Construction and Development of pSB111 Bar Plasmid Vector for Activation Tagging

Abstract

To get desired traits and to know the precise expression patterns of genes in the plant, Agrobacterium tumefaciens has been widely employed in the generation of transgenic plants using plasmid vectors. Many methods are come forward to establish the gene expression of unknown genes but in these techniques gene alteration is the main drawback or sometimes it is lethal to organisms also, to overcome these problems activator tag method developed to know the specific expression of genes at different stages of growth by activating the genes of an organism. A reporter gene with a modest promoter is included in the activator tag vector. The basic principle behind is the expression of reporter gene of activator vector by elucidating the tagged gene expression. Using the Trans configuration of vir genes from the plasmid Agrobacterium tumefaciens to transfer right and left sequence bordered T-DNA into the nuclear genome of plants, we designed a vector molecule that promotes expression of a specific gene at more than four times its normal expression and is useful for efficient transformation to higher plants. In this study we modified activator vector by inserting the 4x activator which shows four times effectiveness than normal activator to get the more promising results. To tag and know the genes and their expression profiles, we produced a binary vector consisting of 1.8 kb GFP cassette as a reporter gene and 1.4 kb tetramer of CaMv35S activator (4X-Ac) cloned at HindIII site of pSB11 bar intermediate vector. The recombinant clone harbouring different expressions units were mobilized into Agrobacterium tumefaciens through DH5α cells triparental mating to produce a super binary vector pSB111-bar-4xAc-GFP. The generated vector is beneficial to produce transgenic lines of different plant species.