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標題: 受文心蘭OMADS1正調控的未知基因之功能性分析
Characterization and Functional Analysis of a Novel Gene Up-regulated by OMADS1 of Oncidium
作者: 戴書瑜
Dai, Shu-Yu
關鍵字: early-flowering
jasmonic acid
出版社: 生物科技學研究所
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摘要: 文心蘭中的OMADS1基因,與阿拉伯芥AGAMOUS-like 6 基因 (AGL6)具有高度相似性,而將之異位表現會造成極度早開花,植株矮小與失去無限花序的性狀。為了深入研究受到OMADS1調控的基因,我們將轉入35S::OMADS1-GR構築體的阿拉伯芥植株以Dexamethasone (DEX)處理,並且做Microarray的分析。在許多受到OMADS1正調控的基因中,挑選了OIG1 (OMADS1-INDUCING GENE 1)來深入研究。OIG1為一功能未知的假設性基因,為了瞭解OIG1在阿拉伯芥中的功能,因此在阿拉伯芥轉基因植株中做了啟動子活性試驗與功能性分析。在OIG1::GUS植株中,小花苞的花萼、花瓣、雄蕊與雌蕊皆偵測到GUS活性;而在成熟花中,GUS的表現在花藥中轉趨微弱。在35S::OIG1轉基因阿拉伯芥中,觀察到早開花以及雄不孕性狀,而OIG1的RNAi植株中則有晚開花的性狀。早開花的天數在異位表現OIG1的植株中隨著表現量增加而遞增;而OIG1的RNAi植株中晚開花的天數亦隨著基因的抑制程度增加而遞增,這些性狀與基於OIG1受OMADS1正調控所做的推測相當符合。在近年文獻中指出,OIG1蛋白可能受到NMT (N-myristoylated transferase)該酵素的修飾。NMT會利用在目標蛋白的N端加上一長鏈脂肪酸以進行荳蔻酸化(myristoylation)的修飾作用。由於NMT突變後也會得到晚開花的性狀,顯示NMT與OIG1在調控開花過程中可能具有關連性。荳蔻酸化的分析也顯示NMT似乎的確能對OIG1做荳蔻酸化的修飾,並使OIG1能結合在某種與過氧化體相似的胞器膜上。在35S::OIG1轉基因阿拉伯芥中的雄不孕性狀,也發現是由花藥不開裂所造成,而且OIG1的表現量越高,不孕的情況也隨之轉趨嚴重。此外,在35S::OIG1轉基因植物中,合成茉莉花酸(Jasmonic acid)所需的酵素其基因表現量也明顯比野生型低,因此我們認為OIG1可能調控了合成茉莉花酸所需的酵素,另外我們也猜測荳蔻酸化的OIG1能結合在某胞器膜上進而去調控開花時間以及茉莉花酸生合成的路徑。
Ectopic expression of Oncidium OMADS1, with extensive homology to the Arabidopsis AGAMOUS-like 6 gene (AGL6), caused extremely early flowering, significantly reduced plant size and the loss of inflorescence indeterminacy. To investigate the target genes regulated by OMADS1, microarray analysis was performed from 35S::OMADS1-GR transgenic Arabidopsis after Dexamethasone (DEX) treatment. One of numerous OMADS1 up-regulated genes, OIG1 (OMADS1-INDUCING GENE 1), was further analyzed. OIG1 is a hypothetical protein with unknown function. To seek the function of OIG1 in Arabidopsis, promoter assay and functional analysis for OIG1 were performed in transgenic Arabidopsis. In OIG1::GUS plants, GUS activity was detected in young flower bud, including sepal, petal, stamen and carpel. In mature flower, the expression of GUS was weakly detected in anthers. The flowering time was promoted progressively in transgenic plants ectopically expressing OIG1 whereas the flowering time was delayed in transgenic plants ectopically expressing RNAi of OIG1. These phenotypes support our speculation for the OIG1 function based on its up-regulation by OMADS1. OIG1 has been reported to be modified by NMT(N-myristoylated transferase) that are enzymes myristoylating proteins by adding a long-chain fatty acid on N-termini of proteins. Mutation in NMT leads to late flowering, suggesting that there may be a connection between NMT and OIG1 in regulating flower transition. In addition, the myristoylation assay revealed that NMT did myristoylate OIG1 and thus made OIG1 be able to bind to membrane of organelles which looked like peroxisomes. Interestingly, the phenotype of male sterility due to the anther-indehiscence was also observed in 35S::OIG1 transgenic Arabidopsis. The degree of the sterility was correlated with the increasing level of OIG1 expression. Also, the expression level of genes which participate in JA(Jasmonic acid) biosynthesis pathway were much lower in 35S::OIG1 transgenic plants than in wild-type. Thus, we proposed that OIG1 may regulate genes involved in JA biosynthesis. On the other hand, we suggested that myristoylated OIG1 on membrane regulates the flowering time and JA pathway.
其他識別: U0005-2807201018083200
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