农杆菌介导的紫穗槐茎段愈伤组织遗传转化研究

Genetic Transformation through Stem Segments-Derived Callus of Amorpha fruticosa by Agrobacterium Tumefaciens

采用农杆菌介导法对紫穗槐茎段诱导的愈伤组织进行遗传转化研究。确定了卡那霉素临界耐受浓度为40mg/L。菌液浓度与侵染时间的正交试验结果表明:侵染菌液OD600为0.8、侵染时间为30min时转化效率最高,达17.95%。GUS染色检测分析表明:含pBI121-GUS质粒DNA农杆菌侵染转化的愈伤组织其抗性不定芽和再生植株根和叶均呈蓝色。转化植株叶PCR检测结果表明外源GUS基因已整合到紫穗槐基因组中。以上结果表明建立了以茎段诱导愈伤组织为转化受体、农杆菌介导的紫穗槐高效遗传转化体系。为了验证其可重复性,又进行了pBI121-GFP基因的转化,T0代再生植株PCR检测整合率达85%,在488nm蓝光源激发下转化株的顶芽产生绿色荧光,说明转入的基因在35S启动子下过量表达。此遗传转化体系可应用于转基因育种。

Genetic transformation was done to induce callus in stem segment of Amorpha fruticosa L. via Agrobacterium tumefaciens-mediated gene transfer. It was found that critical tolerance concentration of Kanamycin was 40 mg/L; The experiment of orthogonal bacterium concentration and infection time showed that infection concentration of bacterium （OD600） was 0.6-0.8,. and conversion efficiency peaked 17.95 % when infection time was 30 min. The DNA containing pBI121-GUS plasmid in Amorpha fruticosa L. infected the invert callus. Gus staining analysis suggested that adventitious buds and regenerated shoots, roots and leaves possessing resistance showed blue; also, the PCR analysis in invert leaves suggested that exogenous GUS gene was integrated into genome of Amorpha fruticosa L. We established an efficient transformation system, in which callus of stem segment is transformation acceptor via Agrobacterium tumefaciens-mediated gene transfer. In order to confirm their repeatability, pBI121-GFP gene was used for transformation. Via PCR analysis of TO generation shoots, the integration rate reached 85 %. Through blue light stimulation, the apical bud in invert shoot showed green fluorescent, indicating that the invert gene under 35S promoter overexpressed. It was concluded that the conformation system can be used for transgenic breeding, which will lay foundation for improving and cultivating new strains of Amorpha fruticosa L.