臺灣博碩士論文加值系統

在被子植物的外觀上，花為其最顯眼的特徵，所有的花依構造都能分為四個部份，由外到內為花萼、花瓣、雄性生殖器官（如：雄蕊）及雌性生殖器官（如：雌蕊）。目前已知花部各器官的發育機制可由ABC model來解釋，調控花部器官發育的基因主要分成A、B、C、D和E五群，A群和E群共同調控最外層的花萼發育，A群、B群和E群共同調控第二層的花瓣發育，B群、C群和E群共同調控第三層的雄蕊發育，C群和E群共同調控第四層的雌蕊發育，D群和E群共同調控胚珠發育。
台灣為蝴蝶蘭原產地之一，其主要分佈於南部及蘭嶼。台灣有兩種蝴蝶蘭原生種：台灣阿媽 (P. amabilis var. formosa)和姬蝴蝶蘭 (P. equestris)。由於蝴蝶蘭顏色多樣，花型優美，花期長，是深具觀賞價值的花卉，因而在切花及觀賞盆栽市場中深受歡迎。而台灣的氣候條件又恰適合其生長，且加上組織培養技術的成熟，使得台灣蝴蝶蘭成為前途看好的新興產業。蝴蝶蘭的花包括了三片花萼、兩片花瓣、一個特化合蕊柱（融合的雄性和雌性生殖器官）以及一片唇瓣（特化的花瓣），其中唇瓣由於正對著蕊柱腔，所以對蘭花的授粉及演化有很大的關連。在栽種蝴蝶蘭的過程常可發現一種體胚變異株，這種變異株合蕊柱上的雄蕊消失，且兩片唇瓣變異成類似唇瓣的構造，稱為三唇瓣變異株。藉由研究正常株蝴蝶蘭及三唇瓣變異株蝴蝶蘭的在花瓣、唇瓣和唇瓣化花瓣的基因表現差異，可找出調控花瓣特化為唇瓣的關鍵因子。

在本篇實驗中，鑑定了兩個E群相關基因，PeMADS8和PeMADS9。由northern blot, real-time quantitative PCR和in situ localization的結果可知，PeMADS9專一性的表現在唇瓣並異位表現在三唇瓣突變株的唇瓣化花中，這種表現方式和之前發現的B群基因PeMADS4非常相似，兩者可能都跟唇瓣發育有關。除此之外，還藉由oligonucleotide microarray在蝴蝶蘭花部的EST資料庫中篩選唇瓣發育相關基因，並用real-time quantitative PCR加以確認後，得到PeEREBP1、PeEREBP2和PebHLHL1三個基因有類似PeMADS4的表現情況。其中PeEREBP1和PeEREBP2的基因序列相近於兩個非生物逆境相關基因，TINY2和CBF1，其表現與否和唇瓣發育的關係還需進一步探討。

Flower is a characteristic mark of angiosperms with four types of organs. From outside to inside, sepals, petals, male reproductive organ and female reproductive organ compose four layers encircled structures. The development mechanism of the four types of floral organs is well described in the well-known ABC model.
Phalaenopsis is a member of the Orchidaceae, its flowers contain three sepals and two petals and highly evolved with a gynostemium or column (a fusion of the male and female reproductive organs, with stamen on the column top) and a highly modified petal, the labellum or lip. Because of lip faces to the pollinia and stigmatic cavity of gynostemium, it is considered to be important for both pollination and evolution of orchids. The peloric mutant of Phalaenopsis with lip-like petals is one of the common somaclonal variants observed in Phalaenopsis equestris, a native Phalaenopsis orchid in Taiwan. In the peloric mutant flower, the appearance of two petals looks like lips and resulting actinomorphic contrast to the zygomorphic nature of the wild type flower, and the column is without stamen on the top. According to the ABC model, the development of the second floral whorl organ including petal and lip are under the regulation of the combinatorial effects of A, B and E class genes.
In this thesis, I identified two E-class related genes including both PeMADS8 and PeMADS9. Collective results of northern blot, real-time quantitative PCR and in situ localization together suggest that PeMADS4 and PeMADS9 were closely related to the lip development since they showed similar expression patterns in the floral lip and both expressions were extended to the peloric mutant lip-like petal. In addition, we also sieved out lip development related genes from EST database by using oligonucleotide microarray analysis and results were confirmed by using real-time quantitative PCR. Three genes were identified including PeEREBP1, PeEREBP2 and PebHLHL1 that all showed similar expression patterns as the PeMADS4. Both sequences of PeEREBP1 and PeEREBP2 were similar to the abiotic stress response genes of TINY2 and CBF1. However, the expression patterns showed that they are related to the lip development. The genuine relationship between these two genes and lip development awaits further research.

摘要 1
Abstract 3
誌謝 5
List of Tables 8
List of Figures 9
List of Appendix Tables 11
List of Appendix Figures 12
Introduction 13
Specific aims 20

Materials and methods

Plant materials 21
RNA preparation 21
Real-time RT-PCR 22
Microarray printing 22
Microarray analysis 23
in situ hybridization 24
RNA blot analysis 25
5′-rapid amplification of PeMADS cDNA ends 26
Sequence alignments and construction of
phylogenetic trees 27

Result

Identification of PeMADS8 and PeMADS9
in P. equestris 28
Phylogenetic relationship of PeMADS8 and
PeMADS9 with E-function genes and
AGL6-like genes 29
Expression patterns of PeMADS2~6
and PeMADS8, PeMADS9 29
in situ localization of PeMADS4
and PeMADS9 transcript 32
Transcription factors involved in orchid
lip-development analyzed by using
oligonucleotide microarray 33
Identification of lip development related
genes in P. equestris 35

Discussion

E class and AGL6-like genes 37
B class genes 38
in situ localization 40
Microarray analysis 41
Lip-development related genes 41
Conclusion 43

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