Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/20246
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dc.contributor黃皓瑄zh_TW
dc.contributorHau-Hsuan Hwangen_US
dc.contributor.author鄭惇方zh_TW
dc.contributor.authorCheng, Tun-Fangen_US
dc.contributor.other生命科學系所zh_TW
dc.date2013en_US
dc.date.accessioned2014-06-06T07:12:46Z-
dc.date.available2014-06-06T07:12:46Z-
dc.identifierU0005-0102201311513200en_US
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dc.identifier.urihttp://hdl.handle.net/11455/20246-
dc.description.abstract農桿菌(Agrobacterium tumefaciens)存在於土壤中,為一種植物性病原菌。農桿菌的Ti質體(tumor-inducing plasmid,Ti plasmid)上含有一段T-DNA (transfer DNA)會藉由感染過程,進而轉移並嵌入植物細胞染色體上。由於T-DNA上帶有生長激素(auxin)和細胞分裂素(cytokinin)的生合成基因(如:iaaH[indole-3-acetamide hydrolase]、iaaM[indoleacetic acid tryptophan monooxygenase]、ipt [isopentenyl transferase])。因此當T-DNA嵌入植物染色體後,這些基因被表現進而生合成植物賀爾蒙,導致植物細胞不正常增生而形成冠癭狀腫瘤(crown gall tumor)。農桿菌細胞膜上的VirA/VirG雙分子磷酸依賴系統(two-component phosphor-relay system),可以感受到植物受傷後分泌的醣類及酚類化合物,進而誘導開啟下游vir基因(virulence gene)的大量表現。這些vir基因產物會幫助T-DNA的產生、轉移並嵌入植物染色體中。在胭脂鹼型(nopaline type)農桿菌的Ti質體上,含有一段與ipt基因序列相似的tzs (trans-zeatin synthesis)基因,可以轉譯產生Tzs蛋白質,促使反玉米素生合成。tzs基因可以受到AS (acetosyringone)的誘導而大量表現,也會受到VirA/VirG雙分子磷酸依賴系統的調控而大量表現。Tzs蛋白質可與位在細胞膜上組成第四型分泌系統(Type IV secretion system,T4SS)的VirB5結合。又已知當tzs基因突變後,會導致農桿菌分泌反玉米素的含量減少、感染植物的能力下降及生長速度遲緩。在感染過程中,額外添加細胞分裂素,則能提升突變株感染植物的效率。並發現突變株培養在19oC、pH5.5的AB-MES培養基中,菌體內Vir蛋白質有較高的累積量,並有較高的virB啟動子活性。若在培養基中額外添加細胞分裂素,則突變株及野生株農桿菌中Vir蛋白質累積量,及virB啟動子活性皆有受到抑制。因此本研究為進一步探討tzs基因及其產物對農桿菌生長趨勢、感染植株能力及vir基因表現的影響。研究結果顯示,在19℃酸性AB-MES培養基中,tzs突變株的數個vir基因和tzs基因啟動子活性皆較野生株高,且在誘導培養的早期與野生株差異最明顯。另外,在AS誘導培養過程中額外添加細胞分裂素,可抑制數個vir基因和tzs基因啟動子活性。並發現當tzs基因功能喪失後,可能不會直接影響農桿菌第四型分泌系統的質體轉移效率,及附著在植物根上的能力。此外進一步構築四株tzs基因功能缺失突變株,並發現四株tzs突變株的表現性狀與tzs缺失株和tzs框移突變株相似,其感染力、生長速率皆低於野生株,而Vir蛋白質累積量則高於野生株。因此由本研究可知tzs基因及其產物可以藉由調控vir基因的表現,促使農桿菌改變生理代謝,進而影響農桿菌感染植物的能力。zh_TW
dc.description.abstractAgrobacterium tumefaciens is a plant pathogen and exists in soil, which contains T-DNA (transfer DNA) in a Ti plasmid (tumor-inducing plasmid). The T-DNA are transferred and integrated into plant chromosome during A. tumefaciens infection process. There are auxin and cytokinin biosynthesis genes on the T-DNA, including iaaH (indole-3-acetamide hydrolase), iaaM (indoleacetic acid tryptophan monooxygenase), ipt (isopentenyl transferase). When the T-DNA integrates into plant chromosome, genes