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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 李昆達(Kung-Ta Lee) | |
dc.contributor.author | You-Sheng Lu | en |
dc.contributor.author | 盧祐盛 | zh_TW |
dc.date.accessioned | 2021-06-17T07:20:22Z | - |
dc.date.available | 2021-07-11 | |
dc.date.copyright | 2019-07-11 | |
dc.date.issued | 2019 | |
dc.date.submitted | 2019-07-08 | |
dc.identifier.citation | Abarca, D., Madueño, F., Martı́nez-Zapater, J. M., & Salinas, J. (1999). Dimerization of Arabidopsis 14‐3‐3 proteins: structural requirements within the N‐terminal domain and effect of calcium. FEBS Lett, 462, 377-382.
Barbier‐Brygoo, H., Ephritikhine, G., Klämbt, D., Maurel, C., Palme, K., Schell, J., & Guern, J. (1991). Perception of the auxin signal at the plasma membrane of N. tabacum mesophyll protoplasts. Plant J, 1, 83-93. Bekheet, S. A. (2006). A synthetic seed method through encapsulation of in vitro proliferated bulblets of garlic (Allium sativum L.). Arab J Biotech, 9, 415-426. Benvenuto, E., Ancora, G., Spanó, L., & Costantino, P. (1983). Morphogenesis and isoperoxidase characterization in tobacco «hairy root» regenerants. Z Pflanzenphysiol, 110, 239-245. Bettini, P. P., Marvasi, M., Fani, F., Lazzara, L., Cosi, E., Melani, L., & Mauro, M. L. (2016). Agrobacterium rhizogenes rolB gene affects photosynthesis and chlorophyll content in transgenic tomato (Solanum lycopersicum L.) plants. J Plant Physiol, 204, 27-35. Bhansali, S., & Kumar, A. (2014). In vitro production of secondary metabolite using Hairy root culture of Eclipta alba (L.) Hassk. Unique J Pharm Biol Sci, 2, 97-98. Biotech, D. (2006). DUALmembrane kit 3 P01001. Zurich, Switzerland. Bonho mMe, V., Laurain-Mattar, D., & Fliniaux, M. A. (2000). Effects of the rolC gene on hairy root: Induction development and tropane alkaloid production by Atropa belladonna. J Nat Prod, 63, 1249-1252. Brückner, A., Polge, C., Lentze, N., Auerbach, D., & Schlattner, U. (2009). Yeast two-hybrid, a powerful tool for systems biology. Int J Mol Sci, 10, 2763-2788. Bulgakov, V. P., Gorpenchenko, T. Y., Veremeichik, G. N., Shkryl, Y. N., Tchernoded, G. K., Bulgakov, D. V. & Zhuravlev, Y. N. (2012). The rolB gene suppresses reactive oxygen species in transformed plant cells through the sustained activation of antioxidant defense. Plant Physiol, 158, 1371-1381. Bulgakov VP, Khodakovskaya MV, Labetskaya NV, Chernoded GK, Zhuravlev YN (1998) The impact of plant rolC oncogene on ginsenoside production by ginseng hairy root cultures. Phytochemistry 49, 1929–1934. Bulgakov VP (2008) Functions of rol genes in plant secondary metabolism. Biotechnol Adv 26:318–324 Casanova, E., Trillas, M.I., Moysset, L. and Vainstein, A. (2005). Influence of rol genes in flori culture. Biotechnol Adv, 23, 3-29. Chilton M-D, Tepfer A, Petit A, David C, Casse-Delbart F, Tempé J (1982) A. rhizogenes inserts T-DNA into the genomes of the host plant root cells. Nature, 295, 432–434. Chung, H. J., Sehnke, P. C., & Ferl, R. J. (1999). The 14-3-3 proteins: cellular regulators of plant metabolism. Trends Plant Sci, 4, 367-371. Ciau-Uitz R, Miranda-Ham mL, Coello-Coello J, Chi B, Pacheco LM, Loyola-Vargas VM (1994) Indole alkaloid production by transformed and non-transformed root cultures of Catharanthus roseus. In Vitro Cell Dev Biol, 30, 84–88 Delbarre, A., Muller, P., Imhoff, V., Barbier-Brygoo, H., Maurel, C., Leblanc, N., ... & Guern, J. (1994). The rolB gene of A. rhizogenes does not increase the auxin sensitivity of N. tabacum protoplasts by modifying the intracellular auxin concentration. Plant physiol, 105, 563-569. Denison, F. C., Paul, A. L., Zupanska, A. K., & Ferl, R. J. (2011, September). 