Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/20581
標題: 利用酵母菌互補試驗探討mcSKD1基因鉀離子吸收機制及細胞耐鹽性
Functional complementation of potassium uptake and salt tolerance by mcSKD1 in yeast
作者: 何孟純
He, Mengchun
關鍵字: heterologous complementaion;酵母菌;mcSKD1;potassium;salt tolerance;ATP-binding domain;互補試驗;mcSKD1基因;鉀離子吸收;耐鹽性
出版社: 植物學系
摘要: 
中文摘要
在耐鹽植物冰花中篩選一鹽誘導基因並進行胺基酸序列比對,發現與老鼠鉀離子吸收相關的SKD1蛋白具70%相似度,命名為冰花mcSKD1基因,推測與鉀離子吸收有關。故利用CY162及HY483二個鉀離子吸收缺乏的酵母菌突變株進行功能測試,將mcSKD1基因全長、5’端刪除約300 bp及僅含ATPase domain序列之基因序列構築在酵母菌表現載體,以構築完成之質體轉殖入此二突變株,在CY162突變株中僅有5’端刪除的mcSKD1基因可互補CY162缺鉀之性狀,而在HY483突變株中三種構築皆可互補HY483缺鉀之性狀,其中以mcSKD1基因全長互補缺鉀性狀之能力最好。
在確定冰花mcSKD1基因具有互補鉀離子吸收缺乏性狀後,為了進一步瞭解mcSKD1基因在參與鉀離子吸收與幫助酵母菌耐鹽能力提昇是否相關,故分別給予100、200及300 mM 的鹽處理進行生長測試,以連續稀釋酵母菌轉殖株的方式觀察生長情況,及以TINA2.09軟體進行量化分析。結果顯示100 mM與300 mM鹽處理時,以mcSKD1基因全長及僅含ATPase domain序列之基因幫助鉀離子吸收增進酵母菌耐鹽性的能力相似,但在300 mM 的鹽逆境下酵母菌轉殖株的生長大幅下降。在200 mM 的鹽處理中,mcSKD1基因全長的酵母菌轉殖株較100 mM 鹽逆境中生長有提昇趨勢;另外在低鉀加上50 mM NaCl之處理中,顯示5’端刪除的mcSKD1基因轉殖株較低鉀處理有明顯的細胞生長提高趨勢。綜合以上結果,鹽逆境下以全長的mcSKD1基因幫助酵母菌細胞耐鹽能力最好,僅含ATPase domain之mcSKD1基因次之,而5’端刪除的mcSKD1基因耐鹽能力最差。
由本論文得知冰花mcSKD1基因具有重建鉀離子吸收缺乏突變株之能力,且鹽逆境下可增加酵母菌細胞對高鹽之耐受性,故mcSKD1基因作用機制可能是經由提高鉀離子吸收能力及所含之ATPase活性提供離子平衡所須之能量來源,進而提升細胞之耐鹽性。

Abstract
A salt-induced gene was isolated from the halophytes Mesembryanthemum crystallinum (ice plant) and the deduced amino acid sequence showed 70% homology to a mouse SKD1 (suppressor of potassium growth defect) gene. It was named mcSKD1. To examine the function of mcSKD1 in the process of potassium uptake, functional complementation of potassium uptake in two yeast mutants, CY162 and HY483, was performed. Sequences of full-length, 5' deletion of 300 bp, and ATPase-containing domain of mcSKD1 was cloned into a yeast expression vector pYES2 and transformed into CY162 and HY483. The result showed that 5' deletion of mcSKD1 was able to complement CY162, whereas all three mcSKD1 constructs were able to complement HY483. The construct containing full-length mcSKD1 had the highest ability to suppress the potassium uptake defective phenotype of HY483.
After mcSKD1 was showed to complement the potassium uptake defective phenotype, the possible role of mcSKD1 participating in the salt tolerance mechanism was further examined. Yeast mutants were treated with 100, 200 and 300 mM NaCl in the presence of galactose and 100 mM potassium. The growth of yeast mutant was quantitated by series dilution test. The data showed that both full-length and ATPase-containing domain of mcSKD1 enhanced the growth of yeast mutant under 100 and 300 mM NaCl. The growth of yeast mutant was significantly retarded in the presence of 300 mM NaCl. Interestingly, the growth of yeast mutant in 200 mM NaCl was increased compared to that of 100 mM NaCl. In the medium containing 5 mM KCl plus 50 mM NaCl, the growth of 5' deletion of mcSKD1 was improved compared to cells grown in the medium containing only 5 mM KCl. In conclusion, the construct containing full-length mcSKD1 had the greatest ability to help yeast cell grown in the low potassium and high sodium environment, the construct containing ATPase domain came next, and the construct containing 5' deletion of mcSKD1 was the lowest.
Using yeast complementation test, we have demonstrated that mcSKD1 possessed a strong ability to complement potassium uptake defective phenotype and increase salt tolerance. The molecular mechanism of SKD1 governing salt tolerance may be through the facilitation of potassium uptake under high saline environment and the ATPase activity in the structure fuels the energy needed for maintenance of ion homeostasis.
URI: http://hdl.handle.net/11455/20581
Appears in Collections:生命科學系所

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