应用绿色荧光蛋白报告基因优化辣椒的遗传转化体系

Improvement of Genetic Transformation System by Using GFP as Reporter Gene on Pepper

以‘中椒5号’甜椒和‘湘研10号’辣椒子叶外植体为试料,绿色荧光蛋白GFP基因为报告基因,通过观察检测愈伤组织的荧光表达与特异PCR扩增鉴定,分析了工程菌液pH值、共培养基中乙酰丁香酮(AS)的浓度、共培养时的温度与共培养时间对农杆菌转化辣椒效率的影响。结果表明,在pH5.2、共培养基中加入AS200μmol·L-1、23℃黑暗条件下,农杆菌与辣椒子叶共培养5d,有利于辣椒子叶的遗传转化,‘中椒5号’、‘湘研10号’愈伤组织荧光表达率均达到了40%。

Effect of transferring efficiency of Agrobacterium tumefaciens pH value、concentration of AS added in co-cultivation medium,temperature during co-cultural stage and days for co-cultivation were studied through PCR amplification and observation of green fluorescent protein expression by used GFP as reporter gene on cotyledon of‘Zhongjiao 5’and‘Xiangyan 10’.The results show that when pH value of Agrobacterium solution is 5.2, transformation efficiency of pepper cotyledon incubated on co-cultivation medium added 200 μmol·L -1 AS increased significantly under 23℃ and 5 days dark co-cultivation.The rate of expression of green fluorescent protein are up to 40% on calli of cotyledon of both‘Zhongjiao 5’and‘Xiangyan 10’.