摘要或動機

在模式植物──阿拉伯芥（Arabidopsis thaliana）中，AtYAK1（Arabidopsis thaliana Yak1-related protein kinase）是目前發現唯一屬於DYRK（Dual specificity Yak1-Related protein Kinase）的蛋白激?。雖然之前研究已證明，不同物種之DYRKs 和細胞的生長與發育過程有關。然而，其在植物中的生理功能卻尚未被明確地研究報導過。在先前的研究中，為瞭解AtYAK1 在阿拉伯芥內作用之位置，前人選取AtYAK1 基因ATG 上游約2.5 kb 的序列（Upstream Element, 2.5KUSE）建構至一含有GUS（β-glucuronidase）報告基因的質體中，並轉形至阿拉伯芥，進行GUS 組織染色分析。但在初步結果中，並沒有在轉殖株觀察到明顯的GUS 表現。進一步分析，我們發現在2.5KUSE 序列末端約0.5 kb 的5’非轉譯區（5’untranslated region, 5’UTR）中，有四組開放讀序框（Upstream Open Reading Frame, uORF）。有趣的是，許多研究也顯示，uORFs 會影響轉譯過程中的再起始（re-initiation）作用而調控該基因的表現。另一方面，前人亦透過構築好的2KUSE 轉殖株（即不含有5’UTR）進行上述GUS 實驗。結果發現，此2KUSE 轉殖株的GUS 表現非常顯著。本實驗即要瞭解AtYAK1 的uORFs 是否也會影響其蛋白質的合成。首先，我們以點突變的方式將四組uORFs 中之ATG 換成TTG，目的為構築不含有uORFs 之5’UTR（mutated uORFs, ΔuORFs）。在進行原生質體短暫表現分析法（protoplast transient assay）及GUS 組織染色分析後，將結果與含有uORFs 的結果作比較：當缺乏uORFs 後，其3’端報告基因的表現量確實比原來顯著。綜合以上，我們認為此uORFs 對於AtYAK1 蛋白質之表現佔有相當重要的影響地位。最後，我們對5’非轉譯區是否存在開放讀序框進行阿拉伯芥全基因組分析，相關結果亦於本研究報告中分析討論。AtYAK1（Arabidopsis thaliana Yak1-related protein kinase）is the first DYRK（Dual specificity Yak1-Related protein Kinase ） family member identified in the model plant ─ Arabidopsis thaliana and exists as one copy gene in Arabidopsis. Previous studies showed that many eukaryotic DYRKs are involved in regulating the growth and development of cells. However, the study of AtYAK1 in Arabidopsis is lacking to date. In order to understand where AtYAK1 expresses and functions in plants, a 2.5 kb fragment which is located upstream from the major ATG of AtYAK1（termed Upstream Element, 2.5KUSE）was previously constructed to drive the expression of a reporter gene, GUS（β-glucuronidase）, in transgenic Arabidopsis. Much to our surprise, no GUS expression signal could be detected in such transgenic plants. When further analyses were performed, we found that there are four upstream open reading frames （uORFs） in the 5’untranslated region ( 5’UTR ) within the 2.5KUSE. Many studies indicating that the uORFs can regulate the translation of downstream ORF encoding the major gene product through the procedure of translation re-initiation. This action represents a mode of translational regulation for gene expression. Indeed, GUS activity could be readily detected in transgenic plants expression 2KUSE::GUS, a construct lacking the 5’UTR of AtYAK1. In this study, I have tried to elucidate whether the uORFs of AtYAK1 can regulate the translation of the downstream major ORF. First, in order to construct a 5’UTR fragment of which uORFs have been mutated（ΔuORFs）, we apply site-directed mutagenesis to substitute ATG with TTG for the four uORFs and examine the expression of GUS driven by this mutated 2.5KUSE. After analyzing the results in both Arabidopsis protoplast transient assay and transgenic Arabidopsis, stronger expression of reporter genes in both systems were observed when the four uORFs were mutated. We have also confirmed that, in transient expression system, the increase of reporter gene activity was not due to the excess accumulation of the corresponding mRNAs. Rather, it is the four uORFs which play an important role in negatively regulating the translation of AtYAK1, possibly via inhibiting the translation re-initiation of major ORF. A genome-wide examination of uORFs in all Arabidopsis genes was also performed to assess the possible contribution of uORF in regulating gene expression.