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研究生:梁玉玲
研究生(外文):Yu-Ling Liang
論文名稱:文心蘭之MADSBox基因及其下游調控基因之功能性分析
論文名稱(外文):Functional analysis of orchid (Oncidium Gower Ramsey) MADS box gene and the downstream genes it regulated
指導教授:楊長賢楊長賢引用關係
指導教授(外文):Chang-Hsien Yang
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
畢業學年度:97
語文別:中文
論文頁數:87
中文關鍵詞:MADS box基因文心蘭
外文關鍵詞:MADS boxOncidiumChaCLGUS
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文心蘭是重要的單子葉觀賞花卉,是台灣第三大之外銷切花,主銷售市場為日本,台灣主要栽培品種為「南茜」 (Oncidium Gower Ramsey),栽培幾乎遍布全省,在切花市場上極具經濟價值,但文心蘭花朵發育相關的研究卻不多,本實驗室許多學長姐多年的研究,主要在選殖及分析文心蘭花朵發育相關之MADS box基因,本研究的目的在接續前人自文心蘭中所選殖到的MADS box基因並對這些基因的功能與特性做進一步的分析。
文心蘭OMADS1基因和阿拉伯芥之 AGL6具有高度相似性,將OMADS1轉入阿拉伯芥後出現植株矮小、提早開花與花序異常的性狀。為進一步討論OMADS1 在阿拉伯芥中調控的下游基因,構築35S::OMADS1-GR 轉基因植物以DEX來誘導,再配合cDNA microarray的方式得到一些受OMADS1 促進與抑制的基因;進一步探討ㄧ個受到OMADS1促進的基因ChaCL (ChaC-like family protein)。為了解ChaCL在阿拉伯芥中表現情形與功能,分別進行promoter分析、在阿拉伯芥中大量表現ChaCL與以RNAi抑制ChaCL表現。發現ChaCL的表現位置在根部髓心、保衛細胞、葉脈、葉毛、葉毛基座細胞、莖頂分生組織、成熟花粉等部位有大量表現。在阿拉伯芥中大量表現ChaCL,35S::ChaCL轉基因植物會出現提早開花的性狀,與野生型相比提早約1~2週,其他外型性狀無顯著差異。35S::ChaCL-RNAi 阿拉伯芥轉基因植株,會出現開花時間延遲的性狀,約延遲3~5天,且營養葉數目較多。以real-time PCR相對定量分析檢測35S::ChaCL轉基因植物中GI, CO, FT, SOC1, LFY, AP3之mRNA表現量發現並無顯著差異,顯示ChaCL促進提早開花的機制可能不是光週期調控路徑。藉由分析OMADS1所調控下游基因的功能與特性,將更了解開花調控機制。(第一章)
文心蘭之OMADS5屬於B class中之PaleoAP3族基因,可轉譯出一個含227個胺基酸的蛋白質。其功能可能是在負調控唇瓣的形成。預期若在文心蘭中大量表現OMADS5,可能會抑制唇瓣的形成,使其轉型為花瓣或花萼,相反的若在文心蘭中抑制OMADS5的表現,則可能會促進唇瓣的形成,使文心蘭的花萼花瓣轉型為唇瓣。本研究已完成35S::OMADS5及35S::OMADS-RNAi的構築及文心蘭之基因轉殖,待轉殖文心蘭植株抽花序後將觀察性狀進一步印證推論。本研究另將全長及去除MADS domain之OMADS5轉殖入阿拉伯芥中大量表現,初步觀察性狀發現轉基因植株與野生型相比並無發現顯著差異。(第二章)
Orchids are among the most important plants in the flower market in Taiwan, however little research on MADS box genes has bene reported. Therefore, the isolation of MADS box genes and further study of their roles on orchid (Oncidium Gower Ramsey) flower development is the goal for this study.
