洁净DNA转化获得2mG2-epsps基因单拷贝整合的抗草甘膦水稻

Generation of Glyphosate-resistant Transgenic Rice Harboring Single Copy of 2mG2-epsps Gene by Clean DNA Transformation

洁净DNA转化是基因枪介导外源基因表达框导入植物的转化技术,能从根本上消除载体框架序列对转基因植株的不利影响。2mG2-epsps基因是具有重要育种价值的草甘膦除草剂抗性基因。以日本晴为材料,研究了草甘膦对水稻愈伤组织生长及分化的影响,采用洁净DNA转化技术将2mG2-epsps基因表达框导入水稻。结果表明：1）草甘膦对水稻愈伤组织的生长及分化有明显的抑制作用,当草甘膦浓度为2mmol/L时,愈伤组织绿苗分化率为18.97%,较对照71.67%显著降低。2）基因枪介导2mG2-epsps基因表达框转化水稻时,经草甘膦筛选获得抗性愈伤后,在植株再生培养基中去除筛选剂利于抗性愈伤的分化,转化率为17.20%。经Southern杂交分析,2mG2-epsps基因表达框均以单拷贝整合到受体基因组。52.17%（12/23）转基因株系可耐受12~50mmol/L的草甘膦。

Clean DNA transformation is the technology of introducing exotic gene expression cassette into plant genome via particle bombardment, which can eliminate the disadvantageous impact of vector backbone sequence on transgenic plant fundamentally. Gene 2mG2-epsps is an important glyphosate herbicide resistance gene with important breeding value. The effect of glyphosate on rice callus growth and differentiation was studied using japonica rice Nipponbare as material, and 2mG2-epsps gene cassette was transformed into rice by clean DNA transformation. Results showed.. 1） The growth and differentiation of rice callus can be notably inhibited by glyphosate. The regeneration frequency of green plantlet decreased significantly to 18.97%, comparing with that of the control 71.67%, at 2 mmol/L glyphosate; 2） During rice transformation with 2mG2-epsps gene expression cassette via particle bombardment, removal of the screening agent from regeneration medium is beneficial to the differentiation of the glyphosate resistant calli, which were screened out under glyphosate selection with transformation frequency at 17.20 %. Southern blot analysis revealed that the 2mG2-epsps gene cassette was all integrated into rice genome in single copy and 52.17% （12/23）transgenie lines can render 12--50 mmol/L glyphosate. The present research provided the foundation for breeding appliance of the glvphosate resistant transgenic rices.