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研究生：	陳佑亦 Chen, You-Yi
論文名稱：	姬蝴蝶蘭D-群MADS-box基因PeMADS7之功能探討 Functional characterization of D-class MADS-box gene PeMADS7 from Phalaenopsis equestris
指導教授：	蔡文杰 Tsai, Wen-Chieh
學位類別：	碩士 Master
系所名稱：	生物科學與科技學院 - 熱帶植物科學研究所 Institute of Tropical Plant Sciences
論文出版年：	2011
畢業學年度：	99
語文別：	英文
論文頁數：	57
中文關鍵詞：	姬蝴蝶蘭、胚珠、MADS-box 、D群基因
外文關鍵詞：	Phalaenopsis equestris, ovules, MADS-box, D-class genes
相關次數：	點閱：139 下載：3
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蝴蝶蘭是世界上重要的園藝作物，其完美的花型具有高度的觀賞價值。蘭花胚珠發育過程受到授粉作用精確的調控，也使得蘭花胚珠發育有別於一般的被子植物。前人研究中，矮牽牛的MADS-box D-群基因FLORAL BINDING PROTEIN7 (FBP7) 與 FBP11對調控胚珠的發育扮演重要的角色，且FBP7與 FBP11也與阿拉伯芥中的SEEDSTICK (STK)功能非常相似，也會專一性的表現在胚珠當中，表示這些基因屬於直系同源的基因。此外，在酵母菌雙雜合系統分析中，矮牽牛的FB7與 FBP11會與矮牽牛中的E-群SEP-like作用。同樣的，阿拉伯芥中的SEEDSTICK，也會與阿拉伯芥中的SEP-like作用。在本研究中，我們從姬蝴蝶蘭中選殖出MADS-box D-群基因PeMADS7，並分析其功能。結果顯示，PeMADS7基因表現於花苞發育時期，同時也高量表現在生殖組織與胚珠發育時期。此外，我們利用酵母菌雙雜合及三雜合系統分析C群、D群和E群蛋白質的相互作用關係。實驗證實PeMADS7蛋白能與姬蝴蝶蘭中的E-群PeSEP蛋白作用。而在轉基因阿拉伯芥中，過量表現PeMADS7造成葉部生長捲曲及花部發育不正常，導致花萼及花瓣變短的表現型。且與野生型比較，轉殖株也延長了開花的壽命。另一方面，也因為長角果內部種子發育呈不規則排列，進而導致長角果發育逐漸彎曲。以上這些研究結果可以提供我們對於蝴蝶蘭D-群基因調控胚珠的發育有更進一步的了解。

Phalaenopsis species is one of the important horticultural plants in the world, and the spectacular flowers have highly ornamental value. In orchids, the ovule development is precisely regulated by pollination events, which is different from other angiosperm. Previous studyies show that D-class MADS box genes FLORAL BINDING PROTEIN7 (FBP7) and FBP11 play crucial roles in petunia ovule identity. In Arabidopsis the SEEDSTICK (STK) specialize ovule development, suggesting that it is an orthologous gene of FBP7 and FBP11. Furthermore, yeast two-hybrid assay showed that FBP7 and FBP11 proteins have ability to interact with E-class SEP-like proteins. Similarly, SEEDSTICK interacts with the SEP-like proteins in Arabidopsis. In this study, PeMADS7, a D-class like-gene from the Phalaenopsis equestris, was cloned and characterized its function. Results showed show that the transcript of PMADS7 began to accumulate in the floral buds and it continued to express through all the flower developmental stages. In addition, PeMADS7 was expressed strongly in developing ovulus. Protein interaction relationships among C-class, D-class and E-class proteins were assessed by using both yeast two hybrid and three hybrid analyses. PeMADS7 protein had ability to interact with E-class PeSEP proteins. Transgenic Arabidopsis plants overexpressing PeMADS7 caused the curly leaves and abnormal flowers with short sepals and petals. In addition, the flower of the transgenic plant showed an enhanced longevity than that of wild-type plant. Furthermore, the siliques were bended because of irregular arrangement of the seeds. These results provide us very useful information on the regulatory mechanism of ovule development in orchids.

