GUS 活性組織化學染色法 - 香蕉果實後熟相關基因MhDnaJ之啟動子活性與基因功能分析

GUS 活性組織化學染色之方法，係修改自 Jefferson (1987) 所提出之方法。植物材料浸泡於含有Triton X-100 之緩衝液 [50 mM NaPO4 (pH 6.8), 1%

Triton X-100]，37℃下處理 2 小時，以緩衝液 (50 mM NaPO4, pH 6.8) 沖洗三次，加入含X-gluc 之緩衝液 [50 mM NaPO4 (pH 6.8), 1 mM X-gluc]，以 25 inch-Hg 真空抽氣 5 分鐘二次，回壓後置於 37℃下反應 24 小時，最後以

70%酒精終止酵素反應及褪去葉綠素，並於室溫下浸泡保存。

(十) 轉殖株之啟動子活性與誘導分析

1. 無菌播種

於無菌操作台，將阿拉伯芥或菸草種子放入微量離心管，加入 70%酒精震盪清洗1 分鐘，再以 1%漂白水 (含 0.05% Tween-20) 震盪清洗 15 分

鐘，以無菌水沖洗漂白水3 次，每次 5 分鐘，加入等體積之無菌水懸浮種

子，播種於含有抗生素 (50 μg/mL Kanamycin) 之 1/2 MS (含 1% sucrose) 固體培養基，培養於25℃、16 小時光照及 8 小時黑暗環境。

2. 不同發育階段之啟動子活性分析

取無菌播種後 5、10、15、20、25、30 天大與開花之菸草轉殖株，進行GUS 活性組織化學染色。

3. 不同非生物性逆境誘導處理

取無菌播種後21 天大菸草 T1 轉殖株，以下所列出之逆境進行處理，

除了高溫、低溫與低溫黑暗處理，其他處理皆於25℃、光照 16 小時、黑暗

8 小時之培養室進行，處理後以 GUS 活性組織化學染色，觀察啟動子表現情形。

(1) 高溫逆境處理

將菸草 T1 轉殖株含培養基，放入生長箱以 37、42、45℃處理 24 小時。

(2) 乾旱逆境處理

將菸草 T1 轉殖株置於乾的濾紙上，處理 3 小時。

(3) 低溫處理

將菸草 T1 轉殖株含培養基，放入冰箱以 4℃處理 24 小時。

(4) 黑暗處理

將菸草 T1 轉殖株含培養基，以鋁箔紙包覆避光處理 24 小時。

(5) 低溫與黑暗處理

將菸草 T1 轉殖株含培養基，以鋁箔紙包覆避光，放入冰箱以 4℃處理 24 小時。

(6) 鹽分逆境處理

將菸草 T1 轉殖株置於以 300 mM NaCl 浸潤之濾紙上，處理 3 小時。

(7) 淹水逆境處理

將菸草 T1 轉殖株自培養基取出，放入小試管以無菌水淹蓋處理 24 小時。

(8) 機械創傷處理

以解剖刀劃傷菸草 T1 轉殖株之葉片製造創傷，再置於以含 1% soucrose 之1/2 MS 液態培養基浸潤之濾紙上，處理 0.5 或 1 小時。

4. 不同生長調節劑誘導處理

生長調節劑誘導處理試驗，係修改自Tsuchisaka 與 Theologis (2004) 及 Wang (2005) 所提出之方法。取無菌播種後 21 天大菸草 T1 轉殖株，置於

以含有以下不同濃度生長調節劑之1/2 MS 液態培養基浸潤之濾紙上進行處

理24 小時，處理皆於 25℃、光照 16 小時、黑暗 8 小時之培養室進行，處

理後以GUS 活性組織化學染色，觀察啟動子表現情形。

(1) Auxin：20 μM indole-3-acetic acid (IAA) (2) Benzyadenine：20 μM BA

(3) Gibberellin：20 μM GA3

(4) Abscisic acid：100 μM ABA (5) Methyl jasmonate：50 μM MeJA (6) Salicylic acid：1 mM SA

(7) Ethylene：50 μM 1-aminocyclopropane-1-carboxylic acid (ACC)

肆、結果

一、香蕉MhDnaJ 基因結構分析

(一) 香蕉 MhDnaJ 基因序列分析

將 λMACS38 基因序列與 pMACS38 cDNA 序列比對，顯示香蕉 MhDnaJ 基因含5 個顯子及 4 個隱子 (圖五)，轉譯起始密碼子於第一個顯子，轉譯終止密碼子於第五個顯子，而顯子與隱子連接處之胺基酸序列，均符合 GT-AG rule (表一)。基因組選殖系 λMACS38 與 pMACS38 分別轉譯出 273

