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研究生:周映孜
研究生(外文):Jou Ying Tzy
論文名稱:鹽逆境下高等植物鉀鈉離子平衡及相關基因表現之分析
論文名稱(外文):Analysis of K+/Na+ ion homeostasis and related gene expression in NaCl stress environments.
指導教授:顏宏真顏宏真引用關係
指導教授(外文):Hungchen E. Yen
學位類別:碩士
校院名稱:國立中興大學
系所名稱:植物學系
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:76
中文關鍵詞:冰花鹽逆境離子平衡
外文關鍵詞:ice plantMesembryanthemum crystallium L.salt stression homeostasis
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當植物受到鹽逆境時,會因為細胞內累積過多的氯化鈉離子,而抑制細胞質酵素活性並造成細胞內外離子的不平衡,影響鉀離子的吸收,進而引發缺鉀及缺水等傷害。為探討植物細胞對鹽逆境下對鉀離子吸收之影響,本篇論文以耐鹽植物冰花(Mesembryanthemum crystallinum L.)及不耐鹽植物阿拉伯芥(Arabidopsis thaliana)之培養細胞為材料,施予鹽逆境並配合不同濃度之鉀離子處理,測量細胞生長狀況及細胞內鉀鈉離子的含量及比值,以評估不同植物間對於鹽逆境下離子平衡的差異性。由細胞在不同鈉鉀離子濃度的培養基中生長的狀況可以發現,雖然冰花細胞所能承受的鹽逆境高於阿拉伯芥細胞,但無論是冰花或是阿拉伯芥細胞,培養在低鉀的環境中會使對鹽逆境的敏感度上升,造成過高的鈉鉀比值。當細胞培養在以氯化鉀造成高鉀環境的培養基時,細胞生長會受到抑制,以非滲透性溶質mannitol或硫酸鉀取代氯化鉀之培養基,會改善生長抑制的情況,推測過高濃度之氯離子會抑制細胞生長。雖然高鉀環境會限制細胞之生長,但是冰花及阿拉伯芥細胞在高鉀高鹽培養初期,都可以藉由培養基所提供的高濃度鉀離子維持較低之鈉鉀比值。
除了評估冰花細胞生長及鈉鉀離子含量外,並利用一鉀離子運輸相關mcSKD1基因,觀察基因表現及細胞離子平衡的關係。由北方墨點法分析結果得知,mcSKD1基因在鹽逆境及缺鉀逆境初期表現量會下降,之後表現量會增加。配合之前的生理研究結果發現,鹽逆境下mcSKD1基因表現,可降低冰花培養細胞中鈉鉀比值,推測此基因參與鈉鉀離子平衡之機制。由mcSKD1基因在組織專一性的表現得知,在未受鹽逆境的環境下,mcSKD1在發育中的子房及根尖的表現,推測mcSKD1基因參與提供新生組織營養的功能。鹽逆境下mcSKD1基因會在根部的表皮、莖部的皮層以及葉部腎型細胞表現,與區隔鈉離子的作用相關;由冰花葉片的鉀鈉元素分析結果得知,當mcSKD1 基因在根部表現時,可提高葉片中鉀離子的含量,表現量不足時,葉片中鉀離子量逐漸下降,推測mcSKD1基因參與冰花耐鹽機制中維持細胞鉀離子平衡的途徑。
When plants under high salinity stress, excess NaCl ions accumulate inside the cells, and as the results, the activities of cytosolic enzymes are inhibited and ion homeostasis is interfered. Moreover, the uptake of essential element potassium is severely affected. To examine the effects of high salt on the uptake of potassium, we used well-defined culture media to culture suspension cells of halophyte Mesembryanthemum crystallinum (ice plant) and glycophyte Arabidopsis thaliana. The growth rates and the contents of Na+ and K+ were measured in cells grown in different combinations of Na+ and K+ concentrations. Although ice plant had higher ability to grow in salt-containing media, yet the sensitivity to high salt increased as the concentration of K+ decreased in the culture media in both plant cells. High KCl-containing media inhibited the growth rate of both cells and replacement of KCl by mannitol or K2SO4 would alleviate the inhibitory effect suggesting that high concentration of Cl- in the culture media caused this growth inhibition. At the initial stage of salt stress, both cells maintained lower sodium/potassium ratios when a high external K+ was supplied.
In addition to the growth and ion content measurements, the expressions of an ice plant gene mcSKD1 that has been related to potassium uptake were also examined. As the results from Northern blotting, the expression of mcSKD1 decreased at the initial stage of high salt or low potassium treatments. The levels of mcSKD1 transcript increased as the stress persisted. The increase of mcSKD1 expression parallels to the decrease of cellular Na+/K+ ratio suggesting that this gene be involved in the regulation of ionic balance in ice plant under salt stress. Tissue-specific expression found mcSKD1 gene was expressed in the developing ovary and root tips of the unstressed plants suggesting this gene is involved in nourishing the young developing tissues. Under salt stress, the expressions of mcSKD1 were found in tissues that are responsible for the compartmentation of excess Na+, such as the epidermal cells of roots, the cortex of stems and the specialized epidermal bladder cells of leaves. Together with the analyses of sodium and potassium ion contents, we showed the expression of mcSKD1 in the roots was parallel to the increase of potassium concentrations in the leaves. The function of mcSKD1 protein may play a part in maintaining the potassium homeostasis in salt-stressed ice plant, an important pathway contributing to the overall mechanism of salt adaptation in this halophyte.
中文摘要………………………………………………………..…………………I
英文摘要………………………………………………………….………………II
壹、 前言:………………………………………………………………………. 1
一、 耐鹽植物冰花……………………………………………………..2
二、 鹽逆境下阿拉伯芥的反應………………………………………..3
三、 鹽逆境下鉀離子在植物細胞內離子平衡上所扮演的角色……..4
四、 冰花耐鹽相關基因mcSKD1………….…………………………..6
貳、 材料與方法:………………………………………………………………….8
一、 實驗材料:…………………………………………………………8
(一) 冰花盆栽……………………………………………………..8
(二) 細胞培養……………………………………………………..8
二、 實驗方法:………………………………………………………..9
(一) 植物體中Na, K, Ca, Mg之測定…………………………….9
(二) Total RNA之萃取…………………………………………..10
(三) 北方墨點法分析……………………………………………11
(四) 利用RT-PCR方法擴增全長SKD1基因…………………..12
(五) PCR產物之選殖……………………………………………13
(六) In situ RT-PCR and In situ hybridization…………………...14
(七) 掃瞄式電子顯微鏡觀察冰花葉表腎型細胞………………18
參、 結果…………………………………………………………………20
肆、 討論……………………………………………………………………29
伍、 參考文獻………………………………………………………………37
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