水稻蔗糖合成酶於細胞核中的功能探討

Abstract

蔗糖合成酶在植物中可催化蔗糖與 UDP 形成果糖及 UDP-glucose 的可逆反應。SuS 主要存在於細胞質中，然而也曾被發現位於細胞膜及其它胞器中。在先前研究中指出，水稻的 SuS (RSuS) 也存在於細胞核中，因此推測 SuS可能為「兼職蛋白質」。本篇研究的目的即是以染色質免疫沈澱 (ChIP) 探討 RSuS 在細胞核中的功能是否藉由與 DNA 之交互作用來完成。自經甲醛處理之水稻白化苗 (Oryza sativa L. cv. Tainung 67) 分離出細胞核，並以 anti-RSuS 多株抗體將與 RSuS 結合之 DNA-蛋白質複合體沈澱下來，接著將沈澱所得之 DNA選殖入質體後，隨機挑選數個重組質體進行定序。截至目前為止，已得到六個候選序列，並以 ChIP-PCR 針對其中四個進一步確認。其中有一位於水稻基因 Os04g0503500 之 3’-非轉譯區的序列，應為 RSuS 的目標序列。根據本篇論文研究結果推測，除了原本所具有的酵素功能之外，RSuS 可能也參與了基因表現之調控。

Sucrose synthase (SuS) catalyzes the reversible conversion of sucrose and UDP into fructose and UDP-glucose in plants. The enzyme is mainly present in the cytoplasm; however, it has also been localized in the plasma membrane and organelles. In the previous study, rice SuS (RSuS) was found to be present in nuclei, suggesting that SuS may be a moonlighting protein. The aim of this study was to gain insights into the roles of RSuS in the nucleus. Chromatin immunoprecipitation (ChIP) was performed to investigate whether RSuS performs its functions through protein-DNA interactions. The nuclei were isolated from formaldehyde fixed rice etiolated seedlings (Oryza sativa L. cv. Tainung 67) and the protein-DNA complexes were immunoprecipitated with anti-RSuS polyclonal antibody, after which the precipitated DNAs were cloned into vector, and randomly selected for sequencing. Six candidates were identified so far and four of them were further validated by ChIP-PCR. Among them, the one that was located in the 3’-untranslated region of rice gene Os04g0503500 was confirmed to be the target for RSuS. The results of this study suggested that RSuS may be involved in the modulation of gene expression besides it catalytic function.