Successful expression of heterologous egfp gene in the mitochondria of a photosynthetic eukaryote Chlamydomonas reinhardtii

Hu, Zhangli; Zhao, Zhonglin; Wu, Zhihua; Zhang, Fan; Wu, Jinxia; Li, Jiancheng

doi:10.1016/j.mito.2011.05.012

Cited by 18 publications

(10 citation statements)

References 23 publications

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“…Later, the same mutant was used to transform the mitochondrion to insert the gene encoding the enhanced green fluorescent protein (egfp) between the cob gene and the left terminal repeat, and its expression was demonstrated by confocal microscopy and Western blot analyses (Hu et al . ). The same group also transformed this mutant to insert a gene encoding zeomycin resistance enzyme in the same site, allowing the growth of the microalgae in media with this antibiotic (Hu et al .…”

Section: Chloroplast and Mitochondrial Transformationmentioning

confidence: 97%

“…Mitochondrial transformants obtained from rob-shaped mutants could be rescued after selection in the dark with a mtDNA fragment covering the deletion, because the mutant could not grow under heterotrophic conditions. Later, the same mutant was used to transform the mitochondrion to insert the gene encoding the enhanced green fluorescent protein (egfp) between the cob gene and the left terminal repeat, and its expression was demonstrated by confocal microscopy and Western blot analyses (Hu et al 2011). The same group also transformed this mutant to insert a gene encoding zeomycin resistance enzyme in the same site, allowing the growth of the microalgae in media with this antibiotic .…”

Section: Chloroplast and Mitochondrial Transformationmentioning

confidence: 99%

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Advances and challenges in genetic engineering of microalgae

Fajardo

Donato

Carrasco

et al. 2019

Reviews in Aquaculture

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Knowledge at the molecular level of microalgae has obtained great scientific interest in recent years, because of their potential for biofuel production, as well as other highly valued molecules. Recent advances in the area of the genetic manipulation of microalgae open an entirely new field of possible applications for these organisms as biorefineries. This review represents a compendium that gathers the main milestones that have marked the area of genetic engineering in microalgae during the last decade, from classical techniques for the transformation and expression of transgenes, to the most avant‐garde techniques currently used for gene silencing and DNA edition, as well as a discussion about the future perspectives in this field.

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Section: Chloroplast and Mitochondrial Transformationmentioning

confidence: 97%

Section: Chloroplast and Mitochondrial Transformationmentioning

confidence: 99%

Advances and challenges in genetic engineering of microalgae

Fajardo

Donato

Carrasco

et al. 2019

Reviews in Aquaculture

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“…Mitochondrial transformants are selected by complementation of the respiration deficient mutant dum1 (Boynton and Gillham, 1996; Yamasaki et al, 2005; Hu et al, 2011). …”

Section: Molecular Genetics and Genomics Tools For Volvocine Algaementioning

confidence: 99%

“…Green fluorescent protein (GFP) has been codon optimized for use in Chlamydomonas (Fuhrmann et al, 1999) and has been expressed transgenically from the nucleus, chloroplast and mitochondrial genomes (Fuhrmann et al, 1999; Purton, 2007; Hu et al, 2011; Ramesh et al, 2011). Live cell imaging with GFP in algae is a challenge due to high background fluorescence from chlorophyll and other auto-fluorescent compounds, but can be effective in cases where the GFP is well expressed or concentrated in a specific subcellular location (Fuhrmann et al, 1999; Yoshihara et al, 2008; Diener, 2009; Hayashi and Shinozaki, 2011; Neupert et al, 2012).…”

Section: Molecular Genetics and Genomics Tools For Volvocine Algaementioning

confidence: 99%

Genomics of Volvocine Algae

Umen

Olson

2012

Advances in Botanical Research

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Volvocine algae are a group of chlorophytes that together comprise a unique model for evolutionary and developmental biology. The species Chlamydomonas reinhardtii and Volvox carteri represent extremes in morphological diversity within the Volvocine clade. Chlamydomonas is unicellular and reflects the ancestral state of the group, while Volvox is multicellular and has evolved numerous innovations including germ-soma differentiation, sexual dimorphism, and complex morphogenetic patterning. The Chlamydomonas genome sequence has shed light on several areas of eukaryotic cell biology, metabolism and evolution, while the Volvox genome sequence has enabled a comparison with Chlamydomonas that reveals some of the underlying changes that enabled its transition to multicellularity, but also underscores the subtlety of this transition. Many of the tools and resources are in place to further develop Volvocine algae as a model for evolutionary genomics.

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“…Besides the Saccharomyces cerevisiae , Chlamydomonas reinhardtii is a good choice for mitochondrial studies, as it can undergo mitochondrial transformation with homologous genes [1], [5], [6] or heterologous protein [7]. It has distinct advantages in comparison to higher plants as it is unicellular, haploid, is amenable to tetrad analysis, and its' three genomes are theoretically subject to specific transformation [8].…”

Section: Introductionmentioning

confidence: 99%

Stable Expression of Antibiotic-Resistant Gene ble from Streptoalloteichus hindustanus in the Mitochondria of Chlamydomonas reinhardtii

Zhang

Zhao

et al. 2012

PLoS ONE

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The mitochondrial expression of exogenous antibiotic resistance genes has not been demonstrated successfully to date, which has limited the development of antibiotic resistance genes as selectable markers for mitochondrial site-directed transformation in Chlamydomonas reinhardtii. In this work, the plasmid pBSLPNCB was constructed by inserting the gene ble of Streptoalloteichus hindustanus (Sh ble), encoding a small (14-kilodalton) protective protein into the site between TERMINVREP-Left repeats and the cob gene in a fragment of mitochondrial DNA (mtDNA) of C. reinhardtii. The fusion DNA-construct, which contained TERMINVREP-Left, Sh ble, cob, and partial nd4 sequence, were introduced into the mitochondria of the respiratory deficient dum-1 mutant CC-2654 of C. reinhardtii by biolistic particle delivery system. A large number of transformants were obtained after eight weeks in the dark. Subsequent subculture of the transformants on the selection TAP media containing 3 ìg/mL Zeomycin for 12 months resulted in genetically modified transgenic algae MT-Bs. Sequencing and Southern analyses on the mitochondrial genome of the different MT-B lines revealed that Sh ble gene had been integrated into the mitochondrial genome of C. reinhardtii. Both Western blot, using the anti-BLE monoclonal antibody, and Zeomycin tolerance analysis confirmed the presence of BLE protein in the transgenic algal cells. It indicates that the Sh ble gene can be stably expressed in the mitochondria of C. reinhardtii.

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Successful expression of heterologous egfp gene in the mitochondria of a photosynthetic eukaryote Chlamydomonas reinhardtii

Cited by 18 publications

References 23 publications

Advances and challenges in genetic engineering of microalgae

Advances and challenges in genetic engineering of microalgae

Genomics of Volvocine Algae

Stable Expression of Antibiotic-Resistant Gene ble from Streptoalloteichus hindustanus in the Mitochondria of Chlamydomonas reinhardtii

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