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標題: | I.阿拉伯芥液胞膜醣轉運蛋白在碳氮平衡之研究 II.阿拉伯芥硝酸鹽分佈與再利用相關基因在全基因組關聯性分析研究之篩選 I. The study of Arabidopsis Tonoplast Sugar Transporters AtTSTs in nitrogen and carbon homeostasis II. Genome-wide association study identifies the QTLs involved in nitrate distribution and remobilization in Arabidopsis thaliana |
作者: | 王孟加 Meng-Jia Wang |
指導教授: | 蔡宜芳 Yi-Fang Tsay |
關鍵字: | 阿拉伯芥,硝酸鹽,轉運蛋白,液胞醣轉運蛋白,硝酸鹽分布,硝酸鹽再利用,全基因組關聯性分析, Arabidopsis,nitrate,transporter,tonoplast sugar transporter,nitrate distribution,nitrate remobilization,genome-wide association study,GWAS, |
出版年 : | 2019 |
學位: | 碩士 |
摘要: | I.硝酸鹽是植物利用的主要氮源,不僅是養份分子,同時也能透過訊號傳遞調控植物生長。磷酸化是硝酸鹽感應系統中的重要反應。以硝酸鹽轉運及感應蛋白CHL1為例,Thr101的磷酸化除了改變CHL1對硝酸鹽吸收的親和性之外,也改變其硝酸鹽感應的反應。有鑑於此,我們利用定量磷酸化蛋白質體分析尋找硝酸鹽調控的蛋白質磷酸化現象,發現兩個液胞醣轉運蛋白TST1/2在硝酸鹽處理後發生磷酸化現象。由於硝酸鹽跟醣類分屬植物體內最豐富的兩個元素-碳跟氮。因此,我們利用這兩個蛋白的突變株來研究TST1/2在碳氮平衡中扮演的角色。結果顯示,在高濃度硝酸鹽處理後,短時間內突變株tst1跟tst2的地上部硝酸鹽濃度都比野生型植物高。此外,在相同條件下,根部的硝酸鹽吸收能力以及硝酸鹽在根部和地上部的分配在突變株內並沒有改變,顯示在短期的高濃度硝酸鹽處理之下,TST1/2可能透過影響貯存或氮同化作用來改變硝酸鹽在地上部的濃度。代謝體學實驗結果則顯示TST1/2分別在不同組織中影響醣類運輸與代謝。而磷酸化對於TST1/2的影響則需要用爪蟾蛙卵表現系統與轉殖植物進行後續分析。
II.硝酸鹽是植物利用的主要氮源,不僅是養份分子,也能透過訊號傳遞調控植物生長。已知有許多硝酸鹽轉運蛋白負責葉片之間的硝酸鹽運輸。如NPF2.13從老葉回收硝酸鹽到新葉再次利用。而NPF1.1與NPF1.2則透過把成熟葉片的硝酸鹽運送到發育中的新葉來重新分配葉片中的硝酸鹽。植物藉由這些轉運蛋白回收硝酸鹽提升氮利用效率。為了尋找其他參與在硝酸鹽再利用或重新分配的調節者,我們利用不同葉片的硝酸鹽濃度與總量進行全基因組關聯性分析,找出47個候選的數量性狀基因座,本研究中分析了其中三個基因座的特徵。ACX1是參與在茉莉酸合成的酵素,結果顯示在ACX1基因座上的單核苷酸多型性(SNP)影響了植物的硝酸鹽分佈與生長形態。兩群具有不同單倍型ACX1的野生型植物顯示不同的新葉硝酸鹽比例與相異的葉柄長度。此外,基因剔除株acx1-1具有較長的葉柄,顯示Q28可能是一個抑制葉柄延長的因子。由於葉柄是葉片主要貯存硝酸鹽的部位,Q28是否透過調節葉柄長度影響葉片之間的硝酸鹽則需要測定不同部位的硝酸鹽濃度進行分析。ZML1,又稱作GATA24,是一個參與在光保護作用的GATA轉錄因子。實驗結果顯示,ZML1的基因剔除株在高濃度硝酸鹽下生長受阻。有趣的是,相同條件下,基因剔除株zml1的老葉具有比例較野生型高的硝酸鹽,而成熟葉與新葉則有較低的硝酸鹽比例,顯示在高濃度硝酸鹽的環境中,ZML可能參與硝酸鹽在葉片之間的分佈並影響植物生長。CIA2已知影響蛋白質進入葉綠體的輸送,實驗結果顯示cia2的成熟葉片在高濃度或低濃度的硝酸鹽環境下都具有較野生型植物高的硝酸鹽。此外,cia2的成熟葉的硝酸鹽比例也較野生型高,表示CIA2可能影響硝酸鹽在葉片中的同化作用或葉片之間的分佈。而這些基因座如何影響硝酸鹽分佈與再利用則需要利用同位素標定追蹤實驗與定量核酸分析來闡明。 I.Nitrate, the major nitrogen source, is also a signaling molecule coordinating plant development. Phosphorylation is a critical step in nitrate signaling, e.g. nitrate transceptor CHL1 changes nitrate uptake activity and sensing modes in response to fluctuating nitrate concentrations in the soil through phosphorylation at residue T101. We performed quantitative phosphoproteomic analysis to search other nitrate-regulated phosphorylation, and found that two tonoplast sugar-proton antiporters TST1 (Sugar Transporter1) and TST2 were phosphorylated in response to nitrate. Mutants were characterized to elucidate the roles of TST1/2 in nitrogen (N)/carbon (C) balance. We found that more nitrate accumulated in shoots of tst1 and tst2 when plants were treated with high nitrate for short period of time. Under the same condition, nitrate uptake and partition showed no difference between WT and