苜蓿遗传转化体系的优化与AtPCS1基因表达载体的构建

通过对6种不同基因型苜蓿愈伤组织的形成能力、愈伤组织的分化能力的比较发现,不同苜蓿品种间的分化率存在显著差异(P<0.05),其中甘农1号分化率最高(42.9%);对甘农1号叶片、下胚轴及子叶等外植体的分化再生能力研究发现,叶片起源的愈伤组织分化时间短,且丛生芽形成数量高于下胚轴和子叶;再生芽可在B5培养基上成苗,并在蔗糖浓度为10 g/L的1/2 MS培养基上顺利生根.据此认为,苜蓿甘农1号叶片是适于做遗传转化的理想材料.利用RT-PCR方法扩增拟南芥螯合肽合成酶(AtPCS1)基因全序列构建了AtPCS1的植物表达载体pBI121-AtPCS1,为下一步AtPCS1基因转化苜蓿奠定了基础.

Study on transformation system of alfalfa and constructed AtPCS1 plant expression vector

Six cultivars of alfalfa with different genotypes were compared with each other in their callus induction capacity and plant regeneration ability.The significant differences were observed(P<0.05) in regeneration ability of these genotypes,and the cultivar Medicago sativa'Gannong No.1' has that the highest regeneration ability.Then the callus induction and plant regeneration ability of different explants as leaf,hypocotyls and cotyledon of Gannong No.1 were studied,and found that the calluses coming from leaf differentiated much earlier and had more regeneration buds than others.The in vitro regenerated plants were successfully rooted in 1/2 MS medium and 10 g/L sugar.The results above suggested that the leaves of the cultivar 'Gannong No.1' would be fit for the ideal explants in transgenic researches.Meantime,the full length of AtPCS1 gene was amplified by RT-PCR from Arabidopsis thaliana(ecotype Columbia).Furthermore,the plant expression vector pBI121-AtPCS1 was constructed,and that were as basic for transgenic alfalfa research in future.