Chinese Journal of Rice Science

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Rapidly Obtaining the Marker-Free Transgenic Rice with Three Target Genes by Co-Transformation and Anther Culture

ZHU Li , FU Ya-ping , LIU Wen-zhen , HU Guo-cheng , SI Hua-min , TANG Ke-xuan , SUN Zong-xiu   

  • Received:1900-01-01 Revised:1900-01-01 Online:2007-09-10 Published:2007-09-10
  • Contact: SUN Zong-xiu

利用共转化和花药培养技术快速获得无选择标记的三价转基因水稻

朱丽1, #;傅亚萍1, #;刘文真1;胡国成1;斯华敏1;唐克轩2;孙宗修1,*   

  1. 1中国水稻研究所 水稻生物学国家重点实验室, 浙江 杭州 310006; 2上海交通大学 农业与生物学院 植物生物技术研究中心, 上海 200030; # 共同第一作者; *通讯联系人, E-mail: sunzx405@tom.com

Abstract: The "double T-DNA" binary vector p13HSR which carried two independent T-DNAs,containing hygromycin phosphotransferase gene(hpt) in one T-DNA region and three target genes(hLF,SB401,RZ10) in the other T-DNA region,was used to generate selectable marker-free transgenic rice by Agrobacterium-mediated transformation.T0 plants with the three target genes and hpt gene were selected for anther culture.It took only one year to obtain double haploid selectable marker-free transgenic plants containing the three target genes through co-transformation followed by anther culture,with an efficiency of 9.87%.RT-PCR analysis indicated that the target genes were inserted into rice genomic DNA and successfully transcripted.It was noted that one or two target genes derived from the binary vector were lost in some transgenic rice plants.

Key words: anther culture, co-transformation, selectable marker-free, transgenic rice, double T-DNAs

摘要: 采用农杆菌介导的水稻转化体系,将包含选择标记潮霉素磷酸转移酶基因(hpt)和目的基因人乳铁蛋白(hLF)、高赖氨酸(SB401)、高甲硫氨酸(RZ10)基因的双TDNA表达载体p13HSR转化水稻。筛选潮霉素磷酸转移酶基因和目的基因都是阳性的转基因植株,按单株进行花药培养,快速得到无选择标记而目的基因阳性的转基因纯合植株,得率为987%。RTPCR检测结果显示外源基因已整合到转基因水稻基因组中并转录。同时观测到三价表达载体在转化过程中部分目的基因丢失。

关键词: 花药培养, 共转化, 无选择标记, 转基因水稻, 双T-DNA结构