阿拉伯芥HIT1基因定序及選殖; Identification, sequencing and cloning of Arabidopsis HIT1 gene

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题名:	阿拉伯芥HIT1基因定序及選殖;Identification, sequencing and cloning of Arabidopsis HIT1 gene
作者:	張貴雲;Kuei-Yun Chang
贡献者:	生命科學研究所
关键词:	抗熱基因;滲透壓逆境;阿拉伯芥;osmotic stress;heat stress;Arabidopsis
日期:	2004-07-08
上传时间:	2009-09-22 10:18:21 (UTC+8)
出版者:	國立中央大學圖書館
摘要:	高溫對植物可以是一種致死性逆境。之前本實驗室已運用功能性遺傳學的策略，篩選出一對高溫逆境過度敏感之突變種植物，取名為hit1(heat intolerant) 。此突變植物在37℃之高溫逆境下無法存活超過4天，野生種則可。本研究之主要目的即在於hit1遺傳位址之辨認及定序。實驗結果顯示，hit1是BAC F11F12中，阿拉伯芥登錄序號為AT1g50500之基因內，由C變成A之點突變。此推論尚須轉殖正常基因回突變株體內進行互補實驗，觀察可否恢復其抗高溫的表現型，作為最後之確認。氨基酸序列比對的結果顯示，AT1g50500與conserve domain KOG2180有極高的相似度，而該conserve domain的代表為late golgi membrane sorting complex, subunit VPS53。Vps53p存在於酵母菌中，需與Vps52p及Vps54p形成聚合體，在細胞中主要是從trans golgi membrane將carboxypeptidase Y（CPY）運至prevacuolar / endosomal compartment（PVC）。文獻指出，CPY之結構相似於熱修克蛋白（HSP）。就此推測，HIT1在植物細胞中有可能是扮演運輸HSP的角色。除此之外，有關生理之觀察及實驗發現，HIT1基因會影響植物體內水分之平衡關係。在高濃度mannitol環境下，野生種較hit1突變植物有較優之生長表現。綜合以上結果，HIT1基因除賦予阿拉伯芥耐熱能力之外，亦涉及阿拉伯芥抵抗滲透壓的能力，其機制則有待更進一步的研究來揭露。 A heat-hypersensitive mutant of Arabidopsis was isolated based on its inability to survive under normally non-lethal high temperature condition. The mutant named hit1(heat-intolerant) is distinguished from wild-type plant via incubation at 37℃ for 4 days. hit1 is unable to survive under such condition for more than 4 days. This program is to sequence genomic DNA and identify the locus on chromosome. Current results suggest that the hit1 locus is on BAC F11F12, within the gene whose login number in the Arabidopsis database is AT1g50500, and the mutation of hit1 is a single nucleotide changing from C to A. Amino acid sequence alignment indicated that AT1G50500 contains conserve domain KOG2180, and is highly similar to the late golgi membrane sorting complex, subunit VPS53. Vps53p exists in the yeast, forming a complex with Vps52p and Vps54p, and the main function of the complex is to transport carboxypeptidase Y (CPY) from trans golgi membrane to prevacuolar / endosomal compartment (PVC). It has been indicated by some documents that CPY and HSP might have similar function in terms of structures. According to these data, it is reasonable to hypothesize that the HIT1 might play a role in transporting HSP in the plant cell. On the other hand, HIT1 could affect water balance in plants. Wild-type plants grow better than hit1 mutant at high mannitol concentrations. Taken together, HIT1 may function in sorting certain proteins whose roles are critical for plants to cope with both heat and osmotic stresses.
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