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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 鄭石通(Shih-Tong Jeng) | |
dc.contributor.author | Chun-Chung Wang | en |
dc.contributor.author | 王群中 | zh_TW |
dc.date.accessioned | 2021-06-16T03:37:11Z | - |
dc.date.available | 2015-08-11 | |
dc.date.copyright | 2015-08-11 | |
dc.date.issued | 2015 | |
dc.date.submitted | 2015-05-14 | |
dc.identifier.citation | Aono, M., Kubo, A., Saji, H., Tanaka, K., and Kondo, N. (1993). Enhanced tolerance to photooxidative stress of transgenic Nicotiana tabacum with high chloroplastic
glutathione reductase activity. Plant Cell Physiol. 34: 129-135. Babitha, K.C., Ramu, S., Pruthvi, V., Mahesh, P., Nataraja, K.N., and Udayakumar M. (2013). Co-expression of AtbHLH17 and AtWRKY28 confers resistance to abiotic stress in Arabidopsis. Transgenic Res. 22: 327-341. Bashir, K., Nagasaka, S., Itai, RN., Kobayashi, T., Takahashi, M., Nakanishi, H., Mor, S., and Nishizawa, N.K. (2007). Expression and enzyme activity of glutathione reductase is upregulated by Fe-deficiency in graminaceous plants. Plant Mol. Biol. 65: 277-284. Besseau, S., Li J., and Palva, E.T. (2012). WRKY54 and WRKY70 cooperate as negative regulators of leaf senescence in Arabidopsis thaliana. J. Exp. Bot. 63: 2667-2679. Boatwright, J.L., and Pajerowska-Mukhtar, K. (2013). Salicylic acid: an old hormone up to new tricks. Mol Plant Pathol. 14: 623-634. Burrell, M.M. (1993). Enzymes of Molecular Biology. Methods in Molecular Biology. 16. Cai, M., Qiu, D., Yuan, T., Ding, X., Li, H., L, D., Xu, C., Li, X., and Wang, S. (2008). Identification of novel pathogen-responsive cis-elements and their binding proteins in the promoter of OsWRKY13, a gene regulating rice disease resistance. Plant Cell Environ. 31: 86-96. Campos, P.S., Quartin, V., José Ramalho, C., Nunes, M.A. (2003). Electrolyte leakage and lipid degradation account for cold sensitivity in leaves of Coffea sp. Plants Plant Physiol 160: 283-292. Chung, H.R., Dunkel, I., Heise, F., Linke, C., Krobitsch, S., Ehrenhofer-Murray A.E., Sperling, S.R., and Vingron, M. (2010). The Effect of Micrococcal Nuclease Digestion on Nucleosome Positioning Data. PLoS ONE 5: e15754. Chi, Y., Yang, Y., Zhou, Y., Zhou, J., Fan, B., Yu, J.Q., and Chen, Z. (2013). Protein–Protein Interactions in the Regulation of WRKY Transcription Factors. Mol. Plant. 6: 287-300. Clarke, S.M., Mur, LA., Wood, J.E., and Scott, I.M.(2004). Salicylic acid dependent signaling promotes basal thermotolerance but is not essential for acquired thermotolerance in Arabidopsis thaliana. Plant J. 38: 432-447. Crane-Robinson, C., Myers, FA., Hebbes, T.R., Clayton, A.L., and Thorne, A.W.(1999). Chromatin Immunoprecipitation Assays in Acetylation Mapping of Higher Eukaryotes. Methods Enzymol. 304: 533-547. Dhingra, T., Mittal, K., and Sarma, G.S. (2014). Analytical Techniques for DNA Methylation – An Overview. CURR PHARM ANAL. 10: 71-85. Dong, J., Chen, C., and Chen, Z. (2003). Expression profiles of the ArabidopsisWRKY gene superfamily during plant defense response. Plant Mol. Biol. 51: 21-37. Downs, CA., and Heckathorn, S.A. (1998). The mitochondrial small heat-shock protein protects NADH:ubiquinone oxidoreductase of the electron transport chain during heat stress in plants. FEBS Lett. 3: 246-250. Duan, G., Li, L., and Liu, Q. (2014). A WRKY transcription factor from Malus domestica negatively regulates dehydration stress in transgenic Arabidopsis Acta Physiol Plant. 