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研究生:簡誌良
研究生(外文):Chih-Liang Chien
論文名稱:文心蘭B群MADS box基因之功能分析與應用
論文名稱(外文):Functional Analysis and the Application of the B class MADS box genes in Oncidium Gower Ramsey
指導教授:楊長賢楊長賢引用關係
口試委員:呂維茗陳榮芳
口試日期:2011-07-07
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生物科技學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2011
畢業學年度:99
語文別:中文
論文頁數:65
中文關鍵詞:文心蘭MADS box 基因擬圓球體
外文關鍵詞:OncidiumMADS box geneprotocorm-like body
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為了瞭解南西文心蘭花器的發育機制,從已知的文心蘭MADS基因中選殖具花被特異表達的B群功能性MADS box 基因,OMADS5及OMADS9進行研究。此二個基因屬paleoAP3的分支,其基因表達量OMADS5 mRNA不在唇瓣表現外,在三唇瓣突變株中的表現量也有明顯的下降;OMADS9 mRNA除了表現在花瓣及唇瓣部位,三唇瓣中唇瓣化的部位也有很高的表現量。由此認為OMADS5在唇瓣的形成可能扮演負調控的角色。為了直接瞭解在文心蘭中OMADS5對唇瓣發育的關係,在本篇研究中我們從文心蘭基因體DNA中藉由基因體步行的方式選殖出約2.3 kb的OMADS5啟動子。此外我們將含有35S::OMADS5的pEpyon-32H藉由農桿菌EHA105轉入45天大的文心蘭擬圓球體(PLBs)中,再將受感染的PLBs放置在含有8 μg/ml hygromycin的G10培養基中篩選。接著萃取非轉殖株及轉殖株PLB的全部DNA,以抗性基因(hptII)和OMADS5基因進行PCR來鑑定轉基因植物。為增加轉基因的成功率,利用不同品系文心蘭PLB進行轉基因測試,並以效率佳者作為35S::OMADS5及35S::OMADS9基因轉殖的材料。為了藉由農桿菌基因轉殖而以基因靜默的方式探討OMADS5及OMADS9的功能並且針對文心蘭花形進行改造,故構築出OMADS5 RANi、OMADS9 RNAi以及OMADS5和OMADS9接上SRDX、VP16蛋白融合序列。我們也從文心蘭基因體中選殖到一段含內含子(intron)的α-tubulin片段,並以此內含子構築出文心蘭RNAi轉接載體。由於文心蘭的新品種難以產生,所以期望利用分子育種的技術來創造出新的花器型態,因此藉由調控B功能性MADS box基因預期可能使花被發生型態上的改變。未來文心蘭除了可以改變其花色和花的大小之外,還可以創造出具有各式各樣不同花形的文心蘭。

To investigate floral formation in Oncidium ‘Gower Ramsey’, two paleoAPETALA3 (paleoAP3) genes, O. Grower Ramsey MADS box gene5 (OMADS5) and OMADS9 were characterized in our laboratory. The OMADS5 mRNA was absent in lips and was significantly down-regulated in lip-like organs in the peloric mutant flowers. In addition, the mRNA for OMADS9 was expressed in lip and petals and a high amount of OMADS9 expression was also observed in lip-like organs of peloric mutant flowers. This suggested a possible negative role for OMADS5 in regulating lip formation. To explore the function of OMADS5 in lip formation, we first cloned OMADS5 promoter with 2.3 kb by genomic walking. In addition, the vector pEpyon-32H containing OMADS5 was transformed into 45-day-old Oncidium protocorm-like body (PLB) via Agrobacterium tumefaciens. The transformants were selected by 8 μg/ml hygromycin G10 medium. To confirm that these plants were transformants, total DNA was isolated and analyzed by PCR from PLBs of four transgenic plants to find the appearance of the antibiotic resistant gene (hptII) and OMADS5 gene. We further used new PLB with high efficiency of transformation to perform the 35S::OMADS5 and 35S::OMADS9 transformation. Furthermore, to modify the floral shape of Oncidium, OMADS5 RNAi and OMADS9 RNAi as well as OMADS5/OMADS9 fused with either SRDX or VP16 sequence were constructed and transformed into Oncidium. In order to obtain a useful RNAi transferred construct in Oncidium, an α-tubulin intron from Oncidium genomic DNA was cloned. New Oncidium varieties are difficult to generate; hence molecular breeding is an attractive approach to create a new flower shape. It is expected that the manipulation of B function MADS box genes in this study will potentially convert perianths into differential shapes.

