臺灣博碩士論文加值系統

植物生長時會遭遇到許多壓力，其中鹽害壓力是指環境中存在過多的無機鹽類，會造成滲透壓改變、離子毒害及氧化壓力等影響。植物的耐鹽境能力是很重要的研究課題。多元胺是一類帶有多個胺基的有機化合物，植物中主要含有putrescine、spermidine及spermine三種多元胺。許多研究指出逆境壓力下多元胺含量會增加，提高逆境的耐受性。除了多元胺之外，近年在橘色花椰菜中發現一個基因並命名為Orange(Or)，Or基因也會對逆境產生反應，且過度表現Or基因會使得類胡蘿蔔素(carotenoids)含量增加，且提高植物的抗氧化能力。但Or基因如何影響植物的類胡蘿蔔素合成尚未有明確的答案也，不清楚Or基因是否受到多元胺的調控，因此，本研究在於探討多元胺代謝是否與Or基因有所相關。實驗證實鹽害壓力會造成水稻鮮重下降、葉綠素及類胡蘿蔔素減少，抗氧化能力、MDA含量及Or基因表現量則會增加。但經過多元胺與類胡蘿蔔素抑制劑處理後除了抗氧化能力MDA提高之外，Or基因表現受到抑制，顯示出多元胺可能參與調控Or基因表現。定序分析發現在不同植物間的Or保守性很高，且都共同有兩個極為保守的zinc finger domain。

Salt stress, one of environment stresses, affects plant physiology and causes some damage to plants such as electrolyte leakage. Plants have evolved many mechanisms to prevent themself from injury by stresses. Polyamines have been known as anti-stress molecules and the major polyamines in higher plants are putrescine, spermidine and spermine. Studies indicated that the synthesis of polyamines increased if plants had been under stresses, probably for the purpose of increasing stress torelance. Recently, a mutant gene called Orange (Or), which accumulated great amount of carotnoids, was found in cauliflower. But how the Or gene affects the carotnoid is still unknown. In this study, 14 days-old seedlings of rice (Oryza sativa) had been treated with various concentrations of Sodium chloride, and then certain characteristics such as fresh weight, chlorophyll, carotnoids, malondialdehyde (MDA), DPPH scavenging activity and Or gene expression were analyzed. Results showed that rice seedings treated with high salt concentrations, fresh weight, DPPH scavenging activity, chlorophyll and carotnoids content were significantly reduced comparing to controls. Whereas, the MDA content and Or gene expression were increased. The result that Or gene expression, chlorophyll and carotnoids content were reduced after treatment with polyamine and carotenoids synthesis inhibitor, MGBG and Fluridone, suggesting Or gene might be regulated by polyamines in controlling synthesis of carotnoids.

摘要 i
Abstract ii
致謝 iii
目次 I
圖與表 III
一、前言 1
1-1環境壓力、逆境與植物生長 1
1-1.1 溫度與光線對植物造成的影響與傷害 1
1-1.2 乾旱與鹽害對植物造成的影響與傷害 2
1-1.3 逆境所造成的傷害指標 2
1-2 植物對於環境壓力的反應 3
1-2.1 滲透壓調節 3
1-2.2抗氧化能力 4
1-2.3 激素與分子訊號 5
1-3多元胺在植物中的生化合成與代謝 6
1-4類胡蘿蔔素在植物體功能 7
1-5類胡蘿蔔素在植物體中的主要生合成路徑 8
1-6 Orange基因之發現與功能 9
1-7研究目的 10
二、材料與方法 12
2-1植物培育及生長條件 12
2-2水稻Or基因之分析 12
2-2.1植物之RNA萃取 12
2-2.2 Reverse Transcription-Polymerase Chain Reaction (RT-PCR) 13
2-2.3 DNA 瓊酯膠體電泳 14
2-3多元胺萃取及分析 16
2-4植物色素萃取、分析與定量 17
2-5丙二醛(Malondialdehyde, MDA)萃取與定量 18
2-6 DPPH自由基清除能力檢測 19
2-7 統計方法 19
三、結果 20
3-1 不同物種間Or序列比對與親緣分析 20
3-2鹽害逆境對植物生理代謝的影響 24
3-2.1鮮重 24
3-2.2 MDA含量 24
3-2.3 清除DPPH自由基能力 24
3-2.4 葉綠素含量 28
3-2.5 類胡蘿蔔素含量 28
3-2.6 Or基因表現量 28
3-2.8 小結 34
3-3鹽害下多元胺及類胡蘿蔔素對水稻的抗逆境生理活性的表現 34
3-3.1鹽害逆境下多元胺及類胡蘿蔔素對MDA含量的影響 34
3-3.2鹽害逆境下多元胺及類胡蘿蔔素之對逆境下DPPH清除能力的影響 35
3-3.4鹽害逆境下多元胺及類胡蘿蔔素抑制劑對葉綠素含量的影響 38
3-3.7鹽害逆境下多元胺及類胡蘿蔔素抑制劑對類胡蘿蔔素含量的影響 38
3-3.6鹽害逆境下多元胺及類胡蘿蔔素對Or基因的影響 38
3-3.7 小結 43
四、討論 44
4-1不同物種間Or序列比對與親緣分析 44
4-2鹽害逆境對植物生理代謝的影響 45
4-3 鹽害下多元胺及類胡蘿蔔素對水稻的抗逆境生理活性的表現 46
4-4 多元胺與Or基因表現 49
五、參考文獻 50
六、附錄 55
附錄一、多元胺在植物體內生合成路徑 55
附錄二、類胡蘿蔔素生合成路徑 56
附錄三、RT-PCR圓葉菸草Or基因mRNA之Coding Sequence片段之RT-PCR 57
圖與表
表一、RT-PCR 引子列表 15
表二、Or基因間的序列相似度 22
表三、鹽害對水稻鮮重之單因子變異數分析(ANOVA) 25
圖一、Or基因序列比對 21
圖二、高等植物中的Or基因系統樹 23
圖三、鹽害對水稻鮮重含量影響 26
圖四、鹽害與水稻MDA含量變化 27
圖五、鹽害與清除DPPH能力 29
圖六、鹽害對水稻葉綠素含量含量影響 30
圖七、鹽害對水稻類胡蘿蔔素含量影響 31
圖八、不同鹽害濃度對於水稻葉片Or基因之RT-PCR分析 32
圖九、水稻Or基因及Ubc基因之RT-PCR結果 33
圖十、鹽害逆境下多元胺及類胡蘿蔔素相關處理後的MDA含量 36
圖十一、鹽害逆境下多元胺及類胡蘿蔔素相關處理後的清除DPPH能力 37
圖十二、鹽害逆境與多元胺及類胡蘿蔔素相關處理後的葉綠素含量 40
圖十三、鹽害逆境與多元胺及類胡蘿蔔素相關處理後的類胡蘿蔔素含量 41
圖十四、鹽害逆境與多元胺及類胡蘿蔔素相關處理後的水稻葉片Or基因之RT-PCR分析表現量 42

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