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棉花学报  2011 

农杆菌介导转化大丽轮枝菌的体系优化

DOI: 1002-7807(2011)06-0507-08, PP. 507-514

Keywords: 农杆菌,大丽轮枝菌,潮霉素,GFP,遗传转化,遗传稳定性,表型变异

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Abstract:

为了揭示大丽轮枝菌的遗传变异和致病机理,以含潮霉素为抗性筛选标记、绿色荧光蛋白GFP为报告基因载体,优化了农杆菌介导转化大丽轮枝菌的遗传转化体系,获得了大丽轮枝菌菌株V991和BP2含绿色荧光蛋白GFP的转化子,转化率达到100×10-6~550×10-6。优化的转化条件为农杆菌以IM液体培养基调节浓度至OD600=0.2并预培养4h,然后与等体积的浓度为106个·mL-1的大丽轮枝菌孢子混匀,每皿取200μL混合液涂于固体IM共培养基的纤维素膜上,25℃共培养60h,转移至固体PDA培养基上以头孢噻肟钠500mg·L-1+潮霉素B50mg·L-1进行筛选。研究发现农杆菌LBA4404和AGL-1比农杆菌SK1044和EHA105更适用于大丽轮枝菌转化。对转化子观察和鉴定发现从表型上,70.8%转化子保持野生型形态,29.2%转化子发生了形态变化;从致病力上,81%转化子的致病力相对于野生型没有发生显著变化,19%转化子的致病力或增强或减弱。

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