14-3-3 proteins in plant physiology. In Seminars in cell & developmental biology (Vol. 22, No. 7, pp. 720-727). Academic Press. DePaolis, A., Sabatini, S., De Pascalis, L., Costantino, P., & Capone, I. (1996). A rolB regulatory factor belongs to a new class of single zinc finger plant proteins. Plant J, 10, 215-223. Dilshad, E., Cusido, R. M., Estrada, K. R., Bonfill, M., & Mirza, B. (2015). Genetic transformation of Artemisia carvifolia Buch with rol genes enhances artemisinin accumulation. PLoS One, 10, e0140266. Doran, P. M. (2009). Application of plant tissue cultures in phytoremediation research: incentives and limitations. Biotechnol Bioeng, 103, 60-76. Estruch, J. J., Parets-Soler, A., Schmülling, T., & Spena, A. (1991a). Cytosolic localization in transgenic plants of the rolC peptide from A. rhizogenes. Plant Mol Biol, 17, 547-550. Estruch, J. J., Schell, J., & Spena, A. (1991b). The protein encoded by the rolB plant oncogene hydrolyses indole glucosides. EMBO J, 10, 3125-3128. Filippini F, Rossi V, Marin O, Trovato M, Costantino P, Downey PM, Lo Schiavo F, Terzi M (1996). A plant oncogene as a phosphatase. Nature, 8, 499–500. Filippini, F., Schiavo, F. L., Terzi, M., Costantino, P., & Trovato, M. (1994). The plant oncogene rolB alters binding of auxin to plant cell membranes. Plant Cell Physiol, 35, 767-771. Flores, H. E., Vivanco, J. M., and Loyola-Vargas, V. M. 1999. Radicle biochemistry: the biology of root-specific metabolism. Trends Plant Sci, 4, 220–226. Fu, H., Subramanian, R. R., & Masters, S. C. (2000). 14-3-3 proteins: structure, function, and regulation. Annu Rev Pharmacol, 40, 617-647. Georgiev, M. I., Ludwig-Muller, J., & Bley, T. (2010). Hairy root culture: copying nature in new bioprocesses. Med Plant Biotechnol, 156-175. Hey, D., Rothbart, M., Herbst, J., Wang, P., Müller, J., Wittmann, D., ... & Grimm, B. (2017). LIL3, a light-harvesting complex protein, links terpenoid and tetrapyrrole biosynthesis in Arabidopsis thaliana. Plant Physiol, 174, 1037-1050. Ho, C. H., Lin, S. H., Hu, H. C., & Tsay, Y. F. (2009). CHL1 functions as a nitrate sensor in plants. Cell, 138, 1184-1194. Hooper, C. M., Castleden, I. R., Tanz, S. K., Aryamanesh, N., & Millar, A. H. (2016). SUBA4: the interactive data analysis centre for Arabidopsis subcellular protein locations. Nucleic Acids Res, 45, D1064-D1074. Jansson, S. (1994). The light-harvesting chlorophyll ab-binding proteins. Biochim Biophys Acta Bioenerg, 1184, 1-19. Kaneyoshi, J. and Kobayashi, S. (1999). Characteristics of transgenic trifoliate orange (Poncirus trifoliata) possessing the rolC gene of Agrobacterium rhizogenes Ri plasmid. J Jpn Soc Hortic Sci 68, 734-738 Kiselev KV, Dubrovina AS, Veselova MV, Bulgakov VP, Fedoreyev SA, Zhuravlev YN (2007) The rolB gene-induced overproduction of resveratrol in V. amurensis transformed cells. J Biotechnol 128, 681–692. doi:10.1016/j.jbiotec.2006.11.008 Koshita, Y., Nakamura, Y., Kobayashi, S., & Morinaga, K. (2002). Introduction of the rolC gene into the genome of the Japanese persi mMon causes dwarfism. J Jpn Soc Hortic Sci, 71, 529-531. Kumari, S., Bayaa, B., Makkouk, K., El-Ahmed, A., El-Heneidy, A., Jamal, M., ... & Choueiri, E. (2009). 10 th Arab Congress of Plant Protection. Lin, T. C. (2015). 利用雙分子螢光互補法研究與 RolB 有交互作用之蛋白. 