Establishment of an efficient genetic transformation method in Dunaliella tertiolecta mediated by Agrobacterium tumefaciens

Abstract

Microalgae are photosynthetic microorganisms widely used for the production of highly valued compounds, and recently they have been shown to be promising as a system for the heterologous expression of proteins. Several transformation methods have been successfully developed, from which the Agrobacterium tumefaciens-mediated method remains the most promising. However, microalgae transformation efficiency by A. tumefaciens is shown to vary depending on several transformation conditions. The present study aimed to establish an efficient genetic transformation system in the green microalgae Dunaliella tertiolecta using the A. tumefaciens method. The parameters assessed were the infection medium, the concentration of the A. tumefaciens and co-culture time. As a preliminary screening, the expression of the gusA gene and the viability of transformed cells were evaluated and used to calculate a novel parameter called Transformation Efficiency Index (TEI). The statistical analysis of TEI values showed five treatments with the highest gusA gene expression. To ensure stable transformation, transformed colonies were cultured on selective medium using hygromycin B and the DNA of resistant colonies were extracted after five subcultures and molecularly analyzed by PCR. Results revealed that treatments which use solid infection medium, A. tumefaciens OD = 0.5 and co-culture times of 72 h exhibited the highest percentage of stable gusA expression. Overall, this study established an efficient, optimized A. tumefaciens-mediated genetic transformation of D. tertiolecta, which represents a relatively easy procedure with no expensive equipment required. This simple and efficient protocol opens the possibility for further genetic manipulation of this commercially-important microalgae for biotechnological applications.

Multiomics approaches and genetic engineering of metabolism for improved biorefinery and wastewater treatment in microalgae

“…sorokiniana , [ 87 ] Schizochytrium , [ 88 ] Isochrysis , [ 89 ] Dunaliella , [ 90 ] Parachlorella kessleri , [ 91 ] Dunaliella bardawil , [ 92 ] D . tertiolecta , [ 93 ] and Nannochloropsis sp. [ 94 ] The heterologous expression of β‐carotene hydroxylase isolated from C .…”

“…The target site is chosen from a targeted DNA sequence directly upstream PAM site (NGG). Cas9, CRISPR associated protein 9; CRISPR, Clustered Regularly Interspaced Short Palindromic Repeats; MN, meganuclease; sgRNA, single guide RNA; TALEN, transcription activator-like effector nuclease; ZFN, zinc finger nucleases transfer in C. reinhardtii, [86] C. sorokiniana, [87] Schizochytrium, [88] Isochrysis, [89] Dunaliella, [90] Parachlorella kessleri, [91] Dunaliella bardawil, [92] D. tertiolecta, [93] and Nannochloropsis sp. [94] The heterologous expression of β-carotene hydroxylase isolated from C. reinhardtii into D. salina, a β-carotene accumulation halotolerant marine microalga, by Agrobacterium-mediated gene transformation using rubisco promoter with the cooperation of chloroplast transit peptide sequence causes a significant accumulation of violaxanthin and zeaxanthin.…”

Grand Challenges in Microalgae Domestication

“…Dunalliella has a main haploid cycle, but sexual reproduction is known. Draft genomic data are available (Polle et al, 2017 ) and nuclear and chloroplastic transformations have been validated recently (Dehghani et al, 2017 ; Norzagaray-Valenzuela et al, 2018 ; Bahador et al, 2019 ; Song et al, 2019 ). Technically both Chlorella and Dunaliella could be amenable to intensive genetic engineering.…”

Two splice variants of the DsMEK1 mitogen-activated protein kinase kinase (MAPKK) are involved in salt stress regulation in Dunaliella salina in different ways

“…Constructed plasmids were transformed into D. salina cells using the Agrobacterium-mediated transformation method as described previously with slight modifications [51]. The empty vector pGreen-0029-Cmr (control), and constructed plasmids were transformed into Agrobacterium tumefaciens GV3101 via the freeze-thaw method.…”