Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/89186
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dc.contributorMenq-Jiau Tsengen_US
dc.contributor曾夢蛟zh_TW
dc.contributor.authorShun-Min Yangen_US
dc.contributor.author楊舜閔zh_TW
dc.contributor.other園藝學系所zh_TW
dc.date2015zh_TW
dc.date.accessioned2015-12-07T06:29:18Z-
dc.identifierU0005-1408201520592800zh_TW
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dc.identifier.urihttp://hdl.handle.net/11455/89186-
dc.description.abstractNobile type Dendrobium is a section of Dendrobium that includes many closely related species and their hybrids. Nobile-type Dendrobiums are cultivars bred mainly from Dendrobium nobile. Nobile type Dendrobium is a new orchid crop to be promoted in Taiwan due to its potentials in economic cultivation. Many plants grown in temperate climates require vernalization and must experience a period of low winter temperature to initiate or accelerate the flowering process. In Taiwan, the culture practice is bringing the pot plants to higher elevation or flower forcing in cold room to satisfy the chilling requirement. The mechanism of vernalization in Dendrobium nobile has been elucidated recently and has revealed ways to regulate flowering of Nobile type Dendrobium. The objective of this study is to develop the innovative biotechnologies for regulating flowering of Nobile type Dendrobium with uniform flowering, low cost, and high marketing value via transformation of vernalization-related genes VRN1 and AGL19. A suitable production system for protocorm-like bodies (PLBs) induction and proliferation of nobile type Dendrobium was established from seeds germinated in vitro. VRN1, AGL19, and FT cDNAs had been cloned from nobile type Dendrobium and verified by DNA sequence analysis. Plant transformed vectors harboring the VRN1, DnAGL19, SOC1, and FT genes under the control of constitutive (using CaMV 35S promoter and constructed into pCAMBIA 2301 and pCAMBIA 1304), tissue specific (using AP1 promoter and constructed into p1304-AP1-IN) or alcohol inducible (using AlcR/AlcA system and constructed into pBJ36-AlcA and pMLBART-AlcR) expression had been constructed. VRN1 and AGL19 genes were co-transformed into the PLB of the nobile type Dendrobium by Agrobacterium-mediated transformation. The regenerated plantlets were selected by antibiotics. The results of GUS activities, PCR and RT-PCR analysis of putative transgenic leaves indicated that the transformed genes were presented in the genome of transformed plantlets, and expressed its mRNA.en_US