36: 541-548. Dufourc, E.J. (2008). The role of phytosterols in plant adaptation to temperature. Plant Signal Behav. 2: 133-134. Dure, L. , Crouch, M., Harada, J., Ho, T.H.D., Mundy, J., Quatrano, R., Thomas, T. and Sung, Z. R. (1989). Common amino acid sequence domains among the LEA proteins of higher plants. Plant Mol. Biol. 12: 475-486. Foyer, C.H., Descourvieres, P., and Kunert, K.J. (1994). Protection against oxygen radicals: an important defence mechanism studied in transgenic plants. Plant Cell Environ. 17: 507-523. Garg, N., and Manchanda, G. (2009). ROS generation in plants: boon or bane? Plant Biosys, 143: 8-96. Gendrel, A.V., Lippman, Z., Martienssen, R., and Colot, V. (2005). Profiling histone modification patterns in plants using genomic tiling microarrays. Nat. Methods 2: 213-218. Giacomelli, J.I., Weigel, D., Chan, R.L., and Manavella, P.A. (2012). Role of recently evolved miRNA regulation of sunflower HaWRKY6. New Phytol. 195: 766-773. Gill, S.S., and Tuteja, N. (2010) . Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiol. Biochem. 48: 909-930. Grunwald, C. (1975).PLANT STEROLS. Plant Physiol. 26:209-36. Kim, KC., Lai, Z., Fan, B., and Chen, Z. (2008). rabidopsis WRKY38 and WRKY62 transcription factors interact with histone deacetylase 19 in basal defense. Plant Cell. 20: 2357-2371. Kimura, M., Yamamoto, Y.Y., Seki, M., Sakurai, T., Abe, T., Yoshida, S., Manabe, K., Shinozaki, K., and Matsui, M. (2003). Identification of Arabidopsis genes regulated by high light-stress using cDNA microarray. Photochem. Photobiol. 77: 226–233. Kiyosue, T., Yamaguchi-Shinozaki, K. and Shinozaki, K.(1994) loning of cDNAs for genes that are early-responsive to dehydration stress (ERDs) in Arabidopsis thaliana L.: identification of three ERDs as HSP cognate genes. Plant Mol Biol. 25: 791-798. Kornyeyev, D., Logan, B.A., Payton, P., Allen, R.D., and Holaday, A.S. (2003). Elevated chloroplastic glutathione reductase activities decrease chilling-induced photoinhibition by increasing rates of photochemistry, but not thermal energy dissipation, in transgenic cotton. Funct. Plant Biol. 30: 101-110. Luo, M., Dennis, E.S., Berger, F., Peacock, W.J., and Chaudhury, A. (2005). MINISEED3 (MINI3), a WRKY family gene, and HAIKU2 (IKU2), a leucine-rich repeat (LRR) KINASE gene, are regulators of seed size in Arabidopsis. Proc Natl Acad Sci U S A. 102: 17531-17536. Hinderhofer, K., and Zentgraf, U. (2001). Identification of a transcription factor specifically expressed at the onset of leaf senescence. Planta 213: 469-473. Howarth, C.J. (2005). Genetic improvements of tolerance to high temperature. In: Ashraf, M., Harris, P.J.C. (Eds.), Abiotic Stresses: Plant Resistance Through Breeding and Molecular Approaches. Howarth Press Inc., New York. Hu, Y., Chen, L., Wang, H., Zhang, L., Wang, F., and Yu, D. (2013). Arabidopsis transcription factor WRKY8 functions antagonistically with its interacting partner VQ9 to modulate salinity stress tolerance. Plant J. 74: 730-745. Hu, Y., Dong, Q., and Yu, D. (2012). Arabidopsis WRKY46 coordinates with WRKY70 and WRKY53 in basal resistance against