共转化法获得无筛选标记的转PEPC、PPDK基因水稻恢复系纯合体

Generating Marker-free Transgenic Homozygous Rice Restorer Lines with PEPC and PPDK Genes by Agrobacterium-mediated Transformation Using Super Binary Vector

用根癌农杆菌共转化的方法将pSB130/CK质粒[目的基因磷酸烯醇式丙酮酸羧化酶基因(PEPC)、磷酸丙酮酸二激酶基因(PPDK)与筛选标记潮霉素抗性基因HPT分别构建于2个T-DNA区]导入水稻恢复系R299,经PCR、Southern检测,筛选到T0代13个转基因植株。对T1～T3代转基因后代进行PCR跟踪鉴定,分离出无潮霉素抗性基因的转基因植株(PEPC+PPDK+HPT-),分离株系比率为15%～21%,分离株系中PEPC+PPDK+HPT-型单株分离频率为7.2%～10.0%。在T3代成功获得了无筛选标记的转基因纯合系3个(06D351,06D352和06D353),PEPC酶活性和光合速率分析结果表明,其PEPC活性比对照提高了1.5～4.9倍,光合速率提高了19%～40%,说明这些转基因纯系材料在水稻高光效育种上具有很大的应用价值。

Transgenic restorer lines of hybrid rice expressing PEPC and PPDK genes from maize were obtained by the method of Agrobacterium tumefociens mediated transformation using super binary vector pSB130/CK. PCR and Southern blotting analysis showed that three transgenic homozygous lines with PEPC and PPDK genes but HPT gene free （06D351, 06D352 and 06D353） were identified in T3 generation. From T1 to T3 generations, the proportions of segregated lines with the target genes but HPT free were 15% ～ 21% and those of segregated plants with the gene type of PEP- C^＋ PPDK^＋ HPT^- were 7.2% - 10.0% among the segregated lines. Primary results showed that the photosynthesis rate and the PEPC enzyme activity of the transgenic homozygous lines increased by 19% -40% and 1.5 to 4.9 times, respectively, compared with those of the wild type, indicating that these transgenic rice lines are of the great potential values in increasing photosynthesis for hybrid rice breeding.