臺灣大學生化科技學系學位論文, 1-46. Lin, Y. C., Li, W., Chen, H., Li, Q., Sun, Y. H., Shi, R., ... & Qu, G. Z. (2014). A simple improved-throughput xylem protoplast system for studying wood formation. Nat. Protoc, 9, 2194. Lohscheider, J. N., Rojas‐Stütz, M. C., Rothbart, M., Andersson, U., Funck, D., Mendgen, K., ... & Adamska, I. (2015). Altered levels of LIL 3 isoforms in Arabidopsis lead to disturbed pigment–protein assembly and chlorophyll synthesis, chlorotic phenotype and impaired photosynthetic performance. Plant Cell Environ, 38, 2115-2127. Lorence, A., Medina-Bolivar, F., & Nessler, C. L. (2004). Camptothecin and 10-hydroxycamptothecin from Camptotheca acuminata hairy roots. Plant Cell Rep, 22, 437-441. Matveeva, T. V., Sokornova, S. V., & Lutova, L. A. (2015). Influence of Agrobacterium oncogenes on secondary metabolism of plants. Phytochem Rev, 14, 541-554. Maurel, C., Barbier-Brygoo, H., Spena, A., Tempe, J., & Guern, J. (1991). Single rol genes from the A. rhizogenes TL-DNA alter some of the cellular responses to auxin in N. tabacum. Plant Physiol, 97, 212-216. Maurel, C., Brevet, J., Barbier-Brygoo, H., Guern, J., & Tempé, J. (1990). Auxin regulates the promoter of the root-inducing rolB gene of A. rhizogenes in transgenic N. tabacum. Mol Gen Genet, 223, 58-64 Maurel, C., Leblanc, N., Barbier-Brygoo, H., Perrot-Rechenmann, C., BouvierDurand, M. and Guern, J. (1994). Alterations of auxin perception in rolB-transformed tobacco protoplasts. Time course of rolB mRNA expression and increase in auxin sensitivity reveal multiple control by auxin. Plant Physiol, 105, 1209-1215. Mauro, M. L., Costantino, P., & Bettini, P. P. (2017). The never ending story of rol genes: a century after. Plant Cell Tissue Organ Cult, 131, 201-212. Mauro, M.L., Trovato, M., DePaolis, A., Gallelli, A., Costantino, P. and Altamura, M.M. (1996). The plant oncogene rolD stimulates flowering in transgenic tobacco plants. Dev Biol 180, 693-700. Moritz, T., & Schmülling, T. (1998). The gibberellin content of rolA transgenic N. tabacum plants is specifically altered. J Plant Physiol, 153, 774-776. Moriuchi, H., Okamoto, C., Nishihama, R., Yamashita, I., Machida, Y., & Tanaka, N. (2004). Nuclear localization and interaction of RolB with plant 14‐3‐3 proteins correlates with induction of adventitious roots by the oncogene rolB. Plant J, 38, 260-275. Mork-Jansson, A., Bue, A. K., Gargano, D., Furnes, C., Reisinger, V., Arnold, J., ... & Eichacker, L. A. (2015a). Lil3 assembles with proteins regulating chlorophyll synthesis in barley. PLoS One, 10, e0133145. Mork-Jansson, A. E., & Eichacker, L. A. (2019). A strategy to characterize chlorophyll protein interaction in LIL3. Plant Methods, 15, 1. Mork-Jansson, A. E., Gargano, D., Kmiec, K., Furnes, C., Shevela, D., & Eichacker, L. A. (2015b). Lil3 dimerization and chlorophyll binding in Arabidopsis thaliana. FEBS Lett, 589, 3064-3070. Nilsson, O., Crozier A, Schmülling T, Sandberg G, Olsson O (1993a) Indole-3-acetic acid homeostasis in transgenic N. tabacum plants expressing the A. rhizogenes rolB gene. Plant J, 3, 681–689. Nilsson, O., Moritz, T., Imbault, N., Sandberg, G., & Olsson, O. (1993b). Hormonal characterization of transgenic N. tabacum plants expressing the rolC gene of A. rhizogenes TL-DNA. Plant Physiol, 102, 363-371. Nilsson, O. and Olsson, O. (1997). Getting to the root: the role of the Agrobacterium rhizogenes rol genes in the formation of hairy roots. Physiol Plant, 100, 463-473. Obsil, T., & Obsilova, V. (2011, September). Structural basis of 14-3-3 protein functions. In Seminars in cell & developmental biology (Vol. 22, No. 