臺灣博碩士論文加值系統

蔗糖合成酶催化蔗糖與 UDP形成果糖與 UDP-glucose 的可逆反應。在水稻中，此酵素是由至少六種 RSus 基因所表現，其中 RSus3 主要表現於發育中的種子。本論文的目的在建構具有不同 RSus3表現量的轉殖水稻，以探討 RSus3 的生理功能。
將預期可造成水稻各組織中恆常性表現 RSus3、於穀實中過量表現 RSus3 及於穀實中抑制 RSus3 表現的三種質體 pActinSuS3、pGluB1SuS3 及 pSuS3RNAi，經由農桿菌法轉殖到水稻。pActinSuS3 及 pGluB1SuS3 的 T0 及 T1 轉殖株，以 Southern 分析後發現，這些轉殖株帶有一個或二個外來的 RSus3 基因，且能夠在葉子偵測到其表現。分析 T1 植株充實期種子發現，轉殖株 RSus3 的表現比未轉殖之台農 67 號水稻早，轉殖株 RSus3 mRNA 的量也比未轉殖之台農 67 號水稻多。此外，轉殖株 T2 種子的千粒重及平均內外穎長有大於未轉殖之台農 67 號水稻的趨勢。

Sucrose synthase catalyzes the reversible conversion of sucrose and UDP into fructose and UDP-glucose. The enzyme is encoded by at least six RSus genes in rice. One of the genes, RSus3, is predominatly expressed in the developing seeds. The objective of this study is to construct transgenic rice plants having different expression levels of RSuS3 in order to investigate the biological function of RSus3.
Plasmids pActinSuS3, pGluB1SuS3 and pSuS3RNAi, which are expected to respectively result in constitutive expression of RSus3 in various rice tissues, over-expression of RSus3 in rice seed, and inhibition of RSus3 expression in rice seed were transformed into rice by the Agrobacterium-mediated method. Southern analysis of the T0 and T1 transgenic plants transformed with pActinSuS3 or pGluB1SuS3 revealed that one or two copies of RSus3 genes were introduced into these transgenic plants. Expression of RSus3 was detected in leaves. Analysis of ripening seeds of the T1 transgenic plants revealed that the expression of RSus3 in the developing seeds of these transgenic plants occurred earlier than the untransformed TNG67 rice plant. The abundance of RSus3 mRNA in the seeds of transgenic plants was also higher than the untransformed plant. Moreover, the weight of 1000 grains and the average length of grains of transgenic T2 seeds are larger than the untransformed TNG67.

目錄..………………………………………………………………I
縮寫表………………………………………………………………VI
摘要…………………………………………………………………VII
Abstract…………………………………………………………VIII

第一章 緒論………………………………………………………1

第一節 水稻………………………………………………………1
1.1 水稻簡介 1
1.2 水稻生長 1
第二節 蔗糖合成酶………………………………………………2
2.1 蔗糖合成酶之生化特性 2
2.1.1 蔗糖合成酶催化之反應 2
2.1.2 蔗糖合成酶之一般生化特性 3
2.1.3 蔗糖合成酶異構酶與其基因 3
2.2 蔗糖合成酶的生理功能 4
2.2.1 參與澱粉之生合成 4
2.2.2 參與細胞壁多醣類之合成 4
2.2.3 參與蔗糖運輸 5
2.2.4 參與缺氧逆境反應 5
第三節 水稻蔗糖合成酶之研究…………………………………5
第四節 本篇論文之目的及方向…………………………………7
4.1 水稻植株中恆常性表現 RSuS3 7
4.2 水稻轉殖株穀實中過量表現 RSuS3 7
4.3 水稻轉殖株穀實中抑制 RSuS3 表現 8

