利用逆向遺傳與非生物逆境處理探究AtGSTU基因之生理功能

2012-08-012024-05-18https://scholars.lib.ntu.edu.tw/handle/123456789/704808摘要：麩胺基硫轉移酶(GSTs; EC 2.5.1.18)是一個在阿拉伯芥的超級家族，它含有54 個成員，在生化上可以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。GST 最有名的研究是對除草劑去毒所扮演的角色。GSTs 的表達可以受到各種不同逆境因子的誘導。GSTs 的合成相信是提供植物對抗氧化逆境的保護。然而GSTs 的生理功能大部份 都屬未知。傳統的分子遺傳研究方法無法找出GSTs 的生理功能，因為基因功能上的重疊性。利用親合管柱色層分析來進行配體魚釣篩選，可以找到與GSTs 結合的脂肪酸衍生物，但是這些物質的生理功能仍然不清。除了酵素催化功能之外，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。在初步實驗中我們發現逆向遺傳分析結合非生物性逆境處理，容許我們發現單一基因變種的有趣外表型。當 AtGSTU17 基因被破壞，他們的外表型與野生型在正常生長情況下並無不同。然在遭受非生物性逆境時，他們比野生型更為耐鹽與耐旱。結合逆向遺傳與逆境處理打開了研究GSTs 生理功能的可能性。在本研究中我們將從Salk Institute 獲得阿拉伯芥GSTU 家族基因T-DNA 插入的變種，或利用RNAi 的技術來製造變種。Tau 群基因有28 個成員是GSTs 家族中最大的一群。同型結合子變種的外表型將利用非生物逆境的處理來加以篩選。目前至少已經獲得一個變異的基因atgstu25 的同型結合子會造成死亡。我們將深入研究這個有趣的基因來了解它在細胞的內在功能。總而言之這個計畫將會了解一個至多個GSTU 基因的生理功能，特別是當他們在遭到環境逆境時所扮演的角色。<br> Abstract: Glutathione s-transferases (GSTs; EC 2.5.1.18) are a superfamily containing 54 members in Arabidopsis thaliana and biochemically can catalyze the nucleophilic addition of the thiol of the reduced form of the tripeptide 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. GST expression is induced by a wide variety of stress stimuli, and the increased GST levels were believed to protect organisms against oxidative stress. However the endogenous function of GSTs is largely unknown. Traditional molecular genetics approach failed to identify the physiological function because functional redundancy of GST genes. Ligand fishing screening by affinity column chromatography had identified many fatty acid derivatives as the conjugates of GSTs, but the physiological function awaits further study. 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 preliminary study, we found that reverse genetic approach in combination with abiotic stress treatment allowed us to find interesting phenotype of single gene mutant. When AtGSTU17 was knocked out and didn’t have clear phenotype difference compared with wild type plant under normal growth condition. However, atgstu17 mutants exhibited drought and salt tolerant phenotypes when subjected to abiotic stress treatment. The combination of reverse genetics and stress treatment open a possibility to study the physiological function of GSTs. In this study we will obtain the T-DNA insertion mutant from the Salk Institute or generate knockdown mutant by RNAi technology of the tau class in the GST family of Arabidopsis thaliana. The tau class gene has 28 members and is the largest class in the GSTs family. Phenotype of homozygous mutant will be screened by applying abiotic stress treatment. At least one mutant gene, atgstu25, was generated and causes lethal phenotype. This interesting gene will be studied extensively to uncover the endogenous function played in the cell. Taken together, this project will shed light of the physiological function of one or more GSTU genes, especially in relation to the role in response to environmental stresses.麩胺基&#63950轉移&#37238變種阿&#63781伯芥生&#63972功能AtGSTU25glutathione s-transferaseknockout mutantArabidopsis thalianaphysiological利用逆向遺傳與非生物逆境處理探究AtGSTU基因之生理功能