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研究生:林亞君
研究生(外文):Ya-Chung Lin
論文名稱:以酵母菌雙雜交法分析冰花鹽誘導蛋白mcSKD1與mcSNF1和mcCPN1之間的交互作用
論文名稱(外文):Yeast two-hybrid analysis of salt-induced mcSKD1 interacting with mcSNF1 and mcCPN1 in ice plant
指導教授:顏宏真顏宏真引用關係
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生命科學系所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
畢業學年度:97
語文別:中文
論文頁數:64
中文關鍵詞:酵母菌雙雜交法冰花蛋白交互作用
外文關鍵詞:yeast two-hybridice plantprotein-protein interaction
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冰花(Mesembryanthemum crystallinum L.)為耐鹽機制的模式植物,常用於探討鹽分逆境下植物所誘發的生理反應,mcSKD1 (suppressor of K+ transport growth defect 1)為冰花一鹽誘導蛋白,具有三個保留區(conserved domain),microtubule interacting and trafficking molecule domain (MIT)、ATPase associated with a variety of cellular activities (AAA)和Vps4 C terminal oligomerisation domain (Vps4_C),與酵母菌中內膜系統的蛋白運送相關的Vps4 (vacuolar protein sorting 4)同源,而mcSKD1分佈於冰花細胞的內質網(endoplasmic reticulum)和高基氏體(Golgi),推測mcSKD1可能參與冰花的內膜系統蛋白運送機制。前人研究已利用酵母菌雙雜交系統篩選出和mcSKD1具有交互作用之蛋白mcSNF1 (sucrose non-fermenting 1)與mcCPN1 (copine 1)。mcSNF1與酵母菌中調控碳代謝相關酵素的SNF1同源,具有三個conserved domains,由N端至C端依序為serine/threonine protein kinases domain (S/T)、ubiquitin-associated domain (UBA)和kinase-associated domain 1 (KA1)。mcCPN1的N端為具有myristoylation修飾位置的Front domain、von Willebrand factor type A domain (vWA)和C端的really interesting new gene domain (RING),之前研究中確定mcCPN1具有E3 ligase的活性,能夠將mcSKD1接上ubiquitin。然而上述三蛋白之間產生交互作用的位置及作用強度仍是未知,故本論文中即利用酵母菌雙雜交法分析三者之間的交互作用關係。將各個全長和domain的序列分別構築於表現載體後轉入酵母菌中,使產生48種的待測組合,利用營養篩選培養基以連續稀釋法進行生長分析,並配合β-galactosidase酵素活性分析,區別三者之間的交互作用的高低。結果顯示,mcSKD1以MIT和Vps4_C domain與mcSNF1的KA1 domain產生極高度的交互作用;mcSKD1同時以MIT和AAA domain與mcCPN1的vWA domain產生高度的交互作用;mcCPN1以Front和RING domain與mcSNF1的KA1 domain產生極高度的交互作用。推測mcSNF1為mcSKD1和mcCPN1之間的媒介蛋白,以KA1 domain將mcSKD1和mcCPN1兩者拉近,使三者交互作用關係更為緊密,而藉由調控內膜系統的蛋白運送以及ubiquitination相關途徑參與植物在鹽逆境下的生理反應。
Ice plant (Mesembryanthemum crystallinum L.) is a model plant for study salt-induced physiological responses. In ice plant, mcSKD1 (suppressor of K+ transport growth defect 1) is a salt-induced protein, it contains three conserved domains, microtubule interacting and trafficking molecule domain (MIT), ATPase associated with a variety of cellular activities (AAA), and Vps4 C terminal oligomerisation domain (Vps4_C). McSKD1 share homology to yeast Vps4 (vacuolar protein sorting 4) which involves in vesicle trafficking mechanism, and mcSKD1 also locates in endoplasmic reticulum to Golgi network. Thus, mcSKD1 may participate in protein sorting mechanism. McSNF1 and mcCPN1 were indentified interacting with mcSKD1 by yeast two-hybrid system in previous study. McSNF1 share homology to yeast SNF1 which involves in carbon metabolism pathway, and it contains three conserved domains, serine/threonine protein kinases domain (S/T), ubiquitin-associated domain (UBA), and kinase-associated domain 1 (KA1). McCPN1 contains Front domain, von Willebrand factor type A domain (vWA), and really interesting new gene domain (RING). In previous study, it has been shown that mcCPN1 has E3 ligase activity and conjugates ubiquitin chain to mcSKD1. However, the interaction regions and degree of interaction between mcSKD1, mcSNF1, and mcCPN1 are still unknown. In this study, I use yeast two-hybrid to analyze the interaction network among these three proteins. The full-length genes and domain fragments were constructed into expressing vectors, and transformed into yeast cells, respectively. I obtained 48 different combinations, and analyzed them with serial dilution assay and β-galactosidase assay to distinguish the interaction levels between mcSKD1, mcSNF1, and mcCPN1. The results showed that MIT, Vps4_C, and KA1 domains are the regions of very high degree of interaction between mcSKD1 and mcSNF1; MIT, AAA, and vWA domains are the regions of high degree of interaction between mcSKD1 and mcCPN1; Front, RING, and KA1 domains are the regions of very high degree of interaction between mcCPN1 and mcSNF1. Those results suggested that the KA1 domain of mcSNF1 mediates the interaction between mcSKD1 and mcCPN1, and brought the protein complex together to function in protein sorting and ubiquitination pathway under salt stress.
目次:

中文摘要. . . . . . . . . . . . . . . . . . . . . . . . . .i
英文摘要. . . . . . . . . . . . . . . . . . . . . . . . . ii
壹、前人研究. . . . . . . . . . . . . . . . . . . . . . . .1
一、植物耐鹽機制. . . . . . . . . . . . . . . . . . . . . .1
二、模式植物冰花(Mesembryanthemum crystallinum L.) . . . . 1
三、冰花鹽誘導蛋白mcSKD1. . . . . . . . . . . . . . . . . .1
四、與冰花鹽誘導蛋白mcSKD1具有交互作用之mcSNF1. . . . . . .3
五、與冰花鹽誘導蛋白mcSKD1具有交互作用之mcCPN1. . . . . . .5
六、實驗動機與策略. . . . . . . . . . . . . . . . . . . . .6
貳、材料與方法. . . . . . . . . . . . . . . . . . . . . . .8
一、菌種與質體(Strains and plasmids) . . . . . . . . . . . 8
二、構築質體(Plasmid construction) . . . . . . . . . . . . 8
三、酵母菌之轉型作用(Transformaion of yeast) . . . . . . .11
四、酵母菌雙雜交系統(Yeast two-hybrid) . . . . . . . . . .12
五、連續稀釋法(Serial dilution assay) . . . . . . . . . . 12
六、β-galactosidase酵素活性試驗(β-galactosidase assay) . 13
参、結果. . . . . . . . . . . . . . . . . . . . . . . . . 14
肆、討論. . . . . . . . . . . . . . . . . . . . . . . . . 21
伍、參考文獻. . . . . . . . . . . . . . . . . . . . . . . 25
陸、附錄. . . . . . . . . . . . . . . . . . . . . . . . . 52
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