綠竹蔗糖合成酶與染色質之交互作用探討

Abstract

蔗糖是高等植物中最主要的光合作用產物與醣類運輸形式。蔗糖合成酶 (sucrose synthase, SuS) 在植物中可催化蔗糖與UDP形成果糖及UDPG的可逆反應，是參與蔗糖代謝的重要酵素之一。SuS 先前被認為是屬於細胞質蛋白質，但近來研究顯示，SuS 除了主要存在細胞質外，也被發現存在於細胞膜與其他胞器中。本篇論文即對生長中的綠竹筍，探討 SuS 之細胞內定位。將綠竹筍之細胞質與細胞核蛋白質，以 anti-rice SuS3 多株抗體進行免疫沉澱分析及western，在細胞質與細胞核兩種蛋白質分劃中都偵測到SuS的訊號。此外，也發現細胞核內的 SuS 結合於染色質上，推測SuS可能為「兼職蛋白質」，以直接或間接的方式與 DNA 產生交互作用。所以接下來想進一步利用 chromatin immunoprecipitation-sequencing (ChIP-Seq) 探討 SuS 在綠竹筍細胞核中與 DNA 間之交互作用。因此，本篇論文即針對綠竹筍，建立了染色質免疫沉澱 (ChIP) 的實驗方法。將經甲醛處理之綠竹筍分離出細胞核，並以 anti-rice SuS3 多株抗體將與 SuS 結合之 DNA-蛋白質複合體沉澱下來，並純化出適合進行次世代定序的 DNA 片段。

Sucrose is a major end product of photosynthesis and functions as a primary transport sugar in most higher plants. Sucrose synthase (SuS) is a key enzyme in plant sucrose metabolism. The enzyme catalyzes the reversible conversion of sucrose and UDP into UDPG and fructose. SuS was thought to be a cytosolic protein; however, recent evidences indicate that SuS may be localized in the plasma membrane and organelles in addition to the cytoplasm. In this study, the subcellular localization of SuS in developing bamboo shoots was investigated. The proteins were detected in both the cytoplasm and the nucleus by using western analysis and immunoprecipitation . Moreover, SuS proteins in the nucleus were found to be associated with chromatin, suggesting that SuS is a moonlighting protein and may directly or indirectly interact with DNA. To employ the chromatin immunoprecipitation-sequencing (ChIP-Seq) technology in studying the interactions of SuS with DNA, a ChIP protocol suitable for bamboo shoot sample was developed. The nuclei were isolated from formaldehyde-fixed bamboo shoot and the protein-DNA complexes were immunoprecipitated with anti-rice SuS3 polyclonal antibodies. Precipitated DNA fragments suitable for next-generation sequencing was obtained.