臺灣博碩士論文加值系統

控制金魚草兩側對稱之基因所編碼之蛋白質分別屬於TCP基因家族和MYB基因家族，當基因突變，會改變花朵的對稱形態。本研究目的為探討自朵麗蝶蘭幼嫩花序選殖之TCP和RAD基因與控制蘭花花朵兩側對稱之關係。利用Doritaenopsis Sogo Vivien ‘F858’和Phalaenopsis Little Mary之野生型和三唇瓣(peloric)突變種進行分析。在形態上，突變種於0.2 cm花苞時期即可觀察到翼瓣有唇瓣化突起，突變之Dtps. Sogo Vivien ‘F858’有時會有花粉塊缺失，而Phal. Little Mary之三唇瓣突變花朵無花粉塊。經南方雜合分析，推測TCP和RAD於Dtps. Sogo Vivien ‘F858’基因組DNA中皆具有2個以上拷貝數，而TCP和RAD於Phal. Little Mary基因組DNA中拷貝數分別有1個和4個以上。分別取根尖、葉片及全開花朵進行RT-PCR分析，結果顯示組織各部位中全開花朵之TCP基因表現量最高，三唇瓣突變種之表現量較野生型低，RAD基因表現在組織各部位之間沒有明顯的差異，但野生型之表現量較突變種高。花發育各時期之TCP表現量以0.6 cm花苞表現量較高。以原位雜合(in situ hybridization)分析TCP和RAD基因之表達情形，顯示其主要表達於花序分生組織和花原體中。

The genes controlling floral symmetry in Antirrhinum encode proteins belonging to the TCP and MYB gene families. The purpose of this study was to clone the TCP and RAD genes and to compare their expression in relation to morphological changes in Doritaenopsis. Petals of the peloric mutants presented labellum-like protuberance at 0.2 cm flower bud. Occasionally peloric mutants lack pollinia. There were at least two copies of TCP and RAD in the Dtps. Sogo Vivien ‘F858’ genome, and at least one copy and four copies of TCP and RAD in Phal. Little Mary genome, respectively. RT-PCR analysis showed that TCP was preferentially expressed in flower, and weaker in root tips and leaves. Peloric mutants showed similar expression pattern but with lower level than the wild-type. Expression level of RAD was higher in wild-type than the mutants. In situ hybridization analysis showed that TCP and RAD genes were preferentially expressed in florescence meristem and floral primordia.

中文摘要 I
英文摘要 II
誌謝 III
目錄 VI
圖索引 IX
表索引 XII
壹、前言 1
貳、前人研究 2
一、蝴蝶蘭與朵麗蝶蘭之介紹和開花形態 2
二、植物生殖生長 3
三、控制植物對稱性之分子機制 3
(一) 植物對稱性之發育 4
1. 頂端-基部（Apical-Basal, A-B）非對稱性 4
2. 胚內-外（Inside-Outside）非對稱性 5
3. 胚的兩側對稱 7
4. 側器官（lateral organs）非對稱性 8
(二) 花朵對稱性之形態 10
(三) 控制花朵對稱性之相關基因 11
四、控制花朵對稱性之轉錄因子家族 14
(一) TCP基因家族 14
1. TCP基因之結構特色與功能 14
2. TCP基因家族其他成員 15
(1) TB1基因 15
(2) PCFs基因 16
(二) Myb基因家族 16
1. Myb基因之結構特色與功能 17
2. Myb基因家族成員及功能 17
叁、材料與方法 18
一、試驗材料 18
二、試驗方法 18
（一）植株形態觀察 18
1. 花苞各時期及花器各部位形態 18
2. 電子顯微鏡觀察 18
（二）植物基因組DNA萃取 19
（三）植物總量RNA萃取 20
（四）TCP和RAD基因序列分析 21
1. 核酸序列之轉譯 21
2. 胺基酸序列相似性之比較（Blast analysis of NCBI )21
3. 胺基酸多序列比對（Alignment and tree of CLC）21
4. TCP 和RAD胺基酸多序列親緣圖之繪製（Alignment and tree of CLC）21
5. 分析胺基酸之親殊水性（Protein analysis of CLC）21
6. 胺基酸之二級結構圖（Protein analysis of CLC）21
7. conserved domain 和motif位置（Blast analysis of NCBI and protein analysis of CLC） 21
8. MicroRNA 作用序列和位置 22
（五）TCP和RAD基因拷貝數分析 22
1. 限制酶切割反應 22
2. DNA純化及電泳分析 22
3. DNA轉漬及固定 22
4. 探針製備 23
5. 雜合反應 (Hybridization) 23
6. 免疫反應 24
（六）反轉錄聚合酉連鎖反應（RT-PCR）25
（七）RT-PCR之南方墨點分析（Southern blot analysis）25
1. DNA轉漬和固定 25
2. 探針製備 26
3. 雜合反應 26
4. 免疫反應偵測 26
（八）RNA原位雜合（in Situ hybridization）27
1. 樣品製備 27
2. 滲蠟和包埋 27
3. 組織切片和展片 28
4. DIG-labeled RNA探針製備 28
(1) 模板製備 28
(2) 試管內轉錄（in vitro transcripstion）28
(3) 探針品質偵測與定量 29
5. 雜合反應 29
6. 淘洗 30
7. 偵測和呈色 30
（八）農桿菌轉殖菸草和阿拉伯芥 30
1. 載體構築 30
2. 農桿菌媒介之菸草轉殖 31
3. 農桿菌媒介之阿拉伯芥轉殖 31
4. 以PCR鑑定轉殖株 32
肆、結果 33
一、野生型和突變種植株形態 33
二、TCP和RAD基因序列分析 34
三、TCP和RAD基因拷貝數分析 35
四、TCP和RAD基因之表現量分析 36
（一）組織各部位表現量分析 36
（二）花各時期表現量分析 36
（三）花瓣各部位表現量分析 37
五、原位雜合分析 37
六、轉殖植株之形態觀察 37
伍、討論 38
一、野生型和突變種植株形態 38
二、TCP和RAD基因序列分析 39
三、朵麗蝶蘭和蝴蝶蘭TCP和RAD基因之表現分析 40
陸、結論 43
參考文獻 75
附錄 89
附錄1. 控制花朵背腹非對稱性之基因模式圖 89
附錄2 蝴蝶蘭Phalaenopsis Sogo Romantic之野生型和突變種之全 花形態 90
附錄3. pCAMBIA 1305.3載體圖譜 91
附錄4. pCAMBIA 1302載體圖譜 92
附錄5. pH2GW7載體圖譜 93
附錄6. Dtps. Sogo Vivien 和Phal. Little Mary花瓣各部位之比較 94
附錄7. Dtps. Sogo Vivien RAD和其他與細胞形態發育相關基因domain之比較 95
作者簡介96

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