【摘要】 大豆(Glycine max)下胚轴作为大豆遗传转化的外植体材料,能快速高频再生不定芽。然而,在遗传转化过程中褐化影响基因转化效率。在该研究中,我们用含有GUS染色基因和hptII(Hygromycin phosphotransferase II)筛选基因的农杆菌(Agrobacteriumtumefaciens)LBA4404侵染大豆下胚轴,并用组织化学定位法测定了GUS基因的瞬时表达,以确定大豆的优化基因转化条件。结果显示,在共培养基中加入硫代硫酸钠、L_半胱氨酸以及二硫苏糖醇等抗氧化剂,可以有效地抑制大豆下胚轴在组培过程中褐化的发生,并大幅度提高农杆菌在下胚轴的瞬时表达率。这些结果说明抗氧化剂可以降低这种影响并有效提高基因转化效率。更多还原

【Abstract】 Soybean hypocotyls have advantages of higher regeneration frequency and faster formation of adventitious shoots, and often be used in soybean transformation mediated by Agrobacterium tumefaciens. But the browning and necrosis of hypocotyls during co_cultivation greatly affects the efficiency of the transformation.The effects of antioxidants on the transient expression efficiency of GUS (β_glucuronidase) gene in soybean hypocotyls mediated by Agrobacterium tumefaciens were studied by adding antioxidants (thiosulfate, L_cysteine, and dithiothreitol) in the co_cultivation medium,and the transient expression efficiency of the GUS gene was measured by histochemically method. The results showed that the antioxidant in the co_cultivation medium increased the transient expression efficiency up to 23.4% in hypocotyls. Further investigation demonstrated that higher transient expression efficiency was associated with concomitant decreases in the levels of hypocotyl browning. The stable expression of GUS gene in hypocotyls was found in the cambium cells, which was formed as deep blue zone on the hypocotyls. Our results also showed that concentration of Agrobacterium tumefaciens and co_cultivation time were the important factors that affected the transient expression efficiency of GUS gene in hypocotyls. In our experiments, the highest transient expression efficiency of GUS gene obtained by inoculating hypocotyls in Agrobacterium tumefaciens (OD_ 600 0.6) and co_cultivating for 3 days in the darkness. All results suggested that antioxidants could efficiently improve transient expression efficiency of GUS gene in soybean hypocotyls.更多还原