壹、前言...................................................1
貳、材料與方法.............................................7
參、結果
一、文心蘭OMADS5啟動子之選殖與構築........................14
二、35S::OMADS5進行文心蘭擬圓球體之基因轉殖...............14
三、pEpyon-32H-OMADS9載體之構築...........................15
四、文心蘭擬圓球體之GUS短暫表現分析.......................16
五、以新擬圓球體為材料進行35S::OMADS5與35S::OMADS9之基因轉殖........................................................16
六、文心蘭α-tubulin之內含子(intron)選殖與RANi轉接載體pMETA-OnTUi之構築...............................................16
七、文心蘭OMADS5、OMADS9與OnPDS RANi之構築................17
八、OMADS5和OMADS9與SRDX、VP16蛋白融合序列之構築..........18
肆、討論
一、文心蘭OMADS5與OMADS9功能性探討........................19
二、OMADS5與OMADS9基因對文心蘭花形之應用
(一)、在文心蘭中大量表現OMADS5及OMADS9....................20
(二)、利用RANi技術進行文心蘭花形的改造....................22
伍、參考文獻..............................................24
陸、圖表
表1.本篇實驗中進行聚合酶連鎖反應所用之引子................33
圖1.文心蘭OMADS5基因啟動子之選殖..........................35
圖2.文心蘭OMADS5基因啟動子之序列..........................36
圖3.文心蘭OMADS5基因啟動子之構築..........................37
圖4.35S::OMADS5轉型到農桿菌之鑑定.........................38
圖5.35S::OMADS5轉基因擬圓球體(PLB) .......................39
圖6.35S::OMADS5轉基因擬圓球體之鑑定.......................40
圖7.文心蘭OMADS9基因之構築................................41
圖8.文心蘭擬圓球體之GUS短暫表現分析.......................42
圖9.以新PLB進行35S::OMADS5轉基因擬圓球體..................43
圖10.以新PLB進行35S::OMADS9轉基因擬圓球體.................44
圖11.文心蘭α-tubulin內含子(intron)之選殖.................45
圖12.文心蘭α-tubulin內含子之序列.........................46
圖13.文心蘭RNAi轉接載體 pMETA-OnTUi 之構築................47
圖14.OMADS5 RNAi之構築....................................48
圖15.OMADS9 RNAi之構築....................................49
圖16.OnPDS RNAi之構築.....................................50
圖17.35S::OMADS5-SRDX和35S::OMADS5-VP16之構築.............51
圖18.35S::OMADS9-SRDX和35S::OMADS9-VP16之構築.............52
圖19.預期文心蘭花形之轉變.................................53
附圖1.ABCDE model.........................................54
附圖2.pGEM-T Easy載體圖譜.................................55
附圖3.pEpyon-01K雙偶載體圖譜..............................56
附圖4.pEpyon-32H雙偶載體圖譜..............................57
附圖5.pEpyon-11αP雙偶載體圖譜............................58
附圖6.pBlueACTi轉接載體圖譜...............................59
附圖7.文心蘭RNAi轉接載體pMETA-OnTUi之圖譜.................60
附圖8.pEpyon-12H雙偶載體圖譜..............................61
附圖9.文心蘭ConsensusfromContig 26772序列.................62
附圖10.OMADS5與OMADS9在文心蘭各花器中的表現情形...........63
附圖11.OMADS5基因在文心蘭三唇瓣花中的表現情形.............64
附圖12.本篇實驗中所使用的marker...........................65


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