與217 個胺基酸，比較基因序列與相對應之 cDNA 序列，有一個核苷酸差

異，使第 190 個胺基酸由組胺酸變為精胺酸 (H134R)。比較香蕉 MhDnaJ 與其他物種如阿拉伯芥 (AtDnaJ, NP_565163) 、水稻 (OsDnaJ, NP_001060336) 、楊樹 (PtDnaJ, XP_002302268) 、蓖麻 (RcDnaJ, XP_002513851) 、葡萄 (VvDnaJ, XP_002285821) 和玉米 (ZmDnaJ, NP_001140575) DnaJ 胺基酸序列的相似性，介於 65.9% - 71.6% 之間，而

與葡萄相似性最高，為71.6% (表二)。胺基酸序列分析顯示，香蕉、阿拉

伯芥、水稻、楊樹、蓖麻、葡萄和玉米都具有J-protein 特有的 J-domain，

另外還有Nuclear localization signal (NLS) 與 Nuclear export signal (NES) (圖六)。親緣樹狀圖分析結果顯示，香蕉 MhDnaJ 在演化上和水稻與玉米的同源性較高 (圖七)。

(二) 香蕉 MhDnaJ 基因啟動子序列分析

分析已取得之香蕉MhDnaJ 基因啟動子序列，以 PlantCARE 資料庫比 對，預測 TATA box 位於於轉譯起始點上游 287-291 bp，序列為 TAATAA，

CAAT box 則位於轉譯起始點上游 518-522 bp，序列為 CAAAT。根據比對

ggatccagtaaggacaaacccgagcgaccgcccggagcccgaggtattatctccggattt -3261 HSE

aacggactccctacgagcccagctactctttctttggcagcggctcgatgaactagagaa -3201 ggagtttcgcaactcgaagggagagctcggggtggacacatatcgaggatctccgttcac -3141 accagagatacgagatcacgcggttcccccgaatttccagctcacttctatggacgcata -3081

MBS

cgacggctccattgacctagcggaccatgtcgccgctttccgtgcccaactggcactata -3021 AuxRR-core

tggaacgtctgatgctttaacgtgtaggcgtttcccacgacctgaggggtccagcctaca -2961 catggtacggcggcctgaagaccggaacgatcagttcttcggccagctcgccaaggactt -2901 cgagctccacttagtgcccatgcccggccaaagccctccgcggcactgctcctcggactc -2841 aaacaaagagaggacgagcccctctcacatttcgtggatcgctttgccacgcaaatctga -2781 tgcttaccggacactcacccctctctgttagtgcaggcatttatgataggcttgcgacct -2721 tccacattcctctggtccctcgcggagcgacctcccacggtagtgccaaaaatgctccag -2661

MBS HSE

cgggctaactgatacatcatagcggaggcctgggcgcggcgcgaggagaagggacgacct -2601 ccgaccgtgggctccatgagagaggccaagcatccgcggtggctacccgatgtactcatg -2541

HSE

aaaagaaatctaagccctgccttagtctcttctgagcgaaggttcagtatctgcctgacc -2481 cccttttcggctcgcggttggccccagcttaaacccttctgaatctaaatgccttggcca -2421 tggactgcccgggtccacctcagcgaggcctgcccggcctaagttgtgtccctcggcctg -2361

G-box

agccacgtccctcggccatggactgcccgggtccacctcagcgcggcctgcccgcatgag -2301 G-box

ttgtgtccctcggcccgagccacgtccctcggccatgcactgcccgagtccacctcagcg -2241 cggcctacccgcctgagccatgtccctcggtcgcactctgcccaagtccacctcagcgcg -2181 gactatccgcctgagccaagggtcccccgagtccacctctacttggcttgctgaccgagt -2121 tgcgctcctcgactacatgttgcccgggcccatgcctgcgcggctagccccgctgcgccg -2061 tgctcctcggctgcgtgttgcccaagactacacctacgcggcctgcccgcctgcgtcgtg -2001 ttccttagctccatggcctccgagacacacctgcgcggcctgcccacctgcgtcgtgctc -1941 ctcggccccatgacccgagacacacctttgcggccagcccgcctgtgtcgtgctcctcgg -1881 ctacatgttgcccgagaccacgcctgcgcggcctgcccgtctgcgccgtgttcctcggtc -1821 gcacgttgtccgaggccgcaacctctactcggcttcccgacttagttgggctcctcgacc -1761 ccatttcccgagccacacctgagcggccagcacgcctacatcgtgctcctcggctccatg -1701 ttgcccgagaccacacttgcgcggccagtccgcctgcgtcgtgctcctcggttacacatt -1641