dc.description.abstract春石斛(Nobile-type Dendrobium)為石斛蘭屬石斛蘭節(Dendrobium section Dendrobium)內之原種群,以金釵石斛(Den. nobile)為基本種所雜交選育出之品種群的總稱。春石斛是一種具有較高觀賞價值的熱帶蘭中名貴花卉,花色繁多豔麗,花多,花型大,花枝長,在國際花卉市場上佔有重要的地位。台灣地處亞熱帶氣候區,適合春石斛種苗之養成,是台灣極具外銷發展潛力的外銷蘭花,唯品種來源、種苗生產管理技術、低溫催花等技術仍待開發。 最近的研究對春石斛蘭春化調控的途徑有初步的瞭解,本研究以金釵石斛的春化調控的途徑模式為理論基礎,嘗試轉殖VRN1與AGL19等春化作用之相關基因到春石斛蘭。其目的為以基因轉殖技術創新出可調控植株春化作用的技術,開發出花期一致、節省成本、高品質之春石斛蘭。 本研究已完成建立春石斛蘭之種子無菌播種,誘導出擬原球體(PLB)及增生系統。已完成選殖及分析春石斛蘭的VRN1、AGL19及FT等春化作用相關基因。已完成將選殖之VRN1、AGL19、FT cDNA,分別構築到大量表現 (CaMV35S啟動子,構築到pCAMBIA 2013及pCAMBIA 1304)、組織特異表現 (AP1啟動子,構築到p1304-AP1-IN)及化學藥劑(AlcR/AlcA酒精誘導表現系統,構築到pBJ36-AlcA及pMLBART-AlcR)誘導表現之轉殖載體的農桿菌轉殖載體。已進行將攜帶VRN1及AGL19基因之載體,共同轉殖到春石斛蘭之類原球體 (PLB),並進行篩選及誘導轉殖植株再生。轉殖再生培植體葉片之GUS活性、PCR及RT-PCR分析之結果顯示,轉殖之VRN1及AGL19基因已存在於轉殖培植體之基因組,並表現其mRNA。zh_TW
dc.description.tableofcontents中文摘要…………………………………………………………………………………i 英文摘要………………………………………………………………………………ii 目次……………………………………………………………………………………iii 表目次…………………………………………………………………………………v 圖目次…………………………………………………………………………………vi 前言……………………………………………………………………………………1 前人研究………………………………………………………………………………3 一、春石斛簡介………………………………………………………………3 二、影響植物開花的因子………………………………………………………3 (一)、光周期途徑………………………………………………………… 4 (二)、自主成花途徑………………………………………………………4 (三)、春化途徑………………………………………………………………5 (四)、GA誘導途徑…………………………………………………………6 三、人工化學誘導型啟動子……………………………………………………6 (一)、酒精誘導啟動子…………………………………………………… 7 (二)、銅離子誘導型啟動子………………………………………………8 (三)、動物荷爾蒙誘導型啟動子…………………………………………8 (四)、四環黴素誘導型啟動子……………………………………………9 (五)、蛻皮激素誘導啟動子………………………………………………10 三、DnVRN1及DnAGL19基因……………………………………………10 材料與方法……………………………………………………………………………11 一、春石斛低溫春化與春化作用相關基因之選殖…………………………11 (一)、植株材料與春化處理………………………………………………11 (二)、分析與選殖VRN1、AGL19、FT基因之引子…………………11 (三)、春化作用相關基因VRN1、AGL19、FT cDNA選殖…12 二、春化作用相關基因VRN1、AGL19、FT基因轉殖載體之構築…………12 (一)、CaMV35S啟動子………………………………………………… 12 (二)、AP1啟動子…………………………………………………………14 (三)、AlcR/AlcA酒精啟動子……………………………………………14 (四)、實驗方法……………………………………………………………15 三、春石斛之農桿菌基因轉殖與分析……………………………………………17 (一)、轉殖植物材料……………………………………………………… 17 (二)、農桿菌轉殖菌系與轉殖載體………………………………………17 (三)、農桿菌質體冷凍/回潮轉型方法…………………………………18 (四)、擬轉殖植株之檢測…………………………………………………19 結果……………………………………………………………………………………23 一、 春化作用相關基因VRN1、AGL19、FT cDNA選殖………………23 二、 春化作用相關基因VRN1、AGL19、FT 