7, pp. 663-672). Academic Press. Ono, N. N., & Tian, L. (2011). The multiplicity of hairy root cultures: prolific possibilities. Plant Sci, 180, 439-446. Palazon J, Cusido RM, Roig C, Pinol MT (1998a) Expression of the rolC gene and nicotine production in transgenic roots and their regenerated plants. Plant Cell Rep, 17, 384–390 Raskin, I., Ribnicky, D. M., Komarnytsky, S., Ilic, N., Poulev, A., Borisjuk, N. & O'Neal, J. M. (2002). Plants and human health in the twenty-first century. Trends Biotechnol, 20, 522-531. Schmülling, T., Schell, J., & Spena, A. (1988). Single genes from A. rhizogenes influence plant development. EMBO J, 7, 2621-2629. Sevón, N., Oksman-Caldentey, K. M., & Hiltunen, R. (1995). Efficient plant regeneration from hairy root-derived protoplasts of Hyoscyamus muticus. Plant Cell Rep, 14, 738-742. Shkryl, Y. N., Veremeichik, G. N., Bulgakov, V. P., Tchernoded, G. K., Mischenko, N. P., Fedoreyev, S. A., & Zhuravlev, Y. N. (2008). Individual and combined effects of the rolA, B, and C genes on anthraquinone production in Rubia cordifolia transformed calli. Biotechnol. Bioeng, 100, 118-125. Slightom, J.L., Durandtardif, M., Jouanin, L. and Tepfer, D. (1986). Nucleotide sequence analysis of TL-DNA of Agrobacterium rhizogenes agropine type plasmid. Identification of open reading Frames. J Biol Chem, 261, 108-121. Snider, J., Kittanakom, S., Curak, J., & Stagljar, I. (2010). Split-ubiquitin based membrane yeast two-hybrid (MYTH) system: a powerful tool for identifying protein-protein interactions. JoVE (Journal of Visualized Experiments), 36, e1698. Spena, A., Schmülling, T., Koncz, C. and Schell, J.S. (1987). Independent and synergistic activity of rol A, B and C loci in stimulating abnormal growth in plants. EMBO J, 6, 3891-3899. Srivastava, S., & Srivastava, A. K. (2007). Hairy root culture for mass-production of high-value secondary metabolites. Crit Rev Biotechnol, 27, 29-43. Suza, W., Harris, R. S., & Lorence, A. (2008). Hairy roots: from high-value metabolite production to phytoremediation. Electron J Integr Biosci, 3, 57-65. Takahashi, K., Takabayashi, A., Tanaka, A., & Tanaka, R. (2014). Functional analysis of light-harvesting-like protein 3 (LIL3) and its light-harvesting chlorophyll-binding motif in Arabidopsis. J Biol Chem, 289, 987-999. Tanaka, R., Rothbart, M., Oka, S., Takabayashi, A., Takahashi, K., Shibata, M., ... & Tanaka, A. (2010). LIL3, a light-harvesting-like protein, plays an essential role in chlorophyll and tocopherol biosynthesis. PROC. NATL. ACAD. SCI. U.S.A., 107, 16721-16725. Tepfer, D. (1984). Transformation of several species of higher plants by A. rhizogenes : sexual transmission of the transformed genotype and phenotype. Cell, 37, 959-967. Thieme, C. J., Rojas-Triana, M., Stecyk, E., Schudoma, C., Zhang, W., Yang, L., ... & Scheible, W. R. (2015). Endogenous Arabidopsis messenger RNAs transported to distant tissues. Nat Plants, 1, 15025. Tripp, J. D., Lilley, J. L., Wood, W. N., & Lewis, L. K. (2013). Enhancement of plasmid DNA transformation efficiencies in early stationary‐phase yeast cell cultures. Yeast, 30, 191-200. Trovato, M., Maras, B., Linhares, F., & Costantino, P. (2001). The plant oncogene rolD encodes a functional ornithine cyclodeaminase. PROC. NATL. ACAD. SCI. U.S.A., 98, 13449-13453. Van der Salm, T. P., van der Toorn, C. J., Bouwer, R., ten Cate, C. H. H., & Dons, H. J. (1997). Production of ROL gene transformed plants of Rosa hybrida L. and characterization of their rooting ability. Mol Breed, 3, 