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带npt-Ⅱ基因转基因水稻快速检测技术的建立

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

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【作者】 王紫萱易自力王志成蒋建雄覃静萍张昊谭炎宁

【Author】 WANG Zi-Xuan, YI Zi-Li, WANG Zhi-Cheng, JIANG Jian-Xiong, QIN Jing-Ping, ZHANG Hao, TAN Yan-Ning Key Laboratory of Cell Engineering, Hunan Agricultural University, Changsha 410128, China

【机构】 湖南农业大学细胞工程重点实验室湖南农业大学细胞工程重点实验室 长沙410128长沙410128

【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)80mg/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)300mg/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(1/2MS+0.5mg/L6-BA+1.5%蔗糖培养基)200mg/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(1/2MS+0.5mg/L6-BA+1.5%蔗糖培养基)150mg/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。

【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+0.5mg/L 6-BA+1.5% sucrose) which contains G418 with 0, 100, 150, 200 and 250 mg/L concentration. The conclusions indicated 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.

【关键词】 转基因水稻npt-Ⅱ基因G418快速检测
【Key words】 Trangenic ricenpt-Ⅱ geneG418testing quickly
【基金】 湖南省教育厅科学研究重点资助项目(编号:03A016);教育部科学研究重点资助项目(编号:204016)资助~~
  • 【分类号】S511
  • 【被引频次】28
  • 【下载频次】568
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