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|標題:||Functional Analysis and the Agricultural Application of CONSTANS (CO)-like Genes Involved in Regulating the Flowering Time and Various Developmental Processes in Plants
|作者:||楊長賢||關鍵字:||生物技術, 生物科學類;基礎研究;Arabidopsis thaliana;阿拉伯芥;B-box motifs;CONSTANS基因;開花時間;晚開花基因;B-box motifs;CONSTANS;Flowering time;Late-flowering gene||摘要:||
植物開花是一個複雜的機制，許多與開花時間調控的相關基因都已被選殖出來。其中之CONSTANS (CO)為具有B-box motifs之基因，突變後會造成植株的開花時間延遲。除了與開花時間有關外，一些CO-like (COL)基因如STO則被認為可能與耐逆境相關。本實驗室由阿拉伯芥(Arabidopsis thaliana)中選殖到5個與CO相似的COL基因，名為Arabidopsis CO-like Zinc finger gene 1-5 (ACOZ1,2,3,4,5)。大量表現其中之ACOZ1可使晚開花突變co及gi的開花時間回復正常，且會促使CO下游基因FT及SOC1的表現增加，這個結果說明ACOZ1應參與了開花時間的調控，其大量表現時可以取代CO的功能。本計劃主要的目的在探討所有ACOZ等COL基因參與開花及逆境的調控機制，及這些基因在實際農業生物科技上的應用。本計畫將進行兩個實驗來選殖分析受ACOZ1-5等COL基因調控的下游基因，首先將轉殖及篩選含構築體35S::ACOZ1-5: glucocorticoid receptor (GR)之轉殖阿拉伯芥co植株。在dexamethasone (DEX)的誘導下，這些COL基因才有功能，此時將透過對DEX及translational inhibitor cycloheximide (CYC)處理及不處理之35S::ACOZ1-5:GR轉殖植物進行cDNA subtraction hybridization及microarray之實驗，以便直接將受ACOZ1-5等COL基因調控的下游基因選殖出。另外亦將透過對35S::ACOZ1-5轉殖co突變植株(其開花時間正常)進行suppressor mutagenesis的方法，進一步篩選出開花時間延遲的突變植株，以選殖ACOZ1-5等基因調控的下游基因。另外本計劃將用yeast two-hybrid的系統分離出與ACOZ1-5等COL蛋白質直接共同作用的其它可能蛋白質及其相關基因。以上選殖出的基因都將進行其基因及蛋白質表現上的分析，並將進一步透過轉殖這些基因入植物，分析其性狀，進而在基礎科學上了解這些基因在植物開花時間及反應逆境的機制上扮演的角色為何。此外，本計劃更將進一步透過選殖蘭科植物(蝴蝶蘭)及非開花植物如蕨類之CO-like (COL)基因，並做其功能性互補實驗的分析，以探討其在非開花植物中是否存在，及其在演化上功能性差異的變化。本計劃之最終目的是在產業上的實際應用，本計畫選殖出的COL等基因等及其可能的下游基因都將被加以改良，再轉殖到其他重要蔬果作物及觀賞花卉，以達到人為調控開花時間、營養葉產生多寡及增加逆境之耐受性的目地，這對推展台灣的農業生物科技將會有所助益。
Flowering is a complex procress during plant development. Many flowering genes have been isolated and characterized. Mutation in CONSTANS (CO) that contained two zinc finger structures most similar to B-box motifs of GATA1 transcription factors, caused a delay of flowering. The function of some CO-like (COL) gene such as STO has been thought to be involved in stress tolerance. In our laboratory, we have isolated and characterized five B-box containing COL gene, named Arabidopsis CO-like Zinc finger gene 1 to 5 (ACOZ1-5), from Arabidopsis. 35S::ACOZ1 significantly promoted flowering in late-flowering mutants co-3 and gi-1. The expression of FT and SOC1, downstream genes for CO, was up-regulated in co-3 mutants transformed with 35S::ACOZ1. Therefore, ACOZ1 is able to complement CO's function in regulating flower transition by activating FT and SOC1 in Arabidopsis. The goal for this three-year project is to investigate the mechanism regulated by COL genes such ACOZ1-5 and their agricultural application for some important plants. To explore the link between flowering time and stress response in plant, functional analysis of genes in the signal transduction pathway regulated by ACOZ1-5 genes will be investigated. A strategy using construct containing 35S:: ACOZ1-5 genes fused with glucocorticoid receptor (GR) will be transformed into Arabidopsis co mutants. The transgenic plants will produce significantly amont of functional ACOZ only in the presence of dexamethasone (DEX). Genes immediately regulated (up- or down-regulated) by ACOZ1-5 genes will be isolated using cDNA subtraction hybridization and microarray analyses for DEX treated and untreated transgenic plants. Putative downstream target genes will also be isolated using suppressor mutagenesis of 35S::ACOZ1-5 co mutant plants. In addition, proteins interacted with ACOZ1-5 proteins will be identified by yeast two-hybrid system or proteiomic analysis and their corresponding genes will also be isolated. These cloned genes will be tested for their roles in regulating flowering time or in response to stress through transgenic plant analysis. In addition, to investigate the differences and similarities for COL genes between flowering and non-flowering plants, orthologues of COL genes will be cloned and characterized from orchids and ferns. These genes will then be transformed into Arabidopsis gi and co mutants for complementation and functional analysis. The final goal for this project will be the practical application of the genes cloned and characterized in this project. Genes characterized in this project will be used directly in agricultural application by transforming them into some valuable vegetables and flowering plants in controlling and modification of their flowering time and stress response. Academically, the results obtained from this project should lead to a deeper understanding of how flowering time was regulated by the COL genes signal transduction pathway in genomic manner.
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