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研究生： 巫佩珊
Wu, Pei-Shan
論文名稱： 姬蝴蝶蘭花部PeMADS基因啟動子選殖及序列分析
Identification and characterization of promoter of PeMADS genes in Phalaenopsis equestris
指導教授： 陳虹樺
Chen, Hong-Hwa
學位類別： 碩士
Master
系所名稱： 生物科學與科技學院 - 生物學系
Department of Biology
論文出版年： 2003
畢業學年度： 91
語文別： 中文
論文頁數： 82
中文關鍵詞： 啟動子 、蘭花 、花形
外文關鍵詞： promoter, orchid, AP3
相關次數： 點閱：92  下載：1 
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    • 本實驗目的為利用五個調控姬蝴蝶蘭(Phalaenopsis equestris)花部發育相關之轉錄因子PeMADS基因-AP3-like(PeMADS2、PeMADS3、PeMADS4、PeMADS5)及PI-like(PeMADS6)之五端未轉譯區(5’UTR)設計專一性引子，以姬蝴蝶蘭限制酵素基因庫(GenomeWalker library)或微小基因庫(Mini library)選殖出姬蝴蝶蘭五個花部基因轉錄因子之5’端啟動子區域。利用基因啟動子的分析，研究基因功能。此外構築可藉由報導基因表現姬蝴蝶蘭調控花部發育PeMADS基因啟動子之質體，利用農桿菌轉殖法將構築之質體轉殖阿拉伯芥中，利用轉殖植物確認各個基因啟動子之功能，分析五個PeMADS啟動子空間之表現模式。
    最後PeMADS2啟動子共選殖出1,312 bp、PeMADS3啟動子1,300 bp、PeMADS4啟動子418 bp、PeMADS5啟動子自ATG上游共選殖到260 bp及PeMADS6啟動子1,512 bp。其中CArG box在PeMADS2及PeMADS6啟動子各有兩個，在PeMADS4及PeMADS5啟動子各有一個，而PeMADS3啟動子上並未發現有CArG box。最後將較長片段之PeMADS2、PeMADS3及PeMADS6啟動子片段接上GUS報導基因以農桿菌法轉殖入阿拉伯芥中，以偵測啟動子之表現情形。

    Previously, five B-class of MADS box genes were identified in Phalaenopsis equestris including four AP3-like genes, PeMADS2, PeMADS3, PeMADS4, PeMADS5 and a PI-like gene, PeMADS6. In this research, the promoters of the fine PeMADS genes were isolated and characterized. Both PCR-based method and mini-library screening were used for cloning the PeMADS promoters.
    Results showed that the cloned promoter regions upstream of the transcription start site (TSS) of PeMADS2, PeMADS3, PeMADS4, and PeMADS6 genes were 1,312 bp, 1,300 bp, 418 bp and 1,512 bp, respectively. So far, only 260-bp sequence was identified upstream from the ATG of PeMADS5 gene. Two putative CArG box motifs were identified in the promoters of PeMADS2 and PeMADS6, and one in the promoter regions of PeMADS4 and PeMADS5. However, no CArG box can be identified in the PeMADS3 promoter region. To gain insight into the molecular basis of the regulation of PeMADS promoters, PeMADS promoter::GUS gene fusions were performed by means of the Agrobacterium-mediate transformation system in Arabidopsis. Results of these studies will provide knowledge to floral organ-, development stage-specific regulation in P. equestris.

