Development and analysis of a highly flexible multi-gene expression system for metabolic engineering in Arabidopsis seeds and other plant tissues

Authors: Shockey, Jay; Mason, Catherine; Gilbert, Matthew; Cao, Heping; LI, XIANGJUN - University Of Nebraska; CAHOON, EDGAR - University Of Nebraska; Dyer, John

Submitted to: Plant Molecular Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/29/2015
Publication Date: 8/9/2015
Citation: Shockey, J., Mason, C., Gilbert, M., Cao, H., Li, X., Cahoon, E., Dyer, J. 2015. Development and analysis of a highly flexible multi-gene expression system for metabolic engineering in Arabidopsis seeds and other plant tissues. Plant Molecular Biology. 89(1-2):113-126.
DOI: https://doi.org/10.1007/s11103-015-0355-5

Interpretive Summary: Genetic engineering of plants, especially oilseeds, is often hampered by a lack of sophisticated tools for introduction of foreign DNA. In the current manuscript, we have assembled and characterized a large, flexible set of DNA molecules of allow for creation of transgenic plants containing up to 18 foreign genes. The relative effectiveness of each of the six molecules that control expression of the foreign DNA in seeds was compared, as were the results that came from generating engineered plants by three different selections methods.

Technical Abstract: Production of novel value-added compounds in transgenic crops has become an increasingly viable approach in recent years. However, in many cases, product yield still falls short of the levels necessary for optimal profitability. Determination of the limiting factors is thus of supreme importance for the long-term viability of this approach. One of the significant challenges to most metabolic engineering projects is the need for strong coordinated co-expression of multiple transgenes. A limited number of strong constitutive promoters have been well-characterized during the 30 years since plant transformation techniques were developed. However, organ- or tissue-specific promoters are relatively poorly characterized in many cases. Oilseeds are one such example. Numerous reports spanning at least twenty years have described the use of certain seed-specific promoters. Multi-gene engineering strategies have often been hampered by sub-optimal expression strength, tissue-specificity, or temporal regulation of particular promoters, or have relied on the use of multiple copies of the same promoter, which can result in DNA instability or transgene silencing. We describe here a highly flexible system of plasmids that allows for expression of one to seven genes per binary plasmid, and up to 18 genes altogether after multiple rounds of transformation. Effective gene-suppression cloning vectors, accomplished using RNA interference (RNAi), are also available for targeting endogenous genes both constitutively and in seeds. This vector system includes six seed-specific promoters and two constitutive promoters. Taken together, this molecular toolkit allows combinatorial cloning for transgene expression in seeds, vegetative organs, or both simulataneously, while also providing the means to coordinately overexpress some genes while silencing others.