基于绿色荧光标记的甘草遗传转化体系的建立

Establishment of Genetic Transformation System of Glycyrrhiza uralensis Fisch.Based on GFP Marker

用根癌农杆菌EHA105菌株介导查尔酮异构酶基因(CHI)转化甘草,建立基于绿色荧光标记的高效遗传转化体系。通过比较子叶、子叶节、去子叶胚3种外植体的再生能力,确定不定芽分化率高达73%的去子叶胚为适宜转化的受体。高效遗传转化体系为:分化和生根培养基中草铵膦除草剂(PPT)筛选压力分别为2.0和1.0mg/L;工程菌浓度OD600=0.5;侵染时间30min;共培养基中乙酰丁香酮(AS)浓度为50mg/L;共培养时间为4d;分化和生根培养基中噻孢霉素(Cef)浓度分别为300和200mg/L。该体系通过检测再生苗根部GFP荧光筛选转化植株,简化了阳性苗的鉴定过程,阳性苗比率可达10%。该体系为研究甘草药用成分代谢途径中相关基因的功能和作用机制奠定了基础。

In this paper, a high efficient genetic transformation system of Glycyrrhiza uralensis Fisch. was established, which was mediated by Agrobacterium tumefaciens strain EHA105 containing chalcone isomerase （CHI） gene and GFP marker. In terms of the differentiation potential of cotyledon, cotyledon node and cotyledon-excised embryo,the cotyledon-excised embryo, with high adventitious bud regeneration rate of 73%, was selected as the suitable transgenic acceptor. The optimum selective pressure of PPT in differentiation and rooting media was 2.0mg/L and 1.0mg/L, respectively. The appropriate concentration of engineering strain was OD600 = 0.5 and the infection time was 30min. Add 50mg/L AS in the co-culture medium and co-culture the infected explants for 4d. The optimal concentrations of Cef in differentiation and rooting media were 300mg/L and 200mg/L, respectively. The positive plantlets reached 10% by GFP screening in the system. This genetic transformation system lays foundation for further researches on the function and mechanism of genes involved in metabolic pathway of medicinal compounds in Glycyrrhiza uralensis Fisch.