    目錄 第一章、前言……………………………………………………………………1 第二章、文獻探討………………………………………………………………3 (一) MADS box基因與植物花器發育調控之關聯…………………………… 3 1. ABC模式(ABC model)……………………………………………………… 3 2. MADS box蛋白質的特性…………………………………………………… 4 3. 植物界的MADS box基因…………………………………………………… 4 4. APETAL 3及基因啟動子之研究…………………………………………… 5 5. 姬蝴蝶蘭花苞序列標籤資料庫之PeMADS box基因之初步分析結果…… 7 (二) 植物基因轉殖之研究…………………………………………………… 8 1. 植物基因轉殖之發展…………………………………………………8 2. 農桿菌基因轉殖法……………………………………………………9 第三章、材料與方法……………………………………………………………12 (一) 材料……………………………………………………………………… 12 1. 植物材料…………………………………………………………………… 12 2. 基因序列…………………………………………………………………… 12 3. 培養基及抽氣溶液………………………………………………………… 12 (二) 研究方法………………………………………………………………… 13 1. 啟動子選殖………………………………………………………………… 13 1.1抽取姬蝴蝶蘭基因組DNA……………………………………………………13 1.2建構姬蝴蝶蘭限制酵素基因庫(GenomeWalker library)……………… 14 1.2.1以限制酵素切基因組DNA…………………………………………………14 1.2.2純化切割之基因組DNA……………………………………………………14 1.2.3接合反應(ligation) ……………………………………………………15 1.3聚合酵素連鎖反應(GenomeWalker PCR) …………………………………15 1.4自洋菜膠回收DNA片段………………………………………………………16 1.5基因組DNA選殖及DNA定序………………………………………………… 16 1.5.1質體接合(ligation) ……………………………………………………16 1.5.2轉型作用(Transformation)…………………………………………… 16 1.5.3微量製備質體…………………………………………………………… 17 1.5.4 DNA自動定序…………………………………………………………… 18 1.5.4.1 Big Dye反應………………………………………………………… 18 1.5.4.2酒精沈澱……………………………………………………………… 18 1.6南方轉漬法(Southern blot)………………………………………………18 1.6.1萃取基因組DNA及切割基因組DNA……………………………………… 18 1.6.2酒精沉澱及洋菜膠電泳………………………………………………… 19 1.6.3 DNA轉漬………………………………………………………………… 19 1.6.4探針(probe)的製備………………………………………………………20 1.6.5南方雜合反應(Hybridization)…………………………………………20 1.7建構姬蝴蝶蘭微小基因庫(Mini library)……………………………… 20 1.7.1自洋菜膠回收DNA片段……………………………………………………20 1.7.2聚合酵素連鎖反應………………….……………………………………20 1.7.3質體接合、轉型作用及篩選藍白菌落 (blue/white colony screening)………………………………………21 1.7.4微小基因庫(Mini-library)篩選……………………………………… 21 1.7.4.1微小基因庫轉漬膜製備……………………………………………… 21 1.7.4.2雜合反應……………………………………………………………… 22 1.8啟動子序列分析…………………………………………………………… 22 1.8.1利用軟體分析啟動子序列……………………………………………… 22 1.8.2 CArG序列分析……………………………………………………………23 2. 姬蝴蝶蘭調控花部發育基因啟動子之功能分析………………………… 23 2.1選殖PeMADS基因啟動子之專一性引子設計……………………………… 23 2.2 T-A選殖法(T-A cloning) ……………………………………………… 23 2.3構築Ti (pBI101)載體………………………………………………………23 2.3.1質體DNA切割及回收………………………………………………………23 2.3.2質體接合(ligation)…………………………………………………… 24 2.3.3轉型作用及微量製備質體……………………………………………… 24 2.4農桿菌轉殖法……………………………………………………………… 24 2.4.1農桿菌勝任細胞製備…………………………………………………… 24 2.4.2電穿孔(electroporation) …………………………………………… 25 2.4.3微量製備質體…………………………………………………………… 25 2.4.4種植野生型阿拉伯芥…………………………………………………… 25 2.4.5基因轉殖(抽氣轉殖) ……………………………………………………26 2.5種植轉殖植株---阿拉伯芥…………………………………………………26 2.6轉殖分析---報導基因………………………………………………………27 第四章、結果……………………………………………………………………28 (一) 五個PeMADS基因啟動子選殖結果及初步序析………………………… 28 1. 