遗传 ›› 2007, Vol. 29 ›› Issue (4): 499-499―507.doi: 10.1360/yc-007-0499

• 技术与方法 • 上一篇    下一篇

带npt-Ⅱ基因转基因水稻快速检测技术的建立

王紫萱, 易自力, 王志成, 蒋建雄, 覃静萍, 张昊, 谭炎宁   

  1. 湖南农业大学细胞工程重点实验室, 长沙 410128

  • 收稿日期:2006-05-12 修回日期:2006-12-18 出版日期:2007-04-10 发布日期:2007-04-10
  • 通讯作者: 易自力

Quick testing-technology of transgenic rice with npt-Ⅱ screen marker gene

WANG Zi-Xuan, YI Zi-Li, WANG Zhi-Cheng, JIANG Jian-Xiong, QIN Jing-Ping, ZHANG Hao, TAN Yan-Ning   

  1. Key Laboratory of Cell Engineering, Hunan Agricultural University, Changsha 410128, China
  • Received:2006-05-12 Revised:2006-12-18 Online:2007-04-10 Published:2007-04-10
  • Contact: YI Zi-Li

摘要:

以转溶菌酶基因水稻纯系材料中花9号(ZH9(R))及其受体品种中花9号(ZH9(CK))为材料, 以ZH9(R)中携带的npt-Ⅱ基因作为辅助筛选标记, 利用抗生素对其进行处理, 建立了一套快速检测带npt-Ⅱ基因转基因水稻的技术体系。使用不同浓度的卡那霉素(Kanamycin, Kan)和G418溶液将ZH9(R)和ZH9(CK)成株离体叶片于室内室温下置于培养皿中浸泡处理, 通过观察叶片变化, 确定G418为检测带npt-Ⅱ基因转基因水稻的最佳抗生素, 将G418(溶液)80 mg/L浓度(处理4天)作为检测该类转基因水稻成株离体叶片的临界浓度。进一步用G418对ZH9(R)和ZH9(CK)种子、幼胚和幼苗进行了不同处理。确定: (1) G418(溶液)300 mg/L浓度(处理7天)作为检测该类转基因水稻种子的临界浓度; (2) G418(1/2 MS+0.5 mg/L 6-BA+1.5%蔗糖培养基)200 mg/L浓度(处理10天)作为检测该类转基因水稻幼胚的临界浓度; (3) G418(1/2 MS+0.5 mg/L 6-BA+1.5%蔗糖培养基)150 mg/L浓度(处理12天)作为检测该类转基因水稻幼苗的临界浓度, 并且通过PCR方法证实了上述结论。将这些结论应用于ZH9(R)转育后代叶片、种子、幼胚和幼苗群体的检测, 检测效果都非常明显。这为带npt-Ⅱ基因转基因水稻建立了一套简便、直观且准确的检测方法。

关键词: G418, 快速检测, npt-Ⅱ基因, 转基因水稻

Abstract:

By using npt-Ⅱgene as assistant selection-marker, treating Japonica rice Zhonghua 9 [ZH9 (CK)] and transferred lysozyme gene rice (the donor rice is Japonica rice Zhonghua 9) [ZH9(R)] with antibiotics, we built a system of quickly testing transgenic rice offspring. Detached leaves of ZH9(R) and ZH9(CK) were treated by Kanamycin (0, 400, 450, 500, 550, 600 and 700 mg/L) and G418 (0, 40, 60, 80, 100, 150 and 200 mg/L) with different concentrations. The result show effect of Kanamycin is not evident and G418 is the best antibiotics to test transgenic rice with npt-Ⅱ gene. The data showed that 80 mg/L G418 (treating 4 d) was optimal to test transgenic rice. Further study was done with seeds, young embryos and seedlings by G418 testing: the alive seeds were cultured in the culture dishes which filled with a series of G418 solution with different concentration of 0, 100, 150, 200, 250, 300 and 350 mg/L; in the tissue culture room, the germinating embryos were inoculated in the culture medium (1/2 MS+0.5 mg/L 6-BA+1.5% sucrose) which contains G418 with 0, 150, 200, 250 and 300 mg/L concentration; the aseptic seedlings were inoculated in the the same culture medium (1/2 MS+0.5

mg/L 6-BA+1.5% sucrose) which contains G418 with 0, 100, 150, 200 and 250 mg/L concentration. The conclusions indi-cated that 300 mg/L (treating 7 d) was the critical concentration to test seeds of transgenic rice; 200 mg/L (treating 10 d) was the critical concentration to test young embryo of transgenic rice; 150 mg/L (treating 12 d) was the critical concentra-
tion to test seedlings of transgenic rice. Two primers were designed based on npt-Ⅱ and lysozyme gene sequences. PCR technology confirmed the above detection system. The preliminary results showed npt-Ⅱwas tightly linked with lysozyme
gene. Above confirmed critical concentrations were applied to test detached leaves, seeds, young embryos and seedlings of transferred generations. The effect was very obvious. It is convenient, intuitionistic, and exact way that aparting the positive plants from the mixture of transgenic positive and negative plants with G418.