Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/36222
標題: I. 阿拉伯芥與DNA甲基化相關基因之選殖與特性分析 II. 鐵砲百合花藥與乾燥相關基因之選殖與特性分析
I.Screening, Identification and Characterization of DNA Methylation Related Genes in Arabidopsis thaliana II.Screening, Identification and Characterization of Desiccation-Associated Genes in Anthers of L. longiflorum
作者: 許一丰
Hsu, Yi-Feng
關鍵字: Lily;百合;Pollen;Desiccation;花粉;乾燥
出版社: 生物科技學研究所
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摘要: 
在動植物裡,DNA甲基化在基因組的修飾與發育過程中,扮演了決定性的角色,而成為一個表觀遺傳學上的重要指標。在植物方面,RdDM (RNA-directed DNA methylation) 路徑是一個由RNA干擾(RNAi)所主導,而造成轉錄基因沉默化的重要機制。本研究透過遺傳篩選的方式,進行DNA去甲基酶突變株的次級抑制子的篩選。結果不僅篩選出三個參與RdDM路徑的新成員,命名為RDM2、RDM3和RDM4,同時也篩選到七個已知參與RdDM路徑上的成員-NRPD1、NRPE1、NRPD2a、AGO4、HEN1、DRD1和HDA6。而本研究將針對三個新成員中的RDM3做進一步探討,結果觀察到在失去RDM3基因功能的突變植株中, DNA發生甲基化的情形明顯降低,且在不破壞小干擾RNA的產生下,減緩了受RdDM影響的目標基因座上基因沉默的發生。透過胺基酸序列分析比對,RDM3基因可轉譯出一轉錄因子KTF1蛋白,此蛋白的N端區域和一轉錄延長因子SPT5蛋白的N端區域相似,且在C端區域富含GW/WG的重複胺基酸序列。此外,本研究結果也顯示KTF1蛋白能與AGO4(ARGONAUTE 4)蛋白共定位於細胞核內點狀區,且KTF1蛋白能與AGO4蛋白及RNA轉錄本結合。因此根據本研究可得知KTF1蛋白如同一個銜接蛋白,它能同時鏈結Pol V產生的轉錄本和AGO4蛋白以及AGO4結合的小干擾RNA,而形成一個RdDM路徑的影響子複合物。這種能同時結合AGO蛋白和小RNA轉錄子的雙重結合作用,可能是RNA沉默影響子複合物所具有的特徵之一。

DNA methylation is a conserved epigenetic mark that is critical for genome maintenance and development in plants and mammals. In plant, RNA-directed DNA methylation (RdDM) is an RNAi-based mechanism for establishing transcriptional gene silencing. From a genetic screen for second-site suppressors of the DNA demethylase mutant ros1, three new components (referred to as RDM2, RDM3 and RDM4) as well as seven known components (NRPD1, NRPE1, NRPD2a, AGO4, HEN1, DRD1, and HDA6) of the RdDM pathway were identified. One of novel RdDM components, RDM3 was chosen for further investigation. Loss-of-function mutations in RDM3 reduce DNA methylation and release the silencing of RdDM target loci without abolishing the siRNA triggers. RDM3 encodes a KTF protein with an N-terminal region similar to the transcription elongation factor SPT5 and a C-terminal region rich in GW/WG repeats. KTF1 colocalizes with ARGONAUTE 4 (AGO4) in punctate nuclear foci and binds AGO4 and RNA transcripts. Our results suggest KTF1 as an adaptor protein that binds scaffold transcripts generated by Pol V and recruits AGO4 and AGO4-bound siRNAs to form an RdDM effector complex. The dual interaction of an effector protein with AGO and small RNA target transcripts may be a general feature of RNA-silencing effector complexes.
URI: http://hdl.handle.net/11455/36222
其他識別: U0005-2402201020354000
Appears in Collections:生物科技學研究所

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