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研究生:王郁雯
研究生(外文):Wang, Yu-Wen
論文名稱:NRT1(POT)轉運蛋白家族的硝酸鹽親和性及受質專一性的決定性結構之研究
論文名稱(外文):Structural Determinants for Nitrate Affinity and Substrate Specificity of NRT1(POT) Family
指導教授:蔡宜芳蔡宜芳引用關係
指導教授(外文):Tsay, Yi-Fang
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:遺傳學研究所
學門:生命科學學門
學類:生物訊息學類
論文種類:學術論文
論文出版年:2003
畢業學年度:91
語文別:中文
論文頁數:59
中文關鍵詞:轉運蛋白區域置換複合體非洲爪蟾硝酸鹽雙胜肽
外文關鍵詞:NRT1 familyCHL1NTL1AtPTR2domain-shuffling chimeranitratepeptideXenopus oocyte
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植物為了適應外界環境中巨大的硝酸鹽濃度變化,而演化出低親和性及高親和性兩種硝酸鹽轉運系統。CHL1,是第一個在阿拉伯芥中被選殖出來的植物硝酸鹽轉運蛋白,具有雙親和性的轉運活性;當第101個胺基酸threonine,T101,被磷酸化時,CHL1負責高親和性的硝酸鹽轉運,相對地,T101被去磷酸化時,則負責低親和性硝酸鹽吸收活性。然而,CHL1上哪一部分的結構,負責雙親和性轉運特性及硝酸鹽的專一性轉運至今仍不清楚。NTL1,為阿拉伯芥的一個CHL1同源基因蛋白,同樣負責硝酸鹽的轉運,但只具有低親和性吸收的活性;另外,AtPTR2為一阿拉伯芥的胜肽轉運蛋白,在酵母菌的表現系統中顯示,其具有高親和性的胜肽吸收活性。而NTL1及AtPTR2和CHL1在蛋白質的一級及二級結構的相似性上,皆屬於NRT1 (PRT / POT)轉運蛋白家族。在本論文中,為了了解轉運蛋白功能與結構之間的關係,我們利用構築蛋白質區域置換之複合體 (domain-shuffling chimera),將CHL1分別與NTL1或AtPTR2作不同區域的置換,以非洲爪蟾卵細胞作為表現系統,利用HPLC及放射性標定的胜肽受質,來偵測這些區域置換複合體的高低親和性硝酸鹽吸收活性,以及受質專一性的轉運情形。實驗的結果發現,將CHL1 TM2-3置換入NTL1的複合體,具有雙親和性的硝酸鹽吸收活性;而置換入包含CHL1 N端到TM4區域的複合體,具有硝酸鹽專一性的吸收活性。另一方面,置換入AtPTR2 N端到TM9及TM7到C端的複合體,具有較明顯的胜肽受質專一性吸收活性。因此,CHL1上負責高親和性及硝酸鹽專一性轉運的必須性結構,分別位於包含T101的TM2-3,及N端到TM4之間的區域;而AtPTR2 的loop6到TM9區域內,則可能含有一些參與胜肽轉運的重要因子。
To counteract the fluctuation of nitrate concentration in the environment, plants have evolved high- and low-affinity uptake systems. CHL1 is the first nitrate transporter cloned from Arabidopsis. CHL1 has a dual-affinity nitrate transport activity, and the switching between the two action modes is dependent on phosphorylation state of threonine residue 101. However, which region(s) of CHL1 is responsible for its nitrate affinity and substrate specificity is unknown. NTL1, a CHL1 homolog, is a nitrate transporter in Arabidopsis, but was shown to mediate only low-affinity nitrate uptake. AtPTR2 gene was cloned from Arabidopsis cDNA library by functionally complementing a yeast peptide transport mutation, and was showed a high-affinity peptide uptake activity. All these three transporters genes encoded 12 putative transmembrane segments, and are belong to NRT(POT) family. To evaluate structural determinants for nitrate affinity and substrate specificity respectively, in this study, we used CHL1, NTL1, and AtPTR2 to construct domain-shuffling chimeras. Using Xenopus oocyte to be the expression system, then we used HPLC and isotope-labeled dipeptide to detect the transport activities of nitrate and peptide, respectively. Finally we conclude that TM2-3 and the region from N terminal to TM4 of CHL1 is essential for high-affinity nitrate uptake and nitrate-specific transport respectively. In the second part, the loop6-TM9 region of AtPTR2 is more likely important to peptide-specific transport.
縮寫表………….……………..i
中文摘要……………………..ii
英文摘要……………….…….iii
前言……………………………..1
材料與方法…………………..8
結果………………………….…20
討論…………………………...27
圖表…………………………….31
參考文獻……………………..51
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