A technical breakthrough close at hand: feasible approaches toward establishing a gene-targeting genetic transformation system in seaweeds

Abstract

The lack of a genetic transformation system that allows integration of a foreign gene into the genome is a serious hindrance to progress in biological research on seaweeds. In contrast, physiological research in seaweeds has a long history of focus on the establishment of cell polarity and multicellularity (Brownlee and Bouget, 1998; Hable and Hart, 2010). Today, nuclear genomes have been recently sequenced for many species of seaweeds and there is now a significant accumulation of genomic information in the form of expressed sequence tag data in public and private databases for analyses (see Mikami, 2014). Although physiological and molecular biological approaches have the potential to allow genome-wide and comprehensive analyses of factors involved in seaweed development, the lack of a genetic transformation system hinders functional identification of genes encoding regulatory components of seaweed development. This is impeding the application of the molecular biological knowledge in industrial and economic activities related to seaweed. Because genetic manipulation systems are indispensable for the functional analysis of genes, it is necessary to develop reverse genetic methods for gene knock-out, knock-in, or replacement in a variety of seaweeds via either homologous recombination or random integration of foreign DNA with a marker to identify its integration site in the genome. To accelerate progress toward establishing genetic transformation methods, there are four important issues to be resolved: (1) creating expression constructs particularly suited to seaweed cells; (2) transferring the expression constructs into cells; (3) ensuring integration of expression constructs into the genome; and (4) selecting genetically transformed cells. It has already been confirmed that combining codon-optimized coding regions and endogenous strong promoters results in high-efficiency expression in seaweed cells (Mikami et al., 2011; Mikami and Uji, 2012; Mikami, 2013). Seaweed genomes generally have high GC content and thus coding sequences should be biased toward higher amounts of G and C. Moreover, the Cauliflower mosaic virus (CaMV) 35S RNA promoter that has been used for foreign gene expression in seaweeds prior to establishment of the transient gene expression has rather low activity in seaweed cells (Mikami et al., 2011; Mikami and Uji, 2012; Mikami, 2013). But importantly, neither the codon optimization nor endogenous promoters yet tested could enhance the expression of foreign genes in seaweeds (Mikami et al., 2011). These findings clearly indicate that codon-optimized coding regions directed by strong endogenous promoters will be required elements of the design of constructs that can be expressed efficiently in seaweed cells (Mikami et al., 2011; Mikami and Uji, 2012; Mikami, 2013). This strategy has already been applied to a wide variety of red seaweeds (Hirata et al., 2011a,b; Son et al., 2011). Moreover, the second requirement for genetic transformation, to transfer expression constructs into cells, has been resolved using particle bombardment technology in red seaweeds (Mikami et al., 2011; Mikami and Uji, 2012; Mikami, 2013) and microinjection in Ectocarpus siliculosus (Farnham et al., 2013). However, neither electroporation nor glass bead methods have yet been successful for transferring DNA constructs into seaweed cells. Our preliminary data indicated that the glass bead method and various gene transfer regents designed for mammalian cells were ineffective for cell wall–less protoplasts and monospores from the red seaweed Pyropia yezoensis. Taken together, the third and fourth issues—to ensure integration of expression constructs into the genome, and select genetically transformed cells, respectively—still have to be resolved to establish nuclear genetic transformation.