植物界中，蘭花是高度演化的一群，目前已有多達3萬種以上，其花朵與授粉者的共演化現象是許多學者的研究對象，其花部形成的複雜性亦是許多模式植物所無法達到。本實驗室蔡文杰學長於先前從台灣的原生種姬蝴蝶蘭(P. equestris)找到五個有關花部發育的B群MADS box基因，其中四個屬於AP3-like基因，PeMADS2, PeMADS3, PeMADS4和 PeMADS5，另外一個為PI-like基因，PeMADS6。這五個基因可分別在蝴蝶蘭的不同花器中表現，調控不同花器的形成。
在我的研究中，為了知道這些PeMADS基因是如何在空間上被限制在特定花器中的表現，我利用過去本實驗室巫佩珊學姐所選殖到的基因啟動子全長PeMADS2有3.2 kb，PeMADS3有1.3 kb，PeMADS6有1.5 kb的啟動子長度及我自己所選殖的PeMADS4和PeMADS5的啟動子序列使之達到3.3 kb及2.1 kb的長度，並分析這些啟動子上受到上游轉錄因子直接結合、調控的CArG box序列。在各啟動子的功能分析上，將各基因啟動子的5’端進行連續刪除並將之接到GUS報導基因之前端，以基因槍送入白花蝴蝶蘭花苞中作暫時性的表現，藉著GUS基因的表現來推斷前端各基因不同長度片段的啟動子是否具有啟動基因表現之功能。在PeMADS2啟動子分析上，具有最短啟動子長度的pBI-Pe2pF1 (300 bp)已有使GUS表現在各花器的功能，並有負向調控基因在花瓣及唇瓣中表現的序列存在(-786~-300)與正向調控基因表現在花瓣(-1,312~-786)及唇瓣(-3,224~-2,232)的序列存在，此與PeMASD2在過去利用北方雜合法的表現模式相符。在PeMADS3的啟動子分析上，具有最短啟動子長度的pBI-Pe3pF1 (107 bp)完全沒有GUS表現在各花器上，而調控GUS表現在花萼、花瓣與唇瓣的序列存在於-565~-107之間，調控在合蕊柱中的表現的序列則存在於-1,007~-565之中，此與PeMASD3在過去利用北方雜合法的表現模式不完全相符，過去指出PeMADS3主要在花瓣和唇瓣中表現。在PeMADS4啟動子分析上，具有最短啟動子長度的pBI-Pe4pF1 (309 bp)已可使GUS表現在花萼、花瓣和唇瓣中，主要增強在唇瓣表現的序列存在於-871~-309之間，在合蕊柱則幾乎完全沒有GUS的表現，此與PeMASD4在過去利用北方雜合法的表現模式不相符，過去指出PeMADS4只在唇瓣和合蕊柱中表現。在PeMADS5啟動子分析上，具有調控GUS基因在花萼、花瓣(-123~0)、唇瓣與合蕊柱(-442~-123)表現的功能，在-866~-442及-442~-123區域具有增加GUS在花萼及花瓣中表現的功能，此與PeMASD5在過去利用北方雜合法的表現模式不完全相符，過去指出PeMADS5主要在花瓣中表現，在花萼及唇瓣中僅有少量表現。在PeMADS6啟動子分析上，具有最短啟動子長度的pBI-Pe6pF1 (206 bp)可使GUS在各花器中表現，在-506~-206的序列會減弱GUS 在各花器中表現而在-806~-506及-1,106~-806之間的序列則能促進GUS在各花器中的表現。此與PeMASD6在過去利用北方雜合法的表現模式相符，在各花器中都有表現。
為了進一步比較各刪除啟動子片段在驅動GUS表現量的差異，我以GUS螢光測定方式(GUS fluorometric assay)來進行GUS表現的定量，在對PeMADS4啟動子的定量分析顯示在最短啟動子長度的pBI-Pe4pF1 (206 bp)可使GUS在各花器中表現，在-2,249~-1,433的序列之間具有增加表現的功能。在對PeMADS6啟動子的定量分析顯示在最短啟動子長度的pBI-Pe6pF1 (206 bp)可使GUS在各花器中表現，在序列-806~-506之間具有抑制GUS 在唇瓣中表現的功能而在序列-1,515~-1,106之間則會促進在唇瓣中的表現。在這研究中的結果可以作為未來以蘭花花苞作暫時性表現的實驗設計，並且在所分析得到的各啟動子片段在各花器表現的功能，亦可做為未來進一步深入研究其中與上游調控分子直接結合的序列位置及組成，對於花部發育時期基因表現的調控將提供莫大的貢獻。

The complex flower organization of orchids offers an opportunity to discover new variant genes and different levels of complexity in the morphogenesis of flowers. Previously, our lab has identified five B-class of MADS box genes from a native Phalaenopsis equestris, including four AP3-like genes (PeMADS2, PeMADS3, PeMADS4, PeMADS5), and a PI-like PeMADS6. Differential expression profiles of these genes are detected in various floral organs, suggesting their distinctive roles in the floral morphogenesis of orchids.