第二章 材料與方法………………………………………………9

第一節 實驗材料與樣品…………………………………………9
1.1 轉殖作物 9
1.2 菌種 9
1.3 質體 9
1.4 藥品 10
第二節 實驗儀器設備……………………………………………11
2.1 水平核酸電泳 11
2.2 蛋白質電泳轉印設備 11
2.3 離心機 11
2.4 其他 11
第三節 實驗流程與方法…………………………………………12
3.1 實驗流程 12
3.2 重組質體構築 12
3.2.1 質體 DNA 之抽取 12
3.2.2 DNA 之定量法 13
3.2.3 DNA 限制酶切割 13
3.2.4 Klenow Fragment DNA 片段 5’ 端補齊 13
3.2.5聚合酶連鎖反應 13
3.2.6 DNA 片段純化 14
3.2.7 膠體中 DNA 片段之回收 14
3.2.8 接合反應 14
3.2.9 Competent cells 之製備 14
3.2.9.1 氯化鈣法 14
3.2.9.2 電轉形法 15
3.2.10 質體之轉形 15
3.2.10.1 氯化鈣法 15
3.2.10.2 電轉形法 15
3.2.11質體快速篩選法 16
3.2.12 DNA 瓊脂糖膠體電泳法 16
3.3 水稻農桿菌基因轉殖 16
3.3.1水稻癒合組織誘導 16
3.3.2農桿菌 competent cells 製備 17
3.3.3農桿菌之電轉形 17
3.3.4農桿菌轉殖水稻癒合組織 17
3.3.4.1農桿菌大量培養 17
3.3.4.2 農桿菌水稻癒合組織共培養 18
3.3.4.3 清洗癒合組織 18
3.3.4.4 第一次篩選 18
3.3.4.5 第二次篩選 18
3.3.4.6 分化與移植 18
3.3.5 水稻種子發芽 18
3.3.6 水稻農藝性狀調查及種子保存 19
3.4 Southern 分析 19
3.4.1 水稻染色體 DNA 之抽取 19
3.4.2 水稻染色體 DNA 限制酶作用 19
3.4.3 水稻染色體 DNA 瓊脂糖膠體電泳分析 20
3.4.4 轉印 20
3.4.5 放射線標定探針製備 20
3.4.6 雜合反應 20
3.5 RNA 之抽取與分析 21
3.5.1 水稻 total RNA 之抽取 21
3.5.2 水稻 total RNA 去 DNA 21
3.5.3 RNA 之定量法 22
3.5.4 反轉錄反應 (Reverse transcription) 22
3.6 Western 分析 22
3.6.1 水稻種子蛋白質抽取 22
3.6.2 蛋白質粗抽液定量 22
3.6.3 蛋白質 SDS-PAGE 電泳 22
3.6.4 蛋白質轉印 23
3.6.5 免疫呈色法 23



第三章 結果與討論……………………………………………25

第一節 質體建構…………………………………………………25
1.1 恆常性表現質體 pActinSuS3 之構築 25
1.2 穀實中過量表現質體 pGluB1SuS3 之構築 25
1.3 穀實中抑制 RSus3 表現之質體 pSuS3RNAi 之構築 25
第二節 以農桿菌法進行台農 67 號水稻轉殖…………………26
2.1 農桿菌法轉殖過程 26
2.2 轉殖效率 27
2.3 轉殖株命名 27
2.4 轉殖株繁殖 27
第三節 轉殖基因之分子檢定……………………………………28
3.1 T0 轉殖株 PCR 檢定 28
3.1.1 hpt II 基因之檢定 28
3.1.2 RSus3 基因之檢定 29
3.2 T0 轉殖株 Southern 分析 29
3.2.1 hpt II 基因之檢定 29
3.2.2 RSus3 基因之檢定 30
3.3 T1 轉殖株 Southern 分析 30
第四節 轉殖基因之表現……………………………………………31
4.1 T0 轉殖株成熟葉 RT-PCR 分析 31
4.2 T1 轉殖株成熟葉 RT-PCR 分析 32
4.3 Glutelin 是否在成熟葉中表現 32
4.4 T1 轉殖株充實期種子分析 32
4.4.1 T1 轉殖株充實期種子 RT-PCR 分析 33
4.4.2 T1 轉殖株充實期種子 western 分析 33
第五節 T1 轉殖株表現型觀察及農藝性狀分析…………………33

第四章 總結……………………………………………………35

第五章 未來展望………………………………………………36
5.1 pSuS3RNAi 轉殖株之分析 36
5.2 轉殖株後代分析 36
5.3 轉殖株種子中澱粉及醣類分析 36
5.4 製備能夠區別水稻內生及外來 RSuS3 之抗體 36
參考文獻……………………………………………………………37
圖與表………………………………………………………………42
附錄…………………………………………………………………67

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