Constructions Of VP7 Gene And CTB-VP7 Fusion Gene Expression Vectors And Transformation In Carrot (Daucus Carota L.)

In this study, VP7 gene and CTB-VP7 fusion gene expression vectors were constructed, and a high-efficient genetic transfomation system of carrot(Daucus carota L.) was established.VP7 gene was amplified from cDNA of rotavirus antigene by PCR and was inserted into expression vector pBI121 containing no GUS gene. On the base of construction of pBI121VP7, we constructed the fusion gene expression vector pBI121CTBVP7 by the same sigle cloning site( NdeI and XbaI) of vector pUC19CTB and pBI121VP7. The expression vector pBI121VP7 and pBI121CTBVP7 were mobilized into Agrobacterium tumefacious LBA4404 by chemically-based DNA direct transformation. We confirmed the correct construction by PCR and restriction enzyme analysis.In this research, hypocotyls were used as the explant and several factors affecting genetic transformation of carrot mediated by Agrobacterium tumefaciens were studied. The results indicated that: (1)Marinated in 1% AgNO3 for 15min was the optimal sterile condition of seeds. (2)2,4-D had great effects on the formation of callus , and the optimal concentration was 0.1mg/L. However, KT had no effects on the shooting. (3)The optimal explant age for transformation was 6 or 7 day after aseptic germination of seeds. (4)Development of non-transformated cell was inhibited completely by 100mg/L Kan. (5)500mg/L Cb could avoid pollution of Agrobacterium tumefaciens and had no distinct effects on the formation of callus and shoot regeneration. (6)The optimal co-cultural condition was 3 day on 28 indark. (7)Effects of different concentration and infecting time of Agrobacterium tumefaciens on transduction was little. (8)The add of acetosyringone had little influence on transformation result.Finally, we gained the transgenic plants. PCR and RT-PCR analyse confirmed that the target gene had been inserted into the carrot genome and transcripted successfully.