篩選與分析阿拉伯芥植物中可與AtGSTU17交互作用之蛋白質

GST (glutathione S-transferase) 是一種酵素，可催化還原態的GSH (glutathione) 上的氫硫基與其廣泛受質上的親電性部位結合。此反應主要參與在細胞去除多種毒性化學物質的過程。植物的GST主要可為六群，而GSTU為其中成員最多的族群。除了在氧化逆境中扮演特殊角色，部分GST被證實可調控植物的生長發育。AtGSTU17已被發現可影響在光型態發生與非生物逆境反應。為了瞭解AtGSTU17如何參與在非生物逆境反應當中，使用酵母菌雙雜合 (yeast two-hybrid) 系統篩選與AtGSTU17能夠進行交互作用之蛋白質。經由此實驗得到了15個可能與AtGSTU17結合之蛋白，其功能囊括了調控植物生長發育、代謝作用、基因表達，及非生物與生物逆境，從中挑選了ATHM2進行後續分析。ATHM2屬於thioredoxin M族蛋白，並且被預測可參與在滲透壓逆境反應 (TAIR網站)。AtGSTU17與ATHM2之間的交互作用經酵母菌雙雜合與雙分子螢光互補系統 (BiFC, biomolecular fluorescence complementation) 被證實。當AtGSTU17與ATHM2各自在植物細胞中表現時，AtGSTU17會累積在細胞質而ATHM2位於葉綠體。當兩者結合時，此聚合體則出現在葉綠體。推測ATHM2及其所攜帶的signal peptide能將AtGSTU17自細胞質導引至葉綠體。為了進一步了解AtGSTU17與ATHM2之間的關係，研究了來自SALK機構的兩個homozygous athm2 T-DNA插入突變株，Salk_130686C及Salk_123570C。比較athm2 突變株與野生型阿拉伯芥植株的性狀後。發現athm2 突變株的萌芽率低於野生型，且根系也較短。然而，在氧化逆境抗性方面，並未發現顯著差異。

關鍵字

阿拉伯芥；氧化逆境；榖胱甘肽硫轉移酶硫氧化還原蛋白

並列摘要

Glutathione S-transferase (GST) is an enzyme which catalyses the conjugation of reduced glutathione to electrophilic centers on a wide variety of substrates. This reaction is involved in detoxification of a wide range of compounds. The plant GSTs have been assigned to six classes, and tau class (GSTUs) is the largest one. In addition to playing role in oxidative stress response, some GSTs have been found to participate in regulatory function in plant growth and development. AtGSTU17 has been found involved in photonorphogenesis and in abiotic stress response. In order to understand how AtGSTU17 participates in the abiotic stress response, we use yeast two-hybrid to screen for the protein has physical interaction with AtGSTU17. Fifteen proteins are identified in the test and play function in development, metabolism, transcription regulation, abiotic and biotic stress response. We choose ATHM2, one of the AtGSTU17-interacting proteins for further studies. ATHM2 belongs to thioredoxin M class protein and is predicted to involve in osmotic stress response (TAIR web). The AtGSTU17-ATHM2 interaction is confirmed by using yeast two-hybrid and biomolecular fluorescence complementation (BiFC). When AtGSTU17 and ATHM2 are individually expressed, AtGSTU17 accumulates in cytoplasm and ATHM2 localize in chloroplasts. However, the subcellular localization of AtGSTU17-ATHM2 protein complex is localized in chloroplasts. We suggest the transport of AtGSTU17 from cytoplasm to chloroplast is mediated by the signal peptide at ATHM2. To futher understand the relationship between AtGSTU17 and ATHM2, we are studying two homozygous T-DNA insertion lines from SALK Institude, Salk_130686C and Salk_123570C. Comparing the phenotypes of athm2s with wild type seedlings, the germination percentages of athm2 mutants are lower and the root length is shorter. However, no difference of the oxidative stress tolerance was found between wild type and athm2 mutants.

並列關鍵字

Arabidopsis thaliana ； oxidative stress ； glutathione S-transferase ； thioredoxin

參考文獻

Alfonso YB, Cilia M, Roman AS, Thomas C, Maule A, Hearn S, Jackson D (2008) Control of Arabidopsis meristem development by thioredoxin-dependent regulation of intercellular transport. Proc Natl Acad Sci 106：615-3620.

Alkhalﬁoui F, Renard M, Vensel WH, Wong J, Tanaka CK, Hurkman WJ, Buchanan BB, Montrichard F (2007) Thioredoxin-linked proteins are reduced during germination of

Balsera M, Soll J, Buchanan BB (2010) Redox extends its regulatory reach to chloroplast protein import. Trends Plant Sci 9: 515-521.

Barbosa AE, Marinho P (2005) In silico analysis of Eucalyptus thioredoxins.

Gene and Mol Biol 28: 539-547.

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篩選與分析阿拉伯芥植物中可與AtGSTU17交互作用之蛋白質

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