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作物学报 ›› 2012, Vol. 38 ›› Issue (07): 1328-1333.doi: 10.3724/SP.J.1006.2012.01328

• 研究简报 • 上一篇    下一篇

芥菜AP1基因体外表达及其与FLC相互作用的验证

汤青林**，许俊强**，宋明*，王志敏   

  1. 西南大学园艺园林学院 / 南方山地园艺学教育部重点实验室 / 重庆市蔬菜学重点实验室，重庆 400715
  • 收稿日期:2011-09-30 修回日期:2012-04-16 出版日期:2012-07-12 网络出版日期:2012-05-11
  • 通讯作者: 宋明, E-mail: swausongm@yahoo.com.cn
  • 基金资助:

    本研究由国家自然科学基金项目(31000908), 中央高校基本科研业务费专项资金(XDJK2009C124, XDJK2012B020), 重庆市自然科学基金(2009BB1307, 2011BA1002)和国家重点基础研究发展计划(973计划)项目(2012CB1139000)资助。

Expression of Floral Meristem Identity Gene AP1 in vitro and Validation of Interaction between AP1 and FLC in Brassica juncea Coss. (Mustard)

TANG Qing-Lin**,XU Jun-Qiang**,SONG Ming*,WANG Zhi-Min   

  1. College of Horticulture and Landscape Architecture, Southwest University / Key Laboratory of Horticulture Science for Southern Mountainous Regions, Ministry of Education / Key Laboratory of Olericulture, Chongqing 400715, China
  • Received:2011-09-30 Revised:2012-04-16 Published:2012-07-12 Published online:2012-05-11
  • Contact: 宋明, E-mail: swausongm@yahoo.com.cn

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摘要： 芥菜花分生组织决定因子AP1与开花路径核心调节子FLC可能存在直接的相互作用，从而调节开花时间。为进一步检测该相互作用，从芥菜“QJ”材料中同源克隆了790 bp的AP1 cDNA序列，该基因编码256个氨基酸。生物信息学分析表明, AP1属MIKC型蛋白，其MADS域含有2个a螺旋和2个b折叠，第1个a螺旋内含有1个不保守氨基酸位点; 而K域含有3个a螺旋，第1、2个a螺旋内各有1个不保守位点，第3个a螺旋内具有4个不保守位点。同时构建了原核表达质粒pET43.1a-AP1，转化宿主菌大肠杆菌BL21，以IPTG诱导该融合蛋白体外表达。利用pET43.1a-AP1融合蛋白序列中6×His 标签与Ni+结合的特点，结合SDS-PAGE分析，证实了体外表达蛋白AP1能与FLC相互作用并形成复合体，该结果为深入研究AP1与FLC互作机制及花分生组织的分子调控奠定了基础。

关键词: 芥菜, AP1基因, FLC, 蛋白互作

Abstract: There exists a possible direct interaction between floral meristem factor AP1 and flowering pathway central regulator FLC in Brassica juncea. Coss. To further prove the interactive mechanism between AP1 and FLC, the protein interaction in vitro was testified in Brassica juncea Coss. (mustard). The cDNA of AP1 gene isolated using homologous cloning techniques from mustard “QJ” was 790 bp, encoding 256 amino acids. AP1 belongs to MIKC type protein with MADS domain and K-box known from analysis software. The conserved MADS domain had two alpha helixes (a) and two beta-sheets (b), and one amino acid site in the first a helix was not conserved. The K-box had three alpha helixes (a), and one amino acid site in the first and second a helixes was not conserved, respectively, but four amino acid sites in the third a helix were not conserved. Furthermore, recombinant plasmid pET43.1a-AP1 was constructed, transformed to E. coli (BL21) and then induced protein expression by IPTG. With the characteristics of 6×His tag in fusion protein of pET43.1a-AP1 which could combine with Ni+, the interaction between AP1 and FLC was analyzed via SDS-PAGE. The results showed that AP1 and FLC could act with each other to combine and form a complex. This research provides theoretical and technical bases for further analyzing the interaction mechanism of AP1-FLC protein complex and the molecular regulation of floral meristem in Brassica juncea.

