Agrobacterium-Mediated Transformation of Three Freshwater Microalgal Strains

Sanitha, Mary; Radha, Sudhakar; Fatima, Anwar Aliya; Devi, Selvaraju Gayathri; Ramya, M.

doi:10.33073/pjm-2014-052

Cited by 16 publications

(9 citation statements)

References 29 publications

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“…Extremely inefficiently transgene expression is commonly observed in microalgae [9], and this appears to be the case for Scenedesmus as indicated by this and previous works. For example, transformation of S. almeriensis yielded little or very low GUS reporter expression even after screening many transformants [26], that of S. bajacalifornicus had only 2.96% of transformants with detectable GUS staining [46] and that of S. obliquus also showed only 1.5% of the transformants with GFP expression [25]. In our case, no GFP expression was observed at neither transcript nor protein level.…”

Section: Discussionmentioning

confidence: 64%

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Evaluation of strategies for improving the transgene expression in an oleaginous microalga Scenedesmus acutus

et al. 2019

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BackgroundGenetic transformation of microalgae has been hampered by inefficient transgene expression, limiting the progress of microalgal biotechnology. Many vector tools and strategies have been developed in recent years to improve transgene expression in the model microalga Chlamydomonas, but these were hardly applied to other microalgae. In this work, naturally-isolated oleaginous microalgae were accessed for genetic transformation, and various expression systems were evaluated in a selected microalga to circumvent inefficient transgene expression.ResultsInitially, a strain of Scenedesmus acutus was selected from the oleaginous microalgal collection based on its highest transformation rate and transgene stability. This strain, which had very low or no GFP reporter expression, was first tested to improve transgene expression by using intron-containing constructs and the transcript fusion using ble::E2A. The intron-containing constructs yielded 2.5–7.5% of transformants with 2–4-fold fluorescence signals, while the majority of the transformants of the transcript fusion had the fluorescence signals up to 10-fold. Subsequently, three UV-induced S. acutus mutants were isolated with moderate increases in the level and frequency of transgene expression (2–3-fold and 10–12%, respectively). Finally, a transcript fusion system was developed using psy white mutants with an expression vector containing PSY::E2A for complementation and light selection. Transformants with green colonies were selected under light exposure, and the transgene expression was detected at protein levels. Although the improvement using PSY::E2A was only minor (1–2-fold increase and ~ 7% of transformants), this system provides an alternative selectable marker that is compatible with large-scale culture.ConclusionsHere, the overall improvement of transgene expression using the Chlamydomonas tools was moderate. The most effective tool so far is the transcript fusion using ble::E2A system. This work demonstrates that, so far, genetic engineering of non-model microalgae is still a challenging task. Further development of tools and strategies for transgene expression in microalgae are critically needed.Electronic supplementary materialThe online version of this article (10.1186/s12896-018-0497-z) contains supplementary material, which is available to authorized users.

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Section: Discussionmentioning

confidence: 64%

“…[44], Chlorella vulgaris [45] and Ankistrodesmus sp. [46] were shown to be transformed based on GUS expression at approximately 21, 30 and 3.5% efficiency, respectively. Taken together, this work provides support for the use of the Agrobacterium -mediated method for transforming other potential industrial microalgae yet to be tested.…”

Section: Discussionmentioning

confidence: 99%

Evaluation of strategies for improving the transgene expression in an oleaginous microalga Scenedesmus acutus

et al. 2019

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“…Thus, the use of D-glucose with AS for pre-induction of Agrobacterium increases the transformation frequency up to 4 times that of Agrobacterium- mediated transformation in Dunaliella sp with or without AS (42 ± 3 per 10 6 cells) [ 11 ]. Despite extensive studies on Agrobacterium- mediated transformation of microalgae, the data inferred on transformation frequencies between investigations vary significantly [ 30 , 35 – 37 ]. In our study, induction of the Vir genes in Agrobacterium strains by D-glucose appears to work synergistically with induction by AS.…”

Section: Discussionmentioning

confidence: 99%

Synergistic Action of D-Glucose and Acetosyringone on Agrobacterium Strains for Efficient Dunaliella Transformation

Srinivasan

Gothandam

2016

PLoS ONE

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An effective transformation protocol for Dunaliella, a β-carotene producer, was developed using the synergistic mechanism of D-glucose and Acetosyringone on three different Agrobacterium strains (EHA105, GV3101 and LBA4404). In the present study, we investigated the pre-induction of Agrobacterium strains harboring pMDC45 binary vector in TAP media at varying concentrations of D-glucose (5 mM, 10 mM, and 15mM) and 100 μM of Acetosyringone for co-cultivation. Induction of Agrobacterium strains with 10 mM D-glucose and 100 μM Acetosyringone showed higher rates of efficiency compared to other treatments. The presence of GFP and HPT transgenes as a measure of transformation efficiency from the transgenic lines were determined using fluorescent microscopy, PCR, and southern blot analyzes. Highest transformation rate was obtained with the Agrobacterium strain LBA4404 (181 ± 3.78 cfu per 106 cells) followed by GV3101 (128 ± 5.29 cfu per 106 cells) and EHA105 (61 ± 5.03 cfu per 106 cells). However, the Agrobacterium strain GV3101 exhibited more efficient single copy transgene (HPT) transfer into the genome of D. salina than LBA4404. Therefore, future studies dealing with genetic modifications in D. salina can utilize GV3101 as an optimal Agrobacterium strain for gene transfer.

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“…It is known that streptomycin affects the growth and the photosynthetic activity of C. vulgaris through the inhibition of the synthesis of chloroplast proteins [23] and that a 40 mg L -1 concentration is enough to completely inhibit its growth [24]. However, C. sorokiniana presents a certain level of tolerance to aminoglycoside antibiotics [25], which relates to the high antibiotic concentration that was required to suppress the algae culture, when compared to reports from other species. Vector design and codon optimization…”

Section: Antibiotic Sensitivity Testmentioning

confidence: 99%

Alpha-tubulin promoter from Chlorella vulgaris allows genetic transformation of green coccoid microalga

Fernández-Rodríguez¹,

Garro-Monge²,

Guerrero-Barrantes³

et al. 2020

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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 transgene expression levels after genetic transformation. This work aims to evaluate a sequence from the 5′ upstream region of the alpha-tubulin gene from Chlorella vulgarisas a promoter for the expression of an antibiotic resistant gene in Chlorella sorokiniana. Using in silicoanalysis it was possible to identify a proximal promoter corresponding to 281 nucleotides upstream of the ATG start codon, which possessed 9 potential cis-regulatory element, including TATA Box and CAAT Box. The proximal promoter sequencewas used to drive the expression of a streptomycin-resistance gene (aada), previous optimization of its codon usage. The codon optimization of the aadasequence allowed it to obtain a GC content of 69.8% (compared to 53% of the original sequence), which increases its similarity with Chlorellasp. 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. sorokinianacells. 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.

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Agrobacterium-Mediated Transformation of Three Freshwater Microalgal Strains

Cited by 16 publications

References 29 publications

Evaluation of strategies for improving the transgene expression in an oleaginous microalga Scenedesmus acutus

Evaluation of strategies for improving the transgene expression in an oleaginous microalga Scenedesmus acutus

Synergistic Action of D-Glucose and Acetosyringone on Agrobacterium Strains for Efficient Dunaliella Transformation

Alpha-tubulin promoter from Chlorella vulgaris allows genetic transformation of green coccoid microalga

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