bHLH 轉錄因子調節植物生長發育及生理反應。水稻之 bHLH 轉錄因子基因家族共有173個成員，然而此些轉錄因子於逆境下之調控角色仍未明瞭。本研究旨在探討水稻 OsbHLH068 轉錄因子於植物逆境下之調控角色。首先利用水稻經非生物逆境處理所得之微矩陣晶片( microarray ) 數據挑選出非生物逆境誘導的 OsbHLH 基因，其中OsbHLH068 為受鹽高度誘導之轉錄因子，且相對應之阿拉伯芥同源基因為 AtbHLH112。經 RT-PCR 及 GUS 分析 OsbHLH068::GUS 及AtbHLH112::GUS 轉殖株並確認此兩基因皆受鹽逆境誘導且表現於管束組織之側根及根原基。而相較於阿拉伯芥野生型 (Col-0)，atbhlh112 突變株較不耐逆境且葉片溫度較低。OsbHLH068 異源互補 ( heterologous expression ) 突變株AtbHLH112P::OsbHLH068-GFP 可功能性互補 atbhlh112 不耐鹽逆境的生理缺陷。35S::OsbHLH068 及 AtbHLH112P::OsbHLH068-GFP 轉植株具有發芽後期生育延遲 ( Post-germination developmental arrest, PGDA ) 之外表型，其在逆境下胚根突破種皮後子葉不完全展開。分析 OsbHLH068 蛋白質之次細胞定位與組織表現位置，發現融合蛋白 OsbHLH068-GFP 及 GFP-OsbHLH068 皆表現於細胞核內，並且表現於地上部及地下部之管束組織中。最後，相較於水稻野生型台農67號，35S::OsbHLH068 之轉殖水稻幼苗在 250 mM 高鹽處理下能於其第一葉之葉鞘分泌出較多的鹽類結晶。綜合以上結果顯示 OsbHLH068 為水稻鹽逆境耐受性之正向調控轉錄因子。

The basic/helix-loop-helix (bHLH) transcription factors (TFs) play important roles in the regulation of diverse developmental and physiological processes in plants. So far, at least 173 OsbHLH genes have been identified in rice. Though the expression levels of several OsbHLH genes were noticed to be up-regulated by salt treatments, such as OsbHLH068 gene; however, the gene functions of OsbHLH068 in salt-stress tolerance of rice are still unknown. In this study, the roles of OsbHLH068 and its Arabidopsis ortholog, AtbHLH112, were investigated under salt stress with reverse-genetics approaches. In Arabidopsis, under salt-treatment, the atbhlh112 mutant displays salt-intolerance and low leaf-temperature phenotypes. These physiological defects in atbhlh112 mutant could be complemented by heterologous expression of OsbHLH068 gene. In addition, heterologous over-expression of OsbHLH068 gene in Arabidopsis causes a post-germination developmental arrest (PGDA) phenotype, which refers to non-fully expanded cotyledon under salt treatment. Histochemical staining of promoter::GUS assay in transgenic Arabidopsis indicated that AtbHLH112 expressed in vascular tissue and trichome. Both OsbHLH068-GFP and GFP-OsbHLH068 fusion proteins are localized in the nucleus. Interestingly, compared with wide type (WT, Tainung67), the phenotypic analysis of over-expressed OsbHLH068 transgenic rice seedling showed increased salt crystals accumulation at the base of 1st leaf sheath under 250 mM NaCl treatment. Taken together, these results suggest that OsbHLH068 is a positive regulator that is involved in stress response of plant salt-stress tolerance.

