臺灣大學: 分子與細胞生物學研究所蔡宜芳鄭令欣Cheng, Ling-HsinLing-HsinCheng2013-03-202018-07-062013-03-202018-07-062010http://ntur.lib.ntu.edu.tw//handle/246246/247364硝酸鹽是植物體內主要儲存氮元素的分子，同時也有調節植物發育的功能。先前的研究中發現CHL1（AtNRT1.1）蛋白具有轉運蛋白及硝酸根感應受器的雙重功能，並發現了一個轉運蛋白-感應功能分離的CHL1基因變種株chl1-9。chl1-9是一個Pro492Leu的點突變株，Pro492是一個在阿拉伯芥NRT1基因族中保存度極高的氨基酸，而這個胺基酸與硝酸根運輸有關卻又不會影響硝酸根感應受器的功能。NRT1.13以及NRT1.14是NRT1家族中少數在第492氨基酸相對位置並非Pro的基因，其中NRT1.13在這個位置是Ser，而NRT1.14則是Ala。在水稻的NRT（PTR）基因族中發現有一基因SP1（Short panicle 1），其第492胺基酸相對位置是Leu。利用角蟾卵吸收的實驗發現SP1並不具硝酸轉運蛋白的功能，而當SP1發生突變時植株會呈現短穗表現型。NRT1.13基因在分類上與SP1（Os11g12740）屬於同一亞族群，因此探究NRT1.13基因是否屬於硝酸根感應受器對於瞭解Pro492的角色有重要的意義。 透過GUS組織化學分析以及定量PCR分析我們發現NRT1.13主要分佈在葉柄、果莢著生處及莖遠端部位的實質細胞內，就細胞層次而言NRT1.13則是表現在細胞膜上。而我們也利用喪失NRT1.13功能之突變株nrt1.13與野生種作表現型比較分析以了解NRT.13在植物活體的功能。我發現野生型植株開花時間平均為20.8日，而突變株nrt1.13平均為22.9日；野生型植株平均於長出12.4片葉片時開花，而突變株則在平均有14.9片葉片時開花，顯示nrt1.13有晚開花的現象。果莢生長點間的平均距離也有差異，野生型為0.92公分，而nrt1.13較短為0.64公分，顯示NRT1.13如同水稻的SP1基因一般具有調節花序生長的功能。除此之外，在觀測nrt1.13突變株的含氮量以及放射性氮-15的分佈測試，我們發現氮-15在遠端節點、莖生葉以及花的部分的含量有顯著下降。另外，nrt1.13突變株的種子休眠程度較野生型高，但此休眠現象可經由外加的硝酸根去除，推論nrt1.13種子含硝酸鹽的量較野生型為低。綜合上述發現，我們認為NRT.13對於植物發育的調節以及酸根離子在生殖組之內的分佈有重要的影響。Nitrate is not only the primary nitrogen source but also a signaling molecule regulating plant development. A study of the uptake- and sensing-decoupled mutant chl1-9 demonstrates that CHL1 (AtNRT1.1) is not only a transporter but also a sensor. Pro492, which is mutated in the chl1-9 mutant, is required for nitrate transport, but not for nitrate sensing. Pro492 was highly conserved among NRT1 (PTR) family of Arabidopsis thaliana, except two members, NRT1.13 and NRT1.14. Pro was substituted by Ser in NRT1.13 and Ala in NRT1.14, respectively. It will be interesting to find out if these two NRT1 transporters are involved in nitrate sensing. Consistently, a rice NRT1 (PTR) transporter SP1 (Short Panicle 1) with the corresponding Pro492 residue substituted by Leu exhibit no nitrate transport activity when expressed in Xenopus oocyte and display a short panicle phenotype when mutated. Indeed, NRT1.13 (At1g33440) is classified into the same subgroup with SP1 (Os11g12740). This suggested NRT1.13 might also participate in regulating plant development. Histochemical analysis of PNRT1.13-GUS and quantitative PCR analysis showed that NRT1.13 was mainly expressed in parenchyma cell of petiole, spray and distal part of stem. Furthermore, subcellular localization analysis showed that NRT1.13-GFP fusion protein was in plasma membrane. In order to characterize the in vivo function of NRT1.13, several phenotypes between wild type and nrt1.13 mutant was compared. Compared to the flowering time of 20.8 days in wild type, flowering time in nrt1.13 delayed to 22.9 days. And, the number of leaves when bolted increased from 12.4 in wild type to 14.9 in the mutant. The average distance between sliques of nrt1.13 was also decreased from 0.92 of wild type to 0.64 cm, suggesting that similar to rice SP1, NRT1.13 also participate in regulating elongation of inflorescent stem. In addition, the nitrate content and short-term-15N partition of nrt1.13 were monitored to examine if there were any defects of nrt1.13 in nitrogen distribution. It showed a reduced level of the 15N in distal nodes and flowers of nrt1.13. The nitrate content of nrt1.13 cauline leaf was also reduced. Moreover, germination test showed that fresh seeds of nrt1.13 were more dormant than wild type in a nitrate dependent manner, suggesting that seed nitrate content is reduced in nrt1.13. Taken together, these data indicated that NRT1.13 was important for both developmental regulation and nitrate distribution of reproductive tissue.2712309 bytesapplication/pdfen-US阿拉伯芥NRT1.13Arabidopsisthesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/247364/1/ntu-99-R97B43030-1.pdf