探討NPF家族及CLC家族在葉片之硝酸鹽分佈及硝酸鹽的訊息傳遞

Investigation of NPFs and CLCs in Nitrate Signaling and Nitrate Distribution among Leaves

林展伊(Chan-Yi Lin)

指導教授 ： 曲芳華

共同指導教授 ： 蔡宜芳

國立臺灣大學/生物資源暨農學院/森林環境暨資源學研究所/碩士(2017年)

https://doi.org/10.6342/NTU201703166

摘要

氮源在植物的生長中扮演不可或缺的角色，而硝酸鹽是植物最常利用的形式。為了有效地利用硝酸鹽，植物發展出可以有效吸收、轉移和儲存硝酸鹽的各種機制。NPF的成員是植物很重要的硝酸鹽轉運蛋白，然而在53個成員裡，除了少數成員的角色已被確立之外，仍有一大部分尚未被了解。為了進一步探討哪些基因參與在硝酸鹽在地上部的分布，首先利用公開資料庫（MPSS以及e-FP Browser）所提供的基因表達資料篩選出在葉片表達量較高的基因，並選定19個基因剔除變異株進行硝酸鹽含量測定。研究發現，gtr2-1的基因剔除株中年輕葉的硝酸鹽含量比野生株高，在氮15標定硝酸鹽追蹤實驗中，氮15在年輕葉的含量也比野生株高，此外，在低硝酸鹽環境下，GTR2的基因表現量在年輕葉片中高於老葉，且利用GUS報導基因發現GTR2主要表現在根、地上部與地下部連接處、以及年輕葉片，這些證據指出GTR2可能負責降低年輕葉片中的硝酸鹽。另一方面，為了探討細胞內部硝酸鹽含量是否會影響植物感應外在硝酸鹽濃度的變化，在初期硝酸鹽反應實驗中採用了硝酸鹽儲存缺失的基因剔除株clca/clcb，目前的結果顯示內部硝酸鹽的含量可能在根部輕微影響初期硝酸鹽反應，但對地上部卻沒有影響。本研究針對NPF家族，提供硝酸鹽在葉片儲存及分布的系統化分析，並得知GTR2確實參與在年輕葉片硝酸鹽的分布，另外也藉由clca/clcb的基因剔除株了解細胞內部硝酸鹽的含量是否會影響初期硝酸鹽反應。

關鍵字

阿拉伯芥 ； 硝酸鹽 ； 轉運蛋白 ； 硝酸鹽分布 ； 初期硝酸鹽反應

並列摘要

Nitrogen is one of the most important nutrients for plants to grow, and nitrate is the most common form of nitrogen to be absorbed. To utilize nitrate efficiently, higher plants have developed various transporters, including NPFs being responsible for uptake, transport and storage. Of all the 53 genes in NPF, despite some of the members have been well characterized, a lot of them remain unclear. To find if any NPF genes participate in nitrate distribution among leaves, gene expression from public databases, MPSS and e-FP Browser, was analyzed, and nitrate content among leaves of mutants of 19 candidates was measured. The results showed that the nitrate content of young leaf in gtr2-1 was higher than wild type, and more root-fed 15N was transported to young leaf in gtr2-1. The gene expression of GTR2 was higher in young leaf under low nitrate concentration, andβ-glucuronidase reporter analyses indicated that GTR2 were expressed in roots, root-shoot junction, and the young leaves. Taken together, these results suggest that GTR2 might be involved in repressing nitrate allocation to young leaves. In addition, to find out if internal nitrate level would affect primary nitrate response, a nitrate storage defective mutant, clca/clcb, was characterized, and the data showed that CLCa and CLCb might be partly involved in regulating primary nitrate response in roots but not in shoots. This study provides a systematic analysis of the roles of NPFs in nitrate distribution among leaves and found out that GTR2 has a negative impact on nitrate distribution in young leaves. Besides, the study of clca/clcb suggests that external nitrate plays a major role in inducing primary nitrate response.

並列關鍵字

Arabidopsis ； nitrate ； transporter ； nitrate distribution ； primary nitrate response

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