含草甘膦抗性标记的水稻核不育繁殖系载体构建与验证

【目的】使用草甘膦抗性基因替代潮霉素抗性基因,构建核不育繁殖系的转化载体,赋予繁殖系对除草剂草甘膦的抗性,规避现行政策对转基因作物中使用抗生素标记基因的限制。【方法】利用同源重组的方法,构建含草甘膦抗性基因Epsps^#、花粉致死基因ZmAA1、核不育恢复基因OsEat1和红色荧光基因DsRed2的水稻eat1核不育基因繁殖系载体pC3300-Epsps-AA-Eat-Red;采用农杆菌介导法转化水稻。【结果】通过转化籼稻品系9K19-5获得普通核不育繁殖系Eat9K的转化植株318个。表型鉴定表明,单拷贝转化体Eat9K-3的花粉有可育和不育2种类型,种子有具有和不具有荧光信号2种类型,具有荧光信号的种子在发芽期能耐受浓度至少为1 g/L的草甘膦。建立的四引物检测法能够区分野生型Eat1基因和核不育eat1基因。【结论】证明载体pC3300-Epsps-AA-Eat-Red有效,创制了具有除草剂抗性的普通核不育繁殖系新种质,建立了区分野生型Eat1基因和核不育eat1基因的四引物检测法。

Construction and Verification of Vector Containing Glyphosate Resistance Selection Marker for Multiplication of Common Genic Male Sterile Lines in Rice

【Objective】 The hygromycin resistance gene is often used as a selection marker in expression vectors for multiplication of common genic male sterile (CGMS) lines in rice. The glyphosate resistance gene replaces the hygromycin resistance gene as it not only gives the multiplication line glyphosate resistance, but also bypasses the current policy restrictions on the use of antibiotic marker genes in genetically modified crops. 【Methods】 An expression vector pC3300-Epsps-AA-Eat-Red was constructed by using homologous recombination method in order to multiply the rice CGMS line with eat1 mutation gene, which harbors the glyphosate resistance gene Epsps^#, pollen lethal gene ZmAA1, CGMS restorer gene OsEat1 and red fluorescent gene DsRed2. Agrobacterium-mediated transformation method was adopted. 【Results】 Transformation of indica rice line 9K19-5 produced 318 regenerated plants of the CGMS multiplication line Eat9K that could restore the sterility of eat1 and resist glyphosate. The phenotype identification of single copy transformant Eat9K-3 showed that: there were two kinds of pollens in anthers, one was fertile and the other was sterile; the seeds included two types, one was with fluorescence and the other was without fluorescence; the seedlings from fluorescent seeds tolerated at least 1 g/L glyphosate. In addition, this study established a four-primer detection method to distinguish the wild-type gene Eat1 from the genic male sterile gene eat1. 【Conclusion】 This study not only verified the effectiveness of the expression vector, but also created the new germplasm of CGMS multiplication line with herbicide resistance, as well as established a four-primer detection method to distinguish the wild-type gene Eat1 from genic male sterile gene eat1.