草甘膦为筛选标记的水稻高效遗传转化体系的建立

Establishment of an Efficient Genetic Transformation System for Rice with Glyphosate as a Selection Marker

【目的】建立以日本晴愈伤组织为受体、草甘膦抗性基因CP4为选择标记基因的高效水稻遗传转化体系。【方法】以粳稻日本晴的成熟胚为试验材料,通过农杆菌介导法,将含有草甘膦抗性基因CP4的P1300载体转化日本晴的愈伤组织,分别用质量浓度为10、20和40 mg/L的草甘膦进行3轮抗性筛选,诱导分化和再生成苗;利用1000 mg/L草甘膦对移栽成活的阳性苗进行抗性鉴定。【结果】获得145株转基因幼苗,PCR检测阳性株为128株,阳性率达88.27%;经过3轮抗性筛选后,转基因幼苗的阳性率提高;移栽成活的99株阳性苗中,65株表现为抗性,抗性率为65.66%。【结论】成功建立了以草甘膦为筛选标记的水稻转基因体系,该体系为水稻育种提供了材料,并对以抗草甘膦基因作为筛选标记的水稻转基因和一些基因功能验证提供了技术支持。

[Purpose]To establish an efficient rice genetic transformation system with Nipponbare as the receptor and glyphosate resistance gene(CP4)as the selection marker for transformation research.[Method]Using mature embryos of japonica rice Nipponbare as experimental materials,the P1300 vector containing the glyphosate resistance gene CP4 was transformed into the callus of Nipponbare by Agrobacterium-mediated method,with a concentration of 10,20 and 40 mg/L glyphosate were subjected to three rounds of resistance screening,differentiation induction and seedling formation.l 000 mg/L glyphosate was used to identify the resistance of the positive seedlings transplanted alive.[Result]145 transgenic seedlings were obtained.After PCR detection,128 positive strains were obtained,the positive rate was 88.27%.Experiments proved that after three concentration gradients screening,the positive rate of genetically modified seedlings increases.Among the 99 positive seedlings transplanted alive,65 strains showed resistance,and the resistance rate was 65.66%.[Conclusion]We successfully established a rice transgenic system using glyphosate as a screening marker.The system provided materials for rice breeding and provided technical support for rice trans-genes using glyphosate as a screening marker and functional verification of some genes.