pathogen Pseudomonas syringae. Plant Sci. 185-186: 288-297. Hundertmark, M., Dimova, R., Lengefeld, J., Seckler, R., and Hincha, D.K. (2011). The intrinsically disordered late embryogenesis abundant protein LEA18 from Arabidopsis thaliana modulates membrane stability through binding and folding. Biochim Biophys Acta 1808: 446-453. Ishihama, N., and Yoshioka, H. (2013) Post-translational regulation of WRKY transcription factors in plant immunity. Curr Opin Plant Biol. 15: 431-417. Jiang, S., Zhou, X., Song, Y., and Yu, D. (2009). Heterologous expression of OsWRKY23 gene enhances pathogen defense and dark-induced leaf senescence in Arabidopsis. Plant Growth Regul. 58: 181-190. Jiang, Y., and Deyholos, M.K. (2009). Functional characterization of Arabidopsis NaCl-inducible WRKY25 and WRKY33 transcription factors in abiotic stresses. Plant Mol. Biol. 69: 91-105. Jiang, Y., Liang, G., Yang, S., and Yu, D. (2012). Activated expression of WRKY57 confers drought tolerance in Arabidopsis. Mol. Plant 5: 1375-88. Jiang, Y., Liang, G., Yang, S., and Yu, D. (2014). ArabidopsisWRKY57 Functions as a Node of Convergence for Jasmonic Acid– and Auxin-Mediated Signaling in Jasmonic Acid–Induced Leaf Senescence. Plant Cell. 26: 230-245. Journot-Catalino, N., Somssich, I.E., Roby, D., and Kroj, T. (2006). The transcription factors WRKY11 and WRKY17 act as negative regulators of basal resistance in Arabidopsis thaliana. Plant Cell. 18: 3289-3302. Kalde, M., Barth, M., Somssich, I.E., and Lippok, B. (2003). Members of the Arabidopsis WRKY group III transcription factors are part of different plant defense signaling pathways. Mol. Plant Microbe Interact. 16: 295-305. Knoth, C., Ringler, J., Dangl, J.L. and Eulgem, T. (2007). Arabidopsis WRKY70 is required for full RPP4-mediated disease resistance and basal defense against Hyaloperonospora parasitica. Mol. Plant Microbe Interact. 20: 120-128. Li, J., Besseau, S., Törönen, P., Sipari, N., Kollist, H., Holm, L. and Palva, ET. (2013). Defense-related transcription factors WRKY70 and WRKY54 modulate osmotic stress tolerance by regulating stomatal aperture in Arabidopsis. New Phytol. 200: 455-472. Li, X., Xia, T., Huang, J., Guo, K., Liu, X., Chen, T., Xu, W., Wang, X., Feng, S., and Peng, L. (2014). Distinct biochemical activities and heat shock responses of two UDP-glucose sterol glucosyltransferases in cotton. Plant Sci. 8: 219-220. Klickstein, L.B., and Neve, R.L. (1991). Ligation of Linkers or Adapters to Double‐Stranded cDNA. Current Protocols in Molecular Biology 5.6.1-5.6.10. Long, S.P., and Ort, D.R. (2010). More than taking the heat: crops and global change. Curr. Opin. Plant Biol. 13: 241-248. Löw, D., Brändle, K., Nover, L., and Forreiter, C. (2000). Cytosolic heat-stress proteins Hsp17.7 class I and Hsp17. 3 class II of tomato act as molecular chaperones in vivo. Planta. 4: 575-582. Liu, X., and Huang, B. (2000). Heat stress injury in relation to membrane lipid peroxidation in creeping bent grass. Crop Sci. 40: 503-510. Lv, W.T., Lin, B., Zhang, M., and Hua, X.J. (2011). Proline Accumulation Is Inhibitory to Arabidopsis Seedlings during Heat Stress. Plant Physiol. 