in the T-DNA region express and cause high levels of auxin and cytokinin productions, which increases plant cell numbers and sizes abnormally and finally causes crown gall disease. When plant wounded sites secrete monosaccharides and phenolic compounds, the VirA/VirG two component system in A. tumefaciens recognizes those signals, and induces virulence (vir) gene expressions. The Vir proteins involve in T-DNA production, transfer, and integration into plant chromosome. The tzs (trans-zeatin synthesis) gene which shares sequence similarity with the ipt gene only exists in the nopaline-type Ti plasmid. The Tzs protein mediates the biosynthesis of trans-zeatin in A. tumefaciens. The tzs gene expression is induced by acetosyringone (AS) and regulated by the VirA/VirG two component system. The tzs deletion and frame-shift mutants decreased trans-zeatin secretions, reduced virulence on several plant species and showed growth defects during infections. When exogenous cytokinins were added during infections, it restored virulence of the tzs mutants on plants. When grown in acidic AB-MES media with AS at 19oC, the Vir protein accumulations and virB promoter activities increased in the tzs mutants. When exogenous cytokinins were added during AS induction, the Vir protein accumulations and virB promoter activities were repressed. Results of thesis study showed that several vir genes promoter activities increased in the tzs mutants when grown in acidic AB-MES with AS at 19oC in comparison to wild-type, especially at the early stage of AS inductions. When exogenous cytokinins were added during AS inductions, several vir genes and tzs gene promoter activities decreased in the tzs mutant and the wild-type strains. In addition, RSF1010 plasmid transfer efficiencies by the type IV secretion system and bacterial attachment abilities on Arabidopsis roots were not significantly affected by the absence of the Tzs protein. We additionally generated four new tzs mutants, which phenotype are similar with tzs mutant. Four new tzs mutants reduced virulence on potato tuber discs, showed growth defects under AS inductions and increased Vir protein accumulations. Taken together, these data suggest that the tzs gene and it product, cytokinin, may be involved in regulation of vir gene expression, and therefore affect bacterial growth and virulence on plants.en_US
