- 學位論文
利用抑制性扣減雜交及二維電泳尋找紅毛草在乾旱逆境下被誘導表現的基因和蛋白質
Isolation of drought stress induced genes and proteins in natalgrass (Rhynchelytrum repens) by suppression subtractive hybridization and two-dimensional electrophoresis
摘要
在自然界中時常可以看到一些野草，在惡劣的環境中依然可以正常生長，所以本實驗想了解野草和一般不易在逆境中生長的植物，在逆境下的基因表現有何不同，使野草在不良環境中有較佳的耐性。本研究以紅毛草(Rhynchelytrum repens (Willd.) C. E. Hubb.）為實驗材料，研究此植物在乾旱逆境下之特異性基因表現。紅毛草為禾本科植物，常見於台灣中南部之石礫乾河床及河邊砂質土壤上。以PEG 6000 (-1 MPa)處理使植物處於缺水逆境，從生理及分生的方面來觀察紅毛草在缺水逆境下的變化。在生理方面測定了相對水含量、水分潛勢、葉綠素含量等相關指數。利用分子生物學的抑制性扣減雜交以及二維電泳系統技術，尋找在乾旱逆境下被誘導表現的基因和蛋白質。 觀察處理二十四小時內之植物相對含水量變化，由結果得知在處理的前十小時內的相對含水量由80％下降至45％；觀察二十四小時內水分潛勢之變化，由處理前水分潛勢-0.95 MPa左右，經二十四小時之處理後則下降到-2.0 MPa上下，此時植物出現缺水現象。葉綠素含量，經-1 MPa PEG處理一天後葉綠素含量與未處理者無明顯變化。以未處理（零小時）之紅毛草葉片抽取之mRNA為扣減組（driver），處理後二小時及二十四小時後之紅毛草葉片抽取之mRNA為試驗組（tester），進行抑制性扣減雜交，以構成扣減式基因庫，經定序與Blast-x分析後，有33條序列可在資料庫中找到相似的序列，有5條無法找到相似的序列。33條序列在功能上分類可分下列十五群：Transcription factor、detoxification、protein kinase、protease、transposon、amino acid metabolism、carbohydrate metabolism、TCA cycle、transcription、translation、auxin regulation、circadian clock、cytoskeleton、transporter、unknown protein。以二維膠體電泳分析，比較未處理組與處理PEG二十四小時的紅毛草葉片之蛋白質表現之差異，共有六個蛋白質的表現量增加 (DNA-directed RNA polymerase II 19 kDa polypeptide、NBS-LRR type disease resistance protein、tau class GST protein 3、calcineurin B-like protein 3、vetispiradiene synthase和H+-transporting two-sector ATPase delta chain precursor)，但有兩個蛋白質的表現量下降(eukaryotic initiation factor 4B和ADP-glucose pyrophosphorylase)。
並列摘要
Natalgtass, a weed that is widely distributed in the world and resistant to drought stress was subjected to physiological and molecular studies. In the physiological analysis, the relative water content and water potential decreased after 24 hours at treatment with PEG (-1 MPa), while chlorophyll and carotenoid remained similar. In order to identify genes and proteins regulated by drought stress, suppression subtractive hybridization (SSH) and two-dimensional electrophoresis (2-DE) were performed. The cDNAs from natalgrass treated with PEG 6000 (-1 MPa) were used as testers and cDNAs from unstressed natalgrass as drivers for hybridization. We found 33 uniquely expressed sequence tags (ESTs) in the subtractive cDNA library.The predicied proteins of the 33 ESTs are involved in transcription factor, detoxification, protein kinase, protease, transposon, amino acid metabolism, carbohydrate metabolism, TCA cycle, transcription, translation, auxin regulation, circadian clock, cytoskeleton, transporter and unknown protein are considered to be responsible for drought tolerance. In the 2-D gel analysis, 6 proteins were up regulated (DNA-directed RNA polymerase II 19 kDa polypeptide, NBS-LRR type disease resistance protein, tau class GST protein 3, calcineurin B-like protein 3, vetispiradiene synthase and H+-transporting two-sector ATPase delta chain precursor) and 2 proteins were down regulated (eukaryotic initiation factor 4B and ADP-glucose pyrophosphorylase) by the PEG 6000 treatment.
參考文獻
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延伸閱讀
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