臺灣博碩士論文加值系統

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。

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.

Table of Contents
中文摘要 i
Abstract ii
Table of Contents iv
List of Figures and Tables vi
1. Introduction 1
1-1. Nitrate metabolism in plants 1
1-2. Nitrate transport systems in Arabidopsis thaliana. 1
1-3. NPF members have diverse functions in plants. 2
1-4. Nitrate serves as an important nutrient and a signal molecule. 5
1-5. CLC family members are involved in nitrate compartmentalization in intracellular organelles 6
1-6. Nitrate transporters in Poplar 7
1-7. Aim of this research 8
2. Material and method 10
2-1. Plant Material and Growth Condition 10
2-2. Genomic DNA extraction 12
2-3. Quantitative PCR analysis 13
2-4. Nitrate Content Analysis by HPLC 13
2-5. 15NO3- Labeling Assay 14
2-6. GUS staining 15
3. Results 16
3-1. Investigation of NPF genes for nitrate distribution among leaves 16
3-1-1. Bioinformatics research to identify potential candidates for further study. 16
3-1-2. Three more NPF members were selected based on Massively Parallel Signature Sequencing (MPSS) and RT-PCR results presented in a review article. 17
3-1-3. 19 candidates were chosen to measure nitrate content and biomass among leaves 18
3-2. Functional analyses of Arabidopsis GTR2 24
3-2-1. GTR2 was mainly expressed in root, root-shoot junction and young leaves 24
3-2-2. More 15N was accumulated in young leaves of gtr2-1 24
3-2-3. Expression of GTR2 in young leaves is increased at low nitrate condition 25
3-2-4. Nitrate content in young leaves was higher under different nitrate concentrations in gtr2-1 26
3-2-5. Nitrate content dropped faster under starvation in gtr2-1 27
3-2-6. Primary root length in gtr2-1 was longer under low nitrate condition 28
3-3. CLCa and CLCb are responsible for nitrate storage in vacuole and might have impact on nitrate sensing in roots 29
3-3-1. clca and clcb mutants showed a reduction in nitrate content among all leaves. 29
3-3-2. clca/clcb had no dramatic difference compared to wild type in terms of primary nitrate response under both high- and low-affinity nitrate conditions. 30
4. Discussion 35
4-1. Most of the mutants of NPF members showed an identical pattern of nitrate distribution among leaves compared to wild type in this experiment. 35
4-2. GTR2 might play the role of regulating nitrate storage in young leaves 39
4-3. CLCa and CLCb do not affect nitrate sensing in short-term primary nitrate response 41
5. Reference 45
6. Appendix 109

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