植物学报 ›› 2015, Vol. 50 ›› Issue (3): 363-371.DOI: 10.3724/SP.J.1259.2015.00363

• 技术方法 • 上一篇    下一篇

连续光照转暗培养联合药物处理实现衣藻细胞的高水平同步化

刘维仲1*, , 王洁茹1, 胡勇2   

  1. 1山西师范大学生命科学学院, 临汾 041004;
    2.首都师范大学生命科学学院, 北京 100048
  • 收稿日期:2014-12-25 修回日期:2015-03-19 出版日期:2015-05-01 发布日期:2015-04-08
  • 通讯作者: liuwzh@dns.sxnu.edu.cn
  • 基金资助:
    国家自然科学基金(No.31171167)和山西省自然科学基金(No.2009011044-1)

Highly Synchronized Chlamydomonas reinhardtii Cells by Dark-shifting a Continuous-light Culture Combined with Drugs

Weizhong Liu1*, Jieru Wang1, Yong Hu2   

  1. 1College of Life Sciences, Shanxi Normal University, Linfen 041004, China;
    2.College of Life Sciences, Capital Normal University, Beijing 100048, China
  • Received:2014-12-25 Revised:2015-03-19 Online:2015-05-01 Published:2015-04-08

摘要: 单细胞衣藻(Chlamydomonas)是光合作用和植物细胞周期等生物学过程研究的一个重要模式系统, 同步化培养是进行相关研究的必要手段。该研究探索了连续光照转暗培养联合细胞周期阻断剂实现莱茵衣藻(Chlamydomonas reinhardtii)细胞高水平同步化的新方法, 并利用流式细胞术对同步化程度进行了精确的分析。结果表明, 连续光照转暗培养或联合S期阻断剂可以使衣藻细胞同步化到G1期或G1/S期边界; 连续光照转暗培养联合M期阻断剂或者在“加入-释放”S期阻断剂后再加入M期阻断剂可以使衣藻细胞同步化到M期, 同步化水平可达80%。具体的同步化培养步骤要根据研究对象(特别是某些衣藻突变株系)的特性和研究目的确定。

Abstract: Chlamydomonas reinhardtii, a unicellular eukaryotic green alga, has been a powerful model system for understanding several fundamental biological processes such as photosynthesis and plant cell-cycle regulation. An artificial cell-cycle synchronization system is necessary for these studies. Here, we investigated a new method for synchronizing Chlamydomonas cell-wall-less cells, with which a high level of synchrony can be achieved. The degree of synchrony was analysed by flow cytometry. The cells are grown asynchronously in continuous light, then grown for 24 h in the dark (dark-shifting a continuous-light culture). At the end of the dark period, most cells (>80%) are arrested in the G1 phase. If necessary, an S-phase blocking agent can be used to treat the dark-shifted cells. When the dark-shifted cells are treated with M-phase blocking agent alone or with the S-phase and M-phase blocking agent sequentially, a cell population with more than 80% of cells at the M phase can be achieved. A specific synchronous procedure needs to be selected according to the properties of a certain Chlamydomonas strain (especially certain mutants) and the requirement of research.