臺灣博碩士論文加值系統

蝴蝶蘭屬 (Phalaenopsis)的花形漂亮，花色豔麗，花期長，多為觀賞佈置等商業用途。台灣因地理環境因素適合栽種蝴蝶蘭，不僅為我國外銷出口重要花卉之一，更使得台灣享有「蝴蝶蘭王國」的美名。蘭花主要是以雜交育種方式來增添其多樣性，近年來有不少研究利用基因轉殖技術或誘變方法改變蝴蝶蘭花色及外表形態，但卻少有針對植株大小做改變。在前人研究中發現，轉殖GA2-oxidase 基因的作物具有矮小迷你的外表形態，並有多根、多分蘗的情形出現；蝴蝶蘭花期長，但也因幼年期與生殖週期較長，培育過程中往往需要耗費較多人力物力照顧，相對提高種植成本。在過去研究中發現 MADS-box 基因具有調控花器與分生組織的功能，使得作物有早開花的情形發生，因此本試驗以攜帶矮小基因 (Ubi-GA2ox6)及早開花基因 (Ubi-MADS14)之農桿菌對蝴蝶蘭進行感染，建立基因轉殖系統。 在研究中共使用 6550 棵未轉殖之擬原球體以每盤30 – 40 棵為單位，分別進行農桿菌感染，最後得到 3 棵 GA2ox6 再生植株。這 3 棵再生殖株與同時期的未轉殖株相比，具有矮小、根多、葉片小且葉片較深等外表型態。擷取其葉片與根尖進行 GUS 活性分析，在組織中出現很明顯的藍色。進一步抽取其 DNA 和 RNA 利用 PCR 進行分析，發現這 3 棵再生植株皆具有 GUS、Hyg 及目標基因- GA2ox6，因此確定為 GA2ox6 轉殖株。在南方點墨實驗結果中得知，轉殖株 2 號具有兩個copy，轉殖株 1 號及 3 號僅有一個。

The flowers of Phalaenopsis orchid have beautiful shape, brightly colors with long flowering period, are mostly used for commercial purposes, such as ornamental layout. Due to the semi-tropical location and climate condition, Taiwan becomes the most appropriate location for planting orchids and this makes orchids the most important export flowers in Taiwan, and consequently makes Taiwan as the "Orchid Kingdom" in the world. The diversities of orchid were mainly crossbred in the past; however many studies using transgenic or mutagenesis methods to change the color and/or appearance of the orchid morphology have been reported recently. Among them, few were used to change the plant architectures. In order to minimize and create novel orchid architectures, a rice OsGA2ox6 gene, involved in GA biosynthesis and when it over-expressed it could produce semi-dwarf plants with multiple shoots and roots, was used in this study. In addition to the long flowering period, the Phalaenopsis orchids have at least 2 to 3 year of growth period to reach the flowering stage, therefore more planting costs were needed. In order to shorten the time need before flowering, a MADS-box gene, MADS14, involved in the regulation of floral initiation and early flowering, was used under the control of a ubiquitin promoter as well. In this study, totally 6550 protocorm-like bodies (PLBs) were transformed by pCAM1301、 pCAM1301-Ubi-GA2ox6 and pCAM1301-Ubi-MADS14 vectors respectively. After hygromycin selection, three regenerated orchid plants from pCAM1301-Ubi-GA2ox6 were obtained and named as GA-1、GA-2 and GA-3, respectively. Comparing to the non-transgenic orchid, these regenerated orchids show dwarfism, multiple shoots and roots, and have smaller and dark-green leaf. All three regenerated orchids show GUS activity in examined leaf and root tissues. Genomic PCR analysis demonstrated that these three regenerated orchids contain the GUS and hptII genes and the GA2ox6 transgene as well. RNA expressions of the GA2ox6 transgene in these three regenerated orchids were detected by RT-PCR and showed the same expression levels among them. Southern blot assay showed these three regenerated orchids were independent transgenic orchid lines with one copy of T-DNA insertion in both GA-1 and GA-3, and two copies of T-DNA insertion in GA-2 line.

