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中国农学通报

中国农学通报 ›› 2012, Vol. 28 ›› Issue (31): 15-20.

所属专题： 生物技术； 园艺

• 林学 园艺 园林 • 上一篇    下一篇

根癌农杆菌介导的巴西橡胶树胚性悬浮细胞的遗传转化

于波 吴华玲 黄天带 戴雪梅   

  • 收稿日期:2012-02-07 修回日期:2012-05-18 出版日期:2012-11-05 发布日期:2012-11-05
  • 基金资助:

    巴西橡胶树胚性悬浮细胞遗传转化体系的建立和焦磷酸酶在乳管中的高效表达

Agrobacterium tumefaciens-mediated transformation using embryogenic suspension cultures in rubber tree (Hevea brasiliensis Mu?ll. Arg)

  • Received:2012-02-07 Revised:2012-05-18 Online:2012-11-05 Published:2012-11-05

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858

摘要：

为了建立橡胶树胚性悬浮细胞遗传转化体系，用根癌农杆菌EHA105（携带pCAMBIA1301，含有gusA和hpt Ⅱ）侵染胚性悬浮细胞团，用GUS检测的方法分析了共培养时间和菌液浓度对转化效率的影响；同时，测定了胚性细胞团对潮霉素敏感性。结果表明适宜的共培养时间为3天，适宜的菌液浓度为OD600=0.4~0.6。潮霉素的致死剂量为15 mg/L。使用大约1 g鲜重的胚性细胞团，经过转化和选择培养，获得53块抗性愈伤组织，经诱导获得7个体细胞胚。GUS检测和PCR鉴定证实了gusA基因已经整合到橡胶树基因组中。本研究为根癌农杆菌介导的橡胶树胚性悬浮细胞遗传转化体系建立奠定了基础。

关键词: 吸肥规律, 吸肥规律

Abstract:

In order to establish transformation system based on embryogenic suspension cultures in rubber tree (Hevea brasiliensis Mu?ll. Arg), cell aggregates from embryogenic suspension cultures were co-cultivated with the A. tumefaciens strain EHA105 harboring a binary vector pCAMBIA1301 with gusA and hpt II genes. Effect of co-cultivation time and A. tumefaciens concentration on transient expression of gusA was examined by GUS assay. The sensitivity of cell aggregates to hygromycin (Hyg) added to the medium was also tested in order to determine the optimal concentration of this antibiotic in the selective medium. The results suggested that 3 days was an optimal co-cultivation time, a desired OD600 of strain EHA105 was 0.4-0.6, and 15 mg/L concentration of Hyg was found to be enough to inhibit the growth of cell aggregates. A total of 53 Hyg-resistant embryogenic calli were obtained after transformation and co-cultivation. At last, 7 somatic embryos were further formed. GUS assay and PCR analysis indicated that gusA had been integrated into the genome of rubber tree. In all, this study laid the groundwork for efficient Agrobacterium tumefaciens mediated transformation using embryogenic suspension cultures in rubber tree.

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