Plastid marker gene excision by the phiC31 phage site-specific recombinase

Kittiwongwattana, Chokchai; Lutz, Kerry Ann; Clark, Mark A.; Maliga, Pál

doi:10.1007/s11103-007-9140-4

Cited by 68 publications

(70 citation statements)

References 35 publications

(38 reference statements)

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“…CTAB purified (36) total cellular DNA was digested with the BamHI restriction enzyme and probed with rrn16, aadA, and bar probes (39). Blots were prepared as described (40).…”

Section: Methodsmentioning

confidence: 99%

Cell-to-cell movement of mitochondria in plants

Gurdon

Sváb

Feng

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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We report cell-to-cell movement of mitochondria through a graft junction. Mitochondrial movement was discovered in an experiment designed to select for chloroplast transfer from Nicotiana sylvestris into Nicotiana tabacum cells. The alloplasmic N. tabacum line we used carries Nicotiana undulata cytoplasmic genomes, and its flowers are male sterile due to the foreign mitochondrial genome. Thus, rare mitochondrial DNA transfer from N. sylvestris to N. tabacum could be recognized by restoration of fertile flower anatomy. Analyses of the mitochondrial genomes revealed extensive recombination, tentatively linking male sterility to orf293, a mitochondrial gene causing homeotic conversion of anthers into petals. Demonstrating cell-to-cell movement of mitochondria reconstructs the evolutionary process of horizontal mitochondrial DNA transfer and enables modification of the mitochondrial genome by DNA transmitted from a sexually incompatible species. Conversion of anthers into petals is a visual marker that can be useful for mitochondrial transformation.

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“…CTAB purified (36) total cellular DNA was digested with the BamHI restriction enzyme and probed with rrn16, aadA, and bar probes (39). Blots were prepared as described (40).…”

Section: Methodsmentioning

confidence: 99%

Cell-to-cell movement of mitochondria in plants

Gurdon

Sváb

Feng

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…Plastid transformation vector pSS33 is a pPRV111A plasmid derivative that carries a spectinomycin resistance (aadA) marker gene (Zoubenko et al, 1994) and a bar au gene flanked by the phiC31 phage INT attB/attP target sites (Kittiwongwattana et al, 2007). The vector map is shown in Figure 1A, and the DNA sequence has been deposited in GenBank.…”

Section: Construction Of the Nt-pss33 And Nt-pss42 Transplastomic Plantsmentioning

confidence: 99%

“…The vector map is shown in Figure 1A, and the DNA sequence has been deposited in GenBank. The bar au gene is expressed in the PrrnPclpP::aadA::TrbcL cassette and is identical with the bar au genes in plasmids pCK2 and pMBC12 (Kittiwongwattana et al, 2007;Lutz et al, 2007). Plastid transformation vector pSS42 is identical with the pSS33 plasmid other than in the pSS42 vector, where the bar au gene is flanked with the P1 phage Cre site-specific recombinase loxP target sites (Corneille et al, 2001).…”

Section: Construction Of the Nt-pss33 And Nt-pss42 Transplastomic Plantsmentioning

confidence: 99%

“…Excision of the marker gene is achieved by introducing the site-specific recombinase gene into the plant nucleus, where it is transcribed, its mRNA is translated on cytoplasmic ribosomes, and the engineered recombinase is imported into chloroplasts where the recombinase efficiently excises the marker genes. Recombinases tested for plastid marker excision are P1 phage CRE recombinase (Corneille et al, 2001;Hajdukiewicz et al, 2001;Lutz et al, 2006a;Tungsuchat et al, 2006) and the phiC31 phage integrase (INT; Kittiwongwattana et al, 2007) enzymes.…”

