WU Xiaoting,SHAO Shuaixu,LI Ying,et al.Cloning of BrCDF1 gene and protein subcellular localization and identification of its interaction with BrFKF1 in non-heading Chinese cabbage[J].Journal of Nanjing Agricultural University,2017,40(5):812-819.[doi:10.7685/jnau.201612049]





Cloning of BrCDF1 gene and protein subcellular localization and identification of its interaction with BrFKF1 in non-heading Chinese cabbage
吴小婷1 邵帅旭1 李英1 张昌伟1 侯喜林1 沈振国2 刘同坤1
1. 南京农业大学作物遗传与种质创新国家重点实验室/农业部华东地区园艺作物生物学 与种质创制重点实验室, 江苏 南京 210095;
2. 南京农业大学生命科学学院, 江苏 南京 210095
WU Xiaoting1 SHAO Shuaixu1 LI Ying1 ZHANG Changwei1 HOU Xilin1 SHEN Zhenguo2 LIU Tongkun1
State Key Laboratory of Crop Genetics and Germplasm Enhancement/Key Laboratory of Biology and Germplasm Enhancement of Horticultural Crop in East China Ministry of Agriculture, Nanjing Agricultural University Nanjing 210095, China;
2. College of Life Sciences Nanjing Agricultural University Nanjing 210095, China
Brassica rapa ssp. chinensisBrCDF1subcellular localizationyeast two hybridbimolecular fluorescence complementation
[目的]本文旨在揭示CDF1基因在不结球白菜(Brassica rapa ssp.chinensis)抽薹开花过程中的调控机制,验证其是否与FKF1蛋白发生互作。[方法]以‘苏州青’为材料,采用同源克隆方法获得BrCDF1基因全长序列,并与其他物种进行氨基酸序列比对;将BrCDF1与报告基因YFP融合构建亚细胞定位载体pEarlyGate101-BrCDF1-YFP,采用农杆菌介导的方法将其瞬时表达于本氏烟(Nicotiana benthamiana)叶片中;激光共聚焦显微镜观察,利用酵母双杂交和双分子荧光互补技术验证不结球白菜BrCDF1和BrFKF1蛋白是否存在互作。[结果]BrCDF1基因含有876 bp开放阅读框,编码292个氨基酸。与甘蓝型油菜(Brassica napus)、野甘蓝(原变种)(Brassica oleracea var.oleracea)、萝卜(Raphanus sativus)的氨基酸序列比对结果显示:BrCDF1在进化过程中的保守程度分别为91.11%、88.25%、84.76%,保守性较高。BrCDF1蛋白定位于细胞核中,BrCDF1和BrFKF1之间存在相互作用。[结论]BrCDF1定位于细胞核,并且与BrFKF1互作,在进化过程中保守性较高,并可能参与了不结球白菜的光周期开花途径。
[Objectives]The paper aims to elucidate the flowering regulatory mechanism of transcription factor CDF1 in the flowering process of Brassica rapa ssp. chinensis and verify if CDF1 interacts with the FKF1 protein.[Methods]A full-length sequence of BrCDF1 was isolated by homologous cloning. The subcellular localization vector pEarlyGate101-BrCDF1-YFP,which was constructed by fusion of BrCDF1 and YFP,was transiently expressed in Nicotiana benthamiana leaves by Agrobacterium tumefaciens-mediated method and detected by confocal microscopy. The interaction between BrCDF1 and BrFKF1 in Brassica rapa was studied by yeast two-hybrid and bimolecular fluorescence complementation methods.[Results]The BrCDF1 gene contains an open reading frame of 876 bp in length encoding a protein of 292 amino acids. The alignment of Brassica napus,Brassica oleracea var. oleracea and Raphanus sativus showed that the conservatism of BrCDF1 was 91.11%,88.25% and 84.76% respectively in the evolutionary process,which was highly conservative comparing with other species. The detection of yellow fluorescence signal by confocal showed that BrCDF1 was located in nucleus. And there was an interaction between BrCDF1 and BrFKF1.[Conclusions]In this study,BrCDF1 is located in nucleus and interacts with BrFKF1. BrCDF1 is highly conserved during evolution. BrCDF1 possibly plays a role in photoperiod pathway of flowering in non-heading Chinese cabbage.


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更新日期/Last Update: 2017-09-23