࢂྕࡋၨଯǶҗ่

݀ᡉҢӧ΋૓ᕉნΠ 35S::OsADF11 ᙯ෗ਲ਼ᆶഁғࠠϐယྕคܴᡉৡຯ (კΜ΋

(C) ) ǴԶа 200 mM NaCl ೀ౛ 24 λਔࡕǴ35S::OsADF11 ᙯ෗ਲ਼ϐယྕ߾ࢂၨഁ

ғࠠࣁଯ (კΜ΋ (D) ) Ƕ

37 Β

Βǵġ НዿԼ୏ೈқѐᆫӝӢηৎ௼୷Ӣϐ GFP ᑼӝೈқܭНዿচғ፦ᡏϐԛಒ झۓՏ

ҁጇፕЎ߯ճҔ PEG ᙯ෗ (PEG transfection method) Нዿচғ፦ᡏس಍ࣴز Нዿ OsADF ৎ௼୷ӢϐԛಒझۓՏǴচ౛ࣁճҔଯᅖ೸ᓸΠНዿচғ፦ᡏಒझϐ झ໯բҔஒѦࣚϐటᙯ෗Εϐ፦ಈ֎ΕಒझϣǴԶԜ֖ԖҞ኱୷Ӣϐ፦ಈҗܭҁ يڀԖ௴୏ηԶளаܭಒझϐύ߄ၲ٠ౢғҞ኱ೈқ፦ᆶ኱૶ೈқ (ӵ GFPǵYFP

฻) ϐᑼӝೈқǴӆճҔ੝ۓݢߏϐႜ৔ᐟวځᑻӀ߄౜٠٬ҔӅ೫ขᡉ༾᜔ᢀჸ ځᑻӀ߄౜аΑှځ߄౜೽ՏǶҁჴᡍܭ CYH8 ၩᡏ΢ࡌҥ OsADF1ǵOsADF2ǵ OsADF3ǵOsADF4ǵOsADF5ǵOsADF6ǵOsADF7ǵOsADF10ǵOsADF11 ᆶ GFP ϐᑼӝೈқǴаҏԯ ubiquitin promoter բࣁ௴୏ηǴᢀჸԜ٤ᑼӝೈқϐۓՏ٠ ϩ݋୷Ӣ߄౜೽ՏǶ

җ่݀ᡉҢჹྣಔϐ CYH8-Ub::GFP ߄౜ܭНዿচғ፦ᡏϐಒझ፦ϷਡϣǴ Զ Ub::OsADF1-GFPǵUb::OsADF2-GFPǵUb::OsADF4-GFPǵUb::OsADF5-GFPǵ Ub::OsADF6-GFP ᑼӝೈқࣣ߄౜ܭНዿচғ፦ᡏޑယᆘᡏύǴԶ

Ub::OsADF2-GFPǵUb::OsADF3-GFPǵUb::OsADF11-GFP ᑼӝೈқ߾ࢂಒझ፦ᆶ ಒझਡϣȐკΜΒȑǶ

38 Ϸᆶ෌ނϐ଍ნऐڙ܄ϐᜢᖄǴᙖҗճҔࣴزၸࡋ߄౜Нዿ OsADF2 Ϸ OsADF11

୷Ӣ౦Տ߄ၲܭߓ܎դ޺ύǴᢀჸځߚғނ଍ნΠޑϸᔈǴа௢ፕځёૈୖᆶϐ ғ౛фૈǶ

२ӃӃჹܭ࿶җၭఎ๵޸ׇݠᗹݤ (Agrobacterial-mediated floral dipping) ౢ ғϐ T2ж 35S::OsADF2 Ϸ 35S ::OsADF11 ᙯ෗ਲ਼຾ՉᙁܰϐᑔᒧǴѐନӧ΋૓ᕉ ნΠਭ୻ᢀჸڀԖୋբҔ (side effect) ϐࠔسǴӵ෌ਲ਼׎ᄊǵғߏᖿ༈ܴᡉᆶځд ࠔسόӕޣǴௗ๱຾Չ଍ნϐ߃ᑔǴࡷᒧрܭ଍ნΠڀԖ࣬ӕϸᔈϐࠔسǶӧҁ ጇፕЎύ 35S::OsADF2 ᙯ෗ਲ਼നࡕ܌ᑔᒧϐࠔسࣁǺOsADF2-3ǵOsADF2-4ǵ OsADF2-6ǴԶ 35S::OsADF11 ᙯ෗ਲ਼നࡕ܌ᑔᒧϐࠔسࣁǺOsADF11-1ǵOsADF11-2ǵ OsADF11-3Ƕ

΋ǵ ௖૸Нዿ OsADF2 Ϸ OsADF11 ୷Ӣᆶ෌ނғߏวػϐᜢ߯

在文檔中 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 (頁 34-52)