dc.description.tableofcontents目錄 中文摘要………………………………………………………………i 英文摘要………………………………………………………………ii 目錄 …………………………………………………………………iii 表目錄…………………………………………………………………vii 圖目錄…………………………………………………………………ix 附錄……………………………………………………………………xi 壹、前言………………………………………………………………1 一、農桿菌……………………………………………………………1 1、農桿菌的感染過程………………………………………………2 1.1、農桿菌接觸並附著於植物細胞………………………………2 1.2、植物受傷訊息誘導致病基因的活化及致病基因的調控……2 1.3、農桿菌單股T-DNA的產生 ……………………………………3 1.4、農桿菌利用第四型分泌系統將T-DNA及Vir蛋白質送入植物 細胞中…………………………………………………………4 1.4.1、第四型分泌系統的組成結構及功能………………………4 1.4.2、T-DNA與Vir蛋白質的轉移…………………………………5 二、細胞分裂素………………………………………………………6 1、細胞分裂素的生合成途徑………………………………………7 2、植物細胞中細胞分裂素的接受體………………………………8 3、細胞分裂素對生物的影響………………………………………9 三、tzs基因相關研究 ………………………………………………9 四、研究目的…………………………………………………………11 貳、材料與方法………………………………………………………12 一、菌種………………………………………………………………12 1、用於偵測vir基因啟動子活性之農桿菌 ………………………12 1.1、含有virpro:GFP (Green Fluorescent Protein)之農桿菌 ……………………………………………………………………12 1.2、含有virpro:LacZ(β-galactosidase)之農桿菌 …………13 2、tzs (trans-zeatin synthesis)基因回復株、互補株和含有tzs 基因的不同品系農桿菌 …………………………………………14 3、用於檢測附著於阿拉伯芥切根能力的農桿菌…………………14 4、用於製備大腸桿菌勝任細胞(competent cell)的菌株………15 5、用於進行IncQ質體RSF1010在農桿菌間轉移效率分析的菌株 15 6、用於構築含有virA、virG、tzs啟動子區域,並以此表現GFP 蛋白質的菌株……………………………………………………15 7、用於構築各式tzs突變株的菌株 ………………………………16 8、tzs 各式突變株的菌株…………………………………………18 9、用於測試農桿菌短暫表現T-DNA能力的農桿菌 ………………18 二、培養基……………………………………………………………19 1、B5培養基…………………………………………………………19 2、523培養基 ………………………………………………………19 3、AB-MES medium培養基 …………………………………………20 4、2YT培養基 ………………………………………………………20 5、LB培養基…………………………………………………………20 6、MS固態培養基……………………………………………………20 7、water agar培養基………………………………………………20 8、CIM固態培養基(Callus-Inducing-Medium) …………………21 三、農桿菌在AB-MES液態培養基中,生長曲線的分析……………21 四、質體的構築………………………………………………………21 1、利用 virA、virG啟動子區域,以表現GFP蛋白質的質體之構築 ……………………………………………………………………21 2、tzs基因突變質體的構築 ………………………………………22 3、限制酵素之切割作用……………………………………………23 4、連接酶之黏合反應………………………………………………23 5、水平膠體之電泳分析……………………………………………24 6、PCR產物之純化 …………………………………………………24 7、水平膠體內DNA片段之萃取 ……………………………………24 8、篩選重組之質體…………………………………………………25 9、以雙交換方式(double crossing over)進行同源基因置換 ……………………………………………………………………25 五、大腸桿菌勝任細胞的製備與熱休克轉型法……………………26 1、大腸桿菌勝任細胞之製備………………………………………26 2、大腸桿菌熱休克轉型(heat-shock transformation) ………26 六、農桿菌勝任細胞的製備與電穿孔轉型法(electroporation) ……………………………………………………………………26 1、農桿菌勝任細胞之製備…………………………………………26 2、農桿菌電穿孔轉型………………………………………………26 七、細菌的接合作用、菌種的保存及細菌內質體DNA的萃取 ……27 1、細菌的接合作用(conjugation) ………………………………27 2、菌種的保存………………………………………………………27 3、細菌質體DNA的萃取 ……………………………………………27 