中文摘要  ii
Abstract  iii
致謝  v
List of Tables  ix
List of Figures  x

1. Introduction
  1.1 Orchid Biology
   1.1.1 Evolution of Orchidaceae  1
   1.1.2 Reproductive biology of orchids  2
   1.1.3 Unique control system of orchid ovule development  3
   1.1.4 Post-pollination changes in Phalaenopsis orchid  4
   1.1.5 Ovule morphology of Phalaenopsis orchid  5

  1.2 MADS-box gene family of the floral organ development  6

  1.3 MADS-box D-class genes 
   1.3.1 D-class gene function in Petunia and Arabidopsis (Dicot plants)  7
   1.3.2 D-class gene function in rice and orchid (Monocot plants)  7

  1.4 The floral quartet model  8

  1.5 Overlapping functions of MADS-box genes in ovule development  9

2. Aim of the study  10

3. Materials and Methods
  3.1 Plant materials and growth conditions  11
  3.2 Scanning electron microscopy  11
  3.3 Sequence alignments and phylogenetic analysis  12
  3.4 Isolation of genomic DNA and Southern blot analysis  12
  3.5 RNA extraction and RT-PCR  13
  3.6 in situ hybridization  14
  3.7 Yeast two and three hybrid analysis  15
  3.8 Arabidopsis transformation  17

4. Results
  4.1 Flower pollination and ovule morphogenesis of Phalaenopsis  18
  4.2 Identification of PeMADS7 MADS-box genes in P. equestris  19
  4.3 Phylogenetic relationship of PeMADS7and other MADS-box genes  20
  4.4 Genomic organization of PeMADS7 genes  20
  4.5 Spatial and temporal expression analysis of PeMADS7 in Phalaenopsis  21
  4.6 in situ hybridization of PeMADS7 transcripts  21
4.7 Analysis of protein-protein interactions among PeMADS7 and C-, E-class 
proteins by using yeast two-hybrid system  22
  4.8 Yeast three-hybrid analysis for examination of bridge protein between 
PeMADS1 and PeMADS7  23
  4.9 Functional analysis of the PeMADS7 gene by ectopic expression in Arabidopsis thaliana  23

5. Discussion
5.1 Phalaenopsis D-class MADS-box genes  26
5.2 PeMADS7 may be required for ovule development in Phalaenopsis  26
5.3 The MADS-transcription factor complex involved in ovule development in P. equestris  28
5.4 Ectopic expression of PeMADS7 in Arabidopsis  29

 6. References  31


List of Tables
Table. 1. List of primers used in this study  39
Table. 2. Comparison of sepal length and petal length in wild-type and transgenic plants overexpressing PeMADS7  40

List of Figures
Figure. 1. Structure of Phalaenopsis equestris flower  41
Figure. 2. Scanning electron micrographs of the developing ovule of Phalaenopsis  42
Figure. 3. Alignment of amino acid sequence of PeMADS7 and other D-class genes  43
Figure. 4. Phylogenetic analyses of C- and D-lineages  44
Figure. 5. Southern bolt analysis of PeMADS7 in P. equestris genome  45
Figure. 6. Expression patterns of PeMADS7 in various organs and floral development stages  46
Figure. 7. Expression patterns of PeMADS7 at various developing ovule stages  47
Figure. 8. Expression patterns of PeMADS7 at different late pod developmental stage and protocorm developmental stages  48
Figure. 9. in situ hybridization of PeMADS7 in longitudinal section in developing floral bud  49
Figure. 10. in situ hybridization of PeMADS7 in cross sections in developing ovules  50
Figure. 11. Analysis of protein-protein interactions between D-class PeMADS7 and E-class PeSEP1 and PeSEP2 proteins by yeast two-hybrid system  51
Figure. 12. Analysis of protein-protein interactions among D-class PeMADS7, C- class PeMADS1, E-class PeSEP1 and PeSEP2 proteins by yeast two-hybrid system  52
Figure. 13. Analysis of protein-protein interactions among D-class PeMADS7, C-class PeMADS1 and E-class PeSEP1 and PeSEP2 proteins by yeast three-hybrid system  53
Figure. 14. PCR analysis of transformed Arabidopsis  54
Figure. 15. Phenotypes of leaves and plant size in wild-type and transgenic Arabidopsis overexpress PeMADS7  55
Figure. 16. Phenotypes of flowers and siliques in wild-type and transgenic Arabidopsis overexpress PeMADS7  56

                                    

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