植物無法隨意地移動，因此植物時時刻刻接受環境中各式各樣的環境逆境。高溫逆境為主要的環境逆境之一，高溫逆境對植物所造成傷害，包括蛋白質變性、膜結構之破壞及光合作用酵素活性之抑制。為了了解植物如何抵禦高溫逆境，以期能運用基因工程去增進作物對熱的耐受性，我們利用正向式遺傳研究法(forward genetics approach) 找尋並研究植物的耐熱基因。以化學突變劑甲基磺酸乙酯 (ethylmethane sulfonate) 為突變劑，本實驗室篩選出兩株對長時間高溫逆境過度敏感之阿拉伯芥突變種植物，分別命名為hit3 (heat intolerant 3) 和et (eighteen)，此研究主要目的即為hit3和et 之生理定性及其基因定位。實驗結果顯示，hit3突變基因位於AGI map第五條染色體20,637 kb-20,684 kb之間；et突變點位於阿拉伯芥登錄序號為At5g10010 之基因內，一個由C 變成A 之鹼基點突變。 ET能轉譯為434個胺基酸但功能未知之蛋白質。以NCBI protein blast、PredictProtein及NetNES 1.1 Server分析發現，ET位於細胞核內，具有細胞核定位訊號序列 (nuclear localization signal sequence, NLS)。 此外，et對短時間熱休克處理、離層酸 (abscisic acid) 和巴拉刈 (paraquat) 也呈現高度敏感性，顯示出ET有可能參與並保護植物對抗多種環境逆境。綜合以上研究結果，ET的生理角色，有可能是藉由調節細胞核內之生理生化反應，以促成植物必備之耐熱反應。而ET基因除參與阿拉伯芥耐熱機制外，亦涉及阿拉伯芥抵抗其他種類逆境之能力。 Plants, being immobile and cannot escape from their habitat, are vulnerable to various environmental stresses. One of the most typical stress plants encounte is high temperature stress. Heat stress can cause serious injuries to plants, including structural damage of membrane and inhibition of enzyme activities that will eventually leading to plant death. In order to identify genetic determinants that are essential for plant heat tolerance, with the ultimate goal of improving crop heat tolerance through genetic engineering, we have used a forward genetic approach to screen for heat-intolerant mutants of Arabidopsis after ethyl-methane sulfonate (EMS) -mutagenesis. Two of the mutants, hit3 and et were isolated because of their inability to tolerate sustained high temperature stress. To identify the mutated loci of hit3 and et, we employed map-based cloning procedures. Results indicated that hit3 is located at a region between AGI map 3,104 kb and 3,143 kb of the chromosome Ⅴ. The et locus is mapped to At5g10010. The mutation of et is a single nucleotide change from C to A. ET encodes a novel protein of 434 amino acids. NCBI protein blast, PredictProtein and NetNES 1.1 Server analysis indicate that ET protein contains a strong nuclear localization signal sequence (NLS) and is predicted to subcellularly localized to nucleus. These result suggest that the physiological role of ET is involved in nuclear function by which plant can survive under heat stress condition. Meanwhile, et was found to exhibit hypersensitivity to heat shock, abscisic acid and paraquat as well, suggesting that ET also participate in protecting plants from other forms of stresses.
