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林业科学 ›› 2009, Vol. 12 ›› Issue (5): 20-26.doi: 10.11707/j.1001-7488.20090503

• 论文 • 上一篇    下一篇

国槐遗传转化体系的优化*

张晓英1.2甘敬3 尹伟伦1 朱祯4 王华芳1   

  1. (1.北京林业大学生物科学与技术学院 北京100083;2.北京市琅山苗圃 北京100039; 3.北京市园林绿化局 北京100029; 4.中国科学院遗传与发育生物学研究所 北京100101)
  • 收稿日期:2008-12-23 修回日期:1900-01-01 出版日期:2009-05-25 发布日期:2009-05-25
  • 通讯作者: 王华芳

An effective Transformation System of Sophora japonica

Zhang Xiaoying1,2,Gan Jing3,Yin Weilun1,Zhu Zhen4,Wang Huafang1   

  1. (1.College of Biological Science and Biotechnology,Beijing Forestry University Beijing 100083;2.Beijing Langshan Nursery Beijing 100039; 3. Beijing Gardening and Greening Bureau Beijing 100029; 4. Institute of Genetics and Developmental Biology, Chinese Academy of Sciences Beijing 100101)
  • Received:2008-12-23 Revised:1900-01-01 Online:2009-05-25 Published:2009-05-25

摘要:

采用根癌农杆菌介导的国槐叶盘转化法,在建立修饰的豇豆胰蛋白酶抑制剂基因(sck)转化体系过程中,对影响农杆菌转化频率的各种因素进行研究。结果表明: 国槐叶片需在黑暗条件下在MS培养基上预培养5天,农杆菌菌株选用GV3101,农杆菌共培养3天较合适; 农杆菌菌液浓度OD600约为0.7,感染时间10 min; 侵染前的菌液和共培养基中添加200 μmol·L-1乙酰丁香酮(AS)对国槐遗传转化有明显的促进作用; 抑制农杆菌生长的抗生素浓度以头孢霉素(Cef)500?mg·L-1最好。PCR和Southern blotting检测证实 sck基因已整合到国槐基因组中。

关键词: 国槐, 根癌土壤农杆菌, 乙酰丁香酮, 遗传转化, 槐尺蠖, 抗虫基因

Abstract:

This paper investigated several factors affecting transformation frequency of Sophora japonica in establishing the modified sck gene transfer system process with Agrobacterium tumefaciens-mediated leaf disc transformation method. The results showed that S. japonica leaves were required to pre-culture on MS medium for 5 d in the dark. Agrobacterium strains, GV3101, were selected, and the Agrobacterium was needed to co-cultivation for 3 d. Agrobacterium bacterium concentration was about 0.7 in OD600, and the infection time was 10 min. Addition of 200μmol·L-1 acetosyringone (AS) to the co-culture medium before infection significantly promoted S. japonica transformation. Antibiotic, 500 mg·L-1 Cef, was best for inhibiting Agrobacterium growth. PCR and Southern blotting confirmed that sck gene had been integrated into the S. japonica genome.

Key words: Sophora japonica, Agrobacterium tumefaciens, acetosyringone, genetic transformation, Semiothisa cinerearia, insect-resistant genes