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

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. A significant challenge to most metabolic engineering projects is the need for strong coordinated co-expression of multiple transgenes. Strong constitutive promoters have been well-characterized during the>30 years since plant transformation techniques were developed. However, organ- or tissue-specific promoters are poorly characterized in many cases. Oilseeds are one such example. Reports spanning at least 20 years have described the use of certain seed-specific promoters to drive expression of individual transgenes. Multi-gene engineering strategies are often hampered by sub-optimal expression levels or improper tissue-specificity of particular promoters, or rely on the use of multiple copies of the same promoter, which can result in DNA instability or transgene silencing. We describe here a flexible system of plasmids that allows for expression of 1-7 genes per binary plasmid, and up to 18 genes altogether after multiple rounds of transformation or sexual crosses. This vector system includes six seed-specific promoters and two constitutive promoters. Effective constitutive and seed-specific RNA interference gene-suppression cloning vectors were also constructed for silencing of endogenous genes. Taken together, this molecular toolkit allows combinatorial cloning for multiple transgene expression in seeds, vegetative organs, or both simultaneously, while also providing the means to coordinately overexpress some genes while silencing others.

Identification of Arabidopsis GPAT9 (At5g60620) as an Essential Gene Involved in Triacylglycerol Biosynthesis

“…The phaseolin promoter from cloning vector pK8 (Shockey et al, 2015) was removed by KpnI and NotI digestion and replaced with a 1,594-bp portion of the 59 upstream region of the AtGPAT9 gene, resulting in the synthesis of cloning vector pK51. The open reading frames of AtGPAT9 and tung tree (Vernicia fordii) GPAT9 (VfGPAT9) were cloned into the NotI and SacII sites of pK51, resulting in plant shuttle plasmids pB447 and pB448, respectively.…”

“…The open reading frames of AtGPAT9 and tung tree (Vernicia fordii) GPAT9 (VfGPAT9) were cloned into the NotI and SacII sites of pK51, resulting in plant shuttle plasmids pB447 and pB448, respectively. The AscI fragments bearing the respective AtGPAT9 promoter:GPAT9 open reading frame:35S terminator cassettes from pB447 and pB448 were purified by gel electrophoresis and ligated into the AscI site of the DsRed-selectable binary vector pB110 (Shockey et al, 2015), resulting in the final plant transformation binary plasmids pE434 and pE437. Binary plasmids were transformed into Agrobacterium tumefaciens strain GV3101 via electroporation and selection on solid medium containing 50 mg mL 21 each kanamycin and gentomycin.…”

Gene editing in plants: assessing the variables through a simplified case study

“…In summary, we have presented here the results of a series of experiments designed to reveal which promoter elements and sgRNA design criteria may help to achieve stable, heritable mutations in Arabidopsis and related plant species, accompanied by analyses that provide an estimate of how often off-target mutations might occur. These tools are freely available to the public and can be easily combined with other existing resources (Shockey et al 2015) to combine gene editing with transgene overexpression and/or partial silencing of endogenous genes.…”

“…In the early stages of our work, we also observed similar results. The first test subject was Arabidopsis thaliana E113, a transgenic line engineered to produce ~ 7-8% α-eleostearic acid in its seeds (Dyer et al 2002;Shockey et al 2015). The marker for selection of transgenic E113 plants is the DsRed fluorescent protein gene.…”

“…The makeup of the basic set of cloning vector and plant transformation binary plasmids has been described previously (Shockey et al 2015). A plasmid bearing the open reading frame for epitope-tagged, plant codon-optimized Staphylococcus aureus Cas9 was generously provided by Dr. Jen Sheen (Harvard Medical School) (Li et al 2013).…”