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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

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