共转化获得无抗性标记的转反义蜡质基因籼稻

Transgenic Plants of Markerless Antisense Waxy Indica Rice Obtained by Agrobacterium-Mediated Co-transformation

【目的】利用转基因技术将反义蜡质基因导入籼稻成熟胚愈伤组织中,改良籼稻稻米直链淀粉含量。【方法】用根癌农杆菌介导的方法将p13W8质粒(含反义蜡质基因)和pCAMBIA1300质粒(含抗潮霉素抗性基因)对2个籼稻品种的成熟胚愈伤组织进行遗传共转化。【结果】供试材料愈伤组织继代培养18～24d后具有较高的转化频率和分化频率,可作为共转化的良好受体;抗性愈伤组织PCR检测结果表明,抗潮霉素抗性基因的阳性检测率为97.6%,反义蜡质基因的阳性检测率为27.1%。在46株转基因潮霉素抗性植株中,反义蜡质基因阳性株为5株,占转基因植株的10.9%。经PCR检测,发现上述4个株系T1群体中发生抗潮霉素抗性基因和反义蜡质基因片段的分离,从286个T1单株中筛选到了39个单株只带反义蜡质基因片段,而无潮霉素抗性基因的转基因植株,占T1单株总数的13.6%。部分转反义蜡质基因植株种子的直链淀粉含量有不同程度的降低。【结论】通过农杆菌介导的遗传共转化方法,将反义蜡质基因Waxy导入籼稻成熟胚愈伤组织,获得了直链淀粉含量明显降低的仅含反义蜡质基因籼稻株系。

[Objective] The antisense Waxy gene was transformed into the mature indica rice embryo calli by the transgenic technology in order to improve the amylose content in indica rice. [Method] The plasmid p13W8 carrying antisense fragment of Waxy gene and plasmid pCAMBIA1300 containing hpt gene were introduced into the calli derived from mature indica rice embryo by Agrobacterium tumefaciens-mediated co-transformation. [Result] The results showed that the calli from the seed embryo and subcultured for 18-24 d had higher transformation and differentiation frequency and could provide the acceptor for the Agrobacterium-mediated co-transformation, hpt-positive frequency was 97.6% and Waxy-positive frequency was 27.1% in the hygromycin-resistant calli by PCR analysis. Five plants showed positive bands for Waxy gene by PCR analysis in 46 transgenic hpt-positive plants, the Waxy-positive plants frequency was 10.9%. The segregation of antisense fragment of Waxy gene and hpt gene was observed by PCR analysis in four Tl population. Thirty-nine plants containing only the antisense fragment of Waxy gene were identified in 286 T1 plants, the Waxy-positive plant frequency was 13.6%. The amylose content of some seeds derived from transgenic plants with only the antisense fragment of Waxy gene was found with different varying degree of reduction. [ Conclusion ] The indica rice plants only with antisense Waxy gene and amylose content decreasing significantly were obtained by Agrobacterium tumefaciens-mediated co-transformation.