目錄
誌謝 i
中文摘要 ii
英文摘要 iii
圖表目錄 2
縮寫字對照表 2
第一章 前言 1
第二章 前人研究 4
一、bHLH 轉錄因子之演化過程與基因註解 4
1. bHLH 轉錄因子蛋白質結構與 DNA 結合活性 4
2. bHLH 轉錄因子系統性分群之演化 5
3. bHLH 轉錄因子之系統性基因功能註解 6
4. OsbHLH068 系統性分群整理 10
二、bHLH 轉錄因子於非生物逆境下之功能 10
三、鹽害逆境下植物之生理反應及機制 14
1. 鹽害逆境造成之毒害 14
2. 耐鹽機制的探討 14
2.1 滲透逆境耐受性 15
2.2 鹽類運送與排除機制 16
2.2.1 鹽類運送 16
2.2.2 鹽濃度訊號感知與鹽類排除 16
2.3 組織耐鹽性 17
2.3.1 胞內分室作用 ( compartmentalization ) 17
2.3.2 離子狀態平衡 ( ion-homeostasis ) 18
2.3.3 組織區隔 ( tissue sequestration ) 19
四、研究目的及實驗架構 20
第三章 材料與方法 21
一、水稻 bHLH X 群基因表現分析 21
1. 利用生物資訊分析OsbHLH X群成員於植物荷爾蒙下之表現情形 21
2. 轉錄因子 OsbHLH068 及鹽害逆境下之 Na+ 轉運蛋白基因表現圖譜分析 21
二、阿拉伯芥與水稻同源bHLH轉錄因子 X 群之分群 21
三、試驗材料 22
1. 轉基因水稻 22
1.1 轉基因水稻種子消毒及栽培過程 22
1.2 轉基因水稻構築及構築流程 22
1.3 轉基因水稻同質結合子植株篩選流程 23
2. 轉基因阿拉伯芥 23
2.1 轉基因阿拉伯芥構築及構築流程 24
2.2 轉基因阿拉伯芥種子消毒及栽培過程 24
2.3 轉基因阿拉伯芥同質結合子植株篩選流程 24
2.4 轉基因阿拉伯芥之分子鑑定 25
四、水稻生長條件與鹽害逆境處理 25
1. 水稻鹽害逆境之逆境處理 25
1.1 轉基因水稻幼苗初步外表型分析及逆境處理條件測試 25
五、阿拉伯芥生長條件與鹽害逆境處理 25
1. 無菌培養條件之鹽害逆境處理 25
1.1 轉基因阿拉伯芥幼苗之基因表現及分子鑑定 26
1.2 轉基因阿拉伯芥苗期逆境處理條件 26
1.2.1 逆境處理下之發芽率分析 26
1.2.2 逆境處理下之根長分析 26
2. 盆栽試驗條件之鹽害逆境處理 27
2.1 轉基因阿拉伯芥之植株外表型鑑定 27
2.2 轉基因阿拉伯芥植株金屬元素含量測定 28
六、水稻及阿拉伯芥轉殖株之分子鑑定 29
1. 植物基因體組DNA萃取 29
2. DNA洋菜瓊脂膠體電泳分析 29
3. DNA-based 基因型判定分析 29
七、水稻及阿拉伯芥轉殖株之基因表現分析 30
1. 基因表現分析 30
1.1 RNA之抽取及製備 30
1.2 RNA洋菜瓊脂膠體電泳分析及OD值測定 30
1.3 RNase free DNaseI處理 30
1.4 cDNA合成之反轉錄反應 31
1.5半定量聚合&;#37238;連鎖反應 (RT-PCR) 31
1.6 即時定量聚合&;#37238;鏈鎖反應(qRT-PCR) 32
2. GUS 基因表達組織部位生化染色分析法 32
2.1 GUS染色方法 32
2.2 逆境下之基因表現組織部位分析 32
八、水稻及阿拉伯芥轉殖流程 33
1. 水稻及阿拉伯芥轉殖 33
1.1 構築黏接反應(Ligation) 33
1.2 大腸桿菌轉形作用( Transformation ) 33
1.2.1 大腸桿菌勝任細胞製備 33
1.2.2 質粒大腸桿菌轉形-熱休克法 34
1.2.3 大腸桿菌質粒DNA小量純化 34
1.2.4 檢測大腸桿菌質粒DNA-酵素截切法 34
1.3 植物材料預備 34
1.4 農桿菌轉形 35
1.4.1 農桿菌菌株GV3101勝任細胞製備 35
1.4.2 質粒農桿菌轉形-電穿孔法 35
1.4.3 轉形後農桿菌質粒檢測 35
1.5 阿拉伯芥轉形-花序感染法 36
第四章 結果 37
一、水稻 bHLH X 群成員之親緣關係 37
1. OsbHLH X群親緣關係 37
二、水稻 bHLH X 群基因表現分析 37
1. 利用生物資訊分析OsbHLH F亞族成員受植物荷爾蒙之誘導表現情形 37
2. OsbHLH068 與 AtbHLH112 於非生物逆境下之基因表現 38
三、OsbHLH068 與 AtbHLH112 於鹽害逆境下之基因表現組織部位分析 41
1. OsbHLH068 基因組織專一性表達位置 41
2. AtbHLH112 基因組織專一性表達位置 41
四、基因轉殖植株之分子鑑定與蛋白質次細胞定位 42
1. 阿拉伯芥轉殖植株之分子鑑定 42
2. 阿拉伯芥轉殖植株之蛋白質次細胞定位 43
五、OsbHLH068 之基因功能性分析 43
1. 阿拉伯芥幼苗之基因功能性分析 43
1.1 種子萌芽率 43
1.2 種子發芽後期生育延遲現象 ( PGDA ) 及存活率 45
1.3 阿拉伯芥幼苗於鹽逆境下之熱紅外線攝相儀 ( Thermograph ) 呈相分析 45
1.4 阿拉伯芥幼苗根長分析 46
2. 阿拉伯芥成株之基因功能性分析 9
2.1 阿拉伯芥成株於鹽逆境下之之熱紅外線攝相儀 ( Thermograph ) 呈相分析 47
2.2 阿拉伯芥成株於鹽逆境下之外表型分析 47
2.3 阿拉伯芥鈉與鉀金屬元素含量測定 48
2.4 阿拉伯芥成株於鹽逆境下之基因表現型分析 48
3. 水稻成株之基因外表型分析 50
3.1 T1轉基因水稻幼苗於高鹽逆境下之反應 50
第五章 討論 51
一、 OsbHLH068 可功能性互補 atbhlh112 生長發育之缺陷 51
二、 OsbHLH068 透由調節氣孔開合提升滲透逆境耐受性 51
三、 OsbHLH068 與 AtbHLH112 參與植物內生 Na+/K+ 離子平衡狀態之調節 55
四、 探討 OsbHLH068 與 AtbHLH112 基因功能之異同與抗鹽逆境原因 61
五、 結語 62
參考文獻 65

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陳依甄。2012。水稻轉錄因子 OsbHLH061 與 OsbHLH068 於非生物逆境下基因表現及功能性分析。碩士論文
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