CGTCA-motif

gcccgagaccacgcctgngcggcctgcctgcctgcgtcacactcctcgccttcggctcca -1581 tgttcccttcggctccatgttccccgagacctcgtccgtgcgccagcccgtctgaagaca -1521 HSE

gaagccatgcatcagactcctcttatacaacaaccgacgcatagctcctttcaggggggg -1461 GATA-motif CGTCA-motif

aatatgataagggtaaaaatggcgatgtgacacgccatgacgtcagccatgtctggacaa -1401 cacactgcccgaggaagtcggcattaacacctaactcaggcttggttcttcacgccacgc -1341 taaccccatgtcagacgctatcagcctgcctcctgcaagcagccacatcaggtaacatca -1281 aactcatctataaataccccagagttctaaacgaacaggggaggaagacaagcacaacgc -1221 actcagaggcatcttttcctccaaaaccctctccacattgctaacttgatcgtcggaggg -1161 gtcgggccgagctcgggaacggattgggtgcaggtgtgagaggtgtgcctcttcccggac -1101 gatgcggcgacgttcctcctgacccgacctcccgacccgaaccaccgtgctcggccgccg -1041 ggagaccccgagggacgtcgcccgagatccccgacatccggacccccgaaccgagcagca 0-981 tcggccccgaggcctcggcttaaaggtgtttacactaacaggatcgaaggcctgtaggag 0-921 ggtccggcgagtggcgtcccaatcgacggcatggacgaagaccacgctctgtatatagcg 0-861

CGTCA-motif

acgagcgcagcgtcaacagcgtcgagttataagtagatctcgaggacgcggtggatcata 0-801 tcaggggtggagatgactgaggaagagccgtcgtcgccggggccgctgctccgccgccga 0-741

CGTCA-motif

agagggctaggtcgtcatggtcttgaatggcgtcgttaccagggggccgctgccgttgct 0-681 gctgctgttgcttgggctcgggctcgaggccgagctctgtgggaagcggaactactcgga 0-621

HSE

ctttctatacgcagaagatattcgattcctgagttgactcggacgagtttggtatgggag 0-561 (Countinued)

CATA-box

gttggatttatctgaaccgtccgtttttcctcggtacacaaataccctattccagcatac 0-501 ABRE

ccattcggttactgtctcggtgattgaaacacgtgatggccccatcttaatcagtagaca 0-441 CGTCA-motif G-box

agtcggccgtcaagtaacgcacaaacgtcgactaaaagatgaagcacgtcccgcttctgc 0-381 agctacggccaactcggacaccaactcatgaagtgttcttcatatcgtgtctttcgtacg 0-321

CGTCA-motif TATA-box

cagtccccacgtcagatcgaaatggttcataataatactcgactctcacgaccttttgag 0-261 gcggaacgagcaacaaagcacagcaccatacagcatttacatcgatcgaggaagcgccac 0-201 gtacgacgtacagacaccaaatgattcggtcctctcgttcctcgggagacattcctcggg 0-141 ggagacatttccgtcacgaatctcccaacctccgagccccggaaaaaactcctctcccgg 00-81 ccatctatttcttcccccaaatcgaaaccgtaacccttaattccaatttccccttctcct 0OO-21 ttcctcctactgctctcaggATGAGCAGTCTGAGAGTGATTTGCCGCCCGCACGTGGTCG 00+40

MOOSOOSOOLOOROOVOOIOOCOOROOPOOHOOVOOV

TCTCCTCTATCTACTGCTGCAGAGGCTACGGTCGCCTAAGGTTCGCGCCCTCCGCCCGGA 0+100 VOOSOOSOOIOOYOOCOOCOOROOGOOYOOGOOROOLOOROOFOOAOOPOOSOOAOOR

ATCCTAATCTCCCAATCTCGCACCCGTCGTATGCATCGTCTCCTTTTGTCCTCGATGACT 00+160 NOOPOONOOLOOPOOIOOSOOHOOPOOSOOYOOAOOSOOSOOPOOFOOVOOLOODOOD

CCCCGAGGATGGGACCGGCGTCGCGCGCTAATTCGAGGAGGACGCTCACTAGGGTCTCCA 0+220 SOOPOOROOMOOGOOPOOAOOSOOROOAOONOOSOOROOROOTOOLOOTOOROOVOOS

ATTGGAACTCCGAGAAGTCGCCTTATGATACCCTTGgtaatttcctcaaagatcatatct 0+280 NOOWOONOOSOOEOOKOOSOOPOOYOODOOTOOL

ttggtcctgcccttggaactcacttagatatagtatccaagatctgatttttctatactt 0+340 caagatccatttatttcagatgaatgagaaaatcacgatagtaagtgtaatattttcctt 0+400 caatcttcaagtttaagggcgatcaggttctctatcatgctttgtttatgtttatatgca 0+460 ttttttgtgtgttcttgatcgttcacgatattaaacatgcagAACTGGAAAGGGATGCTG 0+520 OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOEOOLOOEOOROODOOA

