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標題: | 染色質修飾因子mTRIM28應用於阿拉伯芥表觀遺傳育種的可行性探討 Exploring the Possible Application of a Chromatin-Modifying Factor mTRIM28 in Epigenetic Breeding of Arabidopsis |
作者: | Bo-Yuan Huang 黃柏元 |
指導教授: | 張孟基(Men-Chi Chang) |
關鍵字: | 表觀遺傳育種,表觀遺傳修飾工具,TRIM28,dCAS9,NPBT, Epigentic breeding,Epigenetic modification tools,TRIM28,dCAS9,NPBT, |
出版年 : | 2020 |
學位: | 碩士 |
摘要: | 表觀遺傳學 (Epigenetics) 乃透過DNA甲基化程度或組織蛋白的修飾,在不改變基因序列下調控基因表現並影響作物外表型。基因型相同的個體,若具有不同的表觀遺傳修飾或修飾程度差別會產生不一樣的變異,此現象稱『表觀遺傳變異 (Epigenetic variation)』。在遺傳育種中,可以做為另一路徑提供作物外表型變異的新來源。除了透過自然產生、逆境誘導,現今還可利用分子技術創造表觀遺傳變異個體,並根據表觀遺傳修飾位置是否具有專一性區分為標靶或非標靶表觀遺傳修飾 (Targeted or Non-targeted epigenetic modification)。本研究目的主要在探討利用小鼠染色質修飾因子mTRIM28是否能應用於阿拉伯芥,作為發展新的非標靶表觀遺傳修飾工具。若能利用染色質修飾因子mTRIM28作為非標靶表觀遺傳修飾工具,透過隨機影響基因組中表觀遺傳修飾狀態,便可以創造出具有不同變異的族群 (Epi-mutant pool)。為達此目的,首先根據胺基酸序列比對結果,發現阿拉伯芥中並沒有如mTRIM28具有多功能區域的單一蛋白質,而是由具有不同個別相似功能域之諸多蛋白質。進而透過次細胞表現部位結果顯示mTRIM28的核定位訊號可以透過阿拉伯芥importin α1/importin β轉運至細胞核中,接著以異位表現mTRIM28於阿拉伯芥了解mTRIM28是否可透過表觀遺傳修飾影響生長與發育,透過正常或非生物逆境下外表型分析,並以及酵母菌雙雜交系統篩選是否有可能與mTRIM28結合之蛋白質。推測在植物體中可能因為缺乏KRAB-或KRAB-Like鋅指轉錄因子而無法調控基因表現。故若直接以mTRIM28作為非標靶表觀遺傳學修飾工具較不可行。但未來若能利用dCAS9-KRAB解決植物缺乏KRAB-鋅指轉錄因子問題,並發展為標靶表觀遺傳修飾工具。藉由dCAS9-KRAB透過匯集mTRIM28並與阿拉伯芥中組織蛋白修飾酶、染色質重塑蛋白形成異染色質複合物,在不影響基因序列下精準抑制目標基因表現的標靶表觀遺傳修飾系統,將可成為具有發展潛能的新植物育種技術 (New Plant Breeding Technology, NPBT)。 Epigenetics is used for describing the heritable phenotype changes of crops caused by DNA methylation and histone modification without altering the intrinsic DNA sequences, in which phenotypic variation has formed epigenetic diversity for plant breeding. Depending on different extents of epigenetic modification, the individuals can display variation on phenotypic diversity. This represents a new and alternative tool for modern crop improvement. In addition to natural generation and stress induction, nowadays molecular techniques are used to create epigenetic variation, including the target or non-target epigenetic modifications. Based on this, the purpose of this study is to explore whether we can take the chromatin-modifying factor mTRIM28 (Mus musculus Tripartite Motif Containing 28) as a new molecular tool for non-target epigenetic modifications in Arabidopsis. Once the chromatin-modifying factor mTRIM28 can be used as a non-target epigenetic modification tool, an epi-mutant population with epigenetic variation can be created for changing the epigenetic status in the plant genome. Thus, we conducted an amino acid sequence homologous search, there is no single protein with multifunctional domains like mTRIM28 in Arabidopsis. Instead, the genome in Arabidopsis consists of many different proteins with corresponding similar functional domains of mTRIM28. Next, we showed that the nuclear localization signal of TRIM28 can efficiently transport the protein into the nucleus through the Arabidopsis importin α1/importin β system. Furthermore, phenotypic investigation and yeast two-hybrid analysis were performed to explore whether TRIM28 can cause growth and development abnormalities through epigenetic modification, and screen for proteins that are likely to interact with mTRIM28 in Arabidopsis thaliana. As a result, it suggests that Arabidopsis may lack KRAB- or KRAB-Like zinc finger transcription factors; thus it prevents mTRIM28 from silencing gene expression, making TRIM28 non-target epigenetic modification tool is less feasible. However, from my study for future application of mTRIM28 on crop epigenetic breeding, using dCAS9-KRAB/ TRIM28 will be good for targeted epigenetic medication. We expect that this targeted epigenetic modification system holds up the potential for accurately blocking the target gene expression without altering the DNA sequence and represents a possible New Plant Breeding Technology (NPBT) for crop improvement in near future. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/16235 |
DOI: | 10.6342/NTU202002086 |
全文授權: | 未授權 |
顯示於系所單位: | 農藝學系 |
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