(54.236.58.220) 您好!臺灣時間:2021/02/27 12:13
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果

詳目顯示:::

我願授權國圖
: 
twitterline
研究生:林立菁
研究生(外文):Li-Jing Lin
論文名稱:以酵母菌雙雜交法系統分析水稻結鈣激活酵素與其結合蛋白之交互作用
論文名稱(外文):Analyses of Interactions between OSCKs and OIPs by Yeast Two-hybrid System
指導教授:呂維茗
指導教授(外文):Wei-Ming Leu
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生物科技學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2005
畢業學年度:93
語文別:中文
中文關鍵詞:水稻結鈣激活酵素酵母菌雙雜交法
外文關鍵詞:OSCK1Yeast Two-hybrid System
相關次數:
  • 被引用被引用:0
  • 點閱點閱:177
  • 評分評分:系統版面圖檔系統版面圖檔系統版面圖檔系統版面圖檔系統版面圖檔
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
OSCK1與OSCK2為水稻中於花粉成熟期大量表現的CDPK (calcium-dependent calmodulin-independent protein kinase)基因,由於鈣離子是花粉萌發的必要因素,故預期OSCK基因可能與調控花粉萌發,甚或是先前花粉發育的過程相關。為了探討此二個CDPK的作用機制,先前實驗室利用酵母菌雙雜交法,篩選出五個可能為OSCK1的結合蛋白或下游受質蛋白,命名為OIPs (OSCK1-interacting proteins),分別為OIP1、OIP13、OIP18、OIP28及OIP30。其中OIP18與OIP28為兩個不同的CDPK,另稱之為OSCK4與OSCK3。由於藉由酵母菌雙雜交法所選殖出的基因多非全長,因此本實驗首先探討當雙方蛋白均為全長時,是否仍具有交互作用,並進而檢視其生理意義。依據水稻基因組資料庫的資訊,設計引子進行RT-PCR,分別獲得OIP13、OIP18及OIP28的全長cDNA;而OIP1及OIP30則因資料庫中查無對應序列,故利用5- RACE獲得全長cDNA。由RT-PCR實驗結果可知OIP18 (OSCK4)與OIP28 (OSCK3)亦於花粉大量表現,故花粉中至少有四種CDPK基因表現,其蛋白產物彼此間是否可形成複合體值得探討,故構築一系列質體分別可表現四種CDPK全長蛋白,或僅激活酵素區塊,一一配對進行酵母菌雙雜交測試,初步結果顯示僅OSCK2-F可與自我或OSCK4-F蛋白產生交互作用,暗示少數特定之CDPK蛋白間有可能以複合體方式存在體內。此外,OIP1、13、18與28全長蛋白均無法與OSCK1-F產生交互作用,暗示其可能並非水稻中OSCK1蛋白的結合對象;而OIP30則恰相反,除了與OSCK1-F具交互作用外,與其他的OSCKs (OSCK2-F、3-F、4-F)之交互作用亦極明顯,暗示其具有生物意義。為了深入研究OIP30蛋白,故製備其專一性抗體,以西方點墨法偵測水稻各組織之OIP30蛋白含量,發現其於癒傷組織與花粉中表現量最高,於幼葉與根部則含量偏低,未來將藉由抗體探討OSCK1與OIP30蛋白的結合關係。
OSCK1 and OSCK2 are two CDPK (calcium-dependent calmodulin-independent protein kinase) genes predominantly expressed in mature pollen of rice. As Ca2+ is essential for pollen germination, it is conceivable that CDPKs may play critical roles in pollen germination and/or pollen development. To identify protein substrate for OSCK1 in pollen, we have used the kinase domain of OSCK1 (OSCK1-K) as a bait in yeast two-hybrid screen and obtained five OSCK1-interacting proteins (OIPs), namely OIP1, 13, 18, 28 and 30. As OIP18 and 28 are also CDPK genes, they were renamed as OSCK4 and OSCK3, respectively. In this study, full-length OIP cDNAs were obtained either directly by RT-PCR according to sequence information retrieved from GenBank (for OIP13, 18, 28), or by 5-RACE, in silico sequence assembly, followed by RT-PCR (for OIP1 and 30). Interactions of the full-length form of OIPs with OSCK1 were re-examined by yeast two-hybrid analyses to confirm their biological significance. Since OIP18 (OSCK4) and OIP28 (OSCK3) were detected highly expressed in pollen, similar to OSCK1 and OSCK2, it is interesting to investigate whether their encoded OSCK proteins could form complexes with each other. Paired analyses on four CDPKs revealed interactions only within OSCK2-F itself or between OSCK2-F and OSCK4-F, indicating that some specific CDPKs may exist as multimer in vivo. Except for OIP30, other OIPs (OIP1, 13, 18, 28) all failed to show interactions with OSCK1 in their full-length form and were excluded for further analyses. Oppositely, OIP30 exhibited strong interactions not only with OSCK1-F but also with other OSCKs (OSCK2-F, 3-F, 4-F), suggesting a biological significance. Examinations of various rice tissues revealed that OIP30 protein was most abundant in callus and pollen, but barely detected in young leaves or root.
