Functional characterization of the ethylene responsive factor BkERF3 from Bupleurum kaoi

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研究生：	邱孟凡 Chiu, Meng-Fan
論文名稱：	Functional characterization of the ethylene responsive factor BkERF3 from Bupleurum kaoi 高氏柴胡乙烯反應子BkERF3之調控與功能分析
指導教授：	林彩雲 Lin, Tsai-Yun
口試委員:	呂平江楊明德楊長賢曾夢蛟林彩雲
學位類別：	碩士 Master
系所名稱：	生命科學暨醫學院 - 生物資訊與結構生物研究所 Institute of Bioinformatics and Structural Biology
論文出版年：	2011
畢業學年度：	99
語文別：	英文
論文頁數：	61
中文關鍵詞：	乙烯反應子、柴胡
相關次數：	點閱：2 下載：0
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高氏柴胡是台灣特有的一種中草藥。本研究從高氏柴胡中選殖一個新的乙烯反應子BkERF3進行功能分析。首先利用反轉錄聚合酵素鏈鎖反應 (RT-PCR)分析高氏柴胡乙烯反應子的mRNA，發現此基因主要表現在根部，而MeJA 和氯化鈉的處理可以誘導它的表現。接著以持續表達高氏柴胡乙烯反應子BkERF3的轉殖阿拉伯芥進行分析，發現在阿拉伯芥中持續表達此基因會誘導活性氧化物清除基因「超氧化物歧化酶」的表現，並增加植物對高鹽及乾旱的耐受性。
本研究也分離出高氏柴胡乙烯反應子BkERF3的啟動子區域以進行活性分析。將不同區域的啟動子片段構築於報導基因β-glucuronidase (GUS)之前，再以暫時性表達和穩定性表達兩種方式來進行組織染色與GUS表現量分析。GUS表現量分析顯示高氏柴胡乙烯反應子BkERF3啟動子的強度大約為花椰菜鑲嵌病毒35S啟動子的一半。另外，藉由分析含不同區域啟動子的轉殖植株，發現－764 到－566這段區域對高氏柴胡乙烯反應子BkERF3的表現扮演重要角色。

A novel ethylene response factor (ERF) gene, BkERF3, was isolated from Bupleurum kaoi which is a medicinal herb endemic to Taiwan. Semi-quantitative RT-PCR analysis revealed that BkERF3 is preferentially expressed in roots and can be induced by methyl jasmonate (MeJA) and NaCl treatments. Overexpression of BkERF3 in transgenic Arabidopsis thaliana resulted in elevated mRNA levels of genes coding for the reactive oxygen species (ROS) scavenger superoxide dismutases (SOD) enzymes and enhanced tolerance to salt and drought.
The promoter of the BkERF3 gene was isolated and characterized in this study. A series of constructs containing different promoter regions fused to the β-glucuronidase (GUS) reporter gene were transiently and stably transformed then subject to histochemical and fluorometric assays. Fluorometric assay showed that the BkERF3 promoter processes about half activity of the CaMV35S promoter. Deletion analysis showed that the －764 to －566 region is important to activate the BkERF3 promoter.

摘要               i
Abstract              ii
謝誌              iii
Table of content    iv
List of Tables    vii
List of Figures    viii
List of Appendixes    ix
Abbreviations    x
Introduction    1
Materials and Methods    7
1.Plant growth and tissue culture condition    7
 □Bupleurum kaoi plants    7
 □Bupleurum kaoi cell suspension culture    7
 □Bupleurum kaoi adventitious root suspension culture7
 □Transgenic Arabidopsis thaliana plants and liquid culture    8
2.Hormonal and stress treatments    8
3.Genomic DNA extraction    9
4.Total RNA extraction    10
5.Total protein extraction    11
6.Semi-quantitative RT-PCR    12
7.Cloning of the BkERF3 promoter and identification of the gene transcription start site    13
8.DNA ligation, plasmid transformation and preparation, and DNA sequencing    13
 □DNA ligation    13
 □Competent cell preparation    14
 □Bacterial transformation    14
 □Mini-preparation of plasmid DNA    15
 □Double-stranded DNA sequencing    15
9.Protein expression in E. coli    16
 □pET28a constructs preparation    16
 □His-tag protein expression and purification    16
10.Western blot analysis    17
11.Construction of the BkERF3 promoter and its deletions fused to the GUS reporter    18
12.Large- scale plasmid DNA preparation    19
13.PEG-mediated protoplast transformation and agrobacteria-mediated transformation    20
 □Preparation of B. kaoi protoplast    20
 □PEG-mediated protoplast transformation    21
 □Plant transformation    22
14.Quantification of RLUC and GUS activity in transformed protoplasts    22
 □Renilla luciferase assay    22
 □Measuring β-glucuronidase (GUS) activity    23
15.Histochemical staining and fluorometric quantification of GUS activity in transgenic plants    23
