A Novel Method For The Construction Of Plant Multigene Transformation Vectors

With the rapid development of the genetic engineering, researchers have now shifted from transformation of single-gene to transformation of multigenes to plants to meet their research demands. Therefore new and effective technologies to achieve these are urgently needed. Since sexual crossing between plants carrying different transgenes, sequential retransforma -tion or co-transformation with plasmids containing individual transgenes are very time-consuming, low transformation efficiency and/or requiring the use of different selectable marker genes, it would be advantageous to transfer multigenes that are stacked into a single transformation vector to plant. However, to construct such a vector is technically challenging. Here we report a simple and user friendly method for the construction of a single vector containing 3 or more transgenes for one step transformation of plant.We first constructed an auxiliary vector pCR(-XbaI-35S-MCS-Tnos- SpeI-XbaI-) based on the cloning vector pCR2.1-TOPO (invitrogen). It contains a 35S promoter, a nos terminator (Tnos), and a multi-cloning site (MCS) in between. Restriction sites XbaI was added before the 35S and SpeI-XbaI were added after the Tnos to aid cloning of individual expression cassettes. The genes of interest can be individually subcloned into the MCS of this vector, resulting in the new plasmids pCREC1, pCREC2, pCREC3 etc. As each expression cassette was flanked by two XbaI sites, the EC2 expression cassette can be released by XbaI from the pCREC2, and ligated into pCREC1 that was pre-digested with SpeI because XbaI (T/CTAGA) and SpeI (A/CTAGT) can produce compatible cohesive ends, to generate the plasmid containing 2 expression cassettes EC1 and EC2 pCR(-XbaI-EC1-SpeI/XbaI -EC2-SpeI-XbaI-). However, the newly formed sequence ACTAGA (SpeI/XbaI) was no longer digestible by either enzymes. Because a new SpeI site was introduced following the ligation of EC2, in the same way, the third expression cassette EC3 containing the third gene can then be ligated into this plasmid to produce a plasmid with 3 expression cassette. Following the same rule, several gene cassettes can be strung together into this auxiliary vector. Finally, these stacked gene cassettes can be released by XbaI from this auxiliary vector and ligated into the plant binary vector pCambia2300 pre-digested by XbaI to obtain a plant transformation vector containing multigenes.To confirm the feasibility of the above method, we constructed a transformation vector pCambia3EC containing three transgenes, including two reporter genes (GUS, GFP) and a selectable marker gene (BAR) .We also constructed another transformation vector pCambia4EC containing four genes that are related to the biosynthesis of very long chain polyunsaturated fatty acid EPA (the main ingredient of fish oil). Both PCR and restriction digestion have confirmed that both vectors contain all the genes of interest. Arabidopsis were transformed with the pCambia3EC and pCambia4EC constructs via the Agrobacteria-mediated transformation strategies. 26 Basta herbicide resistant plants and 58 kanamycin resistant plants were recovered from pCambia3EC from pCambia4EC transformed plants, respectively. These putative transformants will be grown on in soil for further confirmation for the existence of the transgenes. Our results demonstrated that using isocaudomers could solve the difficulties finding appropriate unique restriction sites when three or more genes need to be linked or fused to make a multigene construct.