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表一、本論文所使用的阿拉伯芥HAP 基因專一性引子
左欄為 HAP 基因名稱以及 AGI 號碼。中間欄位為引子核苷酸序列，F 表示 正向引子(forward primer)，R 為反向引子(reversed primer)。右欄表示以引子進行 聚合酶連鎖反應所放大的DNA 片段長度。
HAP 基因 引子序列(5’3’) 編碼序列長
度(bp) F CATGCCATGGCGGATACGCCTTCGAG
AT2G38880
(HAP3a) R GGAATTCTTACCAGCTCGGCATTTCTTCA 426 F CATGCCATGGGGGATTCCGACAGGGA
AT5G47640
(HAP3b) R GGAATTCTTAAGTCCTTGTCCTACCGGAG 573 F CATGCCATGGATACCAACAACCAGCAAC
AT3G48590
(HAP5a) R GGAATTCTTAACCTTGGCCGTCGG 705 F CATGCCATGGAGCAGTCAGAAGAGGG
AT1G56170
(HAP5b) R GGAATTCTTAAGACTCATCAGGGTGTTGC 600 F CGGGATCCGAATGGATCAACAAGACCATGC
AT1G08970
(HAP5c) R GCGTCGACTAATTTTCCTGGTCAGGTTGG 696
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表二、阿拉伯芥HAP 基因家族
由左至右欄位內依序為 HAP2、HAP3 和 HAP5 基因家族成員資訊，包括基 因基因編碼(accession number)、AGI(Arabidopsis Genome Initiative)號碼以及基因 別名。基因編碼之右上角若出現「*」的符號，表示本研究作為檢測與 CIA2 是 否有蛋白質間交互作用之HAP 家族成員。
HAP gene family in Arabidopsis
HAP2 family HAP3 family HAP5 family
accession number
(AT4G14540) NF-YB3 BX815011
(AT1G54830) NF-YC3
AY114711
(AT2G34720) NF-YA4 BT029363
(AT1G09030) NF-YB4 AK317038
(AT5G63470) NF-YC4
AY081445
(AT1G54160) NF-YA5 BT005081
(AT2G47810) NF-YB5 BX833675
(AT5G50490) NF-YC5 NM_112256
(AT3G14020) NF-YA6 AY138461
(AT5G47670)
NF-YB6,
L1L BX833348
(AT5G50480) NF-YC6
BT005561
(AT1G30500) NF-YA7 BT025276
(AT2G13570) NF-YB7 DQ056712
(AT5G50470) NF-YC7
BT033080
(AT1G17590) NF-YA8 BX821279
(AT2G37060) NF-YB8 DQ056694
(AT5G27910) NF-YC8
BX822776
(AT5G06510) NF-YA10 AK176827
(AT3G53340) NF-YB10 BT025266
(AT1G07980) NF-YC10
BT004083
(AT2G27470) NF-YB11 BX842121
(AT3G12480) NF-YC11
AK227164
(AT5G08190) NF-YB12 BX833046
(AT5G38140) NF-YC12
AK317460
(AT5G23090) NF-YB13 NM_123691
(AT5G43250) NF-YC13
30 結果，“－”代表陰性結果。AD，代表Gal4 activation domain C端所接合(fusion) 之蛋白種類，其所使用之表現質體為pACT2；BD，代表Gal4 binding domain C端 所接合之蛋白種類其其所使用之表現質體為pAS2-1。N、M和C所代表的CIA2蛋
3-AT selection β-galactosidase assay CIA2:(N+M+C)
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表四、Y2H 之對照組結果
利用 Y2H 檢測 HAP 及 CIA2 蛋白與不同 CIA2 蛋白片段之交互作用實驗中，
其對照組以營養缺失外加10 mM 3-AT 篩選與 β-galactosidase 活性測試方式檢定 結果，其“＋”代表陽性結果，“－”代表陰性結果。AD，代表 Gal4 activation domain C 端所接合(fusion)之蛋白種類，其所使用之表現質體為 pACT2；BD，代表 Gal4 binding domain C 端所接合之蛋白種類其所使用之表現質體為 pAS2-1。表中 pACT2 及 pAS2-1 代表不帶其他序列的質體。“X”則表示不轉形該質體至酵母菌 中。N、M 和 C 所代表的 CIA2 蛋白片段參考表三圖 A。
AD BD SD/-Leu/-Trp/-His +
3-AT selection β-galactosidase assay
pACT2 X － －
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CIA2
LEU2 pACT2 plasmid
TRP1 pAS2-1 plasmid
HAP
圖一、Y2H 系統載體建構策略示意圖
將不同長度之 CIA2 解碼序列以及欲檢測之 HAP 成員解碼序列分別黏合至 pAS2-1(含 Gal4 BD)和 pACT2(含 Gal AD)質體中，並轉形至 CG1945 進行檢測。
TRP
LEU pAS2-1 plasmid
pACT2 plasmid HAP 蛋白之解碼序列
HAP 蛋白之解碼序列 不同長度CIA2 片
段之解碼序列
不同長度CIA2 片 段之解碼序列
33
]
圖二、阿拉伯芥CIA2 CCT-motif與HAP2成員胺基酸比對分析示意圖
使用 Vector NTI 10 軟體比對阿拉伯芥 CIA2 之 CCT motif（CIA2）、阿拉伯 芥十個HAP2 蛋白成員（NFYA1-10）和 Saccharomyces cerevisiae HAP2 (Yeast HAP2)之胺基酸序列，左排括號內顯示各序列排頭之胺基酸序號，其保守度由高 至低分別以黃色、藍色及綠色背景表示。最末行顯示保守之胺基酸。最上方橫線 上文字代表HAP2 各區域片段之蛋白功能。CIA2 的 CCT motif 與 HAP2 成員完 全保守的胺基酸以「*」表示於最上方；而在 HAP2 或 CCT-class proteins 突變分 析指示與HAP3/5 蛋白交互作用或 DNA 結合有關的胺基酸，其在比對結果的對 應位置以「▲」表示於下方。
* *
▲ ▲ ▲▲
HAP3/HAP5
interaction Linker DNA binding
* * * ***
*
34
圖三、HAP5a、HAP5b 和 HAP5c 與 CIA2 蛋白交互作用檢測結果
不同 HAP5 成員與 CIA2 蛋白交互作用檢測組別，分別於 YPD(左上)、營養 缺失外加10 mM 3-AT 之篩選性培養基(中上)和 β-galactosidae 檢測(右上)之實驗 結果。下圖代表培養基上不同實驗組別之位置，組別A1-A3 為可表現 AD:HAP5 雜合蛋白之酵母菌，組別B 為可表現 BD:CIA2 雜合蛋白的酵母菌，組別 C1-C3 為可同時表現AD:HAP5 與 BD:CIA2 雜合蛋白的酵母菌，而組別 PC 為正控制組，
其所使用酵母菌可同時表現AD:SV40 large T-antigen 與 BD:p53 雜合蛋白。