Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/36160
標題: 洋桔梗與阿拉伯芥中與胚珠分化及花朵發育相關之MADS box基因之選殖及功能分析
Molecular Cloning and Characterization of MADS Box Genes Involving in Ovule and Floral Organ Formation from Lisianthus and Arabidopsis
作者: 陳星宇
Chen, Hsing-Yu
關鍵字: lisianthus;洋桔梗;floral organe;ovule;MADS box;花器;胚珠
出版社: 生物科技學研究所
引用: 第一章: Angenent, G.C., and Colombo, L. (1996) Molecular control of ovule development. Trends Plant Sci. 1, 228-232. Angenent, G.C., Franken, J., Busscher, M., van Dijken, A., van Went, J.L., Dons, H.J.M., and van Tunen, A.J. (1995) A novel class of MADS box genes is involved in ovule development in petunia. Plant Cell 7, 1569-1582. Brand-Saberi, B., and Christ, B. (1999) Genetic and epigenetic control of muscle development in vertebrates. Cell Tissue Res. 296, 199-212. Coen, E.S., and Meyerowitz, E.M. (1991) The war of the whorls: genetic interactions controlling flower development. Nature 353, 31-37. Colombo, L., Franken, J., Koetie, E., van Went, J., Dons, H.J.M., Angenent, G.C., and van Tunen, A.J. (1995) The petunia MADS box gene FBP11 determines ovule identity. Plant Cell 7, 1859-1868. Colombo, L., Franken, J., Alexander, R., van der Krol, R., Wittich, P.E., Dons, H.J.M., and Angenent, G.C. (1997a) Downregulation of ovule–specific MADS box genes from petunia results in maternally controlled defects in seed development. Plant Cell 9, 703-715. Colombo, L., van Tunen, A.J., Dons, H.J.M., and Angenent, G.C. (1997b) Molecular control of flower development in Petunia hybrida. Adv. Bot. Res. 26, 229-250. Egea-Cortines, M., Saedler, H., and Sommer, H. (1999) Ternary complex formation between the MADS-box proteins SQUAMOSA, DEFICIENS and GLOBOSA is involved in the control of floral architecture in Antirrhinum majus. EMBO J. 18, 5370-5379. Fan, H.Y., Hu, Y., Tudor, M., and Ma, H. (1997) Specific interactions between the K domains of AG and AGLs, members of the MADS domain family of DNA binding proteins. Plant J. 12, 999-1010. Hayes, T.E., Sengupta, P., and Cochran, B.H. (1988) The human c-fos serum response factor and the yeast factor GRM/PRTF have related DNA-binding specificities. Genes Dev. 2, 1713-1722. Honma, T., and Goto K. (2001) Complexes of MADS-box proteins are sufficient to convert leaves into floral organs. Nature 409, 535-529. Huang, H., Mizukami, Y., Hu, Y., and Ma, H. (1993) Isolation and characterization of the binding sequences for the product of the Arabidopsis floral homeotic gene AGAMOUS. Nucleic Acid Research. 21, 4769-4776. Kruger, J., Aichinger, C., Kahmann, R., and Bolker, M. (1997) A MADS-box homologue in Ustilago maydis regulates the expression of pheromone-inducible genes but is nonessential. Genetics 147, 1643-52. Ma, H., Yanofsky, M.F., and Meyerowitz, E.M. (1991) AGL1-AGL6, an Arabidopsis gene family with similarity to floral homeotic and transcription factor genes. Genes Dev. 5, 484-495. Molkentin, J.D., and Olson, E.N. (1996) Combinatorial control of muscle development by basic helix-loop-helix and MADS-box transcription factors. Proc. Natl. Acad. Sci. U S A 93, 9366-73. Münster, T., Pahnke, J., Di Rosa, A., Kim, J,T., Martin, W., Saedler, H., and Theissen G. (1997) Floral homeotic genes were recruited from homologous MADS-box genes preexisting in the common ancestor of ferns and seed plants. Proc Natl Acad Sci U S A. 94, 2415-20. Ng, M., and Yanofsky, M.F. (2001) Function and evolution of the plant MADS-box gene family. Nat. Rev. Genet. 