植物学报 ›› 2019, Vol. 54 ›› Issue (1): 72-80.DOI: 10.11983/CBB18118

• 技术方法 • 上一篇    下一篇

铁棍山药微型块茎遗传转化体系的建立

李俊华1,2,3,,刘世宇1,,李成龙1,韩林林1,董亚辉1,张晓丽1,2,3,赵喜亭1,2,3,李明军1,2,3,*()   

  1. 1 河南师范大学生命科学学院, 新乡 453007
    2 河南省道地药材保育及利用工程技术研究中心, 新乡 453007
    3 绿色药材生物技术河南省工程实验室, 新乡 453007
  • 收稿日期:2018-05-15 接受日期:2018-09-13 出版日期:2019-01-01 发布日期:2019-07-31
  • 通讯作者: 李俊华,刘世宇,李明军
  • 基金资助:
    国家自然科学基金(81274019);国家中医药管理局中医药行业科研专项子课题(201407005-08);河南省创新型科技人才队伍建设工程(C20130037)

Establishment of a Genetic Transformation System for Dioscorea opposita Using Microtuber

Junhua Li1,2,3,,Shiyu Liu1,,Chenglong Li1,Linlin Han1,Yahui Dong1,Xiaoli Zhang1,2,3,Xiting Zhao1,2,3,Mingjun Li1,2,3,*()   

  1. 1 College of Life Science, Henan Normal University, Xinxiang 453007, China
    2 Engineering Technology Research Center of Nursing and Utilization of Genuine Chinese Crude Drugs of Henan Province, Xinxiang 453007, China
    3 Engineering Laboratory of Green Medicinal Material Biotechnology of Henan Province, Xinxiang 453007, China
  • Received:2018-05-15 Accepted:2018-09-13 Online:2019-01-01 Published:2019-07-31
  • Contact: Junhua Li,Shiyu Liu,Mingjun Li

摘要: 怀山药(Dioscorea opposita)遗传转化是对其进行基因功能分析和遗传改良的基础, 但目前国内外尚未见相关报道。以怀山药优良品种铁棍山药(D. opposita cv. ‘Tiegun’)的微型块茎为受体材料, 对影响遗传转化的因素进行优化, 建立了由根癌农杆菌介导的山药遗传转化体系。过表达质粒载体pCAMBIA1301-DoSERK2GUS标记基因和潮霉素(Hyg)抗性筛选基因, 沉默质粒载体pART27-DoSERK2含卡那霉素(Kan)抗性筛选基因。根癌农杆菌抑制剂特美汀(Tim)的最佳浓度为500 mg·L -1; 再生芽和生根时, Hyg的最佳浓度分别为15和20 mg·L -1, Kan的最佳浓度分别为120和160 mg·L -1。对转化植株进行PCR和GUS组织化学检测, 结果显示外源基因已整合到铁棍山药转基因株系的基因组中并在细胞中表达。该研究建立了一套取材便利的铁棍山药遗传转化方法, 对其它品种山药的转化也具有参考价值。

关键词: 根癌农杆菌, 遗传转化, 微型块茎, 铁棍山药

Abstract: Genetic transformation system is the foundation for gene function study and genetic improvement of Dioscorea opposita. In this study, we established an Agrobacterium-mediated genetic transformation system using microtuber in D. opposita cv. ‘Tiegun’. The transformation was performed with the DoSERK2 gene overexpression plasmid pCAMBIA1301-DoSERK2 that harboring the GUS reporter gene and the hygromycin (Hyg) resistence gene as the selectable marker, and the gene-silencing vector pART27-DoSERK2 that containing kanamycin (Kan) resistence marker. The optimal concentration of timentin (Tim) for Agrobacterium counter selection was 500 mg·L -1. For shoot regeneration and rooting, the optimal concentrations of Hyg were 15 mg·L -1 and 20 mg·L -1, respectively, and the optimal concentration of Kan were 120 mg·L -1 and 160 mg·L -1, respectively. Results of PCR analysis and GUS histochemical staining indicated that the exogenous genes had been integrated into the genome of transgenic plants, and are expressed in the plants. Our study provides a genetic transformation system in D. opposita cv. ‘Tiegun’, which has the advantage that explants are easy available. This method has reference value for the genetic transformation of other varieties of D. opposita.

Key words: Agrobacterium tumefaciens, genetic transformation, microtuber, Dioscorea opposita cv. ‘Tiegun’