mutant, suggesting that the changes in nitrate accumulation may be due to nitrate storage or assimilation. Metabolite profiling showed that TST1/2 affect sugar transport or metabolism in different tissues. Taken together, these results indicated that TST1/2 play a role in N/C balance as nitrate will affect TST1/2 phoshorylation, and in retrun, TST1/2 will affect nitrate and N metabolites levels. The impact of TST1/2 phosphorylation will be elucidated by functional analysis of phosphomimetic form of TST1/2 in Xenopus oocyte and in planta. II. Nitrate, the major nitrogen source, is also a signaling molecule coordinating plant growth. During the development, there were several transporters known to allocate nitrate among leaves. Nitrate/peptide transporter family (NPF) 2.13 remobilizes nitrate from old leaves to young leaves, while NPF1.1 and 1.2 mediate re-distribution from mature leaves to young leaves. These three transporters recycle more nitrate for plants to feed young leaves and promote the nitrogen utilization efficiency. To identify the other genes involved in nitrate distribution and remobilization, we performed genome-wide association study (GWAS) of nitrate concentrations in different leaves. 47 quantitative trait locus (QTLs) associated with nitrate distribution were identified. We report here the characterization of three loci ACX1, ZML1 and CIA2. The natural variation in ACX1, an enzyme involved in JA biosynthesis, affect nitrate concentration as well as the morphology of plants. Two haplotypes, Col-0 haplotype and Nw-0 haplotype were defined by three SNPs associated with the ratio of young leaf nitrate to the total leaf nitrate. The petiole length also differed significantly between two haplotypes. These results indicated that ACX1 might play a regulatory role in the plant growth and nitrate distribution. ZML1, a GATA-type transcription factor, affected leaf biomass at high nitrate, but not at low nitrate. These results suggested that ZML1 might affect nitrate use efficiency specifically under high nitrate condition. CIA2 is known to regulate chloroplast protein import. Our analysis showed that nitrate concentrations of mature leaves in cia2 mutant are increased at both high and low nitrate conditions. However, the biomass of cia2 mutants only increased at low nitrate condition. These results suggest that CIA2 may regulate nitrate assimilation and/or nitrate distribution in the leaves. The effects of these QTLs on nitrate distribution and utilization efficiency will be elucidated by 15NO3 tracing experiment and RT-qPCR. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/78787 |
DOI: | 10.6342/NTU201901085 |
全文授權: | 未授權 |
電子全文公開日期: | 2024-07-10 |
顯示於系所單位: | 分子與細胞生物學研究所 |
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