ATGATGAGAAGATTAAAGCTGCTTATAGAAGATTGGCCAAGTTTTACCACCCAGATGgtt 0+580 DOODOOEOOKOOIOOKOOAOOAOOYOOROOROOLOOAOOKOOFOOYOOHOOPOOD

tctctttcaccttcaatatttatagttgaagtttttctactgattgtttttgatgcagtt 0+640 gttatgctgtcaaattgcttgtaaatttggctacttttcttatcacaaagaccaagtatt 0+700 tccttttattgaatattaaagtttttactgaaactggaatgaattgatttccccttatac 0+760 agatgagctactttttcgtttttcatatatgagcattttctcttaccttcttctgtctta 0+820 acttgatattttccagtaagttcatacttgctagttcatttttgtgtttgtaatatagcc 0+880 caactgatatatgacataatttactaataatttacagtaaaatgattgctcagtgattgc 0+940 catgtgagtgaactagaatctttatctgaaagttaagctacctattcaactaaataaata +1000 aatacttagatactgtttcttaacataagtattttgatttgctaattgtccttctaggtg +1060 caggcattttaaatgctctatcatgcatatagattttgtctgttctgattttgacctatc +1120 tgttctgattgctttatttgtccttgttatagtctagtgctatagatttcttttattaat +1180 ttttgtgatactttatttctcaagtaaaaagactaactttctgtgtgtctcattaaaagg +1240 tatcttaatcttgtgttctcattgtggcatgcttgcatatgatctttgttagatgtatta +1300 ttttgtttagtgaaataatgtataacttttggttttgaattttcttgacacagatctcag +1360 tatcatataattaaactctttattgtctctttcattgtgtattgaactctagaatcattg +1420 ttcgagtttctcgacttgtctcaatctattcttttttcagtatgaaacatgaggtattag +1480 ttttttctgttctttttctttctatttgcacttctaataggttatattataaacaatcat +1540 tagcaattgctctaagtacccagctgatacttttgttcttgtcttctaaagTTTATGATG +1600 OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOVOOYOOD

GTAGAGGAACTCTTGAGGAAGGAGAAACAGCTGAATCCCGATTTATCAAAATTCAAGCTG +1660 GOOROOGOOTOOLOOEOOEOOGOOEOOTOOAOOEOOSOOROOFOOIOOKOOIOOQOOA

CTTATGAGCTTCTAATAGATGATGAGCAGAGGCGACAATACGACAGAGATCACCATGTCA +1720 AOOYOOEOOLOOLOOIOODOODOOEOOQOOROOROOQOOYOODOOROODOOHOOHOOV

ATCCTATGAAGgtacatattataattttgtatattcgtttacattcttaatctaattatt +1780 NOOPOOMOOK

atgcccattttattgactactttttaattaaccattccatgtcttgtaatgagaggttat +1840 gaggttgtttntgctacctcaatcttgccaaaatttctttgtgattattgtttttgtgac +1900 caagatgtgtcaaactgactcttttttatctcattcctcttacaatagaaatgttataga +1960 gatactttaggaatttacaatacccttgtgtatttaaaaagacttgagaaaataaagttg +2020 tgtatttaaaaaggaaaataactaagaagttcatagtataagttttgaatcaaatctaaa +2080 taaatattaaataatataattttgtaattttttttgttataaaattgacgaaggatttat +2140 (Countinued)

ttttgacaataaattcataatgtattgtttacaattaattaataacttagtttttaaaat +2200 caatgaatgcaaaaaaattaataatttagtttctgaaaccaattgatattttcttaatac +2260 cttatcaattgtaggagaattcttaatcagtatcctaaagttgatggaacatcaactgat +2320 gctctaaagttaataaaatattactatttacgtgataggatagcctatagttagtgtttc +2380 agatggaagtatatattatagacattgtagatgttaatataaagtttgtgtcacagggtt +2440 gtaggttcttagttatatcctgttaaatgtcatggaaagtgacccatgaatttgtcaaag +2500 taaaccttaatagagagaaatatgtggtacctaagtttgtaacttgcggagcttttctat +2560 gcccttgagcaactgaacaggtccaaaagatatgctaaaaataacaatagtgaagcttcc +2620 aagttagtcagtttaaagcagttttatttgcttctgttttatgaaattcctacactgtat +2680 atgatctcaaaactatatttatcaggggctaaatataaaaatataaattaatttgaagat +2740 ccaacaatggagcgatagggaacacttatattaataccttctcaacacaattagaaagaa +2800 aaagaataacattgcacataccataataaaacagctcagttaggttatataaaagtaacc +2860 tgtgcatacagaattagcatttgtagttaaccttattaaagagtttatagataatatatt +2920 aaaaagttttactgatttaaacttgagaaagcaatccaaatatgcctgtctaccaaaagt +2980 gcctccctagagaaagaccgatgatttaatccatatagactttttgtgtaataaaagagg +3040 aaggccatatatgtcagcctcacagttggctctgctggccttcaactcttgttaacaaag +3100 tagtttactattatgatggtaaccgagagagagatgcagagatacgacacaaccttcaag +3160 gtgcataaaggaaagcgagggaggcgagagtgcctcttggccccatttcctctaccctga +3220 tatagatacaggttttaaggacatttctcattgccaacactttgattcaactcttttgtg +3280 ttattggaaaatttcaccataaatttaattgtatggacatctttataattaatgctatac +3340 caaaaatgtgaaatatttgtcatatacaggtgcattactatcagcctcttttattttaga +3400 agtaaatggttttctttgtataaattccaacattctttgtgtttgcacctatcagGCTTC +3460 OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOAOOS

