蔡宜芳臺灣大學：植物科學研究所何承訓Ho, Cheng-SyunCheng-SyunHo2007-11-272018-07-062007-11-272018-07-062004http://ntur.lib.ntu.edu.tw//handle/246246/57933為了適應外在離子與營養物含量的不穩定，植物發展出高、低不同親和性的吸收系統。傳統認為這兩個系統是由不同基因所負責調控；但是最新研究發現，阿拉伯芥的硝酸鹽轉運蛋白CHL1是個具雙親和性的轉運蛋白，而且CHL1的雙親和性是由CHL1第101個蘇胺酸 (threonine) 是否磷酸化來調控；磷酸化的CHL1為高親和性的轉運蛋白，去磷酸化的CHL1為低親和性的轉運蛋白。但是目前對於CHL1的磷酸化與去磷酸化在植物生理的重要性卻還不清楚。本實驗利用多種分生與生化的方法觀察植物生長在不同硝酸鹽來源和不同濃度的環境下，在不同時間，植物對硝酸鹽吸收能力的變化、CHL1和其他負責吸收硝酸鹽轉運蛋白基因的表現情形，以及CHL1蛋白質的磷酸化的變化。並進一步比較野生株和chl1缺失株的高、低親和性硝酸鹽吸收能力，來瞭解CHL1在不同情況下對硝酸鹽吸收的貢獻。我們發現：1.) CHL1在高濃度硝酸鹽(12.5 mM)的吸收為最主要的轉運蛋白。CHL1負責的硝酸鹽吸收活性在高濃度硝酸鹽誘導是呈現一個先上升然後下降的表現情況；而在低濃度硝酸鹽(200μM)誘導下，野生株與chl1-5缺失突變株硝酸鹽吸收活性差異不明顯。2.) 在高濃度硝酸銨鹽下，CHL1蛋白是先上升然後下降的表現，且磷酸化強度是隨誘導的時間遞增而增加；但是在低濃度硝酸鹽下，CHL1蛋白與磷酸化表現情形正好與低硝酸鹽濃度表現情況相反。3.) 我們也觀察到外在不同硝酸鹽濃度，會對CHL1 RNA的表現有明顯的影響，但是對高親和性轉運蛋白NRT2:1的RNA表現則影響不大。4.) 在高濃度硝酸鹽(12.5 mM)誘導下CHL1蛋白與CHL1 RNA的表現一致，但在低濃度硝酸鹽(200μM)誘導下，CHL1蛋白與CHL1 RNA表現不一致，因此，我們推測CHL1在低濃度硝酸鹽(200μM)誘導中具有post-transcriptional regulation。5.) 在比較野生株和chl1-5缺失突變株的硝酸鹽吸收活性與其他負責吸收硝酸鹽轉運蛋白基因在不同硝酸鹽濃度的環境下表現，發現當CHL1缺失時，NRT2:1 RNA的表現與在野生株時的表現不同，推測當CHL1缺失時NRT2:1可能扮演互補CHL1功能的角色。CHL1 is a dual-affinity nitrate transporter. Two functional modes of CHL1 are switched by phosphorylation and dephosphorylation at threonine residue 101: when phosphorylated, CHL1 function as a high-affinity nitrate transporter, whereas, when dephosphorylated, it functions as a low-affinity nitrate transporter. However, little is known about how CHL1 phosphorylation is regulated by the N-status of the plant, and what the physiological impacts of CHL1 phosphorylation are. To address these questions, nitrate uptake activity, CHL1 mRNA levels, CHL1 protein levels, as well as CHL1 phosphorylation levels were determined for plants exposed to high- or low- concentration of nitrate in the presence or absence of ammonium. When exposed to high concentration of nitrate, both CHL1 mRNA level, protein level as well as CHL1-mediated nitrate uptake activity showed transient increment followed by graduate decline. However, phosphorylation levels remain high 24 hour after exposing to high-concentration of nitrate. In contrast, when exposed to low-concentration of nitrate, CHL1 phosphorylation level showed transient increment then decline, but the protein levels remain high 24 hours after shift. These data indicated that phosphorylation level is high when the protein level is low. In addition, under certain condition, CHL1 protein level is not proportional to its mRNA level suggesting that CHL1is post-transcriptionally regulated in response to different nitrogen condition.1.中文摘要………………………………………………………….1 2.英文摘要………………………………………………………….2 3.前言……………………………………………………………….3 4.材料與方法……………………………………………………….9 5.結果……………………………………………………………...26 6.討論……………………………………………………………...33 7.圖表………………………………………………........... 39 8.參考文獻………………………………………………………...66546108 bytesapplication/pdfen-US硝酸鹽轉運蛋白磷酸化CHL1phosphorylationotherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/57933/1/ntu-93-R91226028-1.pdf