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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.author | Po-Kai Hsu | en |
| dc.contributor.author | 許博凱 | zh_TW |
| dc.date.accessioned | 2021-07-01T08:13:05Z | - |
| dc.date.available | 2021-07-01T08:13:05Z | - |
| dc.date.issued | 2003 | |
| dc.identifier.citation | 侯信成．（2001）利用生化的方法研究硝酸鹽轉運蛋白CHL1及其同源蛋白AtNRT1:5在植物體內所扮演的角色及其調控機制．國立台灣大學植物科學研究所碩士學位論文．
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/75415 | - |
| dc.description.abstract | 硝酸鹽是植物的主要氮源之一，植物吸收硝酸鹽後，可以在根部利用，也可以儲存在液泡中，或經由木質部將硝酸鹽送至地上部組織利用和儲存。然而硝酸鹽如何被送到葉片的機制，仍有待利用分生的技術加以瞭解。在西元1993年，第一個植物硝酸鹽轉運蛋白CHL1在阿拉伯芥中被分離出來，而阿拉伯芥基因體定序完成後，發現在阿拉伯芥總共有53個CHL1的同源蛋白，而除了硝酸鹽轉運蛋白以外，勝?轉運蛋白也包含在這群同源蛋白之中。在本研究中，我分離了CHL1的同源蛋白基因AtNRT1:9，並且試圖闡明其生理功能。比對CHL1和AtNRT1:9蛋白質序列，兩者具有31.4％的相同性，43.8％的相似性。蛋白質序列疏水性分析顯示AtNRT1:9可能是一個膜蛋白，具有十二個跨膜區域。藉由蛙卵測試AtNRT1:9功能，證實了AtNRT1:9是一個低親和性硝酸鹽轉運蛋白。而在阿拉伯芥葉肉原生質細胞中表現AtNRT1:9-GFP，證明AtNRT1:9確實為一膜蛋白表現在細胞膜上。北方雜合分析顯示，AtNRT1:9主要表現於阿拉伯芥的地上部，且其表現不受硝酸鹽的調控。利用AtNRT1:9promoter::GUS轉殖株中GUS活性表現位置，進一步發現AtNRT1:9的表現位置限定於維管束組織，並且在葉柄及中肋有較高的表現。由以上證據，推測AtNRT1:9可能在木質部薄壁細胞扮演將硝酸鹽由導管卸載到葉部組織的角色。然而，在野生型與atnrt1:9knockout突變株個體，地上部與根部的硝酸鹽含量和葉部的硝酸鹽分佈狀況都無法見到顯著差異。這樣的結果可能歸因於：1）其他CHL1同源蛋白的功能重複，能夠取代AtNRT1:9的功能。2）由於長時間供應硝酸鹽，雖然野生株與突變株可能有微小的差異，卻因為長時期培養造成的累積，使突變株葉部的硝酸鹽分佈已經平衡到與野生型相當，因此沒有看到差異。 | zh_TW |
| dc.description.abstract | For most plants, nitrate is the major nitrogen source. After being taken up, nitrate could be assimilated or stored in root or could be transported to shoot by xylem to be assimilated there. However, little is know at molecular level about the mechanisms of nitrate long-distance transport in higher plant. The first nitrate transporter gene CHL1 (AtNRT1) in Arabidopsis was cloned in 1993. In Arabidopsis genome, there are 53CHL1 homologs. In addition to nitrate transporters, some peptide transporters also belong to this family. In this report, we cloned the gene AtNRT1:9 and elucidated its function in plant. Functional analysis of AtNRT1:9 in Xenopus oocytes showed that AtNRT1:9 is a low-affinity nitrate transporter. Transient expression of AtNRT1:9-GFP in Arabidopsis mesophyll protoplasts suggested that AtNRT1:9 is localized on the plasma membrane. Northern analysis indicated that AtNRT1:9 primarily expressed in shoot and its expression was not regulated by nitrate. The tissue-specific expression pattern of AtNRT1:9 was analyzed using AtNRT1:9 promoter::GUS transgenic plants. The expression AtNRT1:9 was found to be restricted in the vascular bundle, especially in the petiole and midrib. These data indicated that AtNRT1:9 might be involved in nitrate unloading from xylem into the leaf tissue. However, no significant difference of nitrate content in the shoot and root, and of the nitrate distribution in leaf has been found between atnrt1:9 knockout mutant and wild type. These results might be caused by 1) the functional redundancy of other members of the CHL1 family; 2) subtle difference in nitrate distribution was sequestered by long-term balance effect. | en |
