植物学报 ›› 2018, Vol. 53 ›› Issue (1): 59-71.DOI: 10.11983/CBB16257

• 研究报告 • 上一篇    下一篇

优化子叶节转化法培育大豆MtDREB2A转基因植株

吴国栋, 修宇, 王华芳*()   

  1. 北京林业大学生物科学与技术学院, 林木育种国家工程实验室, 林木花卉遗传育种教育部重点实验室, 北京 100083
  • 收稿日期:2016-12-23 接受日期:2017-05-04 出版日期:2018-01-01 发布日期:2018-08-10
  • 通讯作者: 王华芳
  • 基金资助:
    转基因生物新品种培育重大专项(No.2009ZX08009-089B-4)

Breeding of MtDREB2A Transgenic Soybean by an Optimized Cotyledonary-Node Method

Guodong Wu, Yu Xiu, Huafang Wang*()   

  1. Key Laboratory of Genetics and Breeding in Forest Trees and Ornamental Plants of Ministry of Education, National Engineering Laboratory for Tree Breeding, College of Biological Sciences and Biotechnology, Beijing Forestry University, Beijing 100083, China
  • Received:2016-12-23 Accepted:2017-05-04 Online:2018-01-01 Published:2018-08-10
  • Contact: Huafang Wang

摘要: 将正交因素试验与GUS基因组织化学染色等技术相结合, 优化大豆(Glycine max)品种东农50遗传转化体系, 导入抗旱关键基因MtDREB2A。结果表明, 大豆种子表面消毒, NaClO溶液法与Cl2气熏蒸法的去污染率分别达到98.67%和93.33%。子叶节法转GUS基因组织化学染色率(68.33%)显著高于下胚轴法(14.00%)和胚尖法(0.67%) (P<0.05)。种子萌发5天, 农杆菌(Agrobacterium tumefaciens)培养温度25°C, OD600=0.9, 共培养5天的转GUS基因子叶节最高达72.00%; 恢复培养5天, 草丁膦(3 mg·L-1)、头孢噻肟钠(200 mg·L-1)和羧苄青霉素(300 mg·L-1)筛选诱导分化的转GUS基因不定芽最多为3.33%; 优化的大豆遗传转化体系转化效率为1.11%。转MtDREB2A基因大豆东农50植株根系更加密集, 主根长度和侧根数量均显著高于对照(P<0.05), 证实MtDREB2A基因具有促进大豆根系生长的作用, 为利用该基因进行大豆抗旱育种奠定了坚实的基础并提供了理论依据。

关键词: 大豆, 遗传转化体系优化, GUS基因, MtDREB2A基因, 根系改良

Abstract: Orthogonal factorial experiments and histochemical GUS staining were combined to optimize the genetic transformation system of Glycine max cv. ‘Dongnong 50’ and transfer the key gene MtDREB2A for drought resistance into the soybean. Sterile of soybean seeds used as explants with NaClO solution and Cl2 gas fumigation methods reached 98.67% and 93.33% germination, respectively. Histochemical staining rate of the tissues transformed with GUS by the cotyledon node method was 68.33%, significantly higher than that by the hypocotyl (14.00%) and embryo tip (0.67%) methods (P<0.05). The cotyledon node-transformed GUS gene was up to 72.00% in germinated sterile seeds for 5 days, mediated by Agrobacterium tumefaciens cultured at 25°C, OD600 0.9, and co-cultured for 5 days. The shoots were induced and differentiated with cotyledon node-transformed GUS up to 3.33% by optimal recovery culture for 5 days and were screened on culture medium containing phosphinothricin (3 mg·L-1), cefotaxime sodium (200 mg·L-1) and carbenicillin (300 mg·L-1). The transgenic efficiency was 1.11% with the optimized soybean genetic transformation system. The MtDREB2A transgenic plant roots of soybean ‘Dongnong 50’ were more dense and both taproot length and lateral root number were significantly longer and greater than those of the control (P<0.05). The study verified that the MtDREB2A gene plays a role in promoting soybean root growth, which lays a solid foundation and provides a theoretical basis for the gene using in drought resistance breeding of soybean.

Key words: soybean, optimization of gene transformation system, GUS, MtDREB2A, root enhancement