CCAGGCATGGATGGAATGGGTGATGAAAAAGCAAAAAGCATTTGATCAGCGTGGTGATAT +3520 OOQOOAOOWOOMOOEOOWOOVOOMOOKOOKOOQOOKOOAOOFOODOOQOOROOGOODOOM

GGCAATTGCTGCTTGGGCTGAACAGCAGCAACATGAGATGAATCTCCGAGCACGTCGTCT +3580 OOAOOIOOAOOAOOWOOAOOEOOQOOQOOQOOHOOEOOMOONOOLOOROOAOOROOROOL

TTCTCGTTCAAAGgtttataagtacattatattatatacaagaatttgtcttgggactgc +3640 OOSOOROOSOOK

ttgaatgccagcaaatagctaactggcacctcattaattcattactgaatgtgcattctt +3700 gtggagttgattgacccaaagcttttttcataccattaaataaagacaattttttttgac +3760 ataaggactaatgagtagatccattttttaaaaaatgagtgtttaattgtttagtttgac +3820 ttactttcaatcctgtgtttctggtgctttactgatgaacgacactccattttattactt +3880 tgtcatagcttttacctacttcattgttagtcgcagtctgtcatattaactccaagtagt +3940 ataaatcatgatttttgaagtgtgaaaaccatgattactgagtcaattggtctcacgtcg +4000 tcgttcattttgttgaaacactatttcattatttttgaatttttgtttgaaatgcaattt +4060 agatgtttctaactagatactttctgttttcttcgtattttcattttttcttttttgtcg +4120 tcatctgagatgctcccttcatatggctgacactcctgtggtcagggttttactctttta +4180 ctgctctcctttcagtgcttattcggcagcattactttcttcctcttaggttcactagca +4240 cttggtaaatgtcaccatatttcagATTGATCCTGAAGAAGAGAGGAAGATTTTGGCAAA +4300 OOOOOOOOOOOOOOOOOOOOOOOOOOIOODOOPOOEOOEOOEOOROOKOOIOOLOOAOOK

GGAGAAGAAAGCATCCATGGAGTACTTCACCAATACCCTGAGAAGACATACACTTGTTCT +4360 OOEOOKOOKOOAOOSOOMOOEOOYOOFOOTOONOOTOOLOOROOROOHOOTOOLOOVOOL

GAAGAAGAGAGATTTAATGCGGAAAAAGGCAGAGGAAGACAAGAAAAGGGTCATTAGTCA +4420 OOKOOKOOROODOOLOOMOOROOKOOKOOAOOEOOEOODOOKOOKOOROOVOOIOOSOOQ

GCTTTTAGCGGCGGAAGGTCTCGAGCTTGATACAGACGATGATGATGAAGCTGTATGATG +4480 OOLOOLOOAOOAOOEOOGOOLOOEOOLOODOOTOODOODOODOODOOEOOAOOVOO*

AATTGTTCATCATATGGGAAAATTTGTTGTACATACAGAGATCTCTTTATTTCTGGTCAT +4540 GGAAAGCAGGTAATGAAATCTGTCTAATATTTTTCTCTTAACTGTATCCTATGATATATA +4600 TCATTGTTTTATCGGTATAATCTGAAATCAGTACATCTAAATAGAATGATCGAAAATTAG +4660 CATGACCCAAcccagtgtttgtcaagtacaaattgttctgattggtctcaatctttcgta +4720 tgggtgaagattcaattactgaatgtaatatgaccgcactagca .+4764

圖五、香蕉MhDnaJ基因之核苷酸及演繹胺基酸序列

Fig. 5. The nucleotide and deduced amino acid sequences of the MhDnaJ gene from banana. The first nucleotide of putative translation start site is denoted by

+1. The numbers for the nucleotide are shown on the right. Starting at the translation start site nucleotides in the structural gene sequence are indicated by positive numbers, whereas those in the 5’-flanking region are by negative numbers. Lowercase letters indicate introns and part of the flanking regions.

