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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。
The Agrobacterium tumefaciens strain EHA105 was used to mediate chalcone isomerase gene (CHI) into licorice, and a high efficient genetic transformation system based on green fluorescence was established. By comparing the regeneration ability of three explants of cotyledon, cotyledonary node, and cotyledon embryo, it was determined that adventitious buds with 73% adventitious bud differentiation rate were the suitable transformants. The high efficiency genetic transformation system was as follows: the screening pressure of glufosinate herbicide (PPT) in differentiation and rooting medium were 2.0 and 1.0 mg / L respectively; the concentration of engineering bacteria was OD600 = 0.5; the infection time was 30 min; the content of acetosyringone (AS) concentration of 50mg / L; co-culture time of 4d; differentiation and rooting medium in the concentration of 300 and 200mg / L of Cef. The system simplifies the process of identification of positive seedlings by screening GFP fluorescent screening of regenerated shoots roots, the positive rate of up to 10%. The system lays the foundation for studying the function and mechanism of related genes in the metabolic pathway of medicinal components of liquorice.