Key words: Brassica juncea Coss., AP1 gene, FLC, Protein interaction

[1]Suárez-López P, Wheatley K, Robson F, Onouchi H, Valverde F, Coupland G. CONSTANS mediates between the circadian clock and the control of flowering in Arabidopsis. Nature, 2001, 410: 1116–1120

[2]Gustafson-Brown C, Savidge B, Yanofsky M F. Regulation of the Arabidopsis floral homeotic gene APETALA1. Cell, 1994, 76: 131–143

[3]Reyes B, Ana B, Antonio S M, Francisco M. Floral initiation and inflorescence architecture: a comparative view. Ann Bot, 2007, 100: 659–676

[4]Valverde F, Mouradov A, Soppe W, Ravenscroft D, Samach A, Coupland G. Photoreceptor regulation of CONSTANS protein in photoperiodic flowering. Science, 2004, 303: 1003–1006

[5]Corbesier L, Vincent C, Jang S, Fornara F, Fan Q Z, Searle I, Giakountis A, Farrona S, Gissot L, Turnbull C, Coupand G. FT protein movement contributes to long-distance signaling in floral induction of Arabidopsis. Science, 2007, 316: 1030–1033

[6]Abe M, Kobayashi Y, Yamamoto S, Daimon Y, Yamaguchi A, Ikeda Y, Ichinoki H, Notaguchi M, Goto K, Araki T. FD, a bZIP protein mediating signals from the floral pathway integrator FT at the shoot apex. Science, 2005, 309: 1052–1056

[7]Wigge P A, Kim M C, Jaeger K E, Busch W, Schmid M, Lohmann J U, Weigel D. Integration of spatial and temporal information during floral induction in Arabidopsis. Science, 2005, 309: 1056–1059

[8]Parcy F, Bomblies K, Weigel D. Interaction of LEAFY, AGAMOUS and TERMINAL FLOWER1 in maintaining floral meristem identity in Arabidopsis. Development, 2002, 129: 2519–2527

[9]Weigel D, Nilsson O. A developmental switch sufficient for flower initiation in diverse plants. Nature, 1995, 377: 495–500

[10]Liljegren S J, Gustafson B C, Pinyopich A, Ditta G S, Yanofsky M F. Interactions among APETALA1, LEAFY, and TERMINAL FLOWER1 specify meristem fate. Plant Cell, 1999, 11: 1007–1018

[11]Lü J-H(吕晋慧), Wu Y-L(吴月亮), Sun L(孙磊), Zhang Q-X(张启翔). Genetic transformation of Chrysanthemum morifolium cv.‘Yu Ren Mian’with AP1 gene mediated by Agrobacterium tumefaciens. Sci Silav Sin (林业科学), 2007, 43(9): 128–132 (in Chinese with English abstract)

[12]Folter S D, Immink R G H, Kieffer M, Parenicová L, Henz S R, Weigel D, Busscher M, Maarten Kooiker M, Lucia Colombo L, Martin M, Kater M M, Brendan Davies B, Angenent G C. Comprehensive interaction map of the Arabidopsis MADS box transcription factors. Plant Cell, 2005, 17: 1424–1433

[13]Li Z M, Zhang J Z, Li M, Deng X X, Hu C G, Yao J L. PtSVP, an SVP homolog from trifoliate orange (Poncirus trifoliata L. Raf.), shows seasonal periodicity of meristem determination and affects flower development in transgenic Arabidopsis and tobacco plants. Plant Mol Biol, 2010, 74: 129–142

[14]Honma T, Goto K. Complexes of MADS-box proteins are sufficient to convert leaves into floral organs. Nature, 2001, 409, 525–529

[15]Sridhar V V, Surendrarao A, Liu Z C. APETALA1 and SEPALLATA3 interact with SEUSS to mediate transcription repression during flower development. Development, 2006, 133, 3159–3166