39-47. Veena, V., & Taylor, C. G. (2007). Agrobacterium rhizogenes: recent developments and promising applications. In Vitro Cell Dev Biol Plant, 43, 383-403. Venis, M. A., Napier, R. M., Barbier-Brygoo, H., Maurel, C., Perrot-Rechenmann, C., & Guern, J. (1992). Antibodies to a peptide from the maize auxin-binding protein have auxin agonist activity. PROC. NATL. ACAD. SCI. U.S.A., 89, 7208-7212. Wang, G. L., Que, F., Xu, Z. S., Wang, F., & Xiong, A. S. (2015). Exogenous gibberellin altered morphology, anatomic and transcriptional regulatory networks of hormones in carrot root and shoot. BMC Plant Biol, 15, 290. Wang, J. H. (2015). 菸草毛狀根的生長與其尼古丁高量累積之研究. 臺灣大學生化科技學系學位論文, 1-162. Wang, J. H., Lin, H. H., Liu, C. T., Lin, T. C., Liu, L. Y. D., & Lee, K. T. (2014). Transcriptomic analysis reveals that reactive oxygen species and genes encoding lipid transfer protein are associated with N. tabacum hairy root growth and branch development. Mol Plant Microbe Interact, 27, 678-687. White, F. F., Taylor, B. H., Huffman, G. A., Gordon, M. P., & Nester, E. W. (1985). Molecular and genetic analysis of the transferred DNA regions of the root-inducing plasmid of Agrobacterium rhizogenes. J Bacteriol, 164, 33-44. Xing, S., Wallmeroth, N., Berendzen, K. W., & Grefen, C. (2016). Techniques for the analysis of protein-protein interactions in vivo. Plant Physiol, 171, 727-758. Yi, S. H. (2014). 菸草毛狀根中 Root Locus B 蛋白質交互作用之研究. 臺灣大學生化科技學系學位論文, 1-71. Yoo, S. D., Cho, Y. H., & Sheen, J. (2007). Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat Protoc. 2, 1565. Yu, S. C., Dawson, A., Henderson, A. C., Lockyer, E. J., Read, E., Sritharan, G., ... & Zhang, R. (2016). Nutrient supplements boost yeast transformation efficiency. Sci Rep, 6, 35738. Zhou, F., Wang, C. Y., Gutensohn, M., Jiang, L., Zhang, P., Zhang, D., ... & Lu, S. (2017). A recruiting protein of geranylgeranyl diphosphate synthase controls metabolic flux toward chlorophyll biosynthesis in rice. PROC. NATL. ACAD. SCI. U.S.A., 114, 6866-6871. Zybailov, B., Rutschow, H., Friso, G., Rudella, A., Emanuelsson, O., Sun, Q., & van Wijk, K. J. (2008). Sorting signals, N-terminal modifications and abundance of the chloroplast proteome. PLoS One, 3, e1994. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/73163 | - |
dc.description.abstract | 毛狀根為植物受根毛農桿菌感染所生成的特化組織,被視為植物次級代謝物生產的潛力平台。農桿菌TL-DNA上之rolB 基因對毛狀根的生長與代謝物之累積均有重大影響,然而rolB 基因在毛狀根生理中之角色仍然未知。為了解RolB是否與宿主之膜蛋白交互作用,本研究以RolB 蛋白質作為分裂泛素酵母菌雙雜交系統的餌蛋白來篩選阿拉伯芥中可能與RolB具交互作用之膜蛋白質。系統建構過程中,控制組之酵母轉形株均建構與測試完成,且已知的RolB與Nt14-3-3 ωII蛋白質之交互作用可被重現並作為蛋白質交互作用之正控制組。此外,RolB 蛋白質可在篩選系統中大量表現,且無強烈的自活化HIS3報導基因現象,篩選的嚴苛度也以HIS3報導基因之抑制劑濃度調整完畢。轉形效率為1.46 x 105 / μg 之質體。總計130 酵母菌轉形株在二次104篩選池大小之篩選後,β-半乳糖苷酶測試呈現陽性反應,且篩選結果顯示二個位於質粒的蛋白質LIL3:1及AT1G44920可能與RolB有交互作用,且此二基因之酵母菌轉形株之β-半乳糖苷酶活性與陽性控制組相當。LIL3:1被報導可促進葉綠素、α-生育酚及植醇之生合成,AT1G44920為一功能未知之穿膜蛋白質。然而RolB於酵母菌中之表現位置需進一步確認,且篩選反應之轉形效率須優化至106/ μg 之質體。為進一步確定此二個位於質粒的蛋白質與RolB蛋白質是否具有交互作用及其作用位置,本研究建構紅花菸草N. tabacum毛狀根之原生質體分離方法,以及LIL3:1及AT1G44920之青色螢光融合蛋白表現載體。多個分離參數包括組織處理、酵素裂解前處理、纖維素酶及離析酶之比例與濃度,以及酵素液培養時間皆進行測試。利用新調整之組織去皮法及2:1之酵素比,原生質體之產率達2.02 * 105 / 每克之毛狀根根尖。 | zh_TW |