156: 1921-193. Marè, C., Mazzucotelli, E., Crosatti, C., Francia, E., Stanca, A.M., and Cattivelli, L. (2004). Hv-WRKY38: a new transcription factor involved in cold- and drought-response in barley. Plant Mol. Biol. 55: 399-416. Mao, P., Duan, M., Wei, C., and Li, Y. (2007). WRKY62 transcription factor acts downstream of cytosolic NPR1 and negatively regulates jasmonate-responsive gene expression. Plant Cell Physiol. 48: 833-842. Matsushita, A., Inoue, H., Goto, S., Nakayama, A., Sugano, S., Hayashi, N and Takatsuji, H. (2013). Nuclear ubiquitin proteasome degradation affects WRKY45 function in the rice defense program. Plant J. 73: 302-313. Mishra, M.K., Chaturvedi, P., Singh, R., Singh, G., Sharma, L.K., Pandey, V., Kumari, N., and Misra, P. (2013). Overexpression of WsSGTL1 Gene of Withania somnifera Enhances Salt Tolerance, Heat Tolerance and Cold Acclimation Ability in Transgenic Arabidopsis Plants. PLoS One. 8: e63064. Miao, Y., Laun, T.M., Smykowski, A., and Zentgraf, U. (2007). Arabidopsis MEKK1 can take a short cut: it can directly interact with senescence-related WRKY53 transcription factor on the protein level and can bind to its promoter. Plant Mol. Biol. 65: 63-76. Miao, Y., Laun, T., Zimmermann, P., and Zentgraf, U. (2004). Targets of the WRKY53 transcription factor and its role during leaf senescence in Arabidopsis. Plant Mol. Biol. 55: 853-867. Mukhopadhyay, A., Deplancke, B., Walhout, A.J., and Tissenbaum, H.A. (2008). Chromatin immunoprecipitation (ChIP) coupled to detection by quantitative real-time PCR to study transcription factor binding to DNA in Caenorhabditis elegans. Nat. Protoc. 4: 698-709. Nakayama, A., Fukushima, S., Goto, S., Matsushita, A., Shimono, M., Sugano, S., Jiang, CJ., Akagi, A., Yamazaki, M., Inoue, H., and Takatsuji, H. (2013). Genome-wide identification of WRKY45-regulated genes that mediate benzothiadiazole-induced defense responses in rice. BMC Plant Biol. 13: 150. Orlando, V., Strutt, H., and Paro, R. (1997). Analysis of Chromatin Structure by in Vivo Formaldehyde Cross-Linking. Methods. 11: 205-214. Pandey, S.P., and Somssich, I.E. (2009). The role of WRKY transcription factors in plant immunity. Plant Physiol. 150: 1648-1655. Puhakainen, T., Hess, M.W., Mäkelä, P., Svensson, J. and Heino, P. (2004). Overexpression of multiple dehydrin genes enhances tolerance to freezing stress in Arabidopsis. Plant Mol. Biol. 54: 743-753. Roccaro, M., and Somssich, I.E. (2011). Chromatin Immunoprecipitation to Identify Global Targets of WRKY Transcription Factor Family Members Involved in Plant Immunity. Methods Mol. Biol. 712: 45-58. Rushton, P.J., Macdonald, H., Huttly, A.K., Lazarus, CM., and Hooley, R. (1995). Members of a new family of DNA-binding proteins bind to a conserved cis-element in the promoters of alpha-Amy2 genes. Plant Mol. Biol. 29: 691-702. Rushton, P.J., Somssich, IE., Ringler, P., and Shen, Q.J. (2010). WRKY transcription factors. Trends Plant Sci. 15: 247-258. Savchenko, G.E., Klyuchareva, E.A., Abrabchik, L.M., and Serdyuchenko, E.V. (2002). Effect of periodic heat shock on the membrane system of etioplasts. Russ. J. Plant Physiol. 