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研究生:何孟純
研究生(外文):Mengchun He
論文名稱: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
論文名稱(外文):Functional complementation of potassium uptake and salt tolerance by mcSKD1 in yeast
指導教授:顏宏真顏宏真引用關係
指導教授(外文):Hungchen E. Yen
學位類別:碩士
校院名稱:國立中興大學
系所名稱:植物學系
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:73
中文關鍵詞:酵母菌互補試驗mcSKD1基因鉀離子吸收耐鹽性
外文關鍵詞:heterologous complementaionmcSKD1potassiumsalt toleranceATP-binding domain
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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.
目錄:
中文摘要…………………………………………………………………………….Ⅰ
英文摘要……………………………………………………………………………Ⅱ
前言………………………………………………………………………………… 1
壹、前人研究……………………………………………………………………… 2
一、利用酵母菌互補試驗篩選植物相關基因……………………………… 2
二、利用酵母菌互補試驗證明植物相關基因之功能…………………………4
三、酵母菌突變株CY162與鉀離子吸收相關之基因……………………….5
四、真核生物AAA-type ATPase 基因與SKD1基因……………………. . . 6
五、鉀離子吸收機制與細胞耐鹽性之關係…………………………………...8
六、冰花耐鹽相關之mcSKD1基因…………………………………………..10
貳、材料與方法……………………………………………………………………..12
一、實験材料…………………………………………………………………...12
(一)、材料…………………………………………………………………12
(二)、酵母菌菌株…………………………………………………………12
二、構築pYES2-DH1及pYES2-DH1-full之表現載體………………………12
(一)、小量質體DNA的抽取 (Alkali lysis method)…………………….12
(二)、質體的剪切作用 (digestion)……………………………………… 13
(三)、DNA去磷酸化作用 (dephosphorylation)………………………... 13
(四)、DNA片段的回收…………………………………………………. 14
(五)、DNA的黏接作用…………………………………………………. 14
(六)、勝任細胞 (competent cell)的製備………………………………... 15
(七)、轉形作用 (transformation)………………………………………. 15
(八)、質體的篩選 (rapid plasmid screen)………………………………15
(九)、定序………………………………………………………………... 16
三、酵母菌菌株的培養及保存………………………………………………16
(一)、培養………………………………………………………………... 16
(二)、保存………………………………………………………………... 16
四、酵母菌的轉形作用………………………………………………………. 17
五、聚合酶連鎖反應 (Polymerase Chain Reaction, PCR)確認轉形成功……18
六、酵母菌DNA之抽取………………………………………………………18
七、互補低鉀性狀之測試……………………………………………………..19
八、耐鹽性之測試……………………………………………………………..20
九、稀釋測試…………………………………………………………………23
十、轉形酵母菌細胞蛋白質之分析…………………………………………23
(一)、蛋白質之萃取……………………………………………………… 23
(二)、蛋白質電泳 (SDS-PAGE)………………………………………… 24
(三)、銀染………………………………………………………………… 25
參、結果……………………………………………………………………………..24
一、冰花mcSKD1基因序列之分析…………………………………………..26
二、冰花mcSKD1全長基因之構築…………………………………………..26
三、mcSKD1基因進行5’端與大片段刪除之構築……………………………27
四、酵母菌突變株之轉殖實驗………………………………………………..28
五、冰花mcSKD1基因與酵母菌突變株之互補試驗………………………..29
六、利用連續稀釋進行酵母菌轉殖株之互補試驗…………………………..30
七、利用連續稀釋進行酵母菌轉殖株之耐鹽性測試………………………..32
八、利用連續稀釋進行酵母菌轉殖株之低鉀及耐鹽性關係之探討………..34
九、酵母菌中mcSKD1蛋白質之誘導與累積………………………………..35
肆、討論……………………………………………………………………………..37
一、 冰花mcSKD1基因之構築與酵母菌轉殖株之培養……………………..37
二、 冰花mcSKD1基因互補鉀離子吸收缺乏之酵母菌突變株……………..38
三、 冰花mcSKD1基因與酵母菌突變株之耐鹽性關係……………………..40
伍、參考文獻………………………………………………………………………..43
圖表:
表一、各種限制酶剪切之組合……………….…………………………………….50
表二、100X DO solution中的各類胺基酸………………………………………….51
圖一、冰花mcSKD1基因全長以及進行大片段剪切所得基因序列結果之示意圖...
………………………………………………………………………………………..52
圖二、DH1-full與DH1基因序列選用不同限制酶酵素剪切結果之電泳圖…….53
圖三、pYES2-DH1-full之構築………………………………………………….54
圖四、pYES2-DH1之構築…………………………………………………………55
圖五、pYES2-DH1-full (800)之構築………………………………………………56
圖六、pYES2-DH1-full (600)之構築………………………………………………57
圖七、轉殖入含pYES2表現載體之酵母菌突變株 (CY162)生長在SD培養基中之情況……………………………………………………………………………..58
圖八、含mcSKD1基因之酵母菌轉殖株利用PCR確認之電泳圖………………59
圖九、轉殖入pYES2或pYES2-DH1之酵母菌突變株 (CY162)培養在含低鉀的
篩選培養基5天之生長狀況………………………………………………………...60
圖十、轉殖入含mcSKD1構築質體之酵母菌突變株 (HY483)培養在含低鉀的篩選培養基3天之生長狀況…………………………………………………………..61
圖十一、轉殖入含mcSKD1構築質體之酵母菌突變株 (HY483)培養在含鹽的篩選培養基3天之生長狀況…………………………………………………………...62
圖十二、含mcSKD1基因構築之酵母菌轉殖株於低鉀培養基生長3天之稀釋測
試圖………………………………………………………………………………63
圖十三、含mcSKD1基因構築之酵母菌轉殖株於低鉀培養基中進行稀釋測試之
相對生長趨勢圖………………………………………………………………….64
圖十四、含mcSKD1基因構築之酵母菌轉殖株於100 mM NaCl的培養基生長3天之稀釋測試圖……………………………………………………………………..65
圖十五、含mcSKD1基因構築之酵母菌轉殖株於100 mM NaCl培養基中進行稀釋測試之相對生長趨勢圖…………………………………………………………..66
圖十六、含mcSKD1基因構築之酵母菌轉殖株於200 mM NaCl的培養基生長3天之稀釋測試圖……………………………………………………………………..67
圖十七、含mcSKD1基因構築之酵母菌轉殖株於200 mM NaCl培養基中進行稀釋測試之相對生長趨勢圖…………………………………………………………..68
圖十八、含mcSKD1基因構築之酵母菌轉殖株於300 mM NaCl的培養基生長3天之稀釋測試圖…………………………………………………………………..…69
圖十九、含mcSKD1基因構築之酵母菌轉殖株於300 mM NaCl培養基中進行稀釋測試之相對生長趨勢圖…………………………………………………………..70
圖二十、含mcSKD1基因構築之酵母菌轉殖株於低鉀加上50 mM NaCl的培養
基下生長3天之稀釋測試圖………………………………………………………..71
圖二十一、含mcSKD1基因構築之酵母菌轉殖株於低鉀加上50 mM NaCl培養基中進行稀釋測試之相對生長趨勢圖………………………………………………..72
圖二十二、DH1-full與pYES2酵母菌轉殖株於篩選培養基中進行蛋白質誘導之SDS/PAGE圖………………………………………………………………………...73
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