Transient Expression of the GUS Reporter Gene in the Protoplasts and Partially Digested Cells of Ulva lactuca L. (Chlorophyta)

Abstract

The electroporation-mediated DNA transfer and expression of the gene encoding ß-glucuronidase (GUS) in the protoplasts and partially digested cells of a marine green alga Ulva lactuca was studied. In order to obtain protoplasts, vegetative blades of freshly collected plants were digested with a mixture of 1.5% Cellulysin and 1.5% abalone acetone powder for 3 h. Protoplasts were separated from partially digested thalli by sieving and centrifugation. The GUS reporter gene carried on the plasmid pBI121 was introduced into both isolated protoplasts and partially digested cells by electroporation in the presence of polyethylene glycol (PEG-8000). Different parameters of electroporation were varied in order to achieve 50% viability of the protoplasts. Histochemical assays revealed a transient expression of GUS activity in both electroporated protoplasts and partially digested cells 2 to 7 days after electroporation, indicating that an exogenous gene can be introduced into the protoplasm with or without the removal of the cell wall. Subsequent culture led to the regeneration of protoplasts into filamentous or disc-like multicellular plantlets. However, none of these regenerated plants stained positively for GUS activity.Brought to you by |

Genetic Engineering of Algae for Enhanced Biofuel Production

“…Members of the chlorophyte group that have been transformed include C. reinhardtii, which has been transformed using a variety of methods (44,87,88,93,170); Chlorella ellipsoidea (22, 83); Chlorella saccharophila (108); C. vulgaris (30, 69); Haematococcus pluvialis (177,187); V. carteri (81,163); Chlorella sorokiniana (30); Chlorella kessleri (49); Ulva lactuca (76); Dunaliella viridis (180); and D. salina (181,183). Heterokontophytes that have reportedly been transformed include Nannochloropsis oculata (21); diatoms such as T. pseudonana (147), P. tricornutum (2,210,211), Navicula saprophila (45), Cylindrotheca fusiformis (52,148), Cyclotella cryptica (45), and Thalassiosira weissflogii (51); and phaeophytes, such as Laminaria japonica (150) and Undaria pinnatifada (151).…”

Transient Transformation of Red Algal Cells: Breakthrough Toward Genetic Transformation of Marine Crop Porphyra Species

“…Expression of foreign genes in macroalgae has already been attempted using bacterial lacZ (-galactosidase) and uidA (-glucronidase, GUS) genes under direction by promoters of the cauliflower mosaic virus 35S RNA (CaMV 35S) and simian virus 40 (SV40) genes (Kübler et al, 1994;Kuang et al, 1998;Huang et al, 1996;Gan et al, 2003;Jiang et al, 2003). The CaMV 35S and SV40 promoters are typical eukaryotic class II promoters with a TATA box and thus are generally employed to drive transgenes in dicot plant and animal cells, respectively (Kang & An, 2005;Funabashi et al, 2010).…”

Expression of the lacZ reporter gene in sporophytes of the seaweed Laminaria japonica (Phaeophyceae) by gametophyte-targeted transformation

“…To date, in macroalgae transformation-with the exception of kelponly the CaMV 35S and nos promoters from higher plants have been utilized (Kurtzman and Cheney 1991;Kübler et al 1994;Huang et al 1996;Kuang et al 1998). For L. japonica, the CaMV 35S promoter has also been tested for transient expression in bombarded explants, and a possible histo-specificity expression fashion was found (Qin et al 1994).…”

“…For Ulva (green) and Porphyra (red), well-developed techniques are available for protoplast isolation and regeneration (Polne-Fuller and Gibor 1984;Reddy et al 1989). Transformed protoplasts were found to express the reporter gene transiently (Kübler et al 1994;Huang et al 1996), while no stable expression has yet been detected in regenerated plantlets. In contrast, protoplasts from the kelp, L. japonica, have not been regenerated until recently (Sawabe et al 1997).…”