基因轉殖載體之構築……24 三、 春石斛之農桿菌基因轉殖與分析………………………………………27 討論……………………………………………………………………………………77 一、 扇春化作用相關基因VRN1、AGL19、FT cDNA選殖………………77 二、 春化作用相關基因VRN1、AGL19、FT 基因轉殖載體之構築……78 三、 春石斛之農桿菌基因轉殖與分析………………………………………79 四、 擬轉殖株PCR及RT-PCR檢測…………………………………………80 參考文獻………………………………………………………………………………82  表目次 表1、參考已發表之DnVRN1、DnAGL19、DnFT及DnActin等核酸序列所 設計的引子核酸序列………………………………………………………11 表2、檢測VRN1、ACL19、GUS、AlcR及hptII基因使用的引子與PCR所 使用的黏合溫度以及預期片段的長度……………………………………21 表3、除芽與未除芽對春石斛Den. Lucky Girl 及Den. Red Emperor 'Prince' 之小芽苗誘導PLB增生及白化之影響…………………………………65   圖目次 圖1、發表之金釵石斛 (Dendrobium nobile) DnVRN1 cDNA (EF535598.1) 的核酸序列及所設計引子的核酸序列與位置…………………………31 圖2、發表之'南西'文心蘭 (Oncidium 'Gower Ramsey') OnFYF cDNA (HQ591455.1)的核酸序列及所設計引子的核酸序列與位置…………32 圖3、發表之金釵石斛 (Dendrobium nobile) DnFT cDNA 的核酸序列及所 設計引子的核酸序列與位置………………………………………………34 圖4、發表之鐵皮石斛 (Dendrobium officinale) DnActin cDNA (JQ040497.1) 的核酸序列及所設計引子的核酸序列與位置…………………………35 圖5、Den. nobile、Den. Lucky Girl、Den. Red Emperor 'Prince'、Den. To My Kids 'Smile' 等春石斛蘭,經春化處理(13/10℃,D/N)四週後,以RT- PCR分析VRN1、AGL19、FT、Actin等基因表現之情形……………35 圖6、選殖金釵石斛 (Dendrobium nobile)及Dendrobium To my kids 'Smile' VRN1 cDNA 之策略與步驟………………………………………………36 圖7、選殖之VRN1基因與已發表之DnVRNA (EF535598.1) 核酸序列比對 情形……………………………………………………………………………38 圖8、選殖之DnVRN1基因與已發表之DnVRN1 (EF535598.1)轉譯胺基酸 序列比對情形………………………………………………………………39 圖9、選殖之DnAGL19基因與已發表之OnFYF (HQ591455.1)核酸序列比對情 形………………………………………………………………………………40 圖10、選殖之DnAGL19基因與金釵石斛(Den. nobile)之DnAGL19基因核 酸序列比對情形…………………………………………………………41 圖11、選殖之DnAGL19基因與已發表之DnAGL19轉譯胺基酸序列比對情形…42 圖12、選殖之DnFT基因與已發表之DnFT 核酸序列比對情形…………43 圖13、選殖之DnFT基因與已發表之DnFT轉譯胺基酸序列比對情形……44 圖14、自Den. To My Kids 'Smile'選殖之VRN1基因的核酸序列與轉譯胺 基酸序列……………………………………………………………………45 圖15、自Den. nobile 選殖之AGL19基因的核酸序列與轉譯胺基酸序……46 圖16、自Den. nobile 選殖之FT基因的核酸序列與轉譯胺基酸序列………47 圖17、p1304-35S-VRN1及p2301-35S-VRN1載體之構築流程及限制酵素 圖譜…………………………………………………………………………48 圖18、p1304-35S-VRN1與p2301-35S-VRN1載體構築檢測…………………50 圖19、p1304-35S-AGL19及p2301-35S-AGL19載體之構築流程及限制酵 素圖譜………………………………………………………………………51 圖20、p1304-35S-AGL19與p2301-35S-AGL19載體構築檢測………………53 圖21、p1304-35S-FT及p2301-35S-FT載體之構築流程及限制酵素圖譜 ………………………………………………………………………………54 圖22、p1304-35S-FT與p2301-35S-FT載體之構築與檢測…………………56 圖23、p1304-AP1-VRN1載體之構築……………………………………………57 圖24、p1304-AP1-AGL19載體之構築流程……………………………………58 圖25、p1304-AP1-VRN1與p1304-AP1-AGL19載體構檢測…………………59 圖26、pMLBart-AlcR-AlcA-VRN1載體之構築流程…………………………60 圖27、pMLBart-AlcR-AlcA-VRN1載體構檢測。