Multiple Gene Double T-dna Plant Expression Vector Construction And Arabidopsis Genetic Transformation

Transgenic technology was applied widely to modify plants genetically since 1980s. Routine genetic improvement in plants was carried out by genetic manipulation of single gene. For a long time, developing genetically engineered crops with many excellent agricultural traits, such as high yield, good quality, high resistance and multi-resistance was the common objective for biologists. One of the most effective current strategies appears to be multi-gene transformation to introduce and express multiple transgenes in transgenic plants. And the construction of multigenes plant expression vector is one of the main means in this strategy.Although transgenic technology offers significant advantages for modern agriculture, more and more people concern the safety of transgenic plants due to the broad utilization of selectable marker genes such as the antibiotic resistance genes and herbicide resistance genes. Thus, a suite of strategies has been developed to either avoid or get rid of selectable marker genes before transgenic plants are introduced into the field. Among those strategies, the "double T-DNA" binary vector system is a convenient and feasible approach to eliminate selectable marker genes from the transgenic plants after successful selection.In this research, a double T-DNA plant expression vector (2T-bbgdD) harboring multiple genes was constructed based on plasmid pCAMBIA1300 with DNA recombination technology. The vector contained five genes including two abiotic stress resistance genes (DREB1A , d5) and one report gene (gfp) in one T-DNA region and one herbicide resistance gene (bar) and one abiotic stress resistance gene (betA) in another T-DNA region. All of these genes had their own regulative elements. The construct has been verified with the restriction enzymes digestion.The plant expression vector was transferred into Arabidopsis thaliana by Agrobacterium-mcdiatcd transformation. The PCR results of T₁ plants showed that in five transgenic lines, all transgenes present in the two T-DNA regions were transferred together into the Arabidopsis genome. The PCR analyses of the T₂ plants derived from seeds of the above five T₁ lines indicated that we finally obtained five T₂ transgenic lines co-transformed with five target genes and ten T₂ transgenic lines without selective marker gene.The study showed that it was feasible to use this plant expression vector in Agrobacterium-mediated transformation, implying the promising future of this vector in breeding crops with higher abiotic stress resistance or selectable-marker-free transgenic crops which can be easily screened out by the GFP detection and herbicide screening.