| dc.description.provenance | Made available in DSpace on 2021-07-01T08:13:05Z (GMT). No. of bitstreams: 0 Previous issue date: 2003 | en |
| dc.description.tableofcontents | 縮寫表……………………………………………………I
中文摘要……………………………………………………III 英文摘要……………………………………………………IV 前言……………………………………………………1 材料方法……………………………………………………6 一、植物材料……………………………………………………6 二、植物培養液……………………………………………………6 三、引子(primers)……………………………………………………7 四、質體(plasmid)構築……………………………………………………7 五、利用非洲爪蟾(Xenopus laevis)卵做為做為表現系統分析基因功能……………………………………9 六、AtNRT1:9基因表現分析……………………………………………………12 七、農桿菌轉殖阿拉伯芥……………………………………………………16 八、β-glucuronidase活性分析……………………………………………………18 九、轉殖株T-DNA拷貝數分析……………………………………………………18 十、atnrt1:9 Knockout突變株篩選……………………………………………………20 十一、植物硝酸鹽含量分析……………………………………………………21 結果……………………………………………………23 AtNRT1:9的分離與蛋白質序列分析……………………………………………………23 AtNRT1:9硝酸鹽吸收活性分析……………………………………………………23 AtNRT1:9-GFP之表現膜系……………………………………………………24 AtNRT1:9基因表現……………………………………………………25 AtNRT1:9的組織專一性……………………………………………………25 逆轉遺傳學策略(Reverse Genetics Strategy)篩選AtNRT1:9突變株………………………………………26 atnrt1:9表現型分析……………………………………………………28 討論……………………………………………………30 圖表……………………………………………………35 圖一 CHL1與AtNRT1:9蛋白質序列分析……………………………………………………35 圖二 AtNRT1:9蛋白質序列親水性分析……………………………………………………36 圖三 非洲爪蟾卵低親和性硝酸鹽吸收試驗……………………………………………………37 圖四 非洲爪蟾卵高親和性硝酸鹽吸收試驗……………………………………………………38 圖五 326AtNRT1:9-GFP的構築及結構圖……………………………………………………39 圖六 AtNRT1:9-GFP在阿拉伯芥葉肉原生質細胞(protoplast)中的表現………………………………………40 圖七 AtNRT1:9基因北方雜合分析……………………………………………………41 圖八 硝酸鹽誘導基因表現分析……………………………………………………42 圖九 pBI101.1-AtNRT1:9 promoter::GUS T-DNA區域結構圖……………………………………………………43 圖十 AtNRT1:9 promoter::GUS T1轉殖株T-DNA拷貝數分析……………………………………………………44 圖十一 AtNRT1:9 promoter::GUS轉殖株T2子代GUS活性分析……………………………………………………45 圖十二 AtNRT1:9基因結構及T-DNA插入位置圖……………………………………………………46 圖十三 atnrt1:9T-DNA拷貝數分析……………………………………………………47 表一 Ws-2×atnrt1:9第二子代抵抗殺草劑Basta測試……………………………………………………47 圖十四 atnrt1:9 T-DNA插入結構分析……………………………………………………48 圖十五 RT-PCR分析野生型與突變株中AtNRT1:9表現……………………………………………………48 圖十六 長時期供應硝酸鹽的植物組織硝酸言含量測量……………………………………………………49 圖十七 短時期供應硝酸鹽的植物組織硝酸言含量測量……………………………………………………50 圖十八 葉部硝酸鹽的分佈……………………………………………………51 圖十九 葉部硝酸鹽的分佈……………………………………………………52 參考文獻……………………………………………………53 附圖一 RT-PCR分析AtNRT1:9於葉部不同部位的表現狀況……………………………………………………59 附圖二 CHL1同源蛋白演化樹……………………………………………………60 | |
| dc.language.iso | zh-TW | |
| dc.title | 阿拉伯芥硝酸鹽轉運蛋白AtNRT1:9的功能分析 | zh_TW |
| dc.title | Isolation and Characterization of a New Nitrate Transporter AtNRT1:9 in Arabidopsis | en |
| dc.date.schoolyear | 91-2 | |
| dc.description.degree | 碩士 | |
| dc.relation.page | 59 | |
| dc.rights.note | 未授權 | |
| dc.contributor.author-dept | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 植物科學研究所 | zh_TW |
| 顯示於系所單位： | 植物科學研究所 | |
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