………………………………61 圖28、pMLBart-AlcR-AlcA-AGL19載體之構築流程…………………………62 圖29、pMLBart-AlcR-AlcA-AGL19載體構檢測………………………………63 圖30、春石斛Den. Lucky Girl 及Den. Red Emperor 'Prince'之小芽苗,經 過除芽與未除芽,PLB增生的情形………………………………………64 圖31、春石斛蘭Den. Red Emperor 'Prince'之農桿菌感染及再生流程………66 圖32、共同轉殖p1304-35S-VRN1及p1304-35S-AGL19之Den. Red Emperor 'Prince'與共同轉殖p1304-AP1-VRN1及p1304-AP1-AGL19之Den. Red emperor及對照組 (CK)之培植體,進行GUS活性染色分析之 情形…………………………………………………………………………68 圖33、農桿菌法共同轉殖p1304-35S-VRN1及p1304-35S-AGL19之Den. Red Emperor 'Prince'及Den. Red Emperor培植體,經PCR分析 CaMV35S-VRN1、CaMV35S-AGL19、GUS、hptII基因之情形………69 圖34、農桿菌法共同轉殖p2301-35S-VRN1及p2301-35S-AGL19之Den. Red Emperor 'Prince'及Den. Red Emperor培植體,經PCR分析 CaMV35S-VRN1、CaMV35S-AGL19、GUS基因之情形………………70 圖35、農桿菌法共同轉殖p1304-AP1-VRN1及p1304-AP1-AGL19之Den. Red Emperor 'Prince'及Den. Red Emperor培植體,經PCR分析 VRN1-NOS、AGL19-NOS、GUS、hptII基因之情形…………………71 圖36、農桿菌法共同轉殖pMLBart-AlcR-AlcA-VRN1及pMLBart-AlcR-AlcA- AGL19之Den. Red Emperor 'Prince'及Den. Red Emperor培植體,經PCR分析AlcA-VRN1、AlcA-AGL19、AlcR、AlcR-backbone 基因之情形…………………………………………………………………72 圖37、農桿菌法共同轉殖p1304-35S-VRN1及p1304-35S-AGL19之Den. Red Emperor 'Prince'及Den. Red Emperor培植體,經RT-PCR分析 VRN1、AGL19、GUS、hptII mRNA之情形……………………………73 圖38、農桿菌法共同轉殖p2301-35S-VRN1及p2301-35S-AGL19之Den. Red Emperor 'Prince'及Den. Red Emperor培植體,經RT-PCR分析 VRN1、AGL19 mRNA之情形……………………………………………74 圖39、農桿菌法共同轉殖p1304-AP1-VRN1及p1304-AP1-AGL19之Den. Red Emperor 'Prince'及Den. Red Emperor培植體,經RT-PCR分析 VRN1、AGL19、GUS、hptII mRNA之情形……………………………75 圖40、農桿菌法共同轉殖pMLBart-AlcR-AlcA-VRN1及pMLBart-AlcR- AlcA-AGL19之Den. Red Emperor 'Prince'及Den. Red Emperor 體,經RT-PCR分析VRN1、AGL19、AlcR mRNA之情形…………76zh_TW
dc.language.isozh_TWzh_TW
dc.rights同意授權瀏覽/列印電子全文服務,2018-08-20起公開。zh_TW
dc.subject春石斛zh_TW
dc.subject春化基因zh_TW
dc.subject基因轉殖zh_TW
dc.subjectNobile-type Dendrobiumen_US
dc.subjectVernalization geneen_US
dc.subjectGene transformationen_US
dc.title春石斛蘭春化作用相關基因之選殖與轉殖zh_TW
dc.titleCloning and Transformation of Vernalization-Related Genes in Nobile Type Dendrobium Orchiden_US
dc.typeThesis and Dissertationen_US
dc.date.paperformatopenaccess2018-08-20zh_TW
dc.date.openaccess2018-08-20-
item.openairecristypehttp://purl.org/coar/resource_type/c_18cf-
item.openairetypeThesis and Dissertation-
item.cerifentitytypePublications-
item.fulltextwith fulltext-
item.languageiso639-1zh_TW-
item.grantfulltextrestricted-
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