硫代葡萄糖苷 (Glucosinolate) 是十字花科中的重要次生代謝產物，在植物防禦中起著重要作用。阿拉伯芥硫代葡萄糖苷轉運蛋白1 (AtGTR1) 屬於硝酸鹽轉運蛋白家族之成員，主要表現於花絲和維管束中可調控硫代葡萄糖苷在韌皮部中的含量。已知硫代葡萄糖苷由多種氨基酸合成可形成數十種的化學結構，然而目前 AtGTR1 針對受質多元結構之選擇辨識機制和受質運輸效能仍尚待釐清，生化定性和結構分析是回答這些問題的理想工具。在此研究中，我們建立了酵母菌表現系統來生產重組 AtGTR1，經兩步驟層析純化後其蛋白純度可達到90％。以負染色穿透式電子顯微鏡分析 AtGTR1 純化蛋白，顯示其分子構型大小十分均一，尺寸約為 7-9 nm，推測為形成同型二聚體。此外，我們測量了表現 AtGTR1 的酵母菌之硫代葡萄糖苷的轉運活性和受質選擇性，其分析結果表明 AtGTR1 可以轉運多種結構的硫代葡萄糖苷，並且其運輸效率與受質結構有關。本研究結合定點突變技術建立數個 AtGTR1 的突變株，並用以鑑定出受質運輸相關的關鍵位點。定點突變研究亦發現 T105 胺基酸之磷酸化修飾對於 AtGTR1 之生化特性可產生顯著影響。總結本研究在結構與生化功能方面之實驗結果，對於闡明 AtGTR1 的分子機制和硫代葡萄糖苷運輸的調節提供了新的證據。

Glucosinolates are important secondary metabolites in the Brassicaceae and plays an important role in plant defense. Arabidopsis thaliana Glucosinolate Transporter 1 (AtGTR1), a member of the nitrate transporter family, is mainly expressed in filaments and vascular tissues in Arabidopsis and regulates the glucosinolates level in phloem. Hundred types of glucosinolates are synthesized from a variety of amino acids in plants. However, the substrate selective mechanism and transport efficiency of AtGTR1 still remain unclear. Biochemical characterization and structural analysis would be ideal tools to answer these questions. In this study, we established a yeast expression system to produce recombinant AtGTR1, and the protein purity could achieve 90% after a two-step chromatographic purification. Negative-stain transmission electron microscopy analysis showed that purified AtGTR1 formed a homodimer conformation with a molecular size around 7-9 nm. In addition, we measured the glucosinolate transport activity and substrate selectivity of AtGTR1 that express in Saccharomyces cerevisiae. The results showed that AtGTR1 transported various types of substrates, and its transport efficiency is interrelated to the structure of the substrate. This study combined site-directed mutagenesis technology to establish several AtGTR1 mutant strains, and used to identify the key sites related to the substrate transport. Site-directed mutagenesis studies have also found that the phosphorylation modification of T105 amino acid can have a significant impact on the biochemical characteristic of AtGTR1. Taken together, the structural finding and biochemical studies provided the new evidence to clarify the molecular mechanism of AtGTR1 and the regulation of the glucosinolate homeostasis.

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