摘要 1
Abstract 2
前言 3
材料與方法 9
一、以5-RACE (rapid amplification of cDNA ends)選殖全長cDNA 9
二、菌種與質體(Bacterial strains and plasmids) 9
三、培養基及緩衝溶液(Medium、reagent and buffers) 9
四、小量質體DNA萃取(Mini-preparation of plasmid DNA) 10
(一) 煮沸法(Boiling method) 10
(二) Kit抽取法 10
五、大腸桿菌之轉型作用(Transformation of E. coli ) 10
(一) 勝任細胞之製備(Preparation of competent cells) 10
(二) 轉形作用(Transformation) 11
六、酵母菌之轉形作用( Transformation of yeast ) 11
(一) 製備酵母菌之勝任細胞( Preparation of yeast competent cells) 11
(二) 酵母菌之勝任細胞之轉型( Transformation of yeast) 11
七、酵母菌雙雜交法( Yeast two-hybrid system ) 12
八、序列性生長勢分析 (SD plate serial dilution assay) 12
九、以RT-PCR來檢測OIP30基因的表現 13
(一) 植物材料 13
(二) 檢測基因表現 14
(三) 雜交探針之製備 15
(四) 南方墨點法(Southern blotting) 15
(五) 自動放射顯影 16
十、檢測OSCK1及OIP30於水稻之蛋白表現量 16
(一) 植物材料[同方法九、(一)]及水稻轉殖株之小穗花 16
(二) 萃取植物蛋白(Denatured protein) 16
(三) 預先清除雜抗體(Pre-absorption) 16
(四) 西方點墨法(Western blotting) 17
十一、利用蛋白下拉實驗(pull-down assay)分析水稻中OSCK1 與OIP30之交互作用 18
(一) 萃取大量表現OSCK1-(His)6轉殖株之小穗花(spikelet)的蛋白 18
(二) 以鎳金屬親和性管柱層析(Ni-NTA affinity chromatography)進行蛋白下拉實驗(pull-down assay) 18
(三) 西方墨點法 19
結果 20
一、選殖OIP1、OIP13、OIP18、OIP28及OIP30的全長cDNA 20
二、以酵母菌雙雜交法分析OSCK1、OSCK2、OSCK3和OSCK4的交互作用關係 21
(一) 選擇菌種 21
(二) 質體構築 22
(三) 以營養篩選與序列性稀釋菌液生長勢分析蛋白區塊之間的交互作用 22
三、以酵母菌雙雜交法分析OIPs全長蛋白與OSCK1-F或OSCK1-K的交互作用關係 23
(一) 質體構築 23
(二) 以營養篩選與序列性稀釋菌液生長勢分析蛋白區塊之間的交互作用 24
四、以酵母菌雙雜交法分析OIP30與四種OSCK全長蛋白的交互作用關係 24
五、分析OIP30基因及蛋白於不同組織之表現模式及其與OSCK1的結合關係 25
(一) 製備可專一性辨識OIP30蛋白的抗體 25
(二) 檢測OIP30基因及OIP30蛋白在水稻不同生長時期組織的表現量 26
(三) 以西方墨點法分析水稻中OSCK1蛋白的多寡是否影響OIP30蛋白的含量 27
(四) 利用蛋白下拉實驗(pull-down assay)分析水稻中OSCK1 與OIP30之交互作用 27
討論 29
一、探討水稻花粉中四種OSCKs之間是否能形成蛋白複合體 29
二、OIP1和OIP13全長蛋白均無法與OSCK1產生交互作用 30
三、OIP30與四種OSCKs蛋白均具有交互作用 31
四、OIP30蛋白特性之探討 31
五、在植物體中OIP30是否為OSCK1的結合蛋白 34
參考文獻 36
附錄 61
圖表目錄

表一、利用酵母菌雙雜交法中的營養篩選培養基,配合序列性稀釋菌液之生長勢,分析OSCK1、OSCK2、OSCK3和OSCK4的交互作用關係 40
表二、利用酵母菌雙雜交法中的營養篩選方式與序列性稀釋菌液生長勢,分析OIP1F、OP13F及OIP30與OSCK1-F或OSCK1-K之交互作用關係 41
