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研究生:王為莉
論文名稱:持續表現百脈根LjCYC3基因對轉殖矮牽牛的影響
論文名稱(外文):The effect of over expression of Lotus japonicus CYC3 gene in transgenic petunia
指導教授:侯新龍詹明才
學位類別:碩士
校院名稱:國立嘉義大學
系所名稱:農藝學系研究所
學門:農業科學學門
學類:一般農業學類
論文種類:學術論文
畢業學年度:100
語文別:中文
中文關鍵詞:cycloidea矮牽牛TCP花部對稱性
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花卉產業是台灣重要的出口項目,因此選育出具有特殊性狀的新品種,是保持花卉產業競爭力的重要課題。CYC (CYCLOIDEA) 最初在金魚草 (Antirrhinum majus) 被證實為調控花瓣對稱性之基因,其所屬之TCP家族 [TEOSINTE BRANCHED1 (TB1) 、CYC和proliferating cell factor (PCF)] 主要調控細胞增殖及分化表現。在模式植物阿拉伯芥 (Arabidopsis) 與百脈根 (Lotus japonicus) 已經證實經由導入CYC可以改變花部形態。因此我們分析CYC在各物種間的基因相似度,發現豆科CYC基因演化散布在各群中,推測其基因演化具有較高的歧異度。蛋白質定位分析結果顯示CFP-LjCYC3蛋白產物會表現於遠源植物的細胞核中。以輻射對稱花形的矮牽牛 (Petunia)為材料進行農桿菌轉殖法導入LjCYC3 ,以期能使花部形態產生特殊表現型。獲得轉殖株後,利用南方轉漬法 (Southern blot),確認外來基因已嵌入植物染色體組。形態觀察發現轉殖株有明顯的捲曲葉片與花朵。以即時定量聚合酶連鎖反應分析結果證實大量表現LjCYC3的轉殖植物才會產生捲曲形態。同時發現大量表達LjCYC3的轉殖株其植株莖長度、節間長度與葉柄長度,有明顯縮小的現象,但節間數目的差異不顯著。此外,觀察表皮細胞發現這些轉殖植物的排列較緊密,細胞形狀也較小。推測過量表現LjCYC3可能影響到細胞週期(cell cycle)相關基因所導致。而矮牽牛轉殖株花瓣除捲曲表現外並沒有發現對稱性或形狀上的改變。本試驗結果顯示LjCYC3大量表現在矮牽牛可能調控植株細胞發育過程。本試驗中以LjCYC3為外源基因轉入矮牽牛,證實會造成轉殖株莖長度、葉柄長度和節間長度的減少,因而產生植株矮小的形態。而LjCYC3轉殖株的葉片與花瓣都具有捲曲的表現型,且發現細胞均有縮小現象,推測可能是造成轉殖株形態產生變異的原因。
目錄
摘要 I
Abstract II
目錄 III
圖目錄 V
第一章 緒論 1
一、 前言 1
二、 影響花部形態發育的因素 2
三、 影響花部形態對稱性的基因 3
四、 TCP家族在植物中的功能 4
五、 CYC對花部對稱性的作用 7
六、 LjCYC影響植物的生長發育 8
七、 轉殖技術的應用 9
八、 矮牽牛(Petunia hybrida) 9
九、 研究目的 10
第二章 材料與方法 11
一、 材料 11
二、 序列分析 11
三、 製備大腸桿菌(Escherichia coli)勝任細胞(competent cell) 12
四、 大腸桿菌XL1-Blue之轉形 12
五、 質體DNA的純化 13
六、 質體DNA聚合酶連鎖反應 (polymerase chain reaction, PCR) 13
七、 農桿菌(Agrobacterium tumefaciens )轉形作用 14
八、 農桿菌生化測試 (Ketolactose test) 14
九、 矮牽牛農桿菌轉殖試驗 15
十、 矮牽牛DNA純化 16
十一、 南方轉漬法 16
十二、 矮牽牛農桿菌轉殖試驗 19
十三、 RNA電泳 20
十四、 反轉錄聚合酶連鎖反應 (Reverse transcription PCR) 20
十五、 即時定量PCR (quantitative real time polymerase chain reaction) 21
十六、 石蠟切片 21
十七、 蛋白質定位分析 23
第三章 結果 24
一、 百脈根LjCYC3與其他物種間親緣演化分析 24
二、 百脈根LjCYC同源基因胺基酸序列分析 25
三、 百脈根LjCYC3基因轉殖矮牽牛 25
四、 百脈根LjCYC3轉殖矮牽牛之DNA鑑定 26
五、 轉殖LjCYC3矮牽牛外表型的觀察 27
六、 百脈根LjCYC3轉殖矮牽牛基因表現與性狀分析 28
七、 轉殖LjCYC3矮牽牛植株各部位細胞形態分析 29
八、 百脈根LjCYC3蛋白定位分析 30
第四章 討論 31
一、 百脈根LjCYCs與其他物種間胺基酸序列分析 31
二、 百脈根LjCYC3與TCP domain特性分析 32
三、 轉殖LjCYC3矮牽牛基因表現分析 33
四、 轉殖LjCYC3矮牽牛的外表型分析 34
五、 百脈根LjCYC3基因蛋白質定位分析 35
六、 未來展望 36
圖表 37
參考文獻 56
附錄 63


圖目錄
圖 一、不同植物間CYC的親緣演化分析。……………………………………...37
圖 二、Lotus japonicus 內cycloidea胺基酸序列相似度比對。………………..38
圖 三、LjCYC胺基酸序列之親疏水性分析。 ………………………………….....39
圖 四、以限制酶切割35S::CFP-LjCYC3質體DNA。…………………………..40
圖 五、利用Ketolactose測試農桿菌株LBA4404品系。……………………….41
圖 六、矮牽牛轉殖流程。…………………………………………………….........42
圖 七、LjCYC3基因轉殖矮牽牛南方轉漬試驗分析。…………………………...43
圖 八、LjCYC3轉殖矮牽牛表現型之觀察。.…………………………………..44
圖 九、LjCYC3轉殖矮牽牛花朵外表型之觀察。…………………………….…..45
圖 十、LjCYC3轉殖矮牽牛之莖長度。……………………………………….…..46
圖 十一、LjCYC3轉殖矮牽牛之節間數目。……………………………….……..47
圖 十二、LjCYC3轉殖矮牽牛節間長度的比較。………………………….……..48
圖 十三、LjCYC3轉殖矮牽牛葉柄長度的比較。………………………….……..49
圖 十四、LjCYC3轉殖株花苞發育異常。………………………………….……..50
圖 十五、LjCYC3基因於轉殖株葉片組織表現情形。………………….………..51
圖 十六、LjCYC3轉殖株下表皮細胞的形態。…………………………….……..52
圖 十七、LjCYC3轉殖株葉片縱切面組織排列情形。………………….………..53
圖 十八、LjCYC3轉殖株花瓣捲曲部位細胞之觀察。………………….………..54
圖 十九、百脈根LjCYC3蛋白於台灣阿嬤花瓣細胞定位分析。……………..55

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