ABRE: abscisic acid responsive element. AuxRR-core: auxin responsive element. CGTCA-motif: MeJA-responsive element. GATA-motif: part of a light responsive element. G-box: light responsive element. HSE: heat stress responsive element. MBS: MYB binding site involved in drought-inducibility. *:

stop codon.

表一、香蕉MhDnaJ 基因之顯子與隱子連接部位核苷酸及胺基酸序列

Table 1. MhDnaJ splice-junction nucleotide sequences. The size of each exon and intron were determined from the exon-intron boundaries by comparing the nucleotide sequence of genomic DNA with the cDNA sequence and according to the GT/AG rule of splice junctions.

Exon No.

Exon size

(bp) 3’Exon junction 5’Exon junction Exon No.

1 370 CCCTTGgtaatt-- (intron 1, 246 bp) --atgcagAACTGG 2 . ...L E O.L

2 75 CAGATGgtttct-- (intron 2, 1014 bp) --ctaaagTTTATG 3 ..P D VO. Y

3 140 ATGAAGgtacat-- (intron 3, 1724 bp) --tatcagGCTTCC 4 .M

.

K A ..S

4 138 TCAAAGgtttat-- (intron 4, 672 bp) --tttcagATTGAT 5 .SO^..K .I ..D

5 405

*. Bases from exons are shown in capital letters and those from introns in small letters.

--. Indicates the remaining nucleotides in introns.

表二、香蕉、阿拉伯芥、水稻、楊樹、蓖麻、葡萄與玉米DnaJ 胺基酸序列相似性

Table 2. Pairwise comparison (%) of the deduced amino acid sequences among DnaJ from Musa spp. Hsien Jin Chiao (MhDnaJ), Arabidopsis thaliana (AtDnaJ, accession number: NP_565163), Oryza sativa Japonica type (OsDnaJ, accession number: NP_001060336), Populus trichocarpa (PtDnaJ, accession number:

XP_002302268), Ricinus communis (RcDnaJ, accession number:

XP_002513851), Vitis vinifera (VvDnaJ, accession number: XP_002285821), and Zea mays (ZmDnaJ, accession number: NP_001140575). Amino acid sequence were aligned using the AlignX program of Vector NTI 10.0 software.

MhDnaJ

AtDnaJ OsDnaJ PtDnaJ RcDnaJ VvDnaJ ZmDnaJ

MhDnaJ

100

AtDnaJ

68.8 100

OsDnaJ

66.3 61.8 100

PtDnaJ

66.9 67.5 58.0 100

RcDnaJ

66.7 68.2 61.2 72.3 100

VvDnaJ

72.0 70.3 62.5 71.4 70.8 100

ZmDnaJ

68.7 62.9 84.2 59.7 62.9 63.5 100

圖六、不同作物之DnaJ胺基酸序列比較

Fig. 6. Comparison of the amino acid sequences among DnaJ from Musa spp.

Helix I Helix II

Helix III Helix IV HPD

NES NLS

J domain

Hsien Jin Chiao (MhDnaJ), Arabidopsis thaliana (AtDnaJ, accession number:

NP_565163), Oryza sativa Japonica type (OsDnaJ, accession number:

NP_001060336), Populus trichocarpa (PtDnaJ, accession number:

XP_002302268), Ricinus communis (RcDnaJ, accession number:

XP_002513851), Vitis vinifera (VvDnaJ, accession number: XP_002285821), and Zea mays (ZmDnaJ, accession number: NP_001140575). Amino acid sequences were aligned using the AlignX program of Vector NTI 10.0 software.

Shadings indicate the degree of homology at each position: black= 100%; dark gray= 80%; light gray= 60%. Number at the end of each sequence refers to the number of amino acid in the sequence. The black lines indicate J domain, red lines indicate helix (I-IV), green lines indicate HPD motif (HPD), and blue lines indicate nuclear localization signal (NLS) and nuclear export signal (NES) conserved amino acid.

PtDnaJ RcDnaJ AtDnaJ VvDnaJ MhDnaJ OsDnaJ ZmDnaJ

0 5

10 15

圖七、不同作物之DnaJ胺基酸序列親緣性分析

Fig. 7. Phylogenetic analysis of the deduced amino acid sequences (from No.