dc.description.abstract | Hairy root, a syndrome induced by Agrobacterium rhizogenes, can be a potential platform for phytochemical production. rolB on Agrobacterium rhizogenes TL-DNA is crucial for hairy root growth and phytochemical accumulation. However, how rolB affect hairy root physiology remains elusive. In this study, a split-ubiquitin based membrane yeast two-hybrid system (MYTH) was constructed to screen the possible interacting proteins of RolB in Arabidopsis thaliana. Yeast co-transformants were constructed, and the known interaction between Nt14-3-3 ωII and RolB is reproducible as a positive control for protein-protein interaction in control test. RolB can express in MYTH abundantly without strong self-activation of HIS3 reporter gene, and the screening stringency was optimized by adjusting the 3-AT concentration. The transformation efficiency reached 1.46 x 105 /μg of plasmid. Total 130 yeast transformants were found by β-galactosidase assay after two 104-scale screening reactions. Two plastid-localized protein, LIL3:1 and AT1G44920 were identified, and the β-galactosidase activity of LIL3:1 and AT1G44920 yeast candidates were as higher as positive controls. LIL3:1 has been shown to promote the biosynthesis of chlorophyll, α-tocopherol and phytol, and AT1G44920 is a transmembrane protein with unknown function. However, the RolB expression site in yeast needs verification, and the transformation efficiency should reached 106/μg of plasmid. To examine if RolB can interact with LIL3:1 and AT1G44920 in plant cell, the isolation procedure for Nicotiana tabacum hairy root protoplast were established, and the cyan fluorescent protein (CFP) expression vector for LIL3:1 and AT1G44920 were constructed. Several crucial parameters for isolation including the tissue processing, pre-treatments, enzyme ratio and incubation time have been optimized. The modified peeled method has been developed, and the yield of hairy root protoplast reached 2.02 * 105 / g of hairy root tip. | en |
dc.description.provenance | Made available in DSpace on 2021-06-17T07:20:22Z (GMT). No. of bitstreams: 1 ntu-108-R06b22004-1.pdf: 2350311 bytes, checksum: c0d1dcebc6ffece860b96f4682e370ee (MD5) Previous issue date: 2019 | en |
dc.description.tableofcontents | 誌謝 i
摘要 ii Abstract iii Contents v Contents of Tables and Figures vii Abbreviations viii Introduction 1 Hairy root 1 Root loci (rol) genes 2 rolB 4 The interacting proteins of RolB 5 Objectives 7 Materials and methods 9 Routine work 9 Medium 9 DNA agarose electrophoresis 9 DNA purification 9 DNA quantification 10 Polymerase-chain-reaction (PCR) 10 Restriction enzyme digestion 10 Escherichia coli 11 Escherichia coli transformation 11 Escherichia coli plasmid isolation 11 Plasmid preparation for protoplast transformation 12 Endotoxin removement from E. coli plasmid 13 Yeast 13 Yeast routine transformation 13 Yeast strain construction 14 System construction 14 Yeast protein extraction for bait protein expression test 14 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot 15 Bait protein self-activation test 15 3-AT concentration adjustment 16 β-galactosidase assay of control yeast co-transformants 16 Transformation efficiency estimation 16 Screening and positive candidate confirming 17 A. thaliana cDNA library Screening 17 Confirm positive candidates by β-galactosidase X-gal assay 18 Quantification of β-galactosidase activity by Ortho-Nitrophenyl-β-galactoside 18 Hairy root protoplast 19 Establishment of N. tabacum hairy root protoplast isolation 19 Clone LIL3:1 and AT1G44920 to pEarleyGate102 expression vector 20 Results 22 Eight yeast co-transformants CI, CG, EI, EG, BI, BG, B-Nx, B-xN were successfully generated. 22 β-galactosidase assay of control yeast transformants demonstrated that control sets of screening system worked correctly. 