Stable nuclear transformation of rhodophyte species Porphyridium purpureum: advanced molecular tools and an optimized method

Prasad, Binod; Lein, Wolfgang; General, Thiyam; Lindenberger, Christoph; Vadakedath, Nithya

doi:10.1007/s11120-018-0587-8

Cited by 12 publications

(14 citation statements)

References 54 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Cells were harvested by centrifugation (14,000 rpm for 5 min). Total genomic DNA was extracted using the CTAB method [ 68 ]. Total DNA from each species was sequenced using ONT (Oxford Nanopore Technologies, Oxford, England) and Illumina (NovaSeq 6000 system: paired-end TruSeq Nano DNA Prep Kit) platforms.…”

Section: Methodsmentioning

confidence: 99%

Group II intron and repeat-rich red algal mitochondrial genomes demonstrate the dynamic recent history of autocatalytic RNAs

et al. 2022

View full text Add to dashboard Cite

Background Group II introns are mobile genetic elements that can insert at specific target sequences, however, their origins are often challenging to reconstruct because of rapid sequence decay following invasion and spread into different sites. To advance understanding of group II intron spread, we studied the intron-rich mitochondrial genome (mitogenome) in the unicellular red alga, Porphyridium. Results Analysis of mitogenomes in three closely related species in this genus revealed they were 3–6-fold larger in size (56–132 kbp) than in other red algae, that have genomes of size 21–43 kbp. This discrepancy is explained by two factors, group II intron invasion and expansion of repeated sequences in large intergenic regions. Phylogenetic analysis demonstrates that many mitogenome group II intron families are specific to Porphyridium, whereas others are closely related to sequences in fungi and in the red alga-derived plastids of stramenopiles. Network analysis of intron-encoded proteins (IEPs) shows a clear link between plastid and mitochondrial IEPs in distantly related species, with both groups associated with prokaryotic sequences. Conclusion Our analysis of group II introns in Porphyridium mitogenomes demonstrates the dynamic nature of group II intron evolution, strongly supports the lateral movement of group II introns among diverse eukaryotes, and reveals their ability to proliferate, once integrated in mitochondrial DNA.

show abstract

Section: Methodsmentioning

confidence: 99%

Group II intron and repeat-rich red algal mitochondrial genomes demonstrate the dynamic recent history of autocatalytic RNAs

et al. 2022

View full text Add to dashboard Cite

show abstract

“…After transformation of the target cell by the bacterium, the selection is made using an antibiotic that can be selective against the bacterium while selecting the transformed cells. Variable transformation efficiencies have been reported depending on the protocol followed [103,[108][109][110]. Factors such as temperature, pH, and time of virulence gene induction have been reported to have a substantial effect on transformation efficiency [111].…”

Section: Agrobacterium-mediated Transformationmentioning

confidence: 99%

Gene Delivery Technologies with Applications in Microalgal Genetic Engineering

Gutiérrez

Lauersen

2021

Biology

View full text Add to dashboard Cite

Microalgae and cyanobacteria are photosynthetic microbes that can be grown with the simple inputs of water, carbon dioxide, (sun)light, and trace elements. Their engineering holds the promise of tailored bio-molecule production using sustainable, environmentally friendly waste carbon inputs. Although algal engineering examples are beginning to show maturity, severe limitations remain in the transformation of multigene expression cassettes into model species and DNA delivery into non-model hosts. This review highlights common and emerging DNA delivery methods used for other organisms that may find future applications in algal engineering.

show abstract

“…Initially, the efficacy of three Agrobacterium strains, LBA4404, C58C1, and AGL-1, to transfer T-DNA into the genome of E. gracilis was tested. Based on other microalgae species transformation [35,36,44,45], the Agrobacterium culture's optical density was kept at 1.0 (A 600 ). The optical density of E. gracilis was set to 2.0 (A 750 ).…”

Section: Optimization Of Factors In the Atmt Procedures Of E Gracilismentioning

confidence: 99%

“…In this study, we report an efficient A. tumefaciens-mediated transformation (ATMT) approach and molecular toolkits for the genetic engineering of E. gracilis KLEBS strain Z. A. tumefaciens has been efficiently used to transform its natural host plants and other organisms, including microalgae from different lineages [29,[35][36][37][38][39][40][41][42][43][44][45]. Genetic transformation via A. tumefaciens offers several advantages, including the transfer of large DNA fragments, ease of implementation, low level of rearrangement, and the lower tendency of transgene silencing due to gene integration into transcriptionally active regions and a low copy level of the transgene [43,46,47].…”

Section: Introductionmentioning

confidence: 99%

Agrobacterium tumefaciens-Mediated Nuclear Transformation of a Biotechnologically Important Microalga—Euglena gracilis

Becker

Prasad

Ntefidou

et al. 2021

IJMS

Self Cite

View full text Add to dashboard Cite

Euglena gracilis (E. gracilis) is an attractive organism due to its evolutionary history and substantial potential to produce biochemicals of commercial importance. This study describes the establishment of an optimized protocol for the genetic transformation of E. gracilis mediated by Agrobacterium (A. tumefaciens). E. gracilis was found to be highly sensitive to hygromycin and zeocin, thus offering a set of resistance marker genes for the selection of transformants. A. tumefaciens-mediated transformation (ATMT) yielded hygromycin-resistant cells. However, hygromycin-resistant cells hosting the gus gene (encoding β-glucuronidase (GUS)) were found to be GUS-negative, indicating that the gus gene had explicitly been silenced. To circumvent transgene silencing, GUS was expressed from the nuclear genome as transcriptional fusions with the hygromycin resistance gene (hptII) (encoding hygromycin phosphotransferase II) with the foot and mouth disease virus (FMDV)-derived 2A self-cleaving sequence placed between the coding sequences. ATMT of Euglena with the hptII-2A–gus gene yielded hygromycin-resistant, GUS-positive cells. The transformation was verified by PCR amplification of the T-DNA region genes, determination of GUS activity, and indirect immunofluorescence assays. Cocultivation factors optimization revealed that a higher number of transformants was obtained when A. tumefaciens LBA4404 (A600 = 1.0) and E. gracilis (A750 = 2.0) cultures were cocultured for 48 h at 19 °C in an organic medium (pH 6.5) containing 50 µM acetosyringone. Transformation efficiency of 8.26 ± 4.9% was achieved under the optimized cocultivation parameters. The molecular toolkits and method presented here can be used to bioengineer E. gracilis for producing high-value products and fundamental studies.

show abstract

Stable nuclear transformation of rhodophyte species Porphyridium purpureum: advanced molecular tools and an optimized method

Cited by 12 publications

References 54 publications

Group II intron and repeat-rich red algal mitochondrial genomes demonstrate the dynamic recent history of autocatalytic RNAs

Group II intron and repeat-rich red algal mitochondrial genomes demonstrate the dynamic recent history of autocatalytic RNAs

Gene Delivery Technologies with Applications in Microalgal Genetic Engineering

Agrobacterium tumefaciens-Mediated Nuclear Transformation of a Biotechnologically Important Microalga—Euglena gracilis

Contact Info

Product

Resources

About