利用姬蝴蝶蘭限制酵素基因庫及微小基因庫選殖PeMADS2~6基因 啟動子……………………………………………………………………… 28 1.1 PeMADS2之選殖結果及初步序列分析…………………………………… 28 1.2 PeMADS3之選殖結果及初步序列分析…………………………………… 28 1.3 PeMADS4之選殖結果及初步序列分析…………………………………… 29 1.4 PeMADS5之選殖結果及初步序列分析…………………………………… 30 1.5 PeMADS6之選殖結果及初步序列分析…………………………………… 31 2. 五個PeMADS啟動子之序列分析…………………………………………… 31 2.1啟動子之序列分析………………………………………………………… 32 2.2 CArG box 在PeMADS2~6基因啟動子之位置及序列………………………32 2.3啟動子序列之軟體分析結果……………………………………………… 32 (二) 三個PeMADS基因啟動子之功能分析初步結果………………………… 33 1. PeMADS2之選殖結果及轉殖初步分析………………………………………33 2. PeMADS3之選殖結果及轉殖初步分析………………………………………33 3. PeMADS6之選殖結果及轉殖初步分析………………………………………34 第五章、討論……………………………………………………………………35 1. 由五個PeMADS cDNA基因全長，設計引子選殖啟動子……………………35 2. 利用微小基因庫(Mini-library)篩選啟動子…………………………… 36 2.1利用微小基因庫篩選PeMADS4啟動子………………………………………36 2.2利用微小基因庫篩選PeMADS5啟動子………………………………………36 3. 啟動子序列分析及預測…………………………………………………… 37 3.1 PeMADS2及PeMADS6啟動子…………………………………………………38 3.2 PeMADS3A及PeMADS3B啟動子………………………………………………39 3.3 PeMADS4及PeMADS5啟動子…………………………………………………40 4. 結語………………………………………………………………………… 40 第六章、參考文獻………………………………………………………………42 表目錄 表一、PeMADS基因啟動子序列長度及序列分析結果…………………………51 表二、PeMADS基因啟動子之CArG box之序列比較……………………………52 圖目錄 圖一、選殖五個PeMADS基因啟動子所使用之基因專一性引子位置圖………53 圖二、PeMADS2 GenomeWalker聚合酵素連鎖反應結果………………………55 圖三、pGEM-T Easy/ PeMADS2、PeMADS3、PeMADS4、PeMADS5及PeMADS6 promoter質體……………………………………………………………56 圖四、PeMADS3 GenomeWalker聚合酵素連鎖反應結果………………………57 圖五、PeMADS3A及PeMADS3B之序列分析圖……………………………………58 圖六、PeMADS4 GenomeWalker聚合酵素連鎖反應結果………………………59 圖七、PeMADS4基因之南方轉漬結果………………………………………… 60 圖八、PeMADS5 GenomeWalker聚合酵素連鎖反應結果………………………61 圖九、PeMADS5基因之南方轉漬結果………………………………………… 62 圖十、PeMADS5之Mini-library結果………………………………………… 63 圖十一、以5’UTR設計引子所選殖出之PeMADS5啟動子及PeMADS5 5’UTR之 BLASTN比對結果圖.………………………………………………… 64 圖十二、利用Pe5+489r及Pe5+382r引子之PeMADS5 GenomeWalker 聚合酵素連鎖反應結果………………………………………………65 圖十三、PeMADS6 GenomeWalker聚合酵素連鎖反應結果……………………66 圖十四、PeMADS2~6之CArG box之位置圖…………………………………… 67 圖十五、PeMADS2啟動子PCR反應結果…………………………………………69 圖十六、PeMADS3啟動子PCR反應結果…………………………………………70 圖十七、PeMADS6啟動子PCR反應結果…………………………………………71 圖十八、pBI101質體/ PeMADS2、PeMADS3及PeMADS6 promoter質體………72 圖十九、PeMADS5之trans-splicing過程推測圖…………………………… 73 附表目錄 附表一、姬蝴蝶蘭花苞序列表現標籤資料庫PeMADS基因表現結果…………75 附表二、AP3及DOMADS1之CArG box之序列……………………………………76 附圖目錄 附圖一、論文材料，姬蝴蝶蘭…………………………………………………77 附圖二、建構姬蝴蝶蘭限制酵素基因庫(GenomeWalker library)之流程圖78 附圖三、AP3及DOMADS1啟動子之CArG box之位置圖…………………………79 附圖四、PI啟動子………………………………………………………………80 附圖五、pGEM-T Easy質體…………………………………………………… 81 附圖六、pBI101質體……………………………………………………………82

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