| 摘要註: |
隨著蝴蝶蘭的生長發育, 在不同組織內可觀察到不同程度的內生性多(endopolyploidy) 的現象, 而在花苞發育中也有內生性多倍的現象。內生性多倍(endopolyploidy)是因細胞經過內生性複製(endoreduplication)所造成,DNA 在複製後,沒有完成細胞質分裂期(cytokinesis),而使細胞核內 DNA 含量增加,造成多倍體。本實驗室先前的研究結果顯示,Cyclin B1 與內生性多倍相關,並已選殖出Phalaenopsisbellina 及P. aphrodite 的Cyclin B1,且將其命名為Phabe;CycB1 和Phaap;CycB1。因此,本研究的第一個重點為研究台灣白花蝴蝶蘭Phalaenopsis aphrodite subsp. formosana 開花前不同發育時期花苞中的花萼、花瓣及花柄,細胞內生性多倍的比例以及發生的時間,另外並檢測Phaap;CycB1 基因的表現,並進一步探討內生性多倍與Phaap;CycB1基因調控之間的相關性。結果顯示,開花前花苞生長,2 倍細胞的百分比例會越來越低,即多倍的4 倍及8 倍的細胞的百分比例增加,在1 cm 或更小的花苞中,倍體數較恆定,也就是內生性多倍的趨勢較為平穩,只有2 倍細胞及少數經過1 次內生性複製的4 倍細胞存在,但在大於1 cm 的花苞,開始出現經過2 次內生性複製的8 倍細胞,且多倍細胞的總比例顯著並持續增加,此一現象在花萼與花瓣中都類似;而花柄中,其多倍細胞的數量較花萼及花瓣來得多,並且早在花苞為0.6 cm 時的花柄中就出現8 倍細胞,其多倍化出現得更早,表示不同花部組織,可能有不同調控多倍化的機制。Phaap;CycB1基因表現的結果顯示,在花萼、花瓣及花柄組織中都有表現,在花萼和花瓣組織中,Phaap;CycB1 的表現趨勢都呈現兩個波型,分別介於0.5-1.1 cm 和1.4-1.8 cm 時,在第一階段中,Phaap;CycB1 表現旺盛並持續增加,在0.8-1 cm 中有最高的表現,之後基因表現下滑,直到第二階段又再增加;而在花柄中,Phaap;CycB1 的表現量在0.5 cm 中最高,之後逐漸下降,因此,Phaap;CycB1 基因的表現高峰都與多倍化現象出現的時間相同,或者稍早一、二天。此外,為了研究在花苞的發育過程中,細胞分裂(cell division)及擴大(enlargement)的情形,又細分花萼與花瓣的不同部位,調查其中細胞數、細胞大小及倍體數的變化;研究發現,倍體數在細分成6 個小區域的花萼或花瓣中都沒有明顯的差別;細胞數量在花萼上、中、下部分的差別較大,以下半部分的細胞數含量最多,但細胞最小,花萼中間部分的細胞最大。目前已有多個在育種上有重要影響的蝴蝶蘭原生種被發現有內生性多倍的現象,因Cyclin B1 與內生性多倍有相關,所以本論文的第二個重點在於研究這些重要原生種中, Cyclin B1 基因序列,確認其Cyclin B1 基因及重要domain 序列,進而了解其內生性多倍化藉由Cyclin B1 基因調節方式是否相同。結果顯示,P. pulcherima 以及P. venosa 這兩個重要原生種中的Cyclin B1 的核苷酸序列及胺基酸序列的相似度極高,達95%,且Cyclin B1 胺基酸序列上的cyclins signature 及destruction box 等重要domain 都呈現高度保留,與P. aphrodite 及P. bellina 比較後發現,這四個蝴蝶蘭原生種擁有極相似的Cyclin B1 基因。未來希望能進一步探討Cyclin B1 基因調控內生性多倍的機制,希望在分子育種及產業上能有所貢獻。 Endopolyploidy has been found in various organs and tissues during plant development and flower development in Phalaenopsis orchids. Endopolyploidy is due to endoreduplication, in which one or several rounds of DNA synthesis occurs in the absence of cytokinesis, resulting in an increase in the DNA content and polyploidy. In previous study, two kinds of Cyclin B1 were isolated from P. bellina and P. aphrodite and nomenclatured as Phabe;CycB1 andPhaap;CycB1, respectively, and they might be function during endopolyploidization. Therefore, the aim of this study will be focus on the floral development in P. aphrodite subsp.formosana. In sepal and petal of different floral development, the proportion of 2C nuclei decreased, the polyploidy proportion of 4C and 8C increased. There were 4C nuclei after oneround of endoreduplication and 8C nuclei after twice endoreduplication existedsimultaneously. The 8C nuclei had been detected at 1.1 cm sepal and 1.2 cm petal or larger flower bud. In pedicel, polyploidy levels and percentage were higher and more than sepal and petal, 8C nuclei had been showed in pedicel from 0.6 cm flower bud. These results indicate that different endopolyploiy regulated-mechanisms might be existed in different floral tissues. Phaap;CycB1 gene expression was detectable in sepal, petal and pedicel. In sepal and petal,Phaap;CycB1 gene displayed abundantly among 0.5-1.1 cm and 1.4-1.8 cm of floral bud. The peak of Phaap;CycB1 gene expression is highly correlated with endopolyploidization during floral development because of similar timing and pattern.The highest expression of Phaap;CycB1 had showed during 0.8-1 cm of floral buds. In pedicel, the gene expression of Phaap;CycB1 was rich at 0.5 cm. Then, polyploidy level, cell number, and cell size were investigated in different portions of sepal and petal. There were the largest amount of cellnumber and the smallest size at lower region of sepal, no matter left or right sepal. In the middle area of sepal, cell size is the biggest. This information implied that cell divisions were occurred near lower portion of sepal and enlargement continued later on. Endopolyploidy has been reported on seveval wild Phalaenopsis species. Since Cyclin B1 was highly related withendopolyploidy, cloning of Cyclin B1 in these species is necessary. Different Cyclin B1 were cloned and sequenced in P. pulcherima and P. venosa. A highly similarity( 95%) existed among nucleotide and amino acid sequences, and highly conserved Cyclin B1 domain of cyclin signature and destruction box were found. A better understanding on Cyclin B1 gene and endopolyploidy- regulated- mechanism will contribute to molecular breeding and orchid industry. |