臺灣博碩士論文加值系統

隨著全球氣候變遷，因地球暖化導致的強降雨與颱風發生頻率增加，強降雨與颱風帶來的豪雨，使水稻遭遇淹水逆境，嚴重影響產量，而淹水不僅導致缺氧逆境，也會造成滲透逆境。淹水逆境造成細胞缺氧之生理反應與分子機制已被探討，然而較少針對淹水逆境下之滲透調節相關機制進行深入分析，為了解水稻淹水及淹水加上不同滲透逆境下之調控機制。本研究以秈稻IR64-Sub1進行純淹水、淹水外加甘露醇及淹水外加氯化鈉處理，利用次世代定序分析結果進行轉錄體學差異性探討。結果顯示，淹水處理組之差異表現基因數最少，淹水外加甘露醇處理組次之，淹水外加氯化鈉處理組最多，以500倍以上差異倍率分析，結果顯示，三種不同滲透處理之淹水逆境下共同表現78個基因。在純淹水、淹水外加甘露醇與淹水外加氯化鈉處理組，分別具有78、154與407個特異性表現之基因，而基因表現相似程度部分，淹水與淹水外加甘露醇處理組，高於淹水外加氯化鈉處理組。根據基因功能分類分析，具差異表現之基因大多參與，生物性與非生物性逆境、轉錄因子、蛋白質生合成與降解相關之功能。分析參與糖解發酵路徑相關基因表現之結果顯示，蔗糖合成酶 (sucrose synthase, SUS4)、己醣激酶 (hexokinase, HEX9)、甘油醛-3-磷酸脫氫酶 (glyceraldehyde 3-phosphate dehydrogenase, GAPDH)、丙酮酸脫氫酶 (pyruvate dehydrogenase, PDC2)與乙醇脫氫酶 (alcohol dehydrogenase, ADH1)皆被誘導表現，然而於不同滲透處理之淹水逆境下的誘導程度不相同。GAPDH、PDC2與ADH1於淹水外加甘露醇與淹水外加氯化鈉處理組之表現量較淹水處理組高，SUS4在淹水外加氯化鈉處理組之表現量較淹水與淹水外加甘露醇處理組高；蘋果酸脫氫酶 (Malate dehydrogenase, MDH)在淹水外加氯化鈉處理組被誘導表現，而在淹水與淹水外加甘露醇處理組降低表現量。綜合以上實驗結果顯示，水稻IR64-Sub1遭遇不同滲透類型之淹水逆境，具有不同且複雜的調控機制，此一重要結果可提供未來抗逆境之研究參考。

With the climate change in the world, the frequency of heavy rain and typhoon increased by global warming. The heavy rain and typhoon might result in crop yield reduction. Submergence not only causes hypoxic stress but osmotic stress. The physiological responses and molecular mechanism of hypoxia have been well studied, but there are limited studies in osmotic under submergence. To better understand the mechanisms of the osmotic submergence examined the responses of IR64-Sub1 under submergence (Sub), submergence combined with mannitol (Sub+Man) or sodium chloride (Sub+NaCl). Transcriptomic analysis was done by RNA-seq.
Compared to the gene expression profile under control condition, The differentially expressed genes (DEGs) under Sub treatment was much least, followed by Sub+Man, and Sub+NaCl treatment. Under 500 times fold change selection, 78 genes were co-expressed under three different osmotic stress during submergence.While 78, 154 and 407 genes were specifically regulated under Sub, Sub+Man and Sub+NaCl treatment. Compairing the expression profiles, Sub and Sub+Man treatments were more similar than Sub+NaCl treatment. Gene Ontology analysis showed that gene function were related to biotic and abiotic stresses, transcription factors, protein synthesis and protein degradation. Genes were highly enriched involved in glycolysis pathway and fermentation pathway, including Sucrose Synthase4 (SUS4), Hexokinase9 (HEX9), Glyceraldehyde 3-Phosphate Dehydrogenase (GADPH), Pyruvate Dehydrogenase2 (PDC2) and Alcohol Dehydrogenase1 (ADH1) were differential expressed under different osmotic treatments during submergence. The expression level of ADH1, PDC2 and GAPDH in Sub+Man and Sub+NaCl treatments were higher than Sub treatment. The expression level of SUS4 in Sub+NaCl was higher than Sub treatment. The expression level of HEX9 were induced in all treatments. The expression level of malate dehydrogenase (MDH) was induced in Sub+NaCl treatment, but down regulated by Sub and Sub+Man treatments. Based on the results, IR64-Sub1 has complex mechanisms in response to different osmotic treatments during submergence. This results provide a new insight of the submergence responses.

中文摘要……………………………………………………………………i
Abstract…………………………………………………………………..iii
目錄………………………………………………………...………………v


壹、前言……………………………..……………………………...…..…1
一、滲透逆境對作物之生長與代謝反應………………………….……..1
二、淹水逆境對作物生長與代謝反應………………………………...…3
三、複合性逆境之研究現況…………………………………………...…7


貳、材料與方法…………………………………...……………………..10
一、水稻種子消毒與幼苗生長條件…………………………………….10
二、幼苗於不同滲透壓逆境之淹水處理……………………………….10
三、次世代定序之RNA萃取與分析…………………………………...11
四、即時定量聚合酶鏈鎖反應 (real-time quantitative polymerase chain reaction, qRT-PCR)之總RNA萃取與分析……….…………12
五、統計分析…………………………………………………………….14


參、結果………………………………………………………………….15
一、次世代定序之品質檢測…………………………………………….15
二、逆境處理下水稻幼苗地上部差異表現基因分析………………….16
三、逆境處理下差異性基因之功能分類 (Gene Ontology)……………17
四、不同滲透處理之淹水逆境差異表現基因………………………….18
五、於不同滲透逆境下糖解與發酵路徑之差異表現基因…………….19
六、於不同滲透處理之淹水逆境下糖解與發酵路徑之基因表現…….20


肆、討論………………………………………………………………….22


伍、參考文獻…………………………………………………………….27


陸、圖表………………………………………………………………….35


柒、附錄………………………………………………………………….44

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