阿拉伯芥NPF6.4轉運蛋白之運輸活性分析

Abstract

氮源是植物生長和其他生理反應扮演著不可或缺的角色之一，因此揭示植物獲取和利用氮源的方式是必要的。NPF6.4是阿拉伯芥中硝酸鹽轉運蛋白1/胜肽轉運蛋白家族 (NPF) 的成員。在先前的研究中已經確認了NPF6.4參與了阿拉伯芥對丁香假單胞菌感染的防禦反應。然而，目前仍缺乏對NPF6.4運輸功能的了解。 為了調查 NPF6.4的生理作用，使用爪蟾卵母細胞表現系統和酵母菌互補試驗來測定 NPF6.4 的運輸功能。在這項研究中，我們證實 NPF6.4 坐落於細胞膜上。同時，不論是在低或高硝酸鹽濃度下，NPF6.4 在爪蟾卵母細胞表現系統中都會調控硝酸鹽的吸收，而該結果也證實NPF6.4 是一種雙親和性的硝酸鹽轉運蛋白。而NPF6.4硝酸鹽的 Km 在低親和力和高親合力分別為3.6 mM 和201.7 μM。然而，NPF6.4 在酵母互補試驗中沒有表現出吸收雙胜肽的活性。除此之外，NPF6.4 在本研究中還通過酵母互補試驗鑑定為多胺運輸蛋白。 本研究證實，除了NPF6.3/CHL1，NPF6.4是阿拉伯芥NPF中的另一種雙親和性的硝酸鹽轉運蛋白，且NPF可能也具有調控多胺運輸的能力。

Nitrogen source is a necessary key nutrient for plant growth and other physiological reactions, so it is essential to reveal the ways which plants gain and use nitrogen. NPF6.4 is belong to a group of nitrate transporter1/peptide transporter family (NPF) in Arabidopsis thaliana. In our previous study, NPF6.4 was reported to participate in defense responses to Pseudomonas syringae. However, less is known about the transport function of NPF6.4. In order to understand the physiological roles of NPF6.4, we characterized the transport activity of NPF6.4 by the oocyte expression system and the yeast complementation assay. In this research, we confirmed that the expression of NPF6.4 in subcellular localization was in the plasma membrane. NPF6.4 transported nitrate under low and high nitrate concentrations conditions in the oocyte expression system, indicating that NPF6.4 is a dual-affinity nitrate transporter. The Km for nitrate was 3.6 mM and 201.7 M for low-affinity and high-affinity, respectively. However, NPF6.4 showed no dipeptide uptake activity in the yeast complementation assay. In addition, NPF6.4 is also identified as a polyamine transporter in this research by a yeast complementation assay. This study has shown that besides NPF6.3/CHL1, NPF6.4 is another dual-affinity nitrate transporter in the NPF in Arabidopsis, and NPF may have the ability to mediate the transport of polyamines.