低親和性硝酸鹽轉運蛋白CHL1參與高親和性硝酸鹽吸收之研究

Abstract

植物對於硝酸鹽的吸收依其親和性及受硝酸鹽誘導與否，可分為高親和性誘導型，高親和性持續型，低親和性誘導型及低親和性持續型四種硝酸鹽吸收系統。一般認為不同的系統是由不同的轉運蛋白來負責執行。 CHL1原本認為是一個低親和性誘導型硝酸鹽轉運蛋白。本篇論文中針對CHL1基因缺失植株（chl1-5）及野生株作硝酸鹽吸收分析時，發現chl1-5除了在低親和性硝酸鹽的吸收有缺失外在高親和性誘導型及持續型硝酸鹽吸收系統上亦出現缺失。也利用在不同培養基生長後作硝酸鹽吸收測試，發現這個缺失的現象並非在不同培養基生長下造成細胞內pH值不同所引起的。而高親和性吸收的缺失也進一步由氯酸鹽的抗性實驗獲得確認；野生株在10 μM的氯酸鹽處理後會死亡而chl1-5則可存活下來。由以下三個實驗證實這缺失是由CHL1所引起：（一）．在其他五株chl1突變株作硝酸鹽的吸收實驗中則排除為其他相連CHL1基因也缺失所造成的現象。（二）．利用花椰菜鑲嵌紋病毒?動子表現CHL1基因，並將其轉殖入chl1-5的植株中，轉殖植物可回復chl1-5在高親和性硝酸鹽的吸收能力。（三）．利用非洲爪蟾卵將CHL1 mRNA注射入卵中表現作功能試驗證實CHL1是一個雙親和性的轉運蛋白，同時參與高親和性與低親和性硝酸鹽吸收。此外，另一個低親和性持續型硝酸鹽轉運蛋白，NTL1，則只負責低親和性硝酸鹽的吸收。因為以其轉殖反義基因之轉殖植株作硝酸鹽吸收測試時則無此高親和性吸收缺失現象；而且將NTL1表現於非洲爪蟾卵細胞時只測到低親和性吸收的活性。由此可見，CHL1可因外在濃度變化而改變其作用模式來負責多項吸收系統（低親和性誘導型，高親和性誘導型，及部份的高親和性持續型），但NTL1只單純地負責低親和性持續型的吸收。

CHL1 has been reported to be an inducible low affinity nitrate transporter. In this thesis, we found that CHL1 was also involved in high affinity nitrate uptake. A deletion mutant chl1-5 was demonstrated to be defective in high affinity nitrate uptake. This observation was upheld by a chlorosis test on wild type and chl1-5 at 10 μM chlorate. The possibility that this phenomenum may be caused by different cytosolic pH was ruled out because same results were obtained from plants grown in different nitrogen source. When another five chl1 mutants were assayed, it showed that the reduction in high affinity uptake is directly caused by the defect in CHL1 gene. And, a transgenic plant expressing 35S-CHL1 cDNA in the deletion background showed a recovery in uptake. NTL1 is a second nitrate transporter gene of Arabidopsis which is involved in the constitutive low affinity nitrate uptake. The high affinity nitrate uptake activity in an anti-sense mutant of NTL1 is the same as wild type, suggesting that the dual defect in high and low affinity nitrate uptake is specific to chl1, but can not be generalized to other Arabidopsis nitrate transporters. This observation is further supported by the uptake assay using HPLC in the hetero-expression system of Xenopus oocytes. Both low and high affinity uptake activities were observed in CHL1-injected oocyte. In NTL1-injected oocyte, there is only low-affinity uptake activity. Thus, CHL1 is a dual-affinity transporter involved in multiple systems (inducible low-affinity, inducible high-affinity, and some constitutive high-affinity) of nitrate uptake in Arabidopsis, while NTL1 is a pure low-affinity transporter responsible for constitutive low-affinity nitrate uptake.