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园艺学报 ›› 2015, Vol. 42 ›› Issue (4): 791-798.doi: 10.16420/j.issn.0513-353x.2014-0975

• 研究报告 • 上一篇    下一篇

羽衣甘蓝ARC1与Exo70A1蛋白相互作用结构域的分析与鉴定

曹明明,杨 佳,李晓屿,李玉花,蓝兴国   

  1. 东北林业大学生命科学学院,哈尔滨 150040
  • 出版日期:2015-04-25 发布日期:2015-04-25
  • 基金资助:

    中央高校基本科研业务费专项基金项目(DL13CA13);国家基础科学人才培养基金项目(J1210053)

Analysis and Characterization of Interaction Domain Between ARC1 and Exo70A1 from Ornamental Kale(Brassica oleracea var. acephala)

CAO Ming-ming,YANG Jia,LI Xiao-yu,LI Yu-hua,and LAN Xing-guo   

  1. College of Life Sciences,Northeast Forestry University,Harbin 150040,China
  • Online:2015-04-25 Published:2015-04-25

摘要: 为阐明羽衣甘蓝自交不亲和信号传递因子ARC1与下游底物Exo70A1相互作用的结构域,利用酵母双杂交系统进行检测与分析。将羽衣甘蓝BoARC1、BoARC1-N端(1 ~ 279 aa,含有UND结构域)及BoARC1-C端(280 ~ 663 aa,含有U-box结构域和Arm repeat结构域)的序列分别构建到pGBKT7载体中,获得BD-ARC1、BD-ARC1-N和BD-ARC1-C重组质粒。将羽衣甘蓝BoExo70A1、BoExo70A1-Δ1(1 ~ 85 aa)、Exo70A1-Δ2(1 ~ 270 aa)及Exo70A1-Δ3(271 ~ 638 aa,含有Exo70结构域)的序列分别构建到pGADT7载体中,获得AD-Exo70A1、AD-Exo70A1-Δ1、AD-Exo70A1-Δ2和AD-Exo70A1-Δ3重组质粒。将获得的重组质粒分别共转化到酵母Y187菌株中,在双缺陷(-Leu-Trp)酵母培养基中培养与生长。β-gal显色检测结果表明,共转BD-ARC1-N/BoExo70A1-Δ1或BD-ARC1-N/BoExo70A1-Δ2的酵母在滤纸上显示蓝色,这说明BoARC1的N端与BoExo70A1的N端介导了两者的相互作用。

关键词: 羽衣甘蓝, 自交不亲和性, ARC1, Exo70A1, 结构域

Abstract: The yeast-two hybrid system was used to detect the interaction between ARC1 and Exo70A1 from ornamental kale(Brassica oleracea var. acephala). The nucleotide sequences of full-length BoARC1,N terminal of BoARC1(1–279 aa,UND domain),C terminal of BoARC1(280–663 aa,U-box domain and Arm repeat domain)were constructed into the vector pGBKT7,respectively. Thus,the plasmids BD-ARC1,BD-ARC1-N or BD-ARC1-C were obtained and confirmed. On the other hand,the nucleotide sequences of full-length BoExo70A1,BoExo70A1-Δ1(1–85 aa),Exo70A1-Δ2(1–270 aa),Exo70A1-Δ3(271–638 aa,Exo70 domain)were constructed into the vector pGADT7,respectively. And the plasmids AD-Exo70A1,AD-Exo70A1-Δ1,AD-Exo70A1-Δ2 or AD-Exo70A1-Δ3 were also obtained and confirmed. These plasmids were used to co-transform yeast strain Y187,which were then plated onto SD/-Leu-Trp(synthetic dropout glucose agar plates lacking leucine and tryptophan). The β-galactosidaseassays showed that yeast cells co-transformed with the BoARC1-N/AD-Exo70A1-Δ1 plasmids or BoARC1-N/BD-Exo70A1-Δ2 plasmids turned blue. These results indicated that N terminal of BoARC1 and BoExo70A1 mediated interaction between BoARC1 and BoExo70A1.

Key words: Brassica oleracea var. acephala, self-incompatibility, ARC1, Exo70A1;domain

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