49: 349-359. Schぴoffl, F., Prandl, R., and Reindl, A. (1999). Molecular responses to heat stress. In: Shinozaki, K., Yamaguchi-Shinozaki, K. (Eds.), Molecular Responses to Cold, Drought, Heat and Salt Stress in Higher Plants. R.G. Landes Co., Austin, Texas, pp. 81-98. Shang, Y., Yan, L., Liu, Z.Q., Cao, Z., Mei, C., Xin, Q., Wu, FQ., Wang, XF., Du, SY., Jiang, T., Zhang, XF., Zhao, R., Sun, H.L., Liu, R,. Yu, Y.T., and Zhang, D.P. (2010). The Mg-Chelatase H Subunit of Arabidopsis Antagonizes a Group of WRKY Transcription Repressors to Relieve ABA-Responsive Genes of Inhibition. Plant Cell. 22: 1909-1935. Shim, J.S., Jung, C., Lee, S., Min, K., Lee, Y.W., Choi, Y., Lee, J.S., Song, J.T., Kim, J.K., and Choi, Y.D. (2013). AtMYB44 regulates WRKY70 expression and modulates antagonistic interaction between salicylic acid and jasmonic acid signaling. Plant J. 73: 483-495. Stein, A., and Bina, M. (1984). Model Chromatin Assembly System. J. Mol. Biol. 176: 341-363. Sun, J., Jiang, H., Xu, Y., Li, H., Wu, X., Xie, Q., and Li, C. 2007). The CCCH-Type Zinc Finger Proteins AtSZF1 and AtSZF2 Regulate Salt Stress Responses in Arabidopsis Plant Cell Physiol. 48: 1148-1158. Sweeney, P.J., and Walker, J.M. (1993). Proteinase K (EC 3.4.21.14). Methods in Molecular Biology. 16: 305-311. Taji, T., Seki, M., Yamaguchi-Shinozaki, K., Kamada, H., Giraudat, J. and Shinozaki, K. (1999). Mapping of 25 Drought-Inducible Genes, RD and ERD, in Arabidopsis thaliana. Plant Cell Physiol. 40: 119-123. Turck, F., Zhou, A., and Somssich, I.E. (2004). Stimulus-Dependent, Promoter-Specific Binding of Transcription Factor WRKY1 to Its Native Promoter and the Defense-Related Gene PcPR1-1 in Parsley. Plant Cell 16: 2573-2585. Valerio, Orlando. (2000). Mapping chromosomal proteins in vivo by formaldehydecrosslinked- chromatin Immunoprecipitation. Trends Biochem. Sci. 25: 99-104. Wahid, A., Gelani, S., Ashraf, M., and Foolad, M.R. (2007). Heat tolerance in plants: An overview. Environ. Exp. Bot. 61: 199-223. Wang, L., and Li, S. (2006).Salicylic acid-induced heat or cold tolerance in relation to Ca2+ homeostasis and antioxidant systems in young grape plants. Plant Sci. 170: 685-694. Wang, X., and Culver, J.N. (2012). DNA binding specificity of ATAF2, a NAC domain transcription factor targeted for degradation by Tobacco mosaic virus. Plant Biology 12: 157. Weintraub, H., and Groudine, M. (1976). Chromosomal subunits in active genes have an altered conformation. Sci. 193: 848-856. Xu, S., Li, J., Zhang, X., Wei, H., and Cui, L. (2006). Effects of heat acclimation pretreatment on changes of membrane lipid peroxidation, antioxidant metabolites, and ultrastructure of chloroplasts in two cool-season turfgrass species under heat stress. Environ. Exp. Bot. 56: 274-285. Yamaguchi-Shinozaki, K. and Shinozaki, K.(1993).Arabidopsis DNA Encoding Two Desiccation-Responsive rd29 Genes Plant Physiol. 101: 1119-1120. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/54716 | - |
dc.description.abstract | WRKY蛋白質為植物所具有的一個轉錄因子家族,其對於生物性逆境的功能廣為人知。而近期的研究發現,WRKY家族也在植物面對非生物逆境的基因調控上扮演者重要的角色。阿拉伯芥中WRKY家族的兩名成員:AtWRKY54和AtWRKY70已經被證實參與了許多植物的抗菌以及植物葉片老化的相關基因調控,並可能參與植物耐熱反應。在本次的研究中,嘗試利用protein-DNA pull down assay找出AtWRKY54和AtWRKY70所辨認的基因上游啟動子序列,以推測其所調控的基因。並希望藉由染色質免疫沉澱法(Chromatin immunoprecipitation)確認AtWRKY54和AtWRKY70與此基因的關係。在利用大腸桿菌大量表現AtWRKY54或AtWRKY70時,蛋白質表現會出現在包涵體(inclusion body),並在經過不同溫度、不同誘導時間、以及降低誘導物的濃度以表現AtWRKY54和AtWRKY70時,包涵體的情況並沒有因此而降低,以至於無法使用protein-DNA pull down assay尋找AtWRKY54和AtWRKY70所調控的基因。而在利用染色質免疫沉澱法進行釣取,並測試多樣實驗的步驟,也無法獲得下游所調控的基因。