生物體會在各種的環境之下動態地表現各種不同的生理機能，而單純的DNA序列只提供靜態的資訊。在不同的發育時期或部位，基因會受到不同調控，選擇表現或隱藏，而真正表現於表型者則是蛋白質。蛋白體學的焦點主要在於基因組所轉錄出來的蛋白質以及其相互之間的影響。Ubiquitin-proteasome pathway 是一些短半衰期蛋白質重要的分解路徑，在植物中，此一路徑已被發現對於花的發育、日規週期、控制植物生長的生長素及抗病機制的茉莉酸都具有調控功能。調控花部器官發育的基因已經被研究很久，最廣為人知的為阿拉伯芥中的ABC model，而最近又被發現B群基因的表現會受到上游某一抑制子作用而降低，此一抑制子必須經由ubiquitin-proteasome pathway進行分解以使花部發育基因正常表現，花部才有正確的形態。
本論文利用本實驗室建立出來的蝴蝶蘭花苞EST資料庫中找出ubiquitin、ubiquitin activating enzyme (E1)、 ubiquitin conjugating enzyme (E2)、F-box及SKP1各數個，並針對SKP1做進一步確定，希望找出調控花部發育機制之相關基因。首先利用E. coli (DH 5a) 將GST-PeSKP1-1融合蛋白大量表現出來，將此蛋白對兔子進行免疫反應以取得抗體，並利用GST-PeSKP1-1融合蛋白及PeSKP1-1 抗體分別進行GST pull-down assay及共同免疫沉澱實驗，經過蛋白質二維電泳後共發現了15個差異性的蛋白質點，並進行proteomics研究分析，發現Pd04，CI01，CI03，CI06，CI08及CI09這些蛋白質點分別為ubiquitin-specific protease (UBP)，DNA binding protein RAV1，26S proteasome subunit 4，F-box protein，E2及protein kinase。以此方法可以找出和PeSKP1-1有直接或間接結合的蛋白質或可能為花部發育之抑制子，提供利用蛋白質組學探討花部發育分子機制之模式。

All of life ultimately depends on the well-or-suitable readout of specific genes from the genome, with expression patterns depending on the nature of the starting material such as a specific tissue, cell type, or developmental stage. The technologies of proteomics make it possible to generate quantitative protein expression data on a scale and sensitivity comparable to that achieved at the genetic level. Accumulating evidence indicates that the ubiquitin/proteasome pathway in eukaryotes controls the degradation of many cytosolic and nuclear proteins. Genetic approaches elucidating the molecular mechanisms of auxin signaling pathways, photomorphogenesis, jasmonic acid pathway and floral development are linked to the ubiquitin/proteasome pathway. It has been reported that the F-box and SKP1 proteins are components of a ubiquitin ligase that mediates the proteolysis of a repressor of AP3 and PI expression, MADS box transcription factors for floral development. The goal of this study was to identify the negative regulators during floral development. For this purpose, PeSKP1 gene was used as a bait to pull out its interacting components. PeSKP1-1 gene was identified from the Phalaenopsis floral expressed sequence tag (EST) database. PeSKP1-1 gene was expressed in flower buds, pedicles and root, and to a less extent in leaf as analyzed with Northern blots hybridization. The GST-PeSKP1-1 fusion protein was expressed, purified and applied in a GST pull-down assay. The antibody of PeSKP1-1 was produced from rabbit and applied in a Co-Immunoprecipitation (CoIP) assay. Total proteins extracted from stage 2~4 (0.5~2.0 cm) flower buds of Phalaenopsis amabilis var. formosa were used in the pull-down and CoIP assay. Proteins identified from both GST pull-down and CoIP assays were analyzed using 2-dimentional (2D) gel electrophoresis. Fifteen spots were sequenced and annotated to confirm their functions using mass spectrometry and bioinformatics. Ubiquitin-specific protease (UBP)，DNA binding protein RAV1，26S proteasome subunit 4，F-box protein，E2 and protein kinase were identified from Pd04，CI01，CI03，CI06，CI08 and CI09 spots. These results suggest that the proteomics approach facilitate the study of floral protein-protein interaction.

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