臺灣博碩士論文加值系統

穀類種子（如水稻）發芽時，胚會產生植物荷爾蒙吉貝素（GA; gibberellin），並擴散至糊粉層細胞，促使糊粉層產生α-澱粉水解酶，將貯存於胚乳的澱粉分解成葡萄糖，是提供幼苗生長所需的最主要能量。水稻α-澱粉水解酶基因為一基因群，除了植物荷爾蒙吉貝素可以調控α-澱粉水解酶基因的表現之外，目前已知缺糖亦能誘導表現，α-澱粉水解酶基因的啟動子上含有受糖調控的DNA序列（sugar response sequence，簡稱SRS），包含了3個必須的正向調控序列（GC box, G box,及TATCCA element），其中αAmy3含有兩套TATCCA element直接重覆序列。同時也瞭解水稻中含有TATCCA element的結合蛋白質OsMYBS，可能參與糖調控αAmy3基因表現的分子機制。為了進一步瞭解OsMYBS的生理功能，本論文利用植物基因轉殖技術在水稻中大量表現OsMYBS蛋白質或者利用RNAi的方法降低內生OsMYBS。本論文成功的得到大量表現的OsMYBS2與OsMYBS3及OsMYBS2-RNAi的轉殖株，分析大量表現OsMYBS2轉殖株，發現OsMYBS2會抑制水稻α-amylase基因的表現。這個結果顯示OsMYBS2在水稻α-amylase基因表現與調控機制上扮演repressor的角色。

Expression of some α-amylase genes in cereals are induced both by gibberellin (GA) and sugar starvation. There is sugar response sequence (SRS) in the promoter upstream of these rice α-amylase genes. The SRS of αAmy3 gene contains three essential elements, GC box, G box, and duplicated TATCCA elements. The protein factors that bind to the TATCCA element have recently been isolated and designated OsMYBS1, OsMYBS2, and OsMYBS3. To study the physiological function of OsMYBSs in rice, Agrobacterium-mediated gene transformation strategy was employed. Both constructs for over-expression and RNA interference of OsMYBSs were made and transformed into rice cells. The result from transient and stable transformants showed that OsMYBS2 is a repressor of the sugar-starvation-induced α-amylase expression.

圖目錄
縮寫表
中文摘要
Abstract
壹、 前言
一、 α-澱粉水解酶基因表現的調控方式
(I) 澱粉與α-澱粉水解酶介紹
(II) 吉貝素對α-澱粉水解酶的調控機制
(III) 滲透壓對α-澱粉水解酶的調控機制
二、 以糖類為訊號，調控與其代謝相關基因的表現
(I) 酵母菌中葡萄糖的調控機制
(II) 高等植物對糖的感應及其訊息傳遞路徑
三、 糖調控α-澱粉水解酶基因的表現
(I) 糖調控α-澱粉水解酶基因的表現
(II) 糖調控αAmy3基因的表現
四、 轉錄層次的調控
(I) 轉錄因子
(II) 啟動子上TATCCA element扮演轉錄活化子的角色
(III) OsMYBS的結構特性與MYB家族在演化上的意義
五、 大量表現與基因靜默法的構築
六、 植物基因轉殖系統
貳、實驗材料與方法
第一部份 構築質體
1. 以PCR分別合成OsMYBS基因片段
1.1. primer的設計、製造與純化
1.2. 以PCR合成DNA片段
1.3. 瓊脂膠體回收限制酵素切過之DNA
2. 接合反應
2.1. 線性載體DNA的去磷作用
2.2. 黏頭端載體DNA與插入DNA片段的接合
2.3. 齊頭端DNA片段的接合
3. 細菌的轉殖作用
3.1. 製備E. coli competent cell (XLI-blue strain)
3.2. 細菌的轉殖(transformation)
3.3. 小量純化細菌plasmid DNA法
3.4. 大量純化細菌plasmid DNA法
4. DNA序列分析
第二部份 水稻基因轉殖
5. 水稻細胞利用農桿菌(Agrobacterium)轉殖的方法
5.1. 誘導水稻未成熟胚(或成熟胚)之癒傷組織
5.2. 製備Agrobacterium competent cells (EHA105 strain)
5.3. 農桿菌的轉殖方法
5.4. 農桿菌的生化檢測( Ketolactose test )
5.5. 水稻的轉殖作用
5.6. 誘導水稻懸浮培養細胞的方法
第三部份 轉殖株分析方法
6. 南方墨點轉印法(Southern blot analysis)
6.1. Genomic DNA的純化
6.2. DNA墨點轉印
6.3. 探針的製備
6.4. 探針雜交
7. 北方墨點轉印法(Northern blot analysis)
7.1. 製備RNase-free溶液
7.2. RNA的純化
7.3. RNA墨點轉印
肆、結果
伍、討論
參考文獻
附錄

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