在前人研究上,已發現多個基因會受到AtWRKY54和AtWRKY70所以影響,並利用PLACE網站分析這些基因是否含有類似W-box的序列,其中有21個基因具有W-box, 並利用即時定量聚合酶連鎖反應,調查植物AtWRKY54大量表現株、AtWRKY70大量表現株、wrky54突變株和wrky70突變株中的這些基因表現,結果發現,wrky54突變株和wrky70突變株裡面的五個基因COR47(Cold regulated gene 47)、ERD7(Early-response to dehydration 7)、LEA14(late-embryogenesis abundant protein)、LTI78(Low-temperature-induce 78)和RD2( dessication responsive protein 2)的RNA表現量明顯低於野生型植物珠(Wild Type)。這些基因都不會被熱所誘導,但是已被報導會被諸如光逆境、鹽逆境、滲透壓逆境、乾旱逆境和冷逆境等各種不同的非生物性逆境所誘導,其中的COR47(Cold regulated gene 47)或和RAB18(Ras-related protein Rab-18)共同大量表現的話會使植物有抗寒的作用。這可能意味著AtWRKY54和AtWRKY70雖然在熱的情況會被抑制,但在一些非熱的非生物逆境上扮演者一定程度的角色。 | zh_TW |
dc.description.abstract | WRKY family, a transcription factor family in plant, is well-known for its regulation in biotic stresses. In recent studies, genes in WRKY family also play roles in abiotic stresses. WRKY54 and WRKY70, two members of WRKY family, are involved in the regulation of various processes including plant defense and senescence, and may also participate in plant thermotolerance. In this study, protein-DNA pull down assays were used to find the promoter sequences that are recognized by WRKY54 or WRKY70 protein and to speculate what genes would directly be regulated by WRKY54 or WRKY70. Chromatin immunoprecipitation was designed to confirm this speculation. Expression of AtWRKY54 or AtWRKY70 by transforming E.coli produces inclusion body, and no detectable AtWRKY54 and AtWRKY70 was found in the soluable phase for further study. This problem obstructs the using of protein-DNA pull down assay. In addition, chromatin immunoprecipitation in several ways was performed to find genes regulated by WRKY54 or WRKY70 without predicted primers, but it fails.
In recent study, wrky54wrky70 double mutant would change several gene expression under osmotic treatment. Analysis promoters of these genes by PLACE to find if promoters of these genes contain W-Box-liked sequence. Six genes with W-Box were analyzed by RT-qPCR in WRKY54 overexpression line, WRKY70 overexpression line, wrky54 T-DNA insertion line and wrky70 T-DNA insertion line. As the result, the gene expression of these five genes, COR47(cold regulated gene 47)、ERD7(early-response to dehydration 7)、LEA14(late-embryogenesis abundant protein)、LTI78(low-temperature-induce 78) and RD2( dessication responsive protein 2), are much lower in wrky54 T-DNA insertion line and wrky70 T-DNA insertion line than wild type. These five genes are not induced by heat but induced by other abiotic stresses including light stress, salt stress, osmotic stress, drought stress and cold stress. It means that WRKY54 and WRKY70 may play roles in several abiotic stresses. | en |
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dc.description.tableofcontents | 謝誌………………………………………………………………………………...……I
目錄……………………………………………………………………………………III 中文摘要……………………………………………………………………………...VII 英文摘要……………………………………………………………………………....IX 前言……………………………………………………………………………………..1 1. 熱逆境……………..………………………………………………………………..1 2. 熱逆境對植物的影響……………..………………………………………………..1 3. 植物對抗熱逆境的機制……………..……………………………………………..1 3.1 熱休克蛋白質的表現……………..…………………………….……………2 3.2 提高細胞膜的穩定度…………………………………………….…………..2 3.3 過氧化物清除…………………….…………………………….…………….3 4. WRKY 轉錄因子……………………………...……………………………………4 4.1 WRKY 轉錄因子的分類………………...……………………………………4 4.2 WRKY 轉錄因子的特性與功能………………...……………………………4 4.3 WRKY 對於生物性逆境的調控………………...……………………………4 4.4 WRKY 對於非生物性逆境的調控……………...