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姓名 陳奕儒(Yi-ru Chen)  查詢紙本館藏   畢業系所 生命科學系
論文名稱 水稻Phosphate starvation up-regulated genes (OsPSU)基因的功能性分析
(Functional analysis of phosphate starvation up-regulated genes, OsPSUs, in Oryza sativa)
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摘要(中) 磷是植物生長發育所需的必要元素,磷的含量會嚴重影響作物的產量。水稻為世界上重要的糧食作物之一,為了開發出能夠耐低磷或能高效率使用磷的水稻以因應未來磷礦不足的情況,本篇論文利用microarray 分析缺磷誘導基因的結果中,挑選出六個與缺磷相關的基因OsPSU1,OsPSU2,OsPSU3,OsPSU4,OsPSU5和OsPSU6 (Phosphate starvation up-regulated genes),探討它們是否參與水稻缺磷反應機制。RT-PCR的分析中,顯示所選之6個OsPSUs均為缺磷誘導表現基因。與野生型水稻(WT)相比,過量表達OsPSUs轉殖水稻小苗葉片累積較多的磷,所以OsPSUs過量表達轉殖稻在磷濃度323 μM以下培養時,生長都比WT來的佳,但磷濃度1618 μM以上培養時,則是呈現比WT更嚴重的磷毒性外表型。過量表達OsPSUs轉殖水稻中,參與磷調控機制中的重要調控因子基因OsPHR2和OsPHO2表現量會大量增加,而且OsPHR2下游一個低親和力的phosphate transporter基因OsPT2的表現亦會增加。這些結果顯示,過量表現OsPSUs能提高缺磷的訊號,進而增加OsPT2的表現,增強了水稻對磷的吸收率與累積,而高磷訊號隨即提高OsPHO2的表現,負回饋抑制磷的吸收率與累積。
摘要(英) Phosphorus is an essential macronutrient for plant development and affects crops productivity seriously. Rice is one of major food crop in the world. The goal of this study, 6 inorganic phosphate (Pi) starvation relative genes (OsPSUs) that were identified by microarray analysis of RNA isolated from Pi-starved rice roots of seedling were investigated for rice response to phosphate starvation. Ectopic expression of each OsPSU transgenic rice lines were generated. Pi levels were higher in leaves of each transgenic line than wild-type plants cultured under normal phosphate concentration. Seedlings of transgenic lines overexpressing OsPSUs grown below in 323 μM Pi exhibit heather phenotype than wild-type. Moreover, each OsPSU transgenic lines displayed more severe Pi toxicity than wild type when they were grown in Pi concentration higher than 1618 μM. Furthermore, overexpression of each OsPSU enhanced the expression of OsPHR2 and OsPHO2, both are involved in Pi utilization pathway. In addition, expression of OsPT2, which encodes a low-affinity phosphate transporter, was also increased in each OsPSU overexpressing lines. These results suggest that all of OsPSUs enhance the rate of phosphate uptake through OsPHR2-OsPT2-mediated pathway.
關鍵字(中) ★ 磷
★ 水稻
關鍵字(英)
論文目次 中文摘要 Ⅰ
Abstract Ⅱ
致謝 Ⅲ
本文目錄 Ⅳ
前言-圖目錄 Ⅷ
表目錄 Ⅸ
圖目錄 Ⅹ

本文目錄
壹、前言 1
一、水稻 1
二、植物大量必需元素-磷 1
三、植物缺磷之信號調控機制 6
四、研究動機與目的 9

貳、材料與方法 10
1. Microarray 10
2. Genomic DNA之抽取 10
3.製備cDNA 10
3.1製備DEPC-treated ddH2O (RNase-free)溶液 10
3.2 RNA 之抽取 11
3.3去除遺傳組DNA之汙染 11
3.4 cDNA之合成 11
3.5以PCR放大合成DNA片段 12
4.植物培養 12
4.1水稻小苗生長條件 12
4.2水稻小苗於不同磷濃度的培養 13
4.3水稻癒傷組織之培養 13
4.4水稻懸浮細胞之培養 13
4.5水稻懸浮細胞於不同磷濃度的培養 13
5.水稻轉殖株分析方法 14
5.1無機磷含量測定 14
5.2葉綠素含量測定 14
5.3 水稻小苗在soil pot之生長測試 15
6.顯著差異之分析方法 15

叁、結果 16
1. OsPSUs基因的特性分析 16
2. 分析OsPSUs基因在水稻中的表現模式 17
2.1 OsPSUs在水稻植株與水稻懸浮細胞中缺磷誘導表現的模式 17
2.2 OsPSUs在水稻不同組織下的表現模式 17
3.過量表達OsPSUs之轉殖植株的分析 18
3.1過量表達OsPSUs之genomic DNA分析 18
3.2過量表達OsPSUs之RNA分析 18
4. 過量表達OsPSUs在缺磷時的生理反應與分子機制分析 18
4.1水稻植株中過量表達OsPSUs與缺磷途徑相關基因的表現模式 18
4.2水稻懸浮細胞中過量表達OsPSUs與缺磷途徑相關基因的表現模式 19
4.3過量表達OsPSUs水稻植株之磷含量分析 20

5. 水稻小苗於不同磷濃度下的外表型分析 21
5.1野生型水稻 (TNG67) 於不同磷濃度下的外表型 21
5.2過量表達OsPSUs水稻植株處理過高磷濃度之外表型分析 21

6. 分別過量表達OsPSU1及OsPSU6在缺磷時的生理分析與分生分析 22
6.1分別過量表達OsPSU1及OsPSU6水稻於不同磷濃度下之外表型分析 22
6.2分別過量表達OsPSU1及OsPSU6水稻於不同磷濃度下之磷及葉綠素含
量分析 23
6.3分別過量表達OsPSU1及OsPSU6水稻與缺磷途徑相關基因的表現模式
24

7. 栽種野生型水稻與過量表達OsPSUs轉殖稻於室外之外表型分析 24
7.1過量表達OsPSUs水稻植株之soil pot分析 24
7.2過量表達OsPSUs之農藝性狀分析 24

肆、討論 26
伍、參考文獻 31
附錄Ⅰ 64
附錄Ⅱ 67
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指導教授 陸重安(Chung-an Lu) 審核日期 2015-1-19
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