臺灣博碩士論文加值系統

空氣污染物主要含有硫化物、氮化物等，這些空氣污染物質最後大部份都會溶解在水中，而形成硫酸鹽類及硝酸鹽類化合物。因此，我們利用控制水溶液中硫酸鹽類及硝酸鹽類的濃度來探討環境中硫化物及氮化物對植物體葉部GSH（glutathione）及胺基酸生合成之影響。
本研究是以菠菜為實驗材料，分別以蒸餾水、0.1、0.3、0.5M的硫酸鈉、硝酸鈉及混合（硫酸鈉+硝酸鈉）水溶液培養24小時，於24小時後再利用高效能液相層析儀HPLC（High Performance Liquid Chromatography）來測定菠菜葉部組織中GSH及胺基酸的含量。
由本實驗結果指出當菠菜經0.1、0.3、0.5M硫酸鈉水溶液培養24小時後，菠菜葉部組織中的GSH及polypeptides含量分別有約69、461及104%的明顯增加；經0.1、0.3、0.5M硝酸鈉水溶液培養24小時後，菠菜葉部組織中的GSH及polypeptides含量分別有約124、226、及271%的明顯增加；經0.1、0.3、0.5M混合（硫酸鈉+硝酸鈉）水溶液培養24小時後，菠菜葉部組織中的GSH及polypeptides含量分別有約307、430、及365%的明顯增加。而經0.1及0.3M硫酸鈉水溶液培養24小時後，菠菜葉中胺基酸總含量增加99及173%；經0.1及0.3M硝酸鈉水溶液培養24小時後，菠菜葉中胺基酸總含量增加70及84%；經0.1及0.3M混合（硫酸鈉+硝酸鈉）水溶液培養24小時後，菠菜葉中胺基酸總含量增加78及91%；但經0.5M硝酸鈉及混合（硫酸鈉+硝酸鈉）水溶液培養24小時後，菠菜葉中胺基酸總含量則分別降低12及32%。
其結果顯示：在碳、氫、氧原子來源充足的情況下，給予充分的水分及光合作用，並增加硫原子及氮原子的來源，可增加GSH及胺基酸在植物體葉部組織中的含量，但GSH及胺基酸生成量增加至一定濃度並到達飽和後，不再隨著硫原子及氮原子濃度的提高而呈線性的增加。
因此推論：GSH及胺基酸在植物體中的生合成量，在到達一定量後，則會發生回饋抑制作用。

Different concentrations of sodium sulfate and nitrate [0.1, 0.3 and 0.5M] on the effects of biosynthesis of amino acids and glutathione [GSH] in spinach leaves were studies by high pressure liquid chromatography, respectively. The spinach leaves were cultured for 24 hours. The concentrations of GSH and polypeptides were increased approximately by 69, 461 and 104% under sodium sulfate, 124, 226 and 271% under sodium nitrate, and 307, 430 and 365%under equal mixtures of sodium sulfate and nitrate as compared with distilled water cultured, respectively. The concentration of amino acids were increased approximately by 99 and 173% under sodium sulfate [0.1 and 0.3M], 70 and 84% under sodium nitrate [0.1 and 0.3M], and 78 and 91% under equal mixtures of sodium sulfate and nitrate [0.1 and 0.3M] as compared with distilled water cultured, respectively. The concentrations of amino acids were decreased approximately 12 and 32% under sodium sulfate and equal mixture of sodium sulfate and nitrate at the concentration of 0.5M. The spinach leaves were deteriorated and the amino acid concentrations cannot be determined properly under 0.5M sodium sulfate. From the above results showed that the levels of amino acid, GSH and polypeptides increased linearly with the increase of the concentration of sulfate, nitrate and equal mixtures of these after due to the spinach leaves under high salt concentration [0.5M] especially for sodium sulfate.
It is inferred that when the salt concentration of sulfate and nitrate reached a certain level [e.g. 0.5M], there may be a feedback inhibition mechanism for the biosynthesis of amino acids and GSH in spinach leaves.

