Design rules for efficient transgene expression in plants

Abstract

SummarySustained expression of transgenes in specified developmental patterns is commonly needed in plant biotechnology, but obstructed by transgene silencing. Here, we present a set of gene design rules, tested on the silencing-susceptible beetle luc and bacterial ims genes, expressed in sugarcane. Designs tested independently or in combination included removal of rare codons, removal of RNA instability sequences, blocking of likely endogenous sRNA binding sites and randomization of non-rare codons. Stable transgene expression analyses, on multiple independent lines per construct, showed greatest improvement from the removal of RNA instability sequences, accompanied by greatly reduced transcript degradation evident in northern blot analysis. We provide a set of motifs that readily can be eliminated concurrently with rare codons and undesired structural features such as repeat sequences, using Gene Designer 2.0 software. These design rules yielded 935-and 5-fold increased expression in transgenic callus, relative to the native luc and ims sequences; and gave sustained expression under the control of sugarcane and heterologous promoters over several years in greenhouse and field trials. The rules can be applied easily with codon usage tables from any plant species, providing a simple and effective means to achieve sustained expression of otherwise silencing-prone transgenes in plants.

Deep Sequencing Reveals Divergent Expression Patterns Within the Small RNA Transcriptomes of Cultured and Vegetative Tissues of Sugarcane

“…Transformation protocols amenable to a variety of commercial cultivars have been developed (Basnayake et al 2011;Jackson et al 2013). Promoters that direct expression to the stem have been characterised (Damaj et al 2010;Kinkema et al 2014;Moyle and Birch 2013a;Moyle and Birch 2013b), and effective strategies have been applied to resist transgene silencing in sugarcane (Chou and Moyle 2014;Jackson et al 2014). In combination, these enabling technologies and molecular tools have facilitated metabolic engineering of the sugar profile (Mudge et al 2013).…”

The Need for Integrated Approaches in Metabolic Engineering

“…However, the influence of these factors in distinct organisms or even protein families might vary. For instance, transgenic expression in eukaryotic systems is burdened by gene-silencing issues or low expression levels (Jackson et al 2014). On the other hand, bacterial host systems are known to commonly produce misfolded proteins (Sander et al 2014).…”

Means to optimize protein expression in transgenic plants

“…One exception has been described [23 ] where the effects of different factors in two genes in sugarcane were analyzed. Removal of RNA instability sequences showed the greatest improvement of transgene expression strength.…”