阿拉伯芥中AtGSTU25生理功能性分析

Physiological Function of AtGSTU25 in Arabidopsis thaliana

詹雯琇(Wun-Shiou Jan)

指導教授 ： 林讚標

國立臺灣大學/生命科學院/植物科學研究所/碩士(2012年)

https://doi.org/10.6342/NTU.2012.03270

摘要

麩胺基硫轉移酶 (glutathione S-transferases; GST)在阿拉伯芥中含有55個成員，可催化還原態的穀胱甘肽，提供成對的電子給各式各樣的受質，而最有名的功能則是扮演對殺草劑具有去毒化的作用，提供植物對氧化逆境之保護。除酵素催化功能外，目前有很少數的GSTs被報導可以充當訊息的分子來影響植物的生長與發育。近年來實驗室的研究發現atgstu17突變株於非生物逆境下比野生型更為耐旱及耐鹽，此突變株伴隨glutathione和abscisic acid的增加。於是我們想知道是否擁有很高酵素活性的AtGSTU25，當基因被破壞掉時是否也會出現與atgstu17類似的性狀。本研究中我們從ABRC獲得阿拉伯芥AtGSTU25之T-DNA插入突變株(Salk_042213)，發現異型合子的果莢有缺粒現象，且無法獲得基因被破壞同型合子之突變株，然而Salk_042213突變株致死情況已連續檢查三代。在互補實驗中，將AtGSTU25 cDNA轉入AtGSTU25的異型合子突變株，我們仍未得到同型合子突變株，顯示出致死的性狀可能歸咎於其他的插入點。我們更進一步的建構AtGSTU25大量表現及RNAi轉植株，發現AtGSTU25主要表現在根部及葉尖。利用原生質體顯示35S:AtGSTU25-GFP表現在細胞質。至於氧化逆境耐受性的實驗中，AtGSTU25的各種變異株與野生型並無不同，推測AtGSTU25 可能不是主要參與細胞內去毒工作。AtGSTU25 RNAi轉殖株顯示出經鹽逆境處理下，萌芽率較野生型要來的低。AtGSTU25大量表現轉殖株於鹽逆境下根部延長受到抑制。AtGSTU25 RNAi轉殖株也展現出提早抽花序且葉片有早老化之現象，綜合以上結果，我們推論AtGSTU25於逆境下扮演調控種子萌芽與根部延長，另外也扮演著延遲花軸生長的時間並抑制葉子老化之角色。

關鍵字

麩胺基硫轉移酶穀胱甘肽氧化逆境 ； 老化 ； 開花

並列摘要

Glutathione S-transferases (GSTs; EC 2.5.1.18) constitute a superfamily containing 55 members in Arabidopsis thaliana and biochemically can catalyze the nucleophilic addition of the thiol of the reduced form glutathione to a wide variety of electrophilic substrates. The best known study was GSTs mainly in relation to their role in the detoxification of herbicides and to protect organisms against oxidative stress. In addition to enzymatic function, only a few GSTs have been reported to serve as signaling molecule to influence plant growth and development. In our previous study, atgstu17 mutants exhibited drought and salt tolerant phenotypes when subjected to abiotic stress treatment. The mechanism causing the phenotype of atgstu17 can be explained mostly by the combined effect of elevated contents of both glutathione and abscisic acid. We wonder whether AtGSTU25 having highest catalytic activity in vitro when silenced could exhibit similar phenotype as that of atgstu17. In this study, AtGSTU25 T-DNA knockout mutant (Salk_042213) was obtained from ABRC and the hemizygote mutants carried empty seeds in siliques which produced wild type and hemizygote plants but lack of homozygote knockout mutants. The lethal phenotype of Salk_042213 mutants were reconfirmed after 3 consecutive generations. In the complementary experiment by introducing the AtGSTU25 cDNA into AtGSTU25 hemizygote mutants, we still couldn’t get the AtGSTU25 homozygote mutant indicating the lethal phenotype was attributed to another locus. We generated AtGSTU25 overexpressors and RNAi knock down mutants for further study. Expression of AtGSTU25 is mainly in the root system and apical part of leaf blade. Localization study using protoplast revealed the cytoplasm distribution of 35S:AtGSTU25-GFP. With respect to the oxidative stress tolerance, AtGSTU25 overexpressor and knockdown lines didn’t show difference from wild type plant indicating maybe AtGSTU25 play no role in cell detoxification. RNAi mutants of AtGSTU25 exhibited lower germination rate than wild type plants under salt stress condition. RNAi mutants of AtGSTU25 also exhibited earlier bolting time and earlier senescence of leaf tissue. Taken together, we inferred that The AtGSTU25 would play roles in regulating the seed germination and root elongation under salt stress condition, and delay bolting time and repress senescence of leaf tissues.

並列關鍵字

Glutathione S-transferases ； glutathione ； oxidative stress ； senescence ； bolting time

參考文獻

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