不同载体和农杆菌对苜蓿瞬时表达影响的研究

Effects of Different Vectors and Agrobacterium tumefaciens on Transient Expression of Alfalfa

苜蓿(Medicago sativa)是重要的牧草类植物,为了在苜蓿中有效地实现外源基因的瞬时表达,该研究以绿色荧光蛋白(GFP)作为报告基因,以本氏烟草(Nicotiana benthamiana)作为对比,研究了2种不同的表达载体(非复制型载体和基于菜豆黄矮病毒的复制型载体)、2种不同类型的根癌农杆菌菌株(Agrobacterium tumefa⁃ciens)(LBA4404和EHA105)对苜蓿叶片瞬时表达水平的影响。结果表明,LBA4404与复制型载体的组合(revector/LBA4404)可在苜蓿叶片中有效地实现GFP的瞬时表达。进一步地,研究了re-vector/LBA4404的浓度和侵染后时间对苜蓿叶片瞬时表达的影响。观察显示,随着re-vector/LBA4404菌悬液浓度或侵染后时间的增加,GFP在苜蓿叶片中的瞬时表达水平呈现出先升后降的趋势。菌悬液浓度为1.0(OD600)时,GFP瞬时表达水平最高;侵染后时间为5~7 d时,GFP瞬时表达水平达到峰值。相较于本氏烟草叶片,在苜蓿叶片中实现最高水平的瞬时转化需要更高浓度的农杆菌和更长的侵染后孵育的时间。

Alfalfa(Medicago sativa)is an important forage plants.In order to effectively express foreign genes in alfalfa,by using the green fluorescent protein(GFP)as report gene and Nicotiana benthamiana as a comparison,the effects of two different vector(s the non-replicating and the BeYDV(bean yellow dwarf virus)-based replicating vectors),and two different Agrobacterium tumefaciens strains(LBA4404 and EHA105)on the transient expression of alfalfa leaves were investigated.The results showed that the combination of LBA4404 with the replicating vector(re-vector/LBA4404)led to effective expression of GFP in alfalfa leaves.By using LBA4404 with the replicating vector,the effects of the concentration of re-vector/LBA4404 and incubation time after infection on the transient expression were also evaluated.It was found that with the increase of concentration of bacterial suspension or time post-infection,the expression level of GFP increased first but then decreased.In the leaves of alfalfa,the highest expression level of GFP was observed when the concentration of LBA4404 strain was 1.0(OD600);and the expression level of GFP reached the peak at 5-7 d after infection.Compared with Nicotiana benthamiana,achieving the highest expression level of GFP in alfalfa might need higher concentration of Agrobacterium tumefaciens and longer incubation time after infection.