Identification of Leaf Promoters for Use in Transgenic Wheat

Abstract

Wheat yields have plateaued in recent years and given the growing global population there is a pressing need to develop higher yielding varieties to meet future demand. Genetic manipulation of photosynthesis in elite wheat varieties offers the opportunity to significantly increase yields. However, the absence of a well-defined molecular tool-box of promoters to manipulate leaf processes in wheat hinders advancements in this area. Two promoters, one driving the expression of sedoheptulose-1,7-bisphosphatase (SBPase) and the other fructose-1,6-bisphosphate aldolase (FBPA) from Brachypodium distachyon were identified and cloned into a vector in front of the GUS reporter gene. Both promoters were shown to be functionally active in wheat in both transient assays and in stably transformed wheat plants. Analysis of the stable transformants of wheat (cv. Cadenza) showed that both promoters controlled gus expression throughout leaf development as well as in other green tissues. The availability of these promoters provides new tools for the expression of genes in transgenic wheat leaves and also paves the way for multigene manipulation of photosynthesis to improve yields.

Feeding the world: improving photosynthetic efficiency for sustainable crop production

“…The role of modelling in enabling novel targets to be identified will also be crucial given the complexity of the processes involved. To achieve the full potential of these opportunities, the use of new tools, which allow the quick, efficient, and cheap insertion of multiple transgenes into plants, will be paramount (Engler et al , 2008, 2009, 2014; Marillonnet and Werner, 2015; Exposito-Rodriguez et al , 2017), as will be the development of new promoters for use in crop plants, which are currently limited (Mukherjee et al , 2015; Alotaibi et al , 2018). If these opportunities are to be fully exploited, regulations governing the use of genetic modification and genome editing technologies will need to be reviewed.…”

A modular cloning toolkit for genome editing in plants

“…pFH54: An Arabidopsis codon optimised Cas9 (pICSL90016) [25] was assembled with an Arabidopsis UBIQUITIN10 promoter-5'UTR (pICSL12015) [25] and a pea 3A 3'UTR-Terminator (pFH44) into the level 1 position 2 acceptor pICH47742 [2] (Addgene ID #48001) using a GG cut-ligation reaction via BsaI-HF®v2. pFH114: The ZmUBIp::BAR:NOSt cassette was amplified using the vector pRRES1.111 [26] with FH443/FH444 primers and the PCR product was assembled into the level 1 position 1 acceptor pICH47732 [2] (Addgene ID #48000) using a GG cut-ligation reaction via BsaI-HF®v2.…”

Remarkable recent changes in genetic diversity of the avirulence geneAvrStb6in global populations of the wheat pathogenZymoseptoria tritici

“…The sgRNA constructs, carrying two sgRNAs each (annotated sequences are available on figshare), were assembled using the pENTR4-sgRNA4 vector as previously described (Zhou et al, 2014). All three sgRNA plasmids were co-delivered along with pCas9- GFP (Zhang et al, 2019) encoding the wheat codonoptimised Cas9 fused in frame at the C-terminus with Green Fluorescent Protein (GFP), and pRRes1.111 (Alotaibi et al, 2018) which directs expression of the bar selectable marker gene into immature wheat embryos of wheat cv. Cadenza using biolistics essentially as described in Sparks and Doherty (2020).…”