Resumo

FERNANDEZ-RODRIGUEZ, Raquel; GARRO-MONGE, Giovanni; GUERRERO-BARRANTES, Maritza  e  GOMEZ-ESPINOZA, Olman. Alpha-tubulin promoter from Chlorella vulgaris allows genetic transformation of green coccoid microalga. Tecnología en Marcha [online]. 2020, vol.33, n.2, pp.27-36. ISSN 0379-3982.  http://dx.doi.org/10.18845/tm.v33i1.5017.

Microalgae have become a feasible platform for high-value recombinant protein production. Although diverse sets of genetic tools for microalgae transformation have been developed, some critical elements, such as endogenous promoters, need to be improved for increasing transgenic expression levels after genetic transformation. This work aimed to evaluate a sequence from the 5′ upstream region of the alpha-tubulin gene from Chlorella vulgaris as a promoter for the expression of an antibiotic resistant gene in Chlorella sorokiniana. Using in silico analysis it was possible to identify a proximal promoter corresponding to 281 nucleotides upstream of the ATG start codon, which possessed 9 potential cis-regulatory elements, including TATA Box and CAAT Box. The proximal promoter sequence was used to drive the expression of a streptomycinresistance gene (aada), previous optimization of its codon usage. The codon optimization of the aada sequence allowed investigators to obtain a GC content of 69,8% (compared to 53% of the original sequence), which increases its similarity with Chlorella sp. genomes (67,2%). Both genetic elements were cloned into the pUC57-Kan vector and transformed into C. sorokiniana cells by electroporation. The microalgae transgenic colonies were identified through culture in selective medium and PCR. Our results proved the capacity of the Chlorella vulgaris alpha-tubulin promoter to express a foreign antibiotic-resistance gene in C. sorokiniana cells. Research of endogenous promoting sequences is essential in order to accomplish an efficient heterologous gene expression, especially in microalgae used for industrial production like those from Chlorella genus.

Palavras-chave : Electroporation; kazusa; streptomycin; transgenic; TATA-box.

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