表三、利用酵母菌雙雜交法中的營養篩選方式與序列性稀釋菌液生長勢,分析OIP30與四種OSCKs全長蛋白的交互作用關係 42
表四、比較OSCKs蛋白間的氨基酸序列相同度(identity)及相似度(similarity) 43
圖一、選殖OIP1 (A)及OIP30 (B)全長cDNA 44
圖二、比較由酵母菌雙雜交法所選殖出的OIPs蛋白與OIPs全長蛋白之間的差異 45
圖三、酵母菌雙雜交法(yeast two-hybrid)的原理、菌種中可用的報導系統及載體 46
圖四、以序列性稀釋菌液分析生長勢或直接塗展菌體方式,分析四種OSCKs 蛋白間之交互作用關係 47
圖五、以序列稀釋菌液生長勢分析OIP1F、OP13F及OIP30與OSCK1-F或OSCK1-K之交互作用關係 50
圖六、以序列稀釋菌液生長勢,分析OIP30與四種OSCKs全長蛋白之交互作用關係 51
圖七、以Ni-NTA親和性管柱層析純化Nus-OIP30蛋白作為抗原 52
圖八、Anti-Nus-OIP30抗體效價之鑑定 53
圖九、以RT-PCR檢查OIP30基因在水稻不同生長時期組織之表現量 54
圖十、檢測OIP30蛋白在水稻不同生長時期組織的含量 55
圖十一、以西方墨點法分析大量表現OSCK1蛋白的轉殖株及OSCK1靜默轉殖株之小穗花中OIP30蛋白的含量 56
圖十二、蛋白下拉實驗分析水稻小穗花樣品中OSCK1 與OIP30是否可形成蛋白複合體 57
圖十三、水稻的OIP30與其他物種的DNA helicases氨基酸序列的比較 58
圖十四、演化樹 60
李鍾財 (2000)。水稻花粉成熟期專一性表現基因OSCK1之選殖與分析。碩士論文。台中:國立中興大學農業生物科技學研究所 (現為生物科技學研究所)。
陳婉潔 (2003)。水稻花粉結鈣激活酵素OSCK1之基因表現、蛋白胞內分佈位置與基因轉殖植物分析。碩士論文。台中:國立中興大學生物科技學研究所。
汪承偉 (2003)。利用酵母菌雙雜交法篩選可與OSCK1結合的水稻花粉蛋白。碩士論文。台中:國立中興大學生物科技學研究所。
童嬿融 (2004)。第一部份:水稻OSCK基因靜默轉殖植物之分析;第二部份:水稻OIP基因表現模式之分析。碩士論文。台中:國立中興大學生物科技學研究所。
Angeletti, P. C., Walker, D., and Panganiban, A. T. (2002). Small glutamine-richprotein/viral protein U-binding protein is a novel cochaperone that affects heat shock protein 70 activity. Cell Stress Chaperones 7, 258-268.
Asano, T., Tanaka, N., Yang, G., Hayashi, N., and Komatsu, S. (2005). Genome-wide identification of the rice calcium-dependent protein kinase and its closely related kinase gene families: comprehensive analysis of the CDPKs gene family in rice. Plant Cell Physiol 46, 356-366.
Asano, T., Kunieda, N., Omura, Y., Ibe, H., Kawasaki, T., Takano, M., Sato, M., Furuhashi, H., Mujin, T., Takaiwa, F., Wu Cy, C.Y., Tada, Y., Satozawa, T., Sakamoto, M., and Shimada, H. (2002). Rice SPK, a calmodulin-like domain protein kinase, is required for storage product accumulation during seed development: phosphorylation of sucrose synthase is a possible factor. Plant Cell 14, 619-628.