中文摘要•••••••••••••••••••••••••••ⅰ
英文摘要•••••••••••••••••••••••••••ⅱ
目錄••••••••••••••••••••••••••ⅲ
圖表目錄•••••••••••••••••••••••••••ⅵ
附錄••••••••••••••••••••••••••ⅷ
縮寫字對照•••••••••••••••••••••••••••ⅸ
前言••••••••••••••••••••••••••1
前人研究•••••••••••••••••••••••••••3
一、蝴蝶蘭簡介•••••••••••••••••••••••••••3
(一)、蝴蝶蘭分類•••••••••••••••••••••••••••3
(二)、蝴蝶蘭生長方式•••••••••••••••••••••••••••4
(三)、蝴蝶蘭生長習性及開花條件•••••••••••••••••••••••••••4
二、蝴蝶蘭市場需求•••••••••••••••••••••••••••5
三、蝴蝶蘭轉殖•••••••••••••••••••••••••••6
(一)、基因槍轉殖•••••••••••••••••••••••••••6
(二)、農桿菌轉殖•••••••••••••••••••••••••••7
四、OsGA2OX6功能分析及研究•••••••••••••••••••••••••••7
五、OsMADS14功能分析及研究•••••••••••••••••••••••••••8
材料與方法•••••••••••••••••••••••••••11
一、儀器及設備••••••••••••••••••••••••••11
二、藥品••••••••••••••••••••••••••11
三、試驗材料••••••••••••••••••••••••••11
(一)、蝴蝶蘭品種來源••••••••••••••••••••••••••11
(二)、農桿菌質體構築••••••••••••••••••••••••••11
四、培養基••••••••••••••••••••••••••12
(一)、微生物培養基••••••••••••••••••••••••••12
(二)、蘭花培養基••••••••••••••••••••••••••12
五、蝴蝶蘭基因轉殖及再生流程••••••••••••••••••••••••••13
(一)、蝴蝶蘭擬原球體再生••••••••••••••••••••••••••13
(二)、農桿菌培養••••••••••••••••••••••••••13
(三)、蝴蝶蘭之基因轉殖••••••••••••••••••••••••••14
(四)、蝴蝶蘭之再生篩選••••••••••••••••••••••••••14
六、蘭花轉殖株基因分析••••••••••••••••••••••••••14
(一)、轉殖株基因組DNA之萃取••••••••••••••••••••••••••14
(二)、聚合酶連鎖反應••••••••••••••••••••••••••15
(三)、南方點墨分析法••••••••••••••••••••••••••15
七、蘭花轉殖株基因偵測表現••••••••••••••••••••••••••17
(一)、轉殖株Total RNA之萃取••••••••••••••••••••••••••17
(二)、反轉錄聚合酶連鎖反應••••••••••••••••••••••••••17
(三)、基因表現量偵測之聚合酶連鎖反應•••••••••••••••••18
八、蘭花轉殖株 GUS 基因偵測表現••••••••••••••••••••••••18
九、蘭花轉殖株處理Gibberellin (GA)•••••••••••••••18
(一)、以Gibberellin處理轉殖株••••••••••••••••••••••••18
(二)、轉殖株擬原球體 (PLB)處理Gibberellin•••19
結果•••••••••••••••••••••••20
一、蝴蝶蘭組織培養再生系統建立••••••••••••••••••••••••••••20
二、蝴蝶蘭轉殖系統之建立•••••••••••••••••••••••••••20
(一)、抗生素篩選•••••••••••••••••••••••••••20
(二)、蝴蝶蘭轉殖篩選流程之建立•••••••••••••••••••••••••••21
(三)、以Hygromycin篩選蝴蝶蘭再生轉殖株•••••••••••22
三、蝴蝶蘭再生植株之外表性狀••••••••••••••••••••••••••22
四、蝴蝶蘭再生植株基因轉殖之偵測•••••••••••••••••••••••••23
(一)、再生植株GUS基因表現•••••••••••••••••••••••••23
(二)、再生植株轉殖基因分析•••••••••••••••••••••••••23
(三)、GA2ox6轉殖株Southern blot分析••••••••••••••23
(四)、再生植株RNA分析表現•••••••••••••••••••••••••24
五、蝴蝶蘭轉殖株處理Gibberellin (GA)之表現••••••25
六、蝴蝶蘭轉殖株 PLB 處理Gibberellin (GA)之表現•••••••••••••••25
討論••••••••••••••••••••••••26
一、蝴蝶蘭組織培養再生系統建立•••••••••••••••••••••••••26
二、蝴蝶蘭轉殖株之外表性狀觀察•••••••••••••••••••••••••26
三、蝴蝶蘭轉殖株及PLB處理Gibberellin之結果分析••••••••••••••••••27
四、結論與檢討•••••••••••••••••••••••••28
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