[16]Li D, Liu C, Shen L S, Wu Y, Chen H Y, Robertson M, Helliwell C A, Ito T, Meyerowitz E, Yu H. A repressor complex governs the integration of flowering signals in Arabidopsis. Dev Cell, 2008, 15: 110–120

[17]Lee J H, Park S H, Lee J S, Ahn J H. A conserved role of SHORT VEGETATIVE PHASE (SVP) in controlling flowering time of Brassica plants. Biochimica et Biophysica Acta, 2007, 1769: 455–461

[18]Mathieu J, Warthmann N, Kuttner F, Schmid M. Export of FT protein form phloem companion cells is sufficient for floral induction in Arabidosis. Curr Biol, 2007, 17: 1055–1060

[19]Yant L, Mathieu J, Schmid M. Just say no: floral repressors help Arabidopsis bide the time. Curr Opin Plant Biol, 2009, 12: 1–7

[20]Sheldon C C, Conn A B, Dennis E S, Peacock W J. Different regulatory regions are required for the vernalization-induced repression of FLOWERING LOCUS C and for the epigenetic maintenance of repression. Plant Cell, 2002, 14: 2527–2537

[21]Kaufmann K, Melzer R, Theigen G. MIKC-type MADS-domain proteins: structural modularity, protein interactions and network evolution in land plants. Gene, 2005, 347: 183–198

[22]Tang Q-L(汤青林), Xu J-Q(许俊强), Song M(宋明), Wang Z-M(王志敏). Determination of interactions between the two determinant transcription factors of flowering signal integrators in vitro in Brassica juncea Coss. (mustard). Acta Hort Sin (园艺学报), 2011, 38(12): 2317–2324 (in Chinese with English abstract)

[23]Yang Y(杨洋), Gao Q-G(高启国), Song M(宋明), Niu Y(牛义), Tang Q-L(汤青林), Zhu L-Q(朱利泉), Wang X-J(王小佳). In vitro study on the interaction s between determinant factors of self-incompatibility in Brassica oleracea. var. capitata. Acta Hort Sin (园艺学报), 2009, 36 (3): 355–362 (in Chinese with English abstract)

[24]Immink R, Kaufmann K, Angenent G C. The ‘ABC’ of MADS domain protein behavior and interactions. Sem Cell Dev Biol, 2010, 21: 87–93

[25]Gregis V, Sessa A, Colombo L, Kater M M. AGL24, SHORT VEGETATIVE PHASE, and APETALA1 redundantly control AGAMOUS during early stages of flower development in Arabidopsis. Plant Cell, 2006, 18: 1373–1382

[26]Pelaz S, Gustafson B C, Kohalmi S E, Crosby W L, Yanofsky M F. APETALA1 and SEPALLATA3 interact to promote flower development. Plant J, 2001, 26: 385–394

[27]Ferrandiz C, Gu Q, Martienssen R, Yanofsky M F. Redundant regulation of meristem identity and plant architecture by FRUITFULL, APETALA1 and CAULIFLOWER. Development, 2000, 127: 725–734

[28]Riechmann J L, Krizek B A, Meyerowitz E M. Dimerization specificity of Arabidopsis MADS domain homeotic proteins APETALA1, APETALA3, PISTILLATA, and AGAMOUS. Proc Natl Acad Sci USA, 1996, 93: 4793–4798

[29]An Y-H(安颖慧), Li X-F(李小方), Zhu J-X(朱金鑫), Shao X-H(邵兴华), Sun Y(孙越), Xiong L-J(熊莉君). The screening of interaction factors with BoCAL and BoAP1 related to curd formation. J Mol Cell Biol (分子细胞生物学报), 2007, 40(2): 130–136 (in Chinese with English abstract)

[30]Ng M, Yanofsky M F. Activation of the Arabidopsis B class homeotic genes by APETALA1. Plant Cell, 2001, 13: 739–754
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