A Novel Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) Promoter for Expressing Transgenes in the Halotolerant Alga Dunaliella salina

Abstract

A major challenge for efficient transgene expression in Dunaliella salina is to find strong endogenous promoters to drive the transgene expression. In the present study, a novel glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter was cloned and used to drive expressions of the bialaphos resistance (bar) gene and of the N-terminal fragment of human canstatin (Can-N). The results showed that the bar gene was transcribed by the GAPDH promoter and integrated into the genome of the transformants of D. salina. Furthermore, the PCR identification, Southern and western blots indicated that Can-N was expressed in transgenic D. salina, demonstrating that the promoter of the D. salina GAPDH gene is suitable for driving expression of heterologous genes in transgenic D. salina.

Genetic Engineering for Microalgae Strain Improvement in Relation to Biocrude Production Systems

“…Some analysis has started in species other than Chlamydomonas including Dunaliella (Jia et al 2012;Lu et al 2011;Park et al 2013), and some crossover is expected from plant gene analysis especially in Arabidopsis. A start has also been made in understanding the role of mRNA regulation (Schulze et al 2010;Wobbe et al 2009) and chromatin remodelling in Chlamydomonas (Strenkert et al 2011(Strenkert et al , 2013.…”

Characterization of novel nitrate reductase-deficient mutants for transgenic Dunaliella salina systems

“…salina (UTEX-LB-1644) purchased from the Culture Collection of Algae at the University of Texas were grown in modified medium at 26°C with 12 h light/day under 60 μM photon·m -2 ·s -1 illumination as previously described (Jia et al 2012). When nitrite or urea was used as the sole nitrogen source, KNO 3 in the medium was replaced by 5 mM NaNO 2 or 2.5 mM urea.…”

“…High-quality genomic DNA was extracted from the transformants using a modified sodium dodecyl sulfate (SDS) method as previously described (Jia et al, 2012). PCR was used to detect the integration of the NR gene into the D. salina genome using the NR5 primer according to the DCA promoter (GenBank: AF541981) and the coding region of the NR cDNA (Table 1).…”

Preparation of recombinant human canstatin using transgenic <italic>Dunaliella salina</italic>

“…In contrast to the published reports [6,8], however, the amount of the purified canstatin was lower in the present study. Therefore, an endogenous promoter of D. salina cells [15] was cloned and inserted in order to improve the expression level of canstatin proteins. Future studies should be done including the construction of high-efficient expression vectors for D. salina and screening of the transformants with stable, unvarying, and high expression of canstatin.…”

“…The inserted canstatin fragments yielded a mature protein of 236 amino acids comprising the 227 amino acids of canstatin, 6 amino acids of histidine, and 3 extra amino acids. The recombinant canstatin has a molecular mass of 26 kDa, which may be due to the recombinant protein expressed in D. salina as a fusion protein with a histidine tag [8,15]. After being purified with a Ni-NTA resin purification kit (Qiagen, Hilden, Germany), a total of 84 mg purified canstatin with a molecular weight of 26 kDa was obtained from the 100 ml cultures of D. salina that was further identified by western blot analysis ( Supplementary Fig.…”