八、IncQ質體RSF1010於農桿菌間的轉移效率分析 ………………28 九、農桿菌在AB-MES液態培養基生長時,Vir蛋白質累積量之分析 ……………………………………………………………………28 1、農桿菌的誘導培養………………………………………………28 2、農桿菌蛋白質的萃取……………………………………………29 3、蛋白質濃度的測定………………………………………………29 4、蛋白質的電泳分析(SDS-polyacrylamide gel [SDS-PAGE] analysis)…………………………v……………………………29 4.1、Tricine-SDS-PAGE(Tricine-SDS-polyacrylamide gel)膠體 的製備 ………………………………………………………29 4.2、蛋白質的電泳分析 …………………………………………30 5、西方墨點法(western blot)……………………………………30 5.1、膠體轉漬 ……………………………………………………30 5.2、以抗體偵測特定蛋白質之累積 ……………………………30 5.3、移除轉漬模上抗體的方法 …………………………………31 十、農桿菌在AB-MES液態培養基生長時,菌體內virPro:LacZ或 tzsPro:LacZ啟動子活性分析 …………………………………31 十一、農桿菌在AB-MES液態培養基生長時,菌體內virPro:GFP或 tzsPro:GFP啟動子活性分析……………………………………32 十二、阿拉伯芥植株的培養…………………………………………32 十三、農桿菌附著於阿拉伯芥切根上的效率分析…………………32 1、農桿菌的培養 …………………………………………………32 2、農桿菌附著於阿拉伯芥切根上的效率分析 …………………32 十四、農桿菌於阿拉伯芥切根共培養時,生長狀態分……………33 十五、農桿菌感染馬鈴薯腫瘤性狀分析……………………………33 十六、農桿菌短暫表現T-DNA的效率分析 …………………………33 参、結果………………………………………………………………35 一、將tzs突變株培養在含AS的酸性AB-MES培養基(minimal medium)中,在受到AS誘導的前期,具有較高的tzs、virB、virD和virE啟動子活性…………………………………………………………………35 二、在酸性AB-MES培養基中加入0.01、2.5或8ppm的kinetin、0.01ppm的zeatin或0.01 ppm的trans-zeatin,可使野生株及tzs突變株內數種vir啟動子活性下降 ………………………………………36 三、在含AS的酸性AB-MES培養基中,額外添加kinetin後,不同品系野生農桿菌A208、A348、LBA4404、EHA101的virB啟動子活性未明顯地受到抑制,且Vir蛋白質累積量也未明顯地受影響 ……………39 四、質體轉移效率及黏附在植物細胞的能力與tzs基因是否突變無關………………………………………………………………………40 五、tzs互補株的感染力恢復,且其Vir蛋白質累積量與野生株相似,但其生長速度卻低於野生株…………………………………………41 六、不同的tzs基因突變株感染植物的能力較野生株差,生長速度也較野生株緩慢、且Vir蛋白質的累積量高於野生株 ………………43 肆、討論………………………………………………………………46 一、農桿菌tzs基因和細胞分裂素,對農桿菌vir基因及tzs基因表現量的影響………………………………………………………………46 二、農桿菌tzs 基因及細胞分裂素,對農桿菌Vir 蛋白質累積、生長及致病力的影響………………………………………………………49 伍、參考文獻…………………………………………………………52 表目錄 表一、土壤農桿菌野生株(wild-type)、tzs基因缺失突變株(△tzs-278)及tzs基因框移突變株(tzs-fs-3)分別培養於含有200 μM乙醯丁香酮的AB-MES培養基(pH值為5.5)中加入0.01ppm的kinetin。並於19℃培養0、4、8、16和40小時後,測量其tzs啟動子之活性。…66 表二、土壤農桿菌野生株(wild-type)、tzs基因缺失突變株(△tzs-278)及tzs基因框移突變株(tzs-fs-3)分別培養於19℃含有200 μM乙醯丁香酮的AB-MES培養基(pH值為5.5)中加入0.01ppm的kinetin。並於19℃培養0、4、8、16和40小時後,測量其tzs、virA、virB、virC、virD、virE、virG啟動子之活性。…………………………67 表三、土壤農桿菌野生株(wild-type)NT1RE、NT1RE(pJK270)、A208和C58分別培養於19℃含有200 μM乙醯丁香酮的AB-MES培養基(pH值為5.5)中加入0.01 ppm的kinetin。並於19℃培養0、4、8、16和40小時後,測量其tzs、virA和virG啟動子之活性。 …………………70 表四、土壤農桿菌野生株(wild-type)、tzs基因缺失突變株(△tzs-278)及tzs基因框移突變株(tzs-fs-3)分別培養於19℃含有200 μM乙醯丁香酮的AB-MES培養基(pH值為5.5)中加入不同濃度的kinetin(2.5或8 ppm)。並於19℃培養0、4、8、16和40小時後,測量其tzs啟動子之活性。