遗传 ›› 2019, Vol. 41 ›› Issue (5): 430-438.doi: 10.16288/j.yczz.18-314

• 研究报告 • 上一篇    下一篇

拟南芥CKI1基因上游转录调控因子筛选及鉴定

刘振宁1,2,3,袁黎2,4,VenkatesanSundaresan2,余小林3()   

  1. 1. 临沂大学农林科学学院,临沂 276000
    2. 加州大学戴维斯分校植物科学系,加利福尼亚州戴维斯 95616,美国
    3. 浙江大学农业与生物技术学院,杭州 310058
    4. 西北农林科技大学园艺学院,杨凌 712100
  • 收稿日期:2018-11-21 修回日期:2019-02-28 出版日期:2019-05-20 发布日期:2019-04-02
  • 通讯作者: 余小林 E-mail:xlyu@zju.edu.cn
  • 作者简介:刘振宁,博士,讲师,研究方向：植物生殖发育。E-mail: liuzhenning@lyu.edu.cn
  • 基金资助:
    国家自然科学基金项目(31700272);国家自然科学基金项目(31460521);国家自然科学基金项目(31872110);山东省自然科学基金项目(ZR2017PC012);浙江省农业新品种选育重大科技专项重点项目子课题(2016C02051-6-1)

Screening and identification of CKI1 upstream transcription regulators in Arabidopsis

Zhenning Liu1,2,3,Li Yuan2,4,Venkatesan Sundaresan2,Xiaolin Yu3()   

  1. 1. College of Agriculture and Forestry Sciences, Linyi University, Linyi 276000, China
    2. Department of Plant Biology, University of California, Davis CA 95616, USA
    3. Institute of Vegetable Sciences, Zhejiang University, Hangzhou 310058, China
    4. College of Horticulture, Northwest A & F University, Yangling 712100, China;
  • Received:2018-11-21 Revised:2019-02-28 Online:2019-05-20 Published:2019-04-02
  • Contact: Yu Xiaolin E-mail:xlyu@zju.edu.cn
  • Supported by:
    National Natural Science Foundation of China(31700272);National Natural Science Foundation of China(31460521);National Natural Science Foundation of China(31872110);the Natural Science Foundation of Shandong Province(ZR2017PC012);the Breeding Project of the Sci-tech Foundation of Zhejiang Province(2016C02051-6-1)

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摘要：

拟南芥CKI1 (cytokinin independent 1)是双组分信号系统中一个组氨酸激酶蛋白,通过作用于下游组氨酸磷酸转移蛋白激活双组分信号通路,在调控胚囊中央细胞命运分化和发育过程中具有重要作用。然而目前对于CKI1基因上游转录调控因子还知之甚少。本研究分析了不同长度的CKI1启动子在拟南芥胚囊中的活性,并利用酵母单杂交技术对CKI1上游转录调控因子进行了筛选和鉴定。结果表明,位于内含子区域中的F5/R2片段表现出与CKI1启动子全长相一致的表达活性。进一步选取3个串联重复的F5/R2片段用于构建诱饵表达载体,同时,选取拟南芥雌蕊构建cDNA文库,通过酵母单杂交筛选获得226个阳性克隆。去除低质量及冗余重复的序列后共获得66条可读序列,其中8条序列对应的基因编码具有DNA结合功能的蛋白。研究结果为进一步揭示CKI1基因的转录调控机制提供了重要参考信息。

关键词: CKI1, 转录调控, 酵母单杂交, 拟南芥

Abstract:

Arabidopsis CKI1 (cytokinin independent 1) is a histidine kinase protein involved in the two-component system, which can activate two-component signaling via the downstream histidine phospho-transfer proteins, playing the essential roles in central cell fate determination and development regulation in embryo sacs. However, studies on CKI1 upstream transcription regulators are still limited. In the present study, promoter activities with varying fragments were investigated, and CKI1 upstream transcription regulators were screened and identified by the yeast-one hybrid technique. Results indicated F5/R2 fragments located in the intron region showed promoter activities in embryo sacs, which is consistent with CKI1 full-length promoters. Then three tandem repeats of F5/R2 fragments were used to construct the bait expression vector, and Arabidopsis pistils were collected for cDNA library construction. Totally, 226 positive clones were screened by the yeast-one hybrid technique, 66 readable sequences were retrieved after removing sequences with low quality and redundant repeats, among which eight proteins could act as DNA-binding proteins. These results provided some important clues to study the molecular function of CKI1 in the transcription regulation network.

Key words: CKI1, transcription regulation, yeast-one hybrid, Arabidopsis thaliana