To dissect the spatial and temporal regulation mechanisms of these PeMADS genes, promoter deletion assay was carried out to verify the controlling cis-elements. In this study, I have isolated and sequenced 3.3-kb and 2.1-kb of sequence 5’ to the PeMADS4 and PeMADS5, respectively, from the BAC clones of P. equestris by DNA walking. Together with the 3.2-kb PeMADS2 promoter, the 1.3-kb PeMADS3 promoter and the 1.5-kb PeMADS6 promoter sequences obtained previously by Pei-Shan Wu (2003), serial deletion fragments of PeMADS promoter regions were cloned and fused to the GUS reporter gene encoding ß-glucuronidase and assayed for their transient expression in various floral organs in the white orchid flower buds. In the PeMADS2 promoter analysis, the pBI-Pe2pF1 (300 bp) was sufficient for PeMADS2 expression in all floral organs of P. equestris. The region between nucleotide -786 and -300 were responsible for the reduced expression in petal and lip and the regions between nucleotide -1,312 to -786, -2,232 to -1,823, and -3,224 to -2,232 had the ability to overcome the repression effect. These results were consistent with the expression patterns of the PeMAD2 assayed by northern blotting. In the PeMADS3 promoter analysis, the region between nucleotide -565 to -107 of PeMADS3 promoter sequence may required for PeMADS3 expression at sepal, petal, and lip. The region between nucleotide -1,007 and -565 activated PeMADS3 expression at column. These results were inconsistent with the northern blotting analysis for the PeMADS3, which was mainly expressed in petal and lip, fewer in column, but not in sepal. In the PeMADS4 promoter, the pBI-Pe4pF1 (309 bp) was sufficient for PeMADS4 expression at the basal level at sepal, petal, and lip. More GUS expression at lip was driven by the region from nucleotide -871 to -309 of PeMADS4 promoter. These results were inconsistent with the northern assay which showed PeMADS4 mainly expresses at lip and column, but not at sepal and petal. In the PeMADS5 promoter, the pBI-Pe5pF1 (123 bp) showed GUS expression at sepal and petal, and the promoter region between nucleotide -442 to -123 make higher expression at petal, and basal expression at lip and column. The region between nucleotide -1,508 to -1,054 had the negative effect and the region between nucleotide -2,062 to -1,508 had the positive effect for GUS expression at sepal. The strong GUS expression at petal by both pBI-Pe5pF3 and pBI-Pe5pF6 constructs was consistent with the northern blot assay. In the PeMADS6 promoter analysis, the pBI-Pe6pF1 (206 bp) was sufficient for PeMADS6 expression in P. equestris. The region between nucleotide -506 to -206 showed negative effect for the GUS expression and the regions between nucleotide -806 to -506 and -1,106 to -806 showed positive effects for the GUS expression at all floral organs, respectively. My results were consistent to the expression pattern of PeMADS6 assayed by northern blotting.
In order to quantify the GUS experiment for various deletion clones of promoter sequences, GUS fluorometric activity assay was performed. In the PeMADS4 promoter analysis by GUS fluorometric assay, the regions from nucleotide -309 to 1 and from nucleotide -2,249 to -1,433 of PeMADS4 promoter sequence showed the basal activity and the enhancing effects for the GUS expression, respectively. In the PeMADS6 promoter analysis, I noticed that the pBI-Pe6pF1 (206 bp) was sufficient for PeMADS6 expression and the region between nucleotide -806 to -506 had the repressed effect for the GUS expression at lip and the region between nucleotide -1,515 to -1,106 had the enhanced effect for the GUS expression at lip. These results can give a model for the study using floral buds as a transient expression host, and the functional analysis of these PeMADS promoters can serve a flower-specific, even floral organs-specific model in this kind of study. These results can also contribute to the study about the flower morphology and to investigate the regulatory pathway between each gene participating in flower development.

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