………………………………………………………………72 表五、土壤農桿菌野生株(wild-type)、tzs基因缺失突變株(△tzs-278)及tzs基因框移突變株(tzs-fs-3)分別培養於19℃含有200 μM乙醯丁香酮的AB-MES培養基(pH值為5.5)中加入0.01 ppm的trans-zeatin。並於19℃培養0、4、8、16和40小時後,測量其virB啟動子之活性。………………………………………………………………74 表六、土壤農桿菌野生株(wild-type)、tzs基因缺失突變株(△tzs-278)及tzs基因框移突變株(tzs-fs-3)分別培養於19℃含有200 μM乙醯丁香酮的AB-MES培養基(pH值為5.5)中加入0.01 ppm的zeatin。並於19℃培養0、4、8、16和40小時後,測量其virA、virB、virE啟動子之活性。……………………………………………………………75 表七、土壤農桿菌野生株(wild-type)NT1RE、NT1RE(pJK270)、A208和C58分別培養於19℃含有200 μM乙醯丁香酮的AB-MES培養基(pH值為5.5)中加入0.01 ppm的zeatin。並於19℃培養0、4、8、16和40小時後,測量其virA啟動子之活性。…………………………………77 表八、土壤農桿菌A348、A208、LBA4404和EHA101分別培養於含有200 μM乙醯丁香酮的AB-MES培養基(pH值為5.5)中加入不同濃度的kinetin(0.01, 2.5, 8 ppm)。並於19℃培養0、16、40和64小時後,測量其virB啟動子之活性。…………………………………………78 表九、土壤農桿菌野生株(wild-type)、tzs基因缺失突變株(△tzs-278)及tzs基因框移突變株(tzs-fs-3)分別培養於含有200 μM乙醯丁香酮的AB-MES培養基(pH值為5.5)中,轉移RSF1010質體之效率。 …………………………………………………………………………79 表十、土壤農桿菌野生株(wild-type)、tzs基因缺失突變株(△tzs-278)、tzs基因回復株及基因互補株分別感染馬鈴薯塊莖後產生腫瘤的效率。………………………………………………………………80 表十一、土壤農桿菌野生株(wild-type)、tzs基因缺失突變株(△tzs-278)、tzs基因回復株及基因互補株分別培養於含阿拉伯芥(Ws)根段的MS培養基(pH值為5.5)上。並於22-24℃培養0、24、48小時後,測量其活菌數。…………………………………………………81 表十二、土壤農桿菌野生株(wild-type)及各種tzs基因突變株分別感染馬鈴薯或阿拉伯芥(Ws)後產生腫瘤及短暫表現T-DNA的效率。……………………………………………………………………82 表十三、土壤農桿菌野生株(wild-type) 及各種tzs基因突變株分別培養於含200 μM乙醯丁香酮的AB-MES液態培養基(pH值為5.5)。並於19℃培養0、16、40、64小時後,測量其活菌數。 ………………83 圖目錄 圖一、當tzs缺失突變株(△tzs-278)及tzs框移突變株(tzs-fs-3)培養於19℃含乙醯丁香酮(AS)的酸性AB-MES液態培養基中,其菌體內tzs啟動子活性較野生株高。 ………………………………………84 圖二、當tzs缺失突變株(△tzs-278)及tzs框移突變株(tzs-fs-3)培養於19℃含乙醯丁香酮(AS)的酸性AB-MES液態培養基中,tzs缺失突變株(△tzs-278)菌體內tzs、virA、virB、virC啟動子活性較野生株高。……………………………………………………………………85 圖三、當tzs缺失突變株(△tzs-278)及tzs框移突變株(tzs-fs-3)培養於19℃含乙醯丁香酮(AS)的酸性AB-MES液態培養基中,tzs缺失突變株(△tzs-278)菌體內virE啟動子活性較野生株高。 …………87 圖四、當野生株(NT1RE)培養於19℃含乙醯丁香酮(AS)的酸性AB-MES液態培養基中,其菌體內tzs啟動子活性較其他三種野生株低;而NT1RE菌體內virA、virG啟動子活性與其他三種野生株相似。 …88 圖五、當tzs缺失突變株(△tzs-278)及tzs框移突變株(tzs-fs-3)培養於19℃含乙醯丁香酮(AS)的酸性AB-MES液態培養基中,tzs缺失突變株內tzs啟動子活性較野生株高。 ………………………………89 圖六、當tzs缺失突變株(△tzs-278)及tzs框移突變株(tzs-fs-3)培養於19℃含乙醯丁香酮(AS)及0.01 ppm的 trans-zeatin的酸性AB-MES液態培養基中,其菌體內virB啟動子活性未明顯地受到影響。……………………………………………………………………90 圖七、當野生株、tzs缺失突變株(△tzs-278)及tzs框移突變株(tzs-fs-3)培養於19℃含乙醯丁香酮(AS)的酸性AB-MES液態培養基中,其菌體內virB和virE啟動子因外加0.01 ppm zeatin後,活性有下降的現象。…………………………………………………………………91 圖八、當野生株(NT1RE[pJK270])培養於19℃含乙醯丁香酮(AS)的酸性AB-MES液態培養基中,菌體內virA啟動子活性較其他三種野生株高。……………………………………………………………………92 圖九、當不同品系的野生株A348、A208、LBA4404和EHA101培養於19℃含乙醯丁香酮(AS)的酸性AB-MES液態培養基中,菌體內virB啟動子活性並未因外加kinetin後,而受到抑制。 ……………………93 圖十、當野生株A208、A348、LBA4404和EHA101培養於19℃含乙醯丁香酮(AS)的酸性AB-MES液態培養基中,菌體內VirD2、VirB2和VirB11蛋白質的累積量未因加入0.01 ppm kinetin而有顯著地改變。…94 圖十一、將tzs框移突變株與阿拉伯芥(Ws)切根共同培養於19℃的1 mM CaCl2和0.4% sucrose液體中,其附著在阿拉伯芥切根上的菌體螢光亮度與野生株相似。………………………………………………95 圖十二、將tzs框移突變株與阿拉伯芥(Ws)切根共同培養於19℃的1 mM CaCl2和0.4% sucrose液體中,其附著在阿拉伯芥切根上的菌體螢光亮度與野生株相似。………………………………………………96 圖十三、將tzs框移突變株與阿拉伯芥(Ws)切根共同培養於25℃的1 mM CaCl2和0.4% sucrose液體中,其附著在阿拉伯芥切根上的菌體螢光亮度與野生株相似。………………………………………………97 圖十四、將tzs框移突變株與阿拉伯芥(Ws)切根共同培養於25℃的1 mM CaCl2和0.4% sucrose液體中,其附著在阿拉伯芥切根上的菌體螢光亮度與野生株相似。………………………………………………98 圖十五、tzs基因互補株(△tzs genomic c-1~3)感染馬鈴薯後,腫瘤產生的效率較tzs突變株增加。 ……………………………………99 圖十六、野生株、tzs基因缺失株(△tzs-278)、tzs基因回復株(wild-type revertant of the △tzs)和tzs基因互補株(△tzs genomic c-1~3)生長曲線分析。……………………………………100 圖十七、野生株(NT1RE[pJK270])、tzs基因缺失株(△tzs-278)、tzs基因回復株和tzs基因互補株培養於19℃含乙醯丁香酮(AS)的酸性AB-MES液態培養基中,菌體內VirD2、VirB2和Tzs蛋白質的累積量分析。……………………………………………………………………101 圖十八、各個tzs基因突變株短暫表現T-DNA的效率和腫瘤產生效率。……………………………………………………………………102 圖十九、各個tzs基因突變株培養於19℃含乙醯丁香酮(AS)的酸性AB-MES液態培養基中,其菌體內Tzs、VirD2、VirB2蛋白質累積量與野生株不同。………………………………………………………………103 圖二十、各個tzs基因突變株培養於19℃含乙醯丁香酮(AS)的酸性AB-MES液態培養基中,其生長速率介於野生株及tzs框移突變株之間。……………………………………………………………………104 附錄 附表一、引子序列表…………………………………………………105 附圖一、用於測試virA或virG啟動子活性使用的pRU1156質體之構築流程。…………………………………………………………………106 附圖二、用於置換野生種農桿菌Tzs基因為Tzs-Early-stop codon所用pJQ200KS質體之構築流程。………………………………………107 附圖三、用於置換野生種農桿菌Tzs基因為Tzs-L32RD33S所用pJQ200KS質體之構築流程。…………………………………………108 附圖四、用於置換野生種農桿菌Tzs基因為Tzs-△B所用pJQ200KS質體之構築流程。…………………………………………………………109 附圖五、用於置換野生種農桿菌Tzs基因為Tzs-A+B+C+D/△E+F+G所用pJQ200KS質體之構築流程。…………………………………………110 附圖六、利用基因同源重組的方式,將構築好的Tzs突變基因利用pJQ200KS質體送入野生種農桿菌中。………………………………111 附圖七、農桿菌tzs終止碼提前突變株(Tzs-Estop) (A)、tzs缺失B段突變株(Tzs-△B) (B)、tzs點突變株(Tzs-L32RD33S) (C)及tzsC端缺失株(Tzs-ABCD) (D)的蛋白質結構圖。……………………………112zh_TW
dc.language.isozh_TWen_US
dc.publisher生命科學系所zh_TW
dc.relation.urihttp://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-0102201311513200en_US
dc.subject農桿菌zh_TW
dc.subjectAgrobacterium tumefaciensen_US
dc.subjecttzs基因zh_TW
dc.subject細胞分裂素zh_TW
dc.subjecttzsen_US
dc.subjectcytokininen_US
dc.title功能分析tzs基因及細胞分裂素對農桿菌生長、感染力及致病基因表現之影響zh_TW
dc.titleFunctional studies of the tzs gene and cytokinin effects on Agrobacterium tumefaciens growth, virulence, and virulence gene expressionsen_US
dc.typeThesis and Dissertationzh_TW
Appears in Collections:生命科學系所
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