Bachvarov, D. R., and Ivanov, I. G. (1983). Large scale purification of plasmid DNA. Prep Biochem 13, 161-166.
Bankaitis, V.A., Malehorn, D.E., Emr, S.D., and Greene, R. (1989). The Saccharomyces cerevisiae SEC14 gene encodes a cytosolic factor that is required for transport of secretory proteins from the yeast Golgi complex. J Cell Biol 108, 1271-1281.
Bauer, A., Chauvet, S., Huber, O., Usseglio, F., Rothbacher, U., Aragnol, D., Kemler, R., and Pradel, J. (2000). Pontin52 and reptin52 function as antagonistic regulators of beta-catenin signalling activity. Embo J 19, 6121-6130.
Birnboim, H. C., and Doly, J. (1979). A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7, 1513-1523.
Chehab, E.W., Patharkar, O.R., Hegeman, A.D., Taybi, T., and Cushman, J.C. (2004). Autophosphorylation and subcellular localization dynamics of a salt- and water deficit-induced calcium-dependent protein kinase from ice plant. Plant Physiol 135, 1430-1446.
Cheng, S.H., Willmann, M.R., Chen, H.C., and Sheen, J. (2002). Calcium signaling through protein kinases. The Arabidopsis calcium-dependent protein kinase gene family. Plant Physiol 129, 469-485.
Chico, J.M., Raices, M., Tellez-Inon, M.T., and Ulloa, R.M. (2002). A calcium-dependent protein kinase is systemically induced upon wounding in tomato plants. Plant Physiol 128, 256-270.
Ellard-Ivey, M., Hopkins, R.B., White, T.J., and Lomax, T.L. (1999). Cloning, expression and N-terminal myristoylation of CpCPK1, a calcium-dependent protein kinase from zucchini (Cucurbita pepo L.). Plant Mol Biol 39, 199-208.
Estruch, J.J., Kadwell, S., Merlin, E., and Crossland, L. (1994). Cloning and characterization of a maize pollen-specific calcium-dependent calmodulin-independent protein kinase. Proc Natl Acad Sci U S A 91, 8837-8841.
Franklin-Tong, V.E. (1999). Signaling and the modulation of pollen tube growth. Plant Cell 11, 727-738.
Harper, J.F., Breton, G., and Harmon, A. (2004). Decoding Ca(2+) signals through plant protein kinases. Annu Rev Plant Biol 55, 263-288.
Hernandez Sebastia, C., Hardin, S.C., Clouse, S.D., Kieber, J.J., and Huber, S.C. (2004). Identification of a new motif for CDPK phosphorylation in vitro that suggests ACC synthase may be a CDPK substrate. Arch Biochem Biophys 428, 81-91.
Hong, Y., Takano, M., Liu, C.M., Gasch, A., Chye, M.L., and Chua, N.H. (1996). Expression of three members of the calcium-dependent protein kinase gene family in Arabidopsis thaliana. Plant Mol Biol 30, 1259-1275.
Hwang, I., Sze, H., and Harper, J.F. (2000). A calcium-dependent protein kinase can inhibit a calmodulin-stimulated Ca2+ pump (ACA2) located in the endoplasmic reticulum of Arabidopsis. Proc Natl Acad Sci U S A 97, 6224-6229.
Kanemaki, M., Kurokawa, Y., Matsu-ura, T., Makino, Y., Masani, A., Okazaki, K., Morishita, T., and Tamura, T.A. (1999). TIP49b, a new RuvB-like DNA helicase, is included in a complex together with another RuvB-like DNA helicase, TIP49a. J Biol Chem 274, 22437-22444.
Lee, S.S., Cho, H.S., Yoon, G.M., Ahn, J.W., Kim, H.H., and Pai, H.S. (2003). Interaction of NtCDPK1 calcium-dependent protein kinase with NtRpn3 regulatory subunit of the 26S proteasome in Nicotiana tabacum. Plant J 33, 825-840.
Legnani, P., and Bianucci, F. (1983). [The use of the LB medium modified by preparation of Leishmania microcultures on glass slides]. Nuovi Ann Ig Microbiol 34, 491-495.
Lu, S.X., and Hrabak, E.M. (2002). An Arabidopsis calcium-dependent protein kinase is associated with the endoplasmic reticulum. Plant Physiol 128, 1008-1021.
