农杆菌介导的胀果甘草愈伤组织遗传转化

Agrobacterium Tumefaciens-mediated Genetic Transformation of Glycyrrhiza inflata Callus

目的:旨在建立一个农杆菌介导的甘草愈伤组织遗传转化的可行方案,并对转化条件进行优化。方法:选择EHA105和LBA4404两种根癌农杆菌菌株,热激法转入含有绿色荧光蛋白GFP基因的植物表达载体pBI121-gfp,挑选转化的农杆菌用于侵染胀果甘草愈伤组织。设置不同培养时间的愈伤组织作为受体材料和农杆菌不同侵染时间两组条件,经共培养后的甘草愈伤组织进行荧光检测。结果:愈伤组织在含有100mg/l卡那霉素的继代培养基上进行筛选培养,得到了具有卡那霉素抗性的转化甘草愈伤组织,转化愈伤组织经继代培养基后在紫外光下仍可见绿色荧光,PCR检测转化愈伤组织基因组中含有gfp基因。结论:试验建立了农杆菌介导的甘草愈伤组织的遗传转化体系,为目的基因导入甘草细胞利用基因工程手段调控甘草次生代谢产物生物合成的研究奠定了基础。

Objective： A feasible scheme of Agrobacterium - mediated genetic transformation of Glycyrrhiza inflata callus was established in this paper, and the transformation conditions were also optimized. Method：Two agrobacterium strains of EHA105 and LBA4404 were used, pBI121 -gfp plasmid was transformed in EI-IA105 and LBA4404 by the thermal shock method. Then Glycyrrhiza inflata callus was infected by Agrobacterium. Different preculture time of callus and different agrobacterium infecting time was setted as two conditions. After coculture, calli were estimated by fluorescence detection. Result：The results showed that green fluorescence protein was expressed in the calli. Screened by the medium containing 100rag/1 Kan, the transformed calli were obtained. Conclusion：A Agrobacterium- mediated ge-netic transformation system of Glycyrrhiztt inflata callus laid a foundation for further research on target gene transformation to the callus and regulation of the pathway of the secondary metabolites biosynthesis of liquorice via genetic engineering mothed.