芥菜AP1基因体外表达及其与FLC相互作用的验证  

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

作　　者：汤青林[1] 许俊强[1] 宋明[1] 王志敏[1] 

机构地区：[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

出　　处：《作物学报》2012年第7期1328-1333,共6页Acta Agronomica Sinica

基　　金：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);国家重点基础研究发展计划(973计划)项目(2012CB1139000)资助

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790bp的AP1cDNA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个螺旋和2个折叠,第1个螺旋内含有1个不保守氨基酸位点;而K域含有3个螺旋,第1、2个螺旋内各有1个不保守位点,第3个螺旋内具有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。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 API 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 （ct） and two beta-sheets （~）, and one amino acid site in the first tx helix was not conserved. The K-box had three alpha helixes （c0, and one amino acid site in the first and second tx helixes was not conserved, respectively, but four amino acid sites in the third tx helix were not conserved. Furthermore, recombi- nant plasmid pET43.1 a-AP1 was constructed, transformed to E. coli （BL21） and then induced protein expression by IPTG With the characteristics of 6xHis 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 pro- tein complex and the molecular regulation of floral meristem in Brassicajuncea.

关 键 词：芥菜 AP1基因 FLC 蛋白互作 

分 类 号：S637[农业科学—蔬菜学]