Validation of a New Multicistronic Plasmid for the Efficient and Stable Expression of Transgenes in Microalgae

Abstract

Low stability of transgenes and high variability of their expression levels among the obtained transformants are still pending challenges in the nuclear genetic transformation of microalgae. We have generated a new multicistronic microalgal expression plasmid, called Phyco69, to make easier the large phenotypic screening usually necessary for the selection of high-expression stable clones. This plasmid contains a polylinker region (PLK) where any gene of interest (GOI) can be inserted and get linked, through a short viral self-cleaving peptide to the amino terminus of the aminoglycoside 3'-phosphotransferase (APHVIII) from Streptomyces rimosus, which confers resistance to the antibiotic paromomycin. The plasmid has been validated by expressing a second antibiotic resistance marker, the ShBLE gene, which confers resistance to phleomycin. It has been shown, by RT-PCR and by phenotypic studies, that the fusion of the GOI to the selective marker gene APHVIII provides a simple method to screen and select the transformants with the highest level of expression of both the APHVIII gene and the GOI among the obtained transformants. Immunodetection studies have shown that the multicistronic transcript generated from Phyco69 is correctly processed, producing independent gene products from a common promoter.

Keywords: 2A; microalgae transformation; multicistronic transcript.; paromomycin.

Figure A1

Detailed sequences flanking the start codon of the APHVIII gene before and after splicing of the first RbcS2 intron in the control pSI103 (A) and Phyco69 (B) plasmids and amino acidic sequence of the proteins generated in the three possible reading frames. A comparison of the efficiency of the transformation of Chlamydomonas with different quantities of each plasmid is also shown (C).

Figure A2

Average survival Chlamydomonas clones transformed with the plasmid Phyco69BLE and cultured with increasing concentrations of paromomycin (A) or phleomycin (B). Values are the average of at least three replicates and bars represent the standard deviation.

Figure A3

Inmunoblot analysis showing the APHVIII protein in the full-length blot. Total protein extracts (60 μg) were fractionated with SDS-PAGE, transferred to a PVDF membrane and probed with rabbit anti-APHVIII polyclonal antibodies and alkaline phosphatase-conjugated goat anti-rabbit IgG, as indicated in 3.4 materials and methods. MW, contains molecular weight markers; #4, #7 and #22 corresponding to three Chlamydomonas transformants and C-, one control untransformed Chlamydomonas culture.

Figure 1

Schematic diagram of the main elements of the three new plasmids generated in this work in comparison with the control pSI103 plasmid (A) and of the translation products resulting from them (B). The blue line “^“ represents the flexible peptide sequence GASGQGASGADIGASGQGASDA. “▼“ denotes the hydrolysis point in the self-cleaving peptide FMDV-2A. Representative examples of the nuclear transformation of Chlamydomonas reinhardtii with the three newly generated plasmids (Phyco69, PhycoC67, and PhycoC67FL) and the control pSI103 plasmid are also shown (C).

Figure 2

Schematic diagram of the selective strategy used to choose Chlamydomonas transformants with high levels of expression of genes APHVIII and ShBLE.

Figure 3

Relative expression of genes ShBLE and APHVIII in a series of Chlamydomonas transformants resistant to paromomycin. Chlamydomonas was transformed with the plasmid Phyco69BLE (A), and a selection of clones resistant to paromomycin and phleomycin (B) were analyzed by PCR using genomic DNA as target to test correct insertion of the BLE-2A-APHVIII cassette (C), and by real-time PCR (D) to test the relative expression level of the genes ShBLE (■) and APHVIII (■). Red arrows indicate the clones which show an important increase (>5 times) in the expression levels of APHVIII and ShBLE in relation with the reference transformed clone (↓), and correspond to the clones circled with a red line in panel B. Blue arrows denote the clones with a small difference in the expression of these genes in relation with the reference transformed clone and correspond to the clones circled with a blue line in panel (B). The expression of all genes was normalized to the ubiquitin ligase (UBQL) house-keeping gene and presented as fold-change relative to the transcript level of the reference transformed clone, which was chosen among those transformants which show low expression levels. Values are the average of three replicates, and bars indicate standard deviation.

Figure 4

Immunoblot analysis showing the APHVIII protein product from C. reinhardtii cells transformed with the plasmid Phyco69BLE. Total soluble cell protein extracts (60 μg) of three Chlamydomonas transformants (#4, #7, #22) and one control untransformed Chlamydomonas culture (C-) were fractionated with SDS-PAGE, transferred to a PVDF membrane and probed with rabbit anti-APHVIII polyclonal antibodies and alkaline phosphatase-conjugated goat anti-rabbit IgG, as indicated in materials and methods. MW lane: Protein molecular weight markers.