 □Histochemical staining of GUS acitivity    24
 □Fluorometric analysis of GUS activity    24
Results    26
1.Strong root preference in the expression of the BkERF3 mRNA    26
2.Effects of hormonal treatments and osmotic stresses on BkERF3 transcript levels in B. kaoi adventitious roots    26
3.Molecular analysis of the transgenic Arabidopsis plants overexpressing BkERF3    27
4.Overexpression of BkERF3 in Arabidopsis plants enhanced tolerance to salt and drought stress    28
5.Overexpression of BkERF3 in Arabidopsis upregulated the expression of stress-related genes    28
6.Sequence analysis of the BkERF3 promoter    29
7.Quantitative assays of the BkERF3 promoter using transient transformation in B. kaoi protoplasts    30
8.Molecular analysis of the transgenic Arabidopsis plants    31
9.Expression analyses of Arabidopsis plants carrying the BkERF3 promoter and GUS fusion constructs    31
10.Effects of hormones and abiotic stresses on transgenic Arabidopsis plants    32
11.Deletion analysis of the BkERF3 promoter in transgenic Arabidopsis plants    32
Discussion    34
References    38
Tables    45
Figures    48
Appendixes    60

                                

Berrocal-Lobo M., Molina A., Solano R. 2002. Constitutive expression of ETHYLENE-RESPONSE-FACTOR1 in Arabidopsis confers resistance to several necrotrophic fungi. The Plant Journal 29: 23-32.
Brown R. L., Kazan K., Mcgrath K. C., Maclean D. J., Manners J. M. 2003. A role for the GCC-box in jasmonate-mediated activation of the PDF1.2 gene of Arabidopsis. Plant Physiology 132: 1020-1032.
Chakravarthy S., Tuori R. P., D’ascenzo M. D., Fobert P. R., Despres C., Martin G. B. 2003. The tomato transcription factor Pti4 regulates defense-related gene expression via GCC box and non-GCC box cis elements. Plant Cell 15: 3033-3050.
Chang S., Puryear J., Cairney J. 1993. A simple and efficient method for isolating RNA from pine trees. Plant Molecular Biology 11: 693-699.
Chen L.-R., Chen Y.-J., Lee C.-Y., Lin T.-Y. 2007. MeJA-induced transcriptional changes in adventitious roots of Bupleurum kaoi. Plant Science 173: 12-24.
Clough S. J., Bent A. F. 1998. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. The Plant Journal 16: 735-743.
Facchini P. J., Bird D. A., St-Pierre B. 2004. Can Arabidopsis make complex alkaloids? TRENDS in Plant Science 9: 116-122.
Fits L. V. D., Memelink J. 2000. ORCA3, a jasmonate-responsive transcriptional regulator of plant primary and secondary metabolism. Science 289: 295-297.
Fits L. V. D., Memelink J. 2001. The jasmonate inducible AP2/ERF domain transcription factor ORCA3 activates gene expression via interaction with a jasmonate-responsive promoter element. The Plant Journal 25: 43-53.
Gao S., Zhang H., Tian Y., Li F., Zhang Z., Lu X., Chen X., Huang R. 2008. Expression of TERF1 in rice regulates expression of stress-responsive genes and enhances tolerance to drought and high-salinity. Plant Cell Reports 27: 1787-1795.
Grace M. L., Chandrasekharan M. B., Hall T. C., Crowe A. J. 2004. Sequence and spacing of TATA box elements are critical for accurate initiation from the beta-phaseolin promoter. The Journal of Biological Chemistry 279: 8102-8110.
Gutterson N., Reuber T. L. 2004. Regulation of disease resistance pathways by AP2/ERF transcription factors. Current Opinion in Plant Biology 7: 465-471.
Hamilton D. A., Schwarz Y. H., Rueda J., Mascarenhas J. P. 2000. Comparison of transient and stable expression by a pollen-specific promoter: the transformation results do not always agree. Sex Plant Reprod 12: 292-295.
Huang Z., Zhang Z., Zhang X., Zhang H., Huang D., Huang R. 2004. Tomato TERF1 modulates ethylene response and enhances osmotic stress tolerance by activating expression of downstream genes. FEBS Letters 573: 110-116.