……………………………5 4.5 WRKY 對於種子發育與老化過程的調控……...……………………………6 4.6 AtWRKY54 與 AtWRKY70……...………………………...…………………6 材料與方法……………………………………………………………………………..8 1. 材料 1.1 實驗菌種……………………………………………………………………….8 1.2 質體設計……………………………………………………………………….8 1.3 試驗植物……………………………………………………………………….8 2. 方法 2.1 聚合脢連鎖反應 PCR……………………………….………………………...8 2.2 DNA 膠體電泳…………………………..……………………………..............9 2.3 從膠體回收 DNA……………………..………………………………………..9 2.4 限制酶切 DNA…………………..……………………………………………..9 2.5 DNA 黏合………………………………………………………………………10 2.6 DNA 黏合後除去鹽類…………………………………………………………10 2.7 Heat shock 質體轉入法……………………...…………………………………10 2.8 質體 DNA 抽取……………………………...…………………………………10 2.9 電穿孔質體轉入法………………….………………….………………………11 2.10 誘導細菌表現蛋白質…………..….………………….………………………11 2.11 從細菌中萃取蛋白質…………………………………………………………12 2.12 測試是否產生 inclusion body…………………………………………………12 2.13 SDS 膠體製作………………………………………………………………...12 2.14 SDS 電泳……………………………………………………………………..13 2.15 西方墨點分析(Western blot) ………………………………….……………..13 2.16 染色質免疫沉澱法,標準(Chromatin immunoprecipitation, standard)……….13 2.17 染色質免疫沉澱法,無引子(Chromatin immunoprecipitation, no primer) …..15 2.18 RNA 抽取……………………………………………………..………………17 2.19 反轉錄 DNA 置備……………………………………………..…………..….17 2.20 即時定量聚合酶連鎖反應 q-PCR……………………………………………18 結果 1. BL21/pET32a 誘導 AtWRKY54 與 AtWRKY70………………………………….19 2. Chromatin IP 用酵素酶切取代超音波震盪的效果……………………………….20 3. AtWRKY54 與 AtWRKY70 表現量的變化影響的下游基因……………………20 4. ChIP 所抓到的 AtWRKY54 與 AtWRKY70 所調控的基因…………………21 討論 1. 用 BL21/pET32a 表現 AtWRKY54 和 AtWRKY70 會造成 inclusion body。而在 BL21/pET21a 表現則會出現小分子量的誘導物……………………………......22 2. Chromatin IP 用酵素酶切取代超音波震盪………………………………………23 3. AtWRKY54 或 AtWRKY70 的突變會使的 COR47、ERD7、LEA14、LTI78、RD2 的表現量下降。…………………………………..…………………………22 參考資料………………………………………………….……………………………26 圖說.........................................39 圖一 質體構築 pET32a.................39 圖二 AtWRKY54 與 AtWRKY70 在轉質體菌中表現的情形……………………..40 圖三 從誘導過後將 BL21/pET32a/AtWRKY54 和 BL21/pET32a/AtWRKY70 打 碎,通過 Column 後所 elute 出來的蛋白質…………………………………………41 圖四 在低溫處理下 BL21/pET32a/AtWRKY54 和 BL21/pET32a/AtWRKY70 所表現 的蛋白質………………………………………………………………………….……42 圖五 在低濃度 IPTG、低溫處理下 BL21/pET32a/AtWRKY54 和 BL21/pET32a/AtWRKY70 所表現的蛋白質………………………………………...43 圖六 質體構築 pET21a……………………………………………………………….44 圖七 在低溫處理下 BL21/pET21a/WRKY54 和 BL21/pET21a/WRKY70 所表現的蛋 白質…………………………………………………………………………………….45 圖八 轉基因植物,AtWRKY54 大量表現株與 AtWRKY70 大量表現株…………46 圖九 ChIP 中用酵素酶切取代超音破震盪後,將切過的 DNA 跑電泳膠圖後的結 果……………………………………………………………………………………….47 圖十 基因表現量分析………………………………………...………………………48 圖十一 ChIP 後跑 qPCR--COR47、ERD7 和 LEA14……………………………….49 圖十二 ChIP 後跑 qPCR—LEA14、LTI78 和 RD2………………………………….50 表一 引子序列表…………………………...…………………………………………51 附錄 附錄一:用 PLACE 找出的 W-BOX 類似區域………………………………………54 附錄二:SDS 膠體製作配方……………………………….…………………………55 | |
dc.language.iso | zh-TW | |
dc.title | AtWRKY54 和 AtWRKY70 基因表現與調控的研究 | zh_TW |
dc.title | Expression and regulation AtWRKY54 and AtWRKY70 | en |
dc.type | Thesis | |
dc.date.schoolyear | 103-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 林讚標(Tsan-Piao Lin),謝旭亮(Hsu-Liang Hsieh) | |
dc.subject.keyword | AtWRKY54,AtWRKY70,包涵體,染色質免疫沉澱法, | zh_TW |
dc.subject.keyword | AtWRKY54,AtWRKY70,Chromatin immunoprecipitation,inclusion body, | en |
dc.relation.page | 55 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2015-05-15 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 植物科學研究所 | zh_TW |
顯示於系所單位: | 植物科學研究所 |
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