植物学报 ›› 2017, Vol. 52 ›› Issue (4): 511-519.DOI: 10.11983/CBB16155

• 技术方法 • 上一篇    下一篇

非洲菊原生质体制备及瞬时转化系统的建立

宋爱华1, 张文斌1, 孙姝兰1, 李凌飞1,2,*(), 王小菁1   

  1. 1华南师范大学生命科学学院, 广东省植物发育生物工程重点实验室, 广州 510631
    2深圳市中国科学院仙湖植物园, 深圳市南亚热带植物多样性重点实验室, 深圳 518004
  • 收稿日期:2016-07-22 接受日期:2016-10-16 出版日期:2017-07-01 发布日期:2017-05-05
  • 通讯作者: 李凌飞
  • 作者简介:

    # 共同第一作者

  • 基金资助:
    基金项目: 国家自然科学基金(No.31372099, No.31601784)、教育部高等学校博士学科点专项科研基金(No.20104407110005)和广东省自然科学基金(No.9251063101000002)

Preparation of Protoplast and Establishment of Transient Expression System in Gerbera hybrida

Aihua Song1, Wenbin Zhang1, Shulan Sun1, Lingfei Li1,2*, Xiaojing Wang1   

  1. 1Provincial Key Laboratory of Biotechnology for Plant Development, School of Life Sciences, South China Normal University, Guangzhou 510631, China
    2Key Laboratory of Southern Subtropical Plant Diversity, Fairy Lake Botanical Garden, Shenzhen & Chinese Academy of Sciences, Shenzhen 518004, China;
  • Received:2016-07-22 Accepted:2016-10-16 Online:2017-07-01 Published:2017-05-05
  • Contact: Li Lingfei
  • About author:

    # Co-first authors

摘要:

非洲菊(Gerbera hybrida)已成为研究复杂花序进化与发育的模式植物。以非洲菊无菌组培苗叶片为材料, 分析不同浓度纤维素酶及离析酶配比对非洲菊原生质体提取效率的影响, 建立了稳定、高效的非洲菊原生质体提取与瞬时转化体系。结果表明, 以2.0%纤维素酶+0.3%离析酶组合, 在0.4 mol·L-1甘露醇溶液中, 酶解4小时, 酶解温度为25°C, 得到产量高达2.2×107个·g-1 FW及活力较高的原生质体, 可直接用于植物蛋白亚细胞定位和蛋白间相互作用实验, 转化效率达80%。该研究建立了非洲菊原生质体制备和瞬时转化体系, 为非洲菊基因功能研究奠定重要的技术基础。

Abstract:

Gerbera hybrida has been used as model plant to study the evolution and development of composite inflorescences. To establish an efficient protoplast isolation and transient transformation system in G. hybrida, leaves of cultivar ‘Shenzhen No. 5’ in vitro seedlings were used as materials and the effects of different concentrations of cellulase R10 and macerozyme were tested to screen a suitable preparation method. The best enzyme solution concentration consisted of cellulase R10 2.0% (w/v), macerozyme 0.3% (w/v), and 0.4 mol·L-1 mannitol and more than 2.2×107∙g-1 FW protoplasts with high activity were obtained after 4 h treatment under 25°C condition. Subcellular localization and protein-protein interaction assays were performed using these protoplasts and revealed 80% transformation rate. Collectively, this study established an efficient protoplast isolation and transient assay system that can provide an important platform to facilitate gene function studies in G. hybrida.