Improved molecular tools for sugar cane biotechnology

Abstract

Sugar cane is a major source of food and fuel worldwide. Biotechnology has the potential to improve economically-important traits in sugar cane as well as diversify sugar cane beyond traditional applications such as sucrose production. High levels of transgene expression are key to the success of improving crops through biotechnology. Here we describe new molecular tools that both expand and improve gene expression capabilities in sugar cane. We have identified promoters that can be used to drive high levels of gene expression in the leaf and stem of transgenic sugar cane. One of these promoters, derived from the Cestrum yellow leaf curling virus, drives levels of constitutive transgene expression that are significantly higher than those achieved by the historical benchmark maize polyubiquitin-1 (Zm-Ubi1) promoter. A second promoter, the maize phosphonenolpyruvate carboxylate promoter, was found to be a strong, leaf-preferred promoter that enables levels of expression comparable to Zm-Ubi1 in this organ. Transgene expression was increased approximately 50-fold by gene modification, which included optimising the codon usage of the coding sequence to better suit sugar cane. We also describe a novel dual transcriptional enhancer that increased gene expression from different promoters, boosting expression from Zm-Ubi1 over eightfold. These molecular tools will be extremely valuable for the improvement of sugar cane through biotechnology.

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).…”

Sugarcane biotechnology: tapping unlimited potential

“…Biotechnology may assist the crop to meet future world energy demands, thereby improving the value and sustainability of the crop. Improvement of sugar yield through selective breeding may have reached its potential; however, there still exists scope for the application of biotechnology to improve sugarcane by enhancing desirable traits and adding novel traits that may add value and improve the sustainability of this crop (Kinkema et al 2014a).…”

“…Constitutive +4.5-fold Kinkema et al (2014a, b) considers the use of simplex (single allele copy from one parent) markers to obtain the genetic map (Wu et al 1992, Al-Janabi et al 1993). More recently, molecular genetic maps, although incomplete, have also been constructed from 10 segregating populations (Zhang et al 2014).…”

Design rules for efficient transgene expression in plants

“…There is a strong post‐transcriptional component to this transgene silencing, demonstrated by nuclear run‐on transcription analysis (Birch et al ., ; Ingelbrecht et al ., ). Transgenes designed to lack rare codons and RNA instability motifs can result in higher levels of expression (Kinkema et al ., ; Mudge et al ., ) and show reduced post‐transcriptional silencing as evidenced by RNA degradation products in northern blots and PCR tests for truncated oligoadenylated transcripts (unpublished results from this laboratory). In the present study, we investigated the contributions of codon optimization, removal of RNA instability motifs and blocking RNAi to this effect, to help provide a set of design rules for efficiently expressed transgenes in plants.…”