Jin L.-G., Li H., Liu J.-Y. 2010. Molecular characterization of three ethylene responsive element binding factor genes from cotton. Journal of Integrative Plant Biology 52: 485-495.
Jin L.-G., Liu J.-Y. 2008. Molecular cloning, expression profile and promoter analysis of a novel ethylene responsive transcription factor gene GhERF4 from cotton (Gossypium hirstum). Plant Physiology and Biochemistry 46: 46-53.
Liang H., Lu Y., Liu H., Wang F., Xin Z., Zhang Z. 2008. A novel activator-type ERF of Thinopyrum intermedium, TiERF1, positively regulates defence responses. Journal of Experimental Botany 59: 3111-3120.
Liu W.-Y., Chiou S.-J., Ko C.-Y., Lin T.-Y. 2011. Functional characterization of three ethylene response factor genes from Bupleurum kaoi indicates that BkERFs mediate resistance to Botrytis cinerea. Journal of Plant Physiology 168: 375-381.
Martinez-Garcia J. F., Monte E., Quail P. H. 1999. A simple, rapid and quantitative method for preparing Arabidopsis protein extracts for immunoblot analysis. The Plant Journal 20: 251-257.
Mcgrath K. C., Dombrecht B., Manners J. M., Schenk P. M., Edgar C. I., Maclean D. J., Scheible W.-R. D., Udvardi M. K., Kazan K. 2005. Repressor- and activator-type ethylene response factors functioning in jasmonate signaling and disease resistance identified via a genome-wide screen of Arabidopsis transcription factor gene expression. Plant Physiology 139: 949-959.
Memelink J., Verpoorte R., Kijne J. W. 2001. ORCAnization of jasmonate-responsive gene expression in alkaloid metabolism. TRENDS in Plant Science 6: 212-219.
Menke F. L. H., Champion A., Kijne J. W., Memelink J. 1999. A novel jasmonate- and elicitor-responsive element in the periwinkle secondary metabolite biosynthetic gene Str interacts with a jasmonate- and elicitor-inducible AP2-domain transcription factor, ORCA2. The EMBO Journal 18: 4455-4463.
Montiel G., Zarei A., Korbes A. P., Memelink J. 2011. The jasmonate-responsive element from the ORCA3 promoter from Catharanthus roseus is active in Arabidopsis and is controlled by the transcription factor AtMYC2. Plant and Cell Physiology 52: 578-587.
Nakano T., Suzuki K., Fujimura T., Shinshi H. 2006. Genome-wide analysis of the ERF gene family in Arabidopsis and rice. Plant Physiology 140: 411-432.
Nandi A., Welti R., Shah J. 2004. The Arabidopsis thaliana dihydroxyacetone phosphate reductase gene SUPPRESSSOR OF FATTY ACID DESATURASE DEFICIENCY1 is required for glycerolipid metabolism and for the activation of systemic acquired resistance. Plant Cell 16: 465-477.
Nishiuchi T., Shinshi H., Suzuki K. 2004. Rapid and transient activation of transcription of the ERF3 gene by wounding in tobacco leaves. The Journal of Biological Chemistry 279: 55355-55361.
Onate-Sanchez L., Anderson J. P., Young J., Singh K. B. 2006. AtERF14, a member of the ERF family of transcription factors, plays a nonredundant role in plant defense. Plant Physiology 143: 400-409.
Park H. C., Kim M. L., Kang Y. H., Jeon J. M., Yoo J. H., Kim M. C., Park C. Y., Jeong J. C., Moon B. C., Lee J. H., Yoon H. W., Lee S. H., Chung W. S., Lim C. O., Lee S. Y., Hong J. C., Cho M. J. 2004. Pathogen- and NaCl-induced expression of the SCaM-4 promoter is mediated in part by a GT-1 box that interacts with a GT-1-like transcription factor. Plant Physiology 135: 2150-2161.
Peebles C. a. M., Hughes E. H., Shanks J. V., San K.-Y. 2009. Transcriptional response of the terpenoid indole alkaloid pathway to the overexpression of ORCA3 along with jasmonic acid elicitation of Catharanthus roseus hairy roots over time. Metabolic Engineering 11: 76-86.
Quan R., Hu S., Zhang Z., Zhang H., Zhang Z., Huang R. 2010. Overexpression of an ERF transcription factor TSRF1 improves rice drought tolerance. Plant Biotechnology Journal 8: 476-488.
Reyes J. C., Muro-Pastor M. I., Florencio F. J. 2004. The GATA family of transcription factors in Arabidopsis and rice. Plant Physiology 134: 1718-1732.