66-273 amino acid of MhDnaJ) among DnaJ from Musa spp. Hsien Jin Chiao (MhDnaJ), Arabidopsis thaliana (AtDnaJ, accession number: NP_565163), Oryza sativa Japonica type (OsDnaJ, accession number: NP_001060336), Populus trichocarpa (PtDnaJ, accession number: XP_002302268), Ricinus communis (RcDnaJ, accession number: XP_002513851), Vitis vinifera (VvDnaJ, accession number: XP_002285821), and Zea mays (ZmDnaJ, accession number:

NP_001140575). Amino acid sequences were analyzed using the AlignX program of Vector NTI 10.0 and the Neighbor-joining program of MEGA4 software. Scale bar below shows the number of amino acid substitution per site.

結果顯示，香蕉MhDnaJ 基因啟動子可能具有多種 cis-acting elements，如 植物荷爾蒙調控因子：植物生長素調控因子 (auxin responsive element, AuxRR-core)、離層酸調控因子 (abscisic acid responsive element, ABRE) 與茉莉酸甲酯調控因子 (MeJA-responsive element, CGTCA-motif)，及非生物性調控因子：光調控因子 (light responsive element, GATA-motif; G-box)、高溫調控因子 (heat stress responsive element, HSE) 與乾旱調控因子 (MYB binding site involved in drought-inducibility, MBS) 等調控相關之保守序列 (表三)。

二、香蕉

MhDnaJ 基因功能分析

(一) 過量表達分析

完成以CaMV 35S 啟動子驅動 MhDnaJ 基因之轉殖質體構築，電穿孔 轉型至農桿菌，以農桿菌媒介轉殖法進行菸草葉圓片轉殖，經抗生素 (kanamycin) 篩選後取得 12 株菸草擬轉殖株，經抽取基因組 DNA，以 PstI

酶切，並以 MhDnaJ 基因片段為探針，進行南方氏雜交分析，雜交條帶為

1.4 Kb，確認轉殖系編號 1、4、6、9、10 與 14 號為轉殖株 (圖八)。

(二) 基因默化分析

經核苷酸序列比對，選取 MhDnaJ 與 AtDnaJ 相同區域，完成以 CaMV 35S 啟動子驅動 small interference RNA (siRNA) 構築之暫時性表現與轉殖質體之構築，以農桿菌媒介轉殖法進行阿拉伯芥花序轉殖，目前以抗生素篩選T1 轉殖株。

三、香蕉 MhDnaJ 蛋白質定位分析

表三、香蕉MhDnaJ 基因之啟動子序列分析

Table 3. Analysis of responsive elements in the promoter region of MhDnaJ gene from banana.

Motif Sequence in MhDnaJ

Position in

MhDnaJ Regulatory function Reference

ABRE CACGTG -471～-466

cis-acting element involved in the abscisic acid involved in auxin responsiveness involved in MeJA- responsiveness

Rouster et al., 1997

GATA-motif GATAAGG -1455～-1449 part of a light

responsive element element involved in light responsiveness involved in the heat stress responsiveness

Amin et al., 1988

MBS CAACTG

TAACTG

-3034～-3029 -2655～-2650

MYB binding site involved in

drought-inducibility

Urao et al., 1993

- The first nucleotide of the putative translation start site is denoted by +1.

Fig. 8. Southern blot analysis of putative Nicotiana tabacum 35Spro::MhDnaJ transformants. (A) Schematic diagram of restriction map of pGKUMhDnaJ plasmid and expected hybridization patterns. NPTII: neomycin phosphotransferase II gene. P: nopaline synthase gene promoter. pA: CaMV 35S polyA terminator. T: nopaline synthase terminator sequence. GUS:

β-glucuronidase gene. LB: Left border. RB: Right border. (B) Genomic DNA (20 μg) digested with PstI was separated by electrophoresis on a 0.7 % agarose gel. Wt: negative control, non-transformant. P: 200 pg plasmid DNA of pGKUMhDnaJ served as positive control.

(B) (A)

1.4 Kb

為瞭解香蕉 MhDnaJ 蛋白質於細胞內表達位置，以 35Spro::MhDnaJ::GFP 融合螢光蛋白表達質體，進行阿拉伯芥原生質體與洋蔥表皮細胞之暫時性蛋白質定位分析，以共軛焦顯微鏡觀察，結果顯示MhDnaJ::GFP 融合蛋白螢光訊號出現於阿拉伯芥原生質體 (圖九) 與洋蔥表皮細胞 (圖十) 的細

