Abstract:Abstract: By using Italian lettuce (Lactuca sativa L. var. ramosa Hort.) as the experimental material, and Agrobacterium tumefaciens mediated leaf disc method for transformation, the cotyledon was transformed by LBA4404 and regenerated via plant tissue culture. The best selection pressure for this transformation regeneration system was 50 mg/L kanamycin, and the best antibacterial agent was 400 mg/L carbenicillin. After the addition of the appropriate antibiotics to the culture medium, the appropriate mass concentrations of the phytohormones were 0.4 mg/L 6-BA and 0.15 mg/L NAA or 0.3 mg/L 6-BA and 0.2 mg/L NAA for callus induction, while the callus induced on the medium containing 0.3 mg/L 6-BA and 0.2 mg/L NAA was better for bud differentiation subsequently. Based on the GenBank sequence database, P1 and 3CD genes of EV71 subtype C4 were designed for future expression in plant and then synthesized. These two genes were subcloned into the same plant expression vector pCAMBIA2301 to construct a co-expression vector named pCAMBIA2301-P1-3CD. Cotyledons of Italian lettuce were transformed via A. tumefaciens mediated leaf disc method. Leaf PCR was used to select the transgenic plants first. The verified positive individuals were further identified by Western-blot. Two transgenic plants were then selected, as the biologically active antigens of EV71 were successfully detected in them. It provides evidence theoretically and practically for further development of oral vaccines against EV71 in plants.
引用本文:
罗 雯, 张同林, 艾佐佐, 刘艳梅, 刘旺喜. 意大利生菜转化体系建立及EV71抗原基因表达初探[J]. 生命科学研究, 2018, 22(3): 208-214.
LUO Wen, ZHANG Tong-lin, AI Zuo-zuo, LIU Yan-mei, LIU Wang-xi. Establishment of Transgenic System of Italian Lettuce and Expression of EV71 Antigens in Lettuce Leaves. Life Science Research, 2018, 22(3): 208-214.