青花菜农杆菌遗传转化影响因素研究

Study on the Factors Affecting the Agrobacterium Mediated Transformation of Brassica oleracea var.botrytis

以青花菜"绿秀"为试材,以带柄子叶为外植体,采用根癌农杆菌LBA4404菌株进行农杆菌遗传转化,研究了影响青花菜遗传转化的主要因素。结果表明:乙酰丁香酮浓度、OD600值、侵染时间等对瞬间表达均有一定的影响。其中,OD600值影响最大,AS浓度影响次之,再次是侵染时间。最适宜的试验条件为:乙酰丁香酮浓度为100μmol/L、OD600值为0.3、侵染时间为5min。抗性植株经PCR检测、Southern杂交和GUS组织化学染色法,表明目的基因已整合到青花菜基因组中并得到了很好的表达。

Taking Brassica oleracea var.botrytis variety ＇Lvxiu＇ as material,cotyledons with petiole as explants,Agrobacterium tumefaciens strain LBA4404 were used to transformation system,and the factors affecting the Agrobacterium mediated transformation of Brassica oleracea var.botrytis were studied.The results showed that the concentration of acetosyringone,OD600 value,infection time had certain effect on transient expression.Among which OD600 value had the most influence,followed by the concentration of acetosyringone and infection time.The optimum condition was 100 μmol/L acetosyringone,OD600 value 0.3,infection time 5 min.The resistant plants demonstrated by PCR,Southern hybrization analysis and GUS staining showed that the target gene had been integrated into the broccoli genome and had been well expressed.