Sakuma Y., Liu Q., Dubouzet J. G., Abe H., Shinozaki K., Yamaguchi-Shinozaki K. 2002. DNA-binding specificity of the ERF/AP2 domain of Arabidopsis DREBs, transcription factors involved in dehydration- and cold-inducible gene expression. Biochemical and Biophysical Research Communications 290: 998-1009.
Seo Y. J., Park J.-B., Cho Y.-J., Jung C., Seo H. S., Park S.-K., Nahm B. H., Song J. T. 2010. Overexpression of the ethylene-responsive factor gene BrERF4 from Brassica rapa increases tolerance to salt and drought in Arabidopsis plants. Molecules and Cells 30: 271-277.
Sohani M. M., Schenk P. M., Schultz C. J., Schmidt O. 2009. Phylogenetic and transcriptional analysis of a strictosidine synthase-like gene family in Arabidopsis thaliana reveals involvement in plant defence responses. Plant biology (Stuttgart, Germany) 11: 105-117.
Svensson J. T., Crosatti C., Campoli C., Bassi R., Stanca A. M., Close T. J., Cattivelli L. 2006. Transcriptome analysis of cold acclimation in barley albina and xantha mutants. Plant Physiology 141: 257-270.
Tamura K., Peterson D., Peterson N., Stecher G., Nei M., and Kumar S. 2011. MEGA5: Molecular Evolutionary Genetics Analysis using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods. Molecular Biology and Evolution doi: 10.1093/molbev/msr121
Trujillo L. E., Sotolongo M., Menendez C., Ochogavia M. E., Coll Y., Hernandez I., Borras-Hidalgo O., Thomma B. P. H. J., Vera P., Hernandez L. 2008. SodERF3, a novel sugarcane ethylene responsive factor (ERF), enhances salt and drought tolerance when overexpressed in tobacco plants. Plant and Cell Physiology 49: 512-525.
Wang H., Huang Z., Chen Q., Zhang Z., Zhang H., Wu Y., Huang D., Huang R. 2004. Ecotopic overexpression of tomato JERF3 in tobacco activates downstream gene expression and enhance salt tolerance. Plant Molecular Biology 55: 183-192.
Wu L., Zhang Z., Zhang H., Wang X.-C., Huang R. 2008. Transcriptional modulation of ethylene response factor protein JERF3 in the oxidative stress response enhances tolerance of tobacco seedlings to salt, drought, and freezing. Plant Physiology 148: 1953-1963.
Xu Z.-S., Chen M., Li L.-C., Ma Y.-Z. 2008. Functions of the ERF transcription factor family in plants. Botany 86: 969-977.
Yang W., Liu X.-D., Chi X.-J., Wu C.-A., Li Y.-Z., Song L.-L., Liu X.-M., Wang Y.-F., Wang F.-W., Zhang C., Liu Y., Zong J.-M., Li H.-Y. 2011. Dwarf apple MbDREB1 enhances plant tolerance to low temperature, drought, and salt stress via both ABA-dependent and ABA-independent pathways. Planta 233: 219-229.
Zhang G., Chen M., Li L., Xu Z., Chen X., Guo J., Ma Y. 2009. Overexpression of the soybean GmERF3 gene, an AP2/ERF type transcription factor for increased tolerances to salt, drought, and diseases in transgenic tobacco. Journal of Experimental Botany 60: 3781-3796.
Zhang H., Li W., Chen J., Yang Y., Zhang Z., Zhang H., Wang X.-C., Huang R. 2007. Transcriptional activator TSRF1 reversely regulates pathogen resistance and osmotic stress tolerance in tobacco. Plant Molecular Biology 63: 63-71.
Zhang H., Liu W., Wan L., Li F., Dai L., Li D., Zhang Z., Huang R. 2010. Functional analyses of ethylene response factor JERF3 with the aim of improving tolerance to drought and osmotic stress in transgenic rice. Transgenic Research 19: 809-818.
Zhang H., Yang Y., Zhang Z., Chen J., Wang X.-C., Huang R. 2008. Expression of the ethylene response factor gene TSRF1 enhances abscisic acid responses during seedling development in tobacco. Planta 228: 777-787.
Zhang X., Zhang Z., Chen J., Chen Q., Wang X.-C., Huang R. 2005. Expressing TERF1 in tobacco enhances drought tolerance and abscisic acid sensitivity during seedling development. Planta 222: 494-501.
Zhou J., Tang X., B.Martin G. 1997. The Pto kinase conferring resistance to tomato bacterial speck disease interacts with proteins that bind a cis-element of pathogenesis-related genes. The EMBO Journal 16: 3207-3218.

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