22 RolB can be expressed in S. cerevisiae DSY1 abundantly 23 RolB protein did not self-activate under in BG transformants and background colonies can be inhibited by 1 mM 3-AT 24 Appropriate 3-AT concentration for screening is 10 mM and the yeast transformation efficiency is 1.46 * 105 / μg of plasmid. 24 Two plastid-localized protein, LIL3:1 and AT1G44920 are found in screening. 25 N. tabacum hairy root protoplast isolation procedure is established and the yield is 2.02 * 105 cells per gram of root tip. 26 pEarleyGate102-LIL3:1 and pEarleyGate102-AT1G44920 vectors for CFP transient expression in hairy root protoplast are constructed. 26 Discussion 28 System construction 28 Screening results 32 LIL3:1 and AT1G44920 33 Establishment of N. tabacum hairy root protoplast isolation procedure 36 Conclusion 40 References 68 Appendix 77 Appendix 1. Overview of the mechanism of MYTH. 77 Table 1. Medium used in this study 42 Table 2. Stock composition for SD medium 43 Table 3. PCR conditions. 45 Table 4. Primers used in this study. 46 Table 5. Purposes of yeast transformants generated in this study 47 Table 6. Sequencing results of positive candidates 48 Table 7. Results of ONPG assay of LIL3:1 and AT1G44920 49 Table 8. Yield of A. thaliana mesophyll protoplast under different isolation conditions 50 Table 9. Yield of N. tabacum hairy root protoplast under different isolation conditions 51 Table 10. The screening parameters of two screening reaction 53 Table 11. Genes on gibberellin, carotenoid, chlorophyll biosynthesis pathway which are down-regulated in N. tabacum hairy root. 54 Figure 1. Establishment of MYTH system of yeast co-transformants construction as shown by colony PCR 56 Figure 2. Establishment of MYTH system of control assay as shown by β-galactosidase assay 57 Figure 3. Establishment of MYTH system of bait protein expression validation as shown by western blot. 58 Figure 4. Establishment of MYTH system of bait protein self-activation test. 59 Figure 5. Establishment of MYTH system of screening stringency adjustment as shown by 3-AT concentration adjustment 60 Figure 6. Establishment of MYTH system of screening pool as shown by transformation efficiency test 60 Figure 7. MYTH system screening of positive interaction yeast transformants confirmation as shown by β-galactosidase assay. 61 Figure 8. MYTH system screening of positive interaction yeast transformants confirmation as shown by Colony PCR. 62 Figure 9. Morphology of N. tabacum hairy root protoplasts…………………...63 Figure 10. Topology of LIL3:1 and AT1G44920 64 Figure 11. LIL3:1 and AT1G44920 CFP expression vector construction as shown by electrophoresis results 65 Figure 12. RolB may affect hairy root physiology through protein-protein interaction with LIL3:1 and cause a lil3:1 mutant-like effect. 67 | |
dc.language.iso | en | |
dc.title | 以分裂泛素酵母菌雙雜交系統篩選與RolB具交互作用之蛋白質 | zh_TW |
dc.title | RolB-interacting protein screening by split-ubiquitin membrane based yeast two-hybrid system | en |
dc.type | Thesis | |
dc.date.schoolyear | 107-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 張英?(Ing-Feng Chang),蔡宜芳(Yi-Fang Tsay),靳宗洛(Tsung-Luo Jinn),楊健志(Chien-Chih Yang) | |
dc.subject.keyword | 毛狀根,根毛農桿菌,rolB,蛋白質交互作用,分裂泛素酵母菌雙雜交系統, | zh_TW |
dc.subject.keyword | hairy root,Agrobacterium rhizogenes,rolB,protein-protein interaction,split-ubiquitn based membrane yeast two-hybrid, | en |
dc.relation.page | 77 | |
dc.identifier.doi | 10.6342/NTU201901286 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2019-07-08 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 生化科技學系 | zh_TW |
顯示於系所單位: | 生化科技學系 |
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