Chloroplast Dual Divergent Promoter Plasmid for Heterologous Protein Expression in Tetraselmis suecica (Chlorophyceae, Chlorodendrales). Issue 4 (23rd May 2020)

• Record Type: Journal Article
• Authors: Gutiérrez, Carla L.; Muñoz, Carla; San Martín, Margarita; Cadoret, Jean‐Paul; Henríquez, Vitalia
• Editors: Herron, M.
• Is Part Of: Journal of phycology. Volume 56:Issue 4(2020:Aug.)
• Page Start: 1066
• Page End: 1076
• Publication Date: 2020-05-23
• Subjects: cat gene -- chloroplast -- divergent promoters -- Tetraselmis suecica -- tGFP; Algae -- Periodicals; 579.8
• DOI: 10.1111/jpy.13013
• Languages: English
• ISSNs: 0022-3646

Abstract

Abstract : The eukaryotic green microalga Tetraselmis suecica is commonly used for aquaculture purposes because of its high stress tolerance and ease of culture in a wide spectrum of environments; they are therefore suitable candidates for biotechnology applications. To date, no data are available regarding chloroplast transformation vectors based on specific endogenous promoters and homologous targeting regions. We report on the identification of Tetraselmis suecica genes encoding the ribulose bisphosphate carboxylase/oxygenase large subunit protein, the photosystem II D1 protein and the ATP synthase CF1‐beta subunit protein together with their untranslated regions (5′UTR, 3′UTR). The full‐length ORFs of the putative genes with their regulatory sequences were obtained. We were also able to identify the downstream 3′ end of the large subunit ribosomal RNA gene (23S) along with the 5S RNA end‐to‐end with the psb A gene on the complementary strand. The intergenic region between these genes appears to be a good target site for the integration of target proteins. Moreover, we identified a back‐to‐back promoter region among the rbc L and atp B genes. To assess the bidirectionality activities of both promoters, a dual reporter vector was constructed for Tetraselmis suecica transformation containing the cat and TurboGFP genes driven by the 5′ rbc L/5′ atp B divergent promoter. The vector included the 23S‐5S and psb A nucleotide sequences as flanking regions. These flanking regions provided suitable insertion sites within the chloroplast genome for cassette integration via homologous recombination. Simultaneous expression of the chloramphenicol‐resistant conferring gene and the gene coding for TurboGFP driven by 5′ rbc L/5′ atp B showed a potent natural bidirectional promoter as a reliable genetic tool.