2, 186-95. Parenicova, L., de Folter, S., Kieffer, M., Horner, D.S., Favalli, C., Busscher, J., Cook, H.E., Ingram, R.M., Kater, M.M., Davies, B., Angenent, G.C., and Colombo, L. (2003) Molecular and phylogenetic analyses of the complete MADS-box transcription factor family in Arabidopsis: new openings to the MADS world. Plant Cell. 15:1538-51. Pinyopich, A., Ditta, G.S., Baumann, E., Wisman, E., and Yanofsky, M.F. (2003) Unraveling the redundant roles of MADS-box genes during carpel and fruit development. Nature 424, 85–88. Purugganan, M.D., Rousley, S.D., and Yanofsky, M.F. (1995) Molecular evolution of flower development: Diversification of the plant MADS-box regulatory gene family. Genetics 140, 345-356. Riechmann, J.L., Krizek, B.A., and Meyerowitz, E.M. (1996a) Dimerization specificity of Arabidopsis MADS domain homeotic proteins APETALA1, APETALA3, PISTILLATA and AGAMOUS. Proc. Natl. Acad. Sci. USA 93, 4793-4798. Riechmann, J.L., Wang, M., and Meyerowitz, E.M. (1996b) DNA- binding properties of Arabidopsis MADS domain homeotic proteins APETALA1, APETALA3, PISTILLATA, and AGAMOUS. Nucleic Acid Res. 24, 3134-3141. Rousley, S.D., Ditta, G.S., and Yanofsky, M.F. (1995) Diverse roles for MADS box genes in Arabidopsis development. Plant Cell 7, 1259-1269. Rutledge, R., Regan, S., Nicolas, O., Fobert, P., Cote, C., Bosnich, W., Kauffeldt, C., Sunohara, G., Seguin, A., and Stewart, D. (1998) Characterization of an AGAMOUS homologue from the conifer black spruce (Picea mariana) that produce floral homeotic conversion when expressed in Arabidopsis. Plant J. 15, 625-634. Savidge, B., Rousley, S.D., and Yanofsky, M.F. (1995) Temporal relationship between the transcription of the two Arabidopsis MADS box genes and the floral organ identity genes. Plant Cell 7, 721-733. Schwarz-Sommer, Z., Hue, I.,Huijster, P., Flor, P.J., Hansen, R., Tetens, F., Lonning, W.E., Saedler, H., and Sommer, H. (1992) chacacterization of Antirrhinum floral homeotic MADS-box gene deficiens-Evidence for DNA binding and autoregulation of its persistent expression throughout flower development. EMBO J. 11, 251-263. Shore, P., and Sharrocks, A.D. (1995) The MADS-box family of transcription factors. Eur. Biochem. 229, 1-13. Tandre, K., Svenson, M., Svensson, M.F., and Engström, P. (1998) Conservation of gene structure and activity in the regulation of reproductivity organ development of conifers and angiosperms. Plant J. 15, 615-623. Theißen, G. (2001) Development of floral organ identity: stories from the MADS house. Curr. Opin. Biol. 4, 75–85. Theissen, G., Becker, A., Rosa, A.D., Kanno, A., Kim J.T., Münster, T., Winter, K.U., and Saedler, H. (2000) A short history of MADS-box genes in plant. Plant Mol. Biol. 42, 115-149. Theissen, G., Kim, J.T., and Saedler, H. (1996) Classification and phylogeny of the MADS-box multigene family suggest defined roles of MADS-box gene subfamilies