利用高效农杆菌遗传转化系统将ACP反义基因片段导入油菜

TRANSFERRING AN ANTISENSE acyl-acyl carrier protein (ACP) DNA FRAGMENT INTO Brassica napus BY HIGHLY EFFICIENT Agrobacterium tumefaciens GENETIC TRANSFORMATION SYSTEM

通过对已有的油菜农杆菌遗传转化体系的改进 ,建立了一种利用农杆菌感染油菜子叶柄的高效遗传转化方法 ,经过农杆菌感染的子叶柄在含有 6 BA 2 .0mg/L ,IBA 0 .15mg/L ,AgNO33 .5mg/L的MS培养基上由卡那霉素抗性愈伤组织分化出抗性幼苗的频率达到 12 .2 8%。利用已经构建的含有油菜Napin启动子加上反义ACP脱饱和酶第一外显子、内含子的基因片段的植物双元表达载体 pNACP ,将该反义 ACP基因片段转入油菜“沪油 15” ;PCR和GUS染色结果表明抗性植株中有 60 %的植株含有导入的目的基因。

An efficient genetic transformation system by means of Agrobacterium tumefaciens-mediated cotyledon petiole of Brassica napus was established,in which cotyledon petioles infected by Agrobacterium tumefaciens differentiated into the resistant seedlings from the calli with kanamycin resistance on the basic MS medium containing 2.0mg/L 6-BA,0.15mg/L IBA and 3.5mg/L AgNO 3,and the differentiation frequency was 12.28%. The explants of Brassica napus variety 'Huyou 15' was transformed with the expression vector pNACP containing Napin promotor of Brassica napus and the first extron and intron of antisense-ACP DNA fragment by Agrobacterium tumefaciens-mediated method. PCR and GUS detection showed that 60% of kanamycin-resistant plants contained target gene.