Submitted to: In Vitro Biology Meeting
Publication Type: Abstract Only
Publication Acceptance Date: 4/20/2011
Publication Date: 7/4/2011
Citation: Dong, N., Mcmahan, C.M. 2011. Using ribosome binding sequences for multiple-gene transformation. In Vitro Biology Meeting. 47:S57-S58.
Technical Abstract: Metabolic engineering of plants usually requires transformation of multiple genes - over expression of the key enzyme gene(s) in the target pathway and reduction of gene(s) expression in non-target pathway(s). Sometimes, it is desireable to over express multiple enzyme genes in the target pathway. Although, a polycistron approach can be used in plant chloroplast transformation, it lacks efficacy for plant nuclei transformation. In Agrobacterium-mediated transformation, each introduced gene needs one promoter and one terminator. The more genes stacked in a construct, the longer the T-DNA, and the lower the transformation efficiency. To reduce the T-DNA size, we have successfully used a 2A peptide strategy to link two genes, shared with one promoter and one terminator. However, the 2A peptide originates from foot-mouth virus and therefore may delay field trial approvals. Here we report a different strategy, using ribosome binding sequences to link the genes for multiple-gene transformation. An NPTII gene, a GUSplus gene, and a BAR gene, were linked by two different plant ribosome binding sequences (one from rice and the other from Arabidopsis), shared with one promoter and one terminator. Most of the transgenic tobacco plants were not only resistant to kanamycin, but also resistant to glufosinate. They showed GUS activity as well. The plant ribosome binding sequences showed similar GUS activity and glufosinate-resistance as the virus ribosome binding sequences or as separate genes. Each has one promoter and one terminator for GUSplus gene and BAR gene. This strategy can also be combined with RNAi approach. We conclude that this strategy is suitable for multiple-gene transformation.