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標題: | 闡述AtNPF8.1/AtPTR1受質專一性的分子特徵 Elucidating the molecular features of AtNPF8.1/AtPTR1 substrate specificity |
作者: | Feng-Chih Kang 康峰誌 |
指導教授: | 王雅筠(Ya-Yun Wang) |
關鍵字: | 受質專一性,轉運蛋白,硝酸鹽,胜肽,NPF,AtPTR1, substrate specificity,transporter,nitrate,peptide,NPF,AtPTR1, |
出版年 : | 2021 |
學位: | 碩士 |
摘要: | 氮是植物生長發育必要的元素(如硝酸鹽或胜肽),在過去的研究中知道有機的氮源(如胜肽)吸收以及運動對植物的生理反應是非常重要的。AtPTR1是硝酸鹽轉運蛋白1 /胜肽轉運蛋白家族(NPF)的一員,可運輸多種胜肽但不能運輸硝酸鹽。在本研究中我們想藉由AtPTR1去了解,胜肽與硝酸鹽的受質專一性是如何決定的。我們挑選了多個點位並做出21種突變來研究其對於硝酸鹽以及雙肽運輸的影響。先用原生質體來檢視突變後的AtPTR1座落位置是否有表現在細胞膜上,發現H534L、H534Y、120-140、268-311座落位置由細胞膜改變為其他位置。使用酵母菌互補分析看雙肽的運輸,發現Y45I、Y346H、C31A、C31S、C318A、C318S、H534K之突變會降低對於雙肽的運輸;Y45IY46I、Y45IY46IY346H則喪失運輸能力。用非洲爪蟾卵母細胞表現系統看硝酸鹽的運輸,發現Y45I、Y46I、Y46A、Y45IY46I、Y346H、Y45IY46IY346H突變蛋白皆不能運輸硝酸鹽。在本研究中未發現可以影響受質決定的突變蛋白,然而這些結果讓我們了解到AtPTR1的座落位置會因特定點位的改變造成而受到影響,有的突變亦會造成其運輸能力的減弱或喪失,這些關於AtPTR1分子特徵的闡述,可作為模型去推論及研究NPF其他轉運蛋白的功能與特性。 Nitrogen (such as nitrate and peptide) are essential for plant growth and development. The uptake and transport of organic N (such as peptides) is important for many physiological responses. In the past, AtPTR1 has been known as a plasma membrane-localized transporter and a member of the Nitrate transporter 1/Peptide transporter Family (NPF). AtPTR1 can transport several di-, tri-peptides, but not nitrate. In our study, we want to understand the difference in substrate specificity between AtPTR1 and other nitrate transporters in NPF. Therefore, we selected multiple sites to study the effect of nitrate and dipeptide transport. At first, we examined whether the localization of mutated AtPTR1 by the protoplast and found that H534L, H534Y, 120-140, and 268-311 has lost the plasma membrane-localized property. In yeast complementation assay, we analyzed the di-peptide uptake ability of mutated AtPTR1. We found Y45I, Y346H, C31A, C31, C318A, C318S, H534K reduced the di-peptide uptake ability, and Y45IY46I, Y45IY46IY346H loss of the di-peptide uptake ability. We also showed that no mutated AtPTR1 proteins can uptake nitrate by using the Xenopus oocyte expression system. In this study, the mutant protein that can affect substrate determination to nitrate haven’t been found. However, we understand some mutations of the specific sites can affect the localization of AtPTR1, and some mutations also can cause the reduction or loss of the di-peptide uptake ability. These findings of the substrate specificity of AtPTR1 can be used as a model to understand and study the functions and properties of other NPF members. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/8300 |
DOI: | 10.6342/NTU202100600 |
全文授權: | 同意授權(全球公開) |
電子全文公開日期: | 2026-02-18 |
顯示於系所單位: | 植物科學研究所 |
文件中的檔案:
檔案 | 大小 | 格式 | |
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U0001-0502202115561900.pdf 此日期後於網路公開 2026-02-18 | 6.89 MB | Adobe PDF |
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