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Study of Plastid Genome Stability in Tobacco Reveals That the Loss of Marker Genes Is More Likely by Gene Conversion Than by Recombination between 34-bp loxP Repeats

et al. 2010

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In transformed tobacco (Nicotiana tabacum) plastids, we flank the marker genes with recombinase target sites to facilitate their posttransformation excision. The P1 phage loxP sites are identical 34-bp direct repeats, whereas the phiC31 phage attB/attP sites are 54-and 215-bp sequences with partial homology within the 54-bp region. Deletions in the plastid genome are known to occur by recombination between directly repeated sequences. Our objective was to test whether or not the marker genes may be lost by homologous recombination via the directly repeated target sites in the absence of site-specific recombinases. The sequence between the target sites was the bar au gene that causes a golden-yellow (aurea) leaf color, so that the loss of the bar au gene can be readily detected by the appearance of green sectors. We report here that transplastomes carrying the bar au gene marker between recombinase target sites are relatively stable because no green sectors were detected in approximately 36,000 seedlings (Nt-pSS33 lines) carrying attB/ attP-flanked bar au gene and in approximately 38,000 seedlings (Nt-pSS42 lines) carrying loxP-flanked bar au gene. Exceptions were six uniformly green plants in the Nt-pSS42-7A progeny. Sequencing the region of plastid DNA that may derive from the vector indicated that the bar au gene in the six green plants was lost by gene conversion using wild-type plastid DNA as template rather than by deletion via directly repeated loxP sites. Thus, the recombinase target sites incorporated in the plastid genome for marker gene excisions are too short to mediate the loss of marker genes by homologous recombination at a measurable frequency.

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“…The natural function of these systems is the control of the precise excision or integration of defined DNA units into the host genome (Gidoni et al 2008;Nandy and Srivastava 2012). The phage mediated recombination was successfully used in plant biotechnology (Kempe et al 2010;Kittiwongwattana et al 2007). The sifting characterised systems used in plants and other organisms are Cre/lox of bacteriophage P1 of Escherichia coli (Hoa et al 2002;Hoess et al 1985;Sternberg and Hamilton 1981), R/RS from the SR1 plasmid of Zygosaccharomyces rouxii, and FLP/FRT from the 2-μm plasmid of Saccharomyces cerevisiae (Akbudak and Srivastava 2011;Hu et al 2008).…”

Section: Introductionmentioning

confidence: 99%

Generating Marker-Free Transgenic Wheat Using Minimal Gene Cassette and Cold-Inducible Cre/Lox System

et al. 2014

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Abstract:Abstract The precise elimination of selectable marker genes is highly desirable, when their function is no longer needed, because their presence raised worldwide public concerns against the release of genetically modified plants. This is the first report of simultaneous application of the minimal gene cassette and cold-inducible Cre/lox recombination system in wheat. The bar selection and cre-recombinase genes were eliminated from T0 and T1 transgenic lines with 44% and 51% efficiency. This approach provides a new, reasonably effective technique to produce selection genefree transgenic wheat lines either immediately after tissue culture, or from the subsequent transgenic generation. The advantage of this method is that it does not require any additional cold treatment during the plant regeneration/growing because the transgene elimination is ensured by the vernalisation. Application of this method prevents gene flow by pollen and seed, because the selection and recombinase genes are eliminated before pollen development, therefore reducing the risk of GM plants. Powered by Editorial Manager® and ProduXion Manager® from Aries Systems CorporationGenerating marker-free transgenic wheat using minimal gene cassette and cold inducible Cre/lox system AbstractThe precise elimination of selectable marker genes is highly desirable, when their function is no longer needed, because their presence raised worldwide public concerns against the release of genetically modified plants. This is the first report of simultaneous application of the minimal gene cassette and cold-inducible Cre/lox recombination system in wheat. The bar selection and cre-recombinase genes were eliminated from T 0 and T 1 transgenic lines with 44% and 51% efficiency. This approach provides a new, reasonably effective technique to produce selection gene-free transgenic wheat lines either immediately after tissue culture, or from the subsequent transgenic generation. The advantage of this method is that it does not require any additional cold treatment during the plant regeneration/growing because the transgene elimination is ensured by the vernalisation. Application of this method prevents gene flow by pollen and seed, because the selection and recombinase genes are eliminated before pollen development, therefore reducing the risk of GM plants.

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Plastid marker gene excision by the phiC31 phage site-specific recombinase

Cited by 68 publications

References 35 publications

Cell-to-cell movement of mitochondria in plants

Cell-to-cell movement of mitochondria in plants

Study of Plastid Genome Stability in Tobacco Reveals That the Loss of Marker Genes Is More Likely by Gene Conversion Than by Recombination between 34-bp loxP Repeats

Generating Marker-Free Transgenic Wheat Using Minimal Gene Cassette and Cold-Inducible Cre/Lox System

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