Martin, M.L., and Busconi, L. (2000). Membrane localization of a rice calcium-dependent protein kinase (CDPK) is mediated by myristoylation and palmitoylation. Plant J 24, 429-435.
Ogawa, N., Yabuta, N., Ueno, Y., and Izui, K. (1998). Characterization of a maize Ca(2+)-dependent protein kinase phosphorylating phosphoenolpyruvate carboxylase. Plant Cell Physiol 39, 1010-1019.
Patharkar, O.R., and Cushman, J.C. (2000). A stress-induced calcium-dependent protein kinase from Mesembryanthemum crystallinum phosphorylates a two-component pseudo-response regulator. Plant J 24, 679-691.
Qiu, X.B., Lin, Y.L., Thome, K.C., Pian, P., Schlegel, B.P., Weremowicz, S., Parvin, J.D., and Dutta, A. (1998). An eukaryotic RuvB-like protein (RUVBL1) essential for growth. J Biol Chem 273, 27786-27793.
Romeis, T., Ludwig, A.A., Martin, R., and Jones, J.D. (2001).
Calcium-dependent protein kinases play an essential role in a plant
defence response. Embo J 20, 5556-5567.
Rottbauer, W., Saurin, A.J., Lickert, H., Shen, X., Burns, C.G., Wo, Z.G., Kemler, R., Kingston, R., Wu, C., and Fishman, M. (2002). Reptin and pontin antagonistically regulate heart growth in zebrafish embryos. Cell 111, 661-672.
Sanders, D., Brownlee, C., and Harper, J.F. (1999). Communicating with calcium. Plant Cell 11, 691-706.
Sanders, D., Pelloux, J., Brownlee, C., and Harper, J.F. (2002). Calcium at the crossroads of signaling. Plant Cell 14, 401-417.
Saijo, Y., Hata, S., Kyozuka, J., Shimamoto, K., and Izui, K. (2000). Over-expression of a single Ca2+-dependent protein kinase confers both cold and salt/drought tolerance on rice plants. Plant J 23, 319-327.
Yalovsky, S., Rodr Guez-Concepcion, M., and Gruissem, W. (1999). Lipid modifications of proteins - slipping in and out of membranes. Trends Plant Sci 4, 439-445.
Zhao, Y., Kappes, B., and Franklin, R. M. (1993). Gene structure and expression of an unusual protein kinase from Plasmodium falciparum homologous at its carboxyl terminus with the EF hand calcium-binding proteins. J Biol Chem 268, 4347-4354.
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top
1. 探討hsa-miR-185在Wnt/b-catenin訊息傳遞路徑中所扮演的角色
2. 探討竹嵌紋病毒核酸複製酶之類解旋酵素活性區與外鞘蛋白質交互作用之生物意義
3. 水稻OSCK1及其交互作用蛋白OIP30於調控花粉發育、花粉萌發與花藥開裂之角色探討
4. A型流行性感冒病毒非結構蛋白1A在活細胞分佈情形之研究
5. 以竹嵌紋病毒非複製酵素的蛋白質及菸草甲基轉移酶(NbMTS1)作為釣餌蛋白質利用酵母菌雙雜交法尋找菸草中具交互作用之蛋白質
6. 活動性全身性紅斑狼瘡病患周邊血液單核球類鐸蛋白受體的表現和功能分析
7. 利用菸草暫時性轉殖系統評估共同表現之輔助蛋白對T-DNA傳送效率之影響
8. 鵝小病毒活毒疫苗株與田間強毒株之全長序列比較
9. 以表現輔助蛋白增進植物農桿菌轉殖法的效率與建立帶有qPN11(S)基因座之TNG67近同源品系與其性狀分析
10. A型流行性感冒病毒NS1蛋白在細胞株分佈情形之研究
11. 盲鰻筒長度、重量及餌料變化對漁獲效率之影響
12. 自水稻突變體分析鑑定出一個受體激活酵素為控制葉色性狀的重要顯性基因
13. 利用酵母菌雙雜合系統尋找與第一型蛋白質磷酸水解酶結合的蛋白質
14. 水稻胚發育相關基因OSGEP1、OSGEP2及OSTF1之選殖與特性分析
15. 一、竹嵌紋病毒戴帽酵素中參與SAM結合與催化甲基轉移能力之氨基酸探討。二、建立竹嵌紋病毒於酵母菌中之完整複製系統。
 
系統版面圖檔 系統版面圖檔