Oncidium OMADS1 showed high homology to Arabidopsis MADS box gene, AGL6. Trangenic Arabidopsis ectopically expressed OMADS1 showed novel phenotypes by significantly reducing plant size, flowering extremely early, and losing inflorescence indeterminancy. To analyze the downstream genes of OMADS1 in Arabidopsis, construct contaning 35S::OMADS1 fused with glucocorticoid receptor (GR) was transformed into Arabidopsis. Genes up- or down-regulated by OMADS1 were identified by using cDNA microarray for DEX treated transgenic plants. One gene ChaCL showed up-regulated by OMADS1 were further characterized. To explore the ChacL expression in Arabidopsis, the promoter region of ChacL was fused to the reporter gene encoding β-glucuronidase (GUS) and introduced into Arabidopsis plant by Agrobacteria-mediated gene transfer. GUS activity was detected in root pith, guard cells, venation, trichome, shoot apical meristem and mature pollen of ChacL::GUS plants. Ectopic expression of ChacL caused the early flowering phenotype. By contrast, late flowering phenotype was observed in 35S::ChaCL-RNAi transgenic Arabidopsis. The expression of flowering time genes such as GI, CO, FT, SOC1 and LFY was however not affected in the 35S::ChacL transgenic plant. This data suggested that ChacL promotes flowering through a pathway independent from photoperiod flowering pathway.(Chapter 1)
To investigate sepal/petal/lip formation in Oncidium Gower Ramsey, one B function paleoAP3 gene OMADS5 was characterized. The OMADS5 encodes a 227 amino acid protein. The mRNA for OMADS5 was strongly detected only in sepals and petals and was absent in the lips. This result revealed a possible negative role for OMADS5 in regulating lip formation. Ectopic expression of OMADS5 may cause the conversion of lip into sepal/petal whereas ectopic expression of OMADS5 RNAi may cause the conversion of sepal/petal into lips in Oncidium. In this study, 35S::OMADS5 and 35S::OMADS5-RNAi constructs were constructed and transformed into Oncidium for functional analysis. Further phenotypic analysis for sepal/petal/lip formation in transgenic plants should reveal the function for OMADS5 in regulating flower development in Oncidium. Furthermore, ectopic expression of either full length or truncated MADS box form of OMADS5 caused no phenotypic change in transgenic Arabidopsis.(Chapter 2)