in the morphological evolution of eukaryotes. J Mol Evol. 43, 484-516 Theissen, G., and Saedler, H. (1995) MADS-box genes in plant ontogeny and phylogeny: Haeckel''s ''biogenetic law'' revisited. Curr. Opin. Genet. Dev. 5, 628-639. Tröbner, W., Ramirez, L., Motto, P., Hue, I., Huijster, P., Lönning, W.E., Saedler, H., Sommer, H., and Schwarz-Sommer, Z. (1992) GLOBOSA: A homeotic gene which interacts with DEFICIENS in control of Antirrhinum floral organogenesis. EMBO J. 11, 4693-4704. Winter, K.U., Becker, A., Münster, T., Kim, J.T., Saedler, H., and Theissen, G. (1999) MADS-box genes reveal that gnetophytes are more closely related to conifers than to flowering plants. Proc. Natl. Acad. Sci. USA 96, 7342-7347. Yabana, N., and Yamamoto, M. (1996) Schizosaccharomyces pombe map1+ encodes a MADS-box-family protein required for cell-type-specific gene expression. Mol. Cell Biol. 16, 3420-8. Yanofsky, M.F., Ma, H., Bowman, J.L., Drews, G.N., Feldmann, K.A., and Meyerowitz, E.M. (1990) The protein encoded by the Arabidopsis homeotic gene agamous resembles transcription factors. Nature 346, 35-39. Yu, D., Kotilainen, M., Pollanen, E., Mehto, M., Elomaa, P., Helariutta, Y., Albert, V.A., and Teeri, T.H. (1999) Organ identity genes and modified patterns of flower development in Gerbera hybrida (Asteraceae). Plant J. 17, 51-62. Zhang, H., and Forde, B.G. (1998) An Arabidopsis MADS box gene that controls nutrient-induced changes in root architecture. 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Schmidt, R.J., Veit, B., Mandel, M.A., Mena, M., Hake, S. and Yanofsky, M.F. (1993) Identification and molecular characterization of ZAG1, the maize homolog of Arabidopsis floral homeotic gene AGAMOUS. Plant Cell 5, 729– 737. Schultz, E.A. and Haughn, G.W. (1993) Genetic analysis of the floral initiation process (FLIP) in Arabidopsis. Development 119, 745–765. Schwarz-Sommer, Z., Hue, I., Huijser, P., Flor, P.J., Hansen, R., Tetens, F., Lonnig, W.E., Saedler, H. and Sommer, H. (1992) Characterization of the Antirrhinum floral homeotic MADS-box gene deficiens – Evidence for DNA binding and autoregulation of its persistent expression throughout flower development. EMBO J. 11, 251–263. Tandre, K., Svenson, M., Svensson, M.E. and Engström, P. (1998) Conservation of gene structure and activity in the regulation of reproductive organ development of conifers and angiosperms. Plant J. 15, 615–623. 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摘要: 
摘要

在前人研究中,從洋桔梗中所選殖的五個基因,分別命名為為EgMADS1,2,3,5,6,分別為D,B,A以及兩個E功能MADS box基因。本實驗中從洋桔梗又再分別選殖出五個不同功能之MADS box基因,分別是EgMADS4,7,8,9,10。EgMADS4與阿拉伯芥的AP3有著59 %的一致性與74%的相似性,其RNA在四個花器都有表現,但在葉子中並不表現,這與目前所知的B功能基因群大不相同。此外,在EgMADS4的蛋白質C端找不到AP3同源基因所該有的euAP3 motif,推測EgMADS4應該是屬於TM6的同源基因。EgMADS7與阿拉伯芥中的PI有56%的一致性與73%的相似性,此基因RNA主要表現在花瓣與雄蕊,屬於功能較明顯的B功能基因,推測應為PI同源基因。EgMADS8與C功能基因蛋白質序列十分相似,與矮牽牛中的AG有71%的一致性與79%的相似性。EgMADS8的RNA專一表現在雄蕊及雌蕊中,為一明顯的C功能基因。EgMADS9與阿拉伯芥中的AP1有73%的一致性與91%的相似性。其RNA在四個花器中均有表現,甚至在葉子中的表現量也很高,與AP1的RNA只在花萼與花瓣表現極為不同,因此應該是FRUITFULL (FUL)的同源基因。EgMADS10並不屬於ABCDE模式其中一員,其序列與金魚草的INCOMPOSITA(INCO)在氨基酸序列上有74%的一致性與86%的相似性。此外,EgMADS10也與阿拉伯芥中的SHORT VEGETATIVE PHASE(SVP)有著64%的一致性與77%的相似性。EgMADS10雖然在葉子及四個花器中均表現其RNA,但在花瓣的表現量相較於其他的花器而言較低。