胞核內，與細胞核染劑 DAPI 螢光訊號位置相同 (圖九、十)，顯示香蕉

MhDnaJ 蛋白質於細胞質表達後，胺基酸序列中的 NLS 能正常的將蛋白質標的至細胞核，使MhDnaJ 蛋白質累積於細胞核中。

四、香蕉

MhDnaJ 基因啟動子活性分析

(一) 菸草轉殖株基因完整性與基因拷貝數之分子驗證

MhDnaJ 啟動子引導報導基因 GUS 之轉殖質體 pBI101-PMhDnaJ，以農

桿菌進行菸草葉圓片轉殖與阿拉伯芥花序轉殖，取得12 株菸草擬轉殖株與

2 株阿拉伯芥 T1 轉殖株。12 株菸草擬轉殖株，經抽取基因組 DNA，以 HindIII 與BamHI 酶切，並以啟動子片段為探針，進行南方氏雜交分析，雜交條帶 為3.5 Kb，確認均為轉殖株 (圖十一)，拷貝數分析結果顯示，轉殖系編號 2、5、9、24 與 33 號之雜交條帶大小分別為 18.4 Kb、7.3 Kb、6.9 Kb、12 Kb 及14.9 Kb，為具外源基因單一拷貝之轉殖株 (圖十二)。

(二) 菸草轉殖株不同發育階段之啟動子活性分析

選取單一拷貝之轉植株Nt-MhDnaJpro::GUS-2 之自交種子進行無菌播 種，觀察菸草T1 轉殖株不同發育天數 (5-30 天) 之啟動子活性，結果顯示啟動子於5-30 天皆無表現 (圖十三)。

進一步進行菸草 R1 轉殖株花序與果實不同生長發育階段之啟動子活

性分析，顯示 MhDnaJ 啟動子於柱頭發育早期 (stages 2-4) 與後期 (stage 8)；雄蕊發育後期 (stages 8-9) 與果實發育早期 (stages 10-11) 表現 (圖十四、十五)。

圖九、香蕉MhDnaJ蛋白質於阿拉伯芥原生質體之細胞內定位

Fig. 9. Subcellular localization of banana MhDnaJ localization in Arabidopsis

leaf mesophyll cell protoplasts. The MhDnaJ::GFP fusion gene inserted into vector and transformed into Arabidopsis thaliana (Col-0) protoplasts by PEG-method for transient expression. (A) MhDnaJ::GFP accumulation emitted green fluorescence. (B) 35S::GFP accumulation emitted green fluorescence. (C) (D) Chlorophyll auto-fluorescent emitted red fluorescence. (E) (F) Nuclei stained with 4',6-diamidino-2-phenylindole (DAPI) emitted blue fluorescent.

(G) Merged image of (A) (C) and (E). (H) Merged image of (B), (D) and (F). (I) (J) Bright field microscopic image. GFP signal were observed by laser scanning confocal microscope (Leica). Bars = 10 μm.

圖十、香蕉MhDnaJ蛋白質於洋蔥表皮細胞之細胞內定位

Fig. 10. Subcellular localization of banana MhDnaJ localization in onion epidermal cells. The MhDnaJ::GFP fusion gene inserted into vector and transformed into onion epidermal cells by particle bombardment for transient expression. (A) MhDnaJ::GFP accumulation emitted green fluorescence. (B) 35S::GFP accumulation emitted green fluorescence. (C) (D) Nuclei stained with 4',6-diamidino-2-phenylindole (DAPI) emitted blue fluorescent. (E) Merged image of (A) and (C). (F) Merged image of (B) and (D). (G) (H) Bright field microscopic image. GFP signal were observed by laser scanning confocal microscope (Leica). Bars = 50 μm.

Probe (3.5 Kb)

圖十一、菸草MhDnaJpro::GUS擬轉殖株南方氏雜交分析

Fig. 11. Southern blot analysis of putative Nicotiana tabacum MhDnaJpro::GUS transformants. (A) Schematic diagram of restriction map of pBI101-PMhDnaJ plasmid and expected hybridization patterns. NPTII:

neomycin phosphotransferase II gene. P: nopaline synthase gene promoter. T:

nopaline synthase terminator sequence. PMhDnaJ: MhDnaJ promoter. GUS:

β-glucuronidase gene. LB: Left border. RB: Right border. (B) Genomic DNA (20 μg) digested with HindIII and BamHI was separated by electrophoresis on a 0.7 % agarose gel. Wt: negative control, non-transformant. P: 200 pg plasmid DNA of pBI101-PMhDnaJ served as positive control.

(B) (A)

3.5 Kb

Signal ( > 6.3 Kb)

Fig. 12. Southern blot analysis for gene copy number of putative Nicotiana tabacum MhDnaJpro::GUS transformants. (A) Schematic diagram of restriction map of pBI101-PMhDnaJ plasmid and expected hybridization patterns. NPTII:

在文檔中香蕉果實後熟相關基因MhDnaJ之啟動子活性與基因功能分析 (頁 40-0)