中文摘要 i
Abstract ii
目錄 iv
第一章 文心蘭OMADS1在阿拉伯芥中直接下游基因之功能性分析
致謝 i
中文摘要 ii
Abstract iii
目錄 v
第一章 文心蘭OMADS1在阿拉伯芥中直接下游基因之功能性分析 1
中文摘要 1
Abstract 2
壹 、前言 3
貳 、材料與方法 10
参 、結果 19
一、經由microarray 的實驗數據找尋阿拉伯芥中受OMADS1調控的直接下游基因 19
二、ChaCL在阿拉伯芥中各部位的表現情形 20
三、ChaCL啟動子之分子選殖、載體構築與表現分析 20
四、35S::ChaCL與35S::ChaCL-RNAi分子選殖與載體構築 20
五、35S::ChaCL轉基因阿拉伯芥會出現早開花性狀而35S::ChaCL-RNAi轉基因阿拉伯芥出現晚開花性狀 21
六、35S::ChaCL轉基因阿拉伯芥中開花調控基因的表現情形 21
七、ChaCL於鹽逆境下表現量提升 21
八、35S::ChaCL-mGFP分子選殖、載體構築與性狀分析 21
九、UP7、LOXL與ATOPT1啟動子之分子選殖、載體構築與表現分析 21
肆 、討論 23
伍 、參考文獻 26
陸 、圖表 33
表1-1. 35S::OMADS-GR轉基因阿拉伯芥中被誘導的基因 33
表1-2. 本章所使用之聚合酶連鎖反應所使用之引子之序列 34
圖1-1.預測ChaCL、UP7、ATOPT1與LOXL在promoter區域之MADS box protein binding site 35
圖1-2. ChaCL::GUS轉基因阿拉伯芥GUS染色圖 36
圖1-3.阿拉伯芥ChaCL基因結構與在阿拉伯芥中表現情形 38
圖1-4.35S::ChaCL 與35S::ChaCL-RNAi分子轉殖與構築 39
圖1-5.35S::ChaCL 與35S::ChaCL-RNAi阿拉伯芥轉基因植株性狀分析 40
圖1-6. 35S::ChaCL 阿拉伯芥轉基因植物中ChaCL與開花調控基因表現量相對定量分析 42
圖1-7.野生型阿拉伯芥在鹽逆境中ChaCL的表現量 42
圖1-8. 35S::ChaCL-mGFP汞燈螢光顯微攝影 45
圖1-9. UP7::GUS轉基因阿拉伯芥GUS染色圖 47
圖1-10. ATOPT1::GUS與LOXL::GUS轉基因阿拉伯芥GUS染色圖 48
圖1-11. ChaCL所參與功能模式圖 49
附圖1-1. 阿拉伯芥中四條開花調控路徑中基因的交互作用 50
附圖1-2. 植物花器形成與ABC model 51
附圖1-3. 植物花器形成與ABCDE model 52
附圖1-4. MADS box 蛋白質形成四聚合體模式(quartet model) 53
附圖1-5. 植物MADS box基因MIKC-type蛋白質結構圖 54
附圖1-6. Gen-KB DNA ladder 55
附圖1-7. pGEM○R-T Easy 載體圖譜及限制酶酵素切位(載體大小3015 bp) 56
附圖1-8. pEpyon01K之圖譜。 57
附圖1-9. pEpyon12K之圖譜。 58
附圖1-10. pBlueACTi 轉接載體圖譜及限制酶酵素切位 59
附圖1-11. pBI-mGFP1載體圖譜及限制酶酵素切位 60
附圖1-12. pBI-mGFP3載體圖譜及限制酶酵素切位 61
附圖1-13. 植物偵測環境逆境訊息傳遞路徑 62
第二章 文心蘭OMADS5基因之功能性分析 63
中文摘要 63
Abstract 64
壹 、前言 65
貳 、材料與方法 67
参 、結果 70
ㄧ、文心蘭OMADS5基因序列來源 70
二、文心蘭花器中OMADS5基因之表現特性 70
三、OMADS5功能性分析之策略 70
四、35S::OMADS5、35S::OMADS5△M構築體之構築與轉殖阿拉伯芥植株之篩選 71
五、35S::OMADS5、35S::OMADS5△M之轉基因阿拉伯芥植物性狀分析 71
六、35S::OMADS5-RNAi構築體之構築與文心蘭轉基因植物之篩選 72
肆 、討論 73
一、35S::OMADS5、35S::OMADS5△M轉基因植物性狀探討 73
二、35S::OMADS5-RNAi文心蘭轉基因植物建構目的與預期性狀 74
三、35S::OMADS5文心蘭轉基因載體構築策略與預期性狀 74
伍 、參考文獻 76
陸 、圖表 78
表2-1.本論文中聚合酶連鎖反應所使用之引子之序列 78
圖2-1. 35S:OMADS5 及OMADS5△M 轉基因植物之構築與鑑定 79
圖2-2. 文心蘭35S::OMADS5-RNAi之構築 81
附圖2-1. 文心蘭OMADS5 cDNA序列與其相對應胺基酸序列 83
附圖2-2. 文心蘭OMADS5 與其他物種植物之演化樹分析 84
附圖2-3. OMADS5、OMADS8、OMADS9在文心蘭中各部位之表現量分析 85
附圖2-4. pBI-mGFP2載體圖譜及限制酶酵素切位 86
附圖2-5. pEpyon 42H載體圖譜及限制酶酵素切位 87
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第二章參考文獻

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