Factors influencing efficiency of transient gene expression in the red macrophyte Porphyra yezoensis

Abstract

Manuscript 2 AbsrtactThe marine red alga Porphyra yezoensis has been proposed as a model plant for physiological and genetic studies in seaweeds because of its biological and economical importance. However, the progress of molecular biological studies using gene transfection and genetic transformation systems has been hindered by difficulties in the expression of foreign genes in P. yezoensis cells. To overcome this situation, we developed a transient gene expression system to monitor gene expression in P. yezoensis cells. An artificial -glucuronidase (GUS) coding region was synthesized to adapt it to the codon usage of P. yezoensis (PyGUS) and then evaluated for efficiency as a reporter of transient gene expression by particle bombardment. We also demonstrated the importance of using the promoter of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene from P. yezoensis for efficient expression of PyGUS, because the cauliflower mosaic virus (CaMV) 35S promoter, which has been successfully used for monitoring gene expression in nuclei and chloroplasts of higher plants, was less active in P. yezoensis cells. Therefore, the lack of knowledge about differences in the regulatory machinery of gene expression between P. yezoensis and terrestrial plants seems to be why experimental systems for monitoring gene expression were previously not developed in P. yezoensis. Establishment of the transient gene expression system in P. yezoensis could facilitate biotechnological developments in this organism.3

Transient Gene Expression System Established in Porphyra yezoensis Is Widely Applicable in Bangiophycean Algae

“…S1d and S1i). Although there are no obvious morphological differences between P. yezoensis and P. tenera, CAPS analysis of the ARP4 loci can distinguish TU-1 and JTW (Park et al 2008). …”

“…Fluorometric GUS assays were performed as described previously (Jefferson et al 1987). Histochemical staining of GUS-expressing cells was performed almost the same as described previously (Fukuda et al 2008). After bombardment, algal materials were incubated at 37 ºC for 6 h in staining solution containing 2 mM 5-bromo-4-chloro-3-indolylglucuronide (X-gluc), 5 mM potassium ferricyanide (K 3 Fe(CN) 6 ), 5 mM potassium ferrocyanide (K 4 Fe(CN) 6 ), 0.5% Triton X-100, 1.5 M sorbitol and 50 mM sodium phosphate, pH 7.0.…”

“…In order to overcome these problems, we have established efficient transient gene expression systems in P. yezoensis. First, a -glucuronidase (GUS) reporter system was developed with a synthetic gene named PyGUS, whose codon usage was optimized to that in P. yezoensis genes, under the direction of endogenous strong promoters such as the PyGAPDH and PyAct1 promoters (Fukuda et al 2008;Takahashi et al 2010). Subsequently, these two alterations were applied to fluorescent protein reporters such as humanized AmCFP and plant-adapted green fluorescent protein [sGFP(S65T); Niwa et al 1999], which allowed successful visualization of the subcellular localization of phosphoinositides and transcription factors in P. yezoensis cells (Mikami et al 2009;Uji et al 2010).…”

Enhanced green fluorescent protein (egfp) gene expression in Tetraselmis subcordiformis chloroplast with endogenous regulators

“…It is generally accepted that the efficiency of regulatory elements (promoters) is higher than foreign promoters for chloroplast transformation (Fukuda et al 2008;Ruhlman et al 2010;Wei et al 2012). Prrn (Svab and Maliga 1993;Sidorov et al 1999), PpsbA (Li et al 2011), TpsbA (Svab and Maliga 1993;Sidorov et al 1999), and TrbcL (Doetsch et al 2001;Cui et al 2014) are commonly used endogenous regulators.…”

Transformation of lacZ using different promoters in the commercially important red alga, Gracilaria gracilis

“…Transient expression of the lacZ gene with the SV40 promoter has also been observed in G. changii (Gan et al, 2003) and Haematococcus pluvialis (Teng et al, 2002). Recently, the use of endogenous promoters, targeted homologous recombination and matrix attachment regions (MARs) to increase reporter gene expression in P. yezoenisis has received much attention (Fukuda et al, 2008;Gong et al, 2007;Liu et al, 2003;Mizukami et al, 2004;Zuo et al, 2007).…”