其中EgMADS1,2,3利用35S啟動子轉入阿拉伯芥中之後,觀察其轉基因植物性狀,發現EgMADS1 轉基因後代出現了捲曲葉,植株極度矮小,極度早開花,花萼轉型為雌蕊並長出柱毛與胚珠,花瓣轉型為雄蕊等等性狀,與其他物種的D功能基因阿拉伯芥中大量表現的性狀類似。在本實驗中也研究了兩個專一表現在阿拉伯芥胚珠中的MADS box基因,AGL11(AGAMOUS-like 11)與AGL13。前人研究中,AGL11的序列與MADS box基因中的D function基因有相當高的相似性,而AGL13的基因序列則與AGL6有較高的相似性。將AGL11與AGL13以大量異位表現之方式轉入阿拉伯芥之後,AGL11的轉基因植物出現提早開花與捲曲葉的性狀,AGL13的轉基因植物則出現了植株極度早開花,捲曲葉,植株極度矮小,終結花,花萼雌蕊化及內側著生胚珠等性狀。AGL13 antisense轉基因植物則出現花苞發育異常及雄蕊花瓣變短之性狀。此外,AGL11之intron 1長約1.6 kb而AGL13的intron 1則約有600 bp,推測此二基因之intron 1中可能有調控基因表現位置專一性相關之區域,本實驗透過將AGL11與AGL13的intron 1或基因的啟動子加intron 1分別接上GUS基因,進行植物基因轉殖及後續GUS活性之分析。實驗結果發現,AGL11與AGL13之intron 1及啟動子皆無單獨調控其基因表現位置專一性之功能,這兩個基因能專一表現在胚珠中應是多個調控區域綜合調控之結果。

Abstract

Five D, B, A and E functional MADS box genes, EgMADS1, 2, 3, 5, 6, have been cloned and characterized from Eustoma grandiflorum (lisianthus) in our laboratory previously. In this study, five more lisianthus MADS box genes, EgMADS4, 7, 8, 9, 10 were investigated. EgMADS4, lacks the C terminal euAP3 motif, is the B functional AP3 homologue in TM6 lineage and shows sequence similar to EgMADS2. EgMADS4 RNA was expression in all four floral organs. EgMADS7 showed sequence similar to B functional PI genes and its RNA was expressed mainly in the petal and stamen. EgMADS8 showed sequence similar to Arabidopsis C functional AG and was specific expressed in the stamen and carpel. EgMADS9 was A functional FUL homologue with the C terminal euFUL motif. EgMADS9 was expressed in leaves and all floral organs. EgMADS10, showed high homology to INCO, the SVP homolog in the Antirrhinum, was expressed in leaves and all floral organs. Ectopic expression of EgMADS1 in transgenic Arabidopsis flowered early and showed novel phenotype by producing curly leaf, terminal flower and extremely small plant size. Conversion of sepal into carpel-like structure with ovule and stigmatic papillae and petal into stamen-like structure were observed in these transgenic plants. Furthermore, two Arabidopsis MADS box genes AGL11 and AGL13, specifically expressed in the ovules, were characterized in this study. AGL11 showed high sequence homology to D function MADS box genes whereas AGL13 was similar to AGL6. Ectopic expression of AGL11 in Arabidopsis produced curly leaves and flowered slightly earlier than wild-type plants. Ectopic expression of AGL13 in Arabidopsis flowered extremely early and showed similar phenotype to that observed in 35S::EgMADS1 transgenic Arabidopsis plants. Ectopically expressing antisense of AGL13 produced abnormal flowers with short stamen and petal in transgenic Arabidopsis plants. To further investigate the role of the large intron 1 in regulating the tissue specific expression for AGL11 and AGL13, intron 1 or intron 1 plus promoter fused with GUS report gene were transferred into Arabidopsis and GUS activity in different tissues of transgenic plants was analyzed. The results indicated that neither the promoter nor the intron 1 alone was able to control the ovules-specific expression for AGL11 or AGL13.
URI: http://hdl.handle.net/11455/36160
其他識別: U0005-2808200716230400
Appears in Collections:生物科技學研究所

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