總目錄
中文摘要………………………………………………………………………Ⅲ
英文摘要………………………………………………………………………Ⅴ
總目錄…………………………………………………………………………Ⅶ
表目錄…………………………………………………………………………Ⅸ
圖目錄…………………………………………………………………………Ⅹ
壹、文獻回顧
ㄧ、前言…………………………………………………………………1
二、碳元素進入植物體之途徑…………………………………………3
三、氮元素進入植物體之途徑…………………………………………5
四、二氧化硫對植物體之毒性…………………………………………9
五、硫元素進入植物體之途徑…………………………………………12
六、GSH（glutathione）簡介…………………………………………18
貳、實驗器材與藥品
一、儀器與器材…………………………………………………………26
二、藥品…………………………………………………………………27
三、反應試劑製備………………………………………………………27
參、實驗部份
ㄧ、實驗原理……………………………………………………………30
二、Glutathione 的定量方法…………………………………………31
三、HPLC（High Performance Liquid Chormato graphy）分析條
件之建立……………………………………………………………31
四、實驗流程……………………………………………………………34
肆、結果與討論
一、GSH 標準曲線………………………………………………………37
二、檢測波長254nm與365nm之比較……………………………………38
三、各種培養方式與對照組之結果比較………………………………43
四、不同濃度的硫酸鈉溶液培養的結果………………………………45
五、不同濃度的硝酸鈉溶液培養的結果………………………………45
六、不同濃度的硫酸鈉＋硝酸鈉混合溶液培養的結果………………45
七、硫酸鈉與硝酸鈉的結果比較………………………………………46
八、硫酸鈉與硫酸鈉＋硝酸鈉的混合液的結果比較…………………47
九、硝酸鈉與硫酸鈉＋硝酸鈉的混合液的結果比較…………………48
十、莖部與葉部組織中胺基酸及GSH 的比較…………………………52
伍、結論………………………………………………………………………54
表目錄
表一 GSH標準品在HPLC中的滯留時間與吸收峰面積………………………37
表二 365nm、254nm波長檢測0.1M、0.3M硫酸鈉培養24小時後菠菜葉中
胺基酸及GSH的含量……………………………………………………39
表三365nm、254nm波長檢測0.1M、0.3M硝酸鈉水培養24小時後菠菜葉中
胺基酸及GSH的含量……………………………………………………41
表四 365nm、254nm波長檢測0.1M、0.3M混合水溶液（硫酸鈉＋硝酸鈉）
培養24小時後菠菜葉中胺基酸及GSH的含量…………………………42
表五 各種不同培養方法培養24 小時後菠菜葉中胺基酸含量……………49
表六 各種不同培養方法培養24小時後菠菜葉中GSH及polypeptides含量…
…………………………………………………………………………50
表七 各種不同培養液培養24小時後菠菜葉部與莖部中胺基酸及GSH含量之
比較……………………………………………………………………52
圖目錄
圖一 自然界碳循環…………………………………………………………4
圖二 卡爾文循環……………………………………………………………6
圖三 植物體內胺基酸的合成………………………………………………7
圖四 酸雨形成的原因………………………………………………………8
圖五 硝酸根離子在植物體中之代謝情形…………………………………10
圖六 S 元素進入植物體之途徑……………………………………………14
圖七 二氧化硫進入植物體之代謝情形……………………………………16
圖八 硫酸根離子進入植物體後之代謝情形………………………………17
圖九 GSH 與GSSG……………………………………………………………19
圖十 GSH 之解毒功能………………………………………………………20
圖十一 Glutamate 之合成…………………………………………………22
圖十二 Serine之合成………………………………………………………23
圖十三 Cysteine之合成……………………………………………………23
圖十四 Glycine 之合成……………………………………………………24
圖十五 Glutathione 之合成………………………………………………25
圖十六 GSH 呈色反應機制…………………………………………………32
圖十七 沖提液中75 g與60 g的sodium acetate對GSH peak之滯留時間的
比較…………………………………………………………………56
圖十八 沖提液中75 g與90 g的sodium acetate對GSH peak之滯留時間的
比較…………………………………………………………………57
圖十九 HPLC分析的流速1.1 ml/min與0.9 ml/min對分析結果的滯留時間
之比較………………………………………………………………58
圖二十 HPLC分析的流速0.9 ml/min與0.7 ml/min對分析結果的滯留時間
之比較………………………………………………………………59
圖二十一 以不同量的1% FDNB，0.1、0.5 ml作Sanger’s reaction，經
HPLC分析之結果比較……………………………………………60
圖二十二 以不同量的1% FDNB，0.1、0.3 ml作Sanger’s reaction，經
HPLC分析之結果比較……………………………………………61
圖二十三 實驗流程…………………………………………………………62
圖二十四 植物前處理步驟流程圖…………………………………………63
圖二十五 Glutathione 及胺基酸定性定量之流程圖……………………64
圖二十六 GSH 之標準曲線…………………………………………………38
圖二十七 各種不同濃度之水溶液培養24小時後菠菜葉中胺基酸含量變化
情形………………………………………………………………49
圖二十八 各種不同濃度之水溶液培養24小時後菠菜葉中胺基酸含量之比
較…………………………………………………………………50
圖二十九 各種不同濃度之水溶液培養24小時後菠菜葉中GSH及
polypeptides含量變化情形……………………………………51
圖三十 各種不同濃度之水溶液培養24小時後菠菜葉中GSH及polypeptides
含量之比較…………………………………………………………51
圖三十一 菠菜葉部及莖部組織中胺基酸含量的比較……………………53
圖三十二 菠菜葉部及莖部組織中GSH 及polypeptides含量的比較……53

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