Efficient plastid transformation in tobacco using the aphA-6 gene and kanamycin selection

Huang, Fengting; Klaus, Sebastian; Herz, Stefan; Zou, Zhurong; Koop, Hans‐Ulrich; Golds, T. J.

doi:10.1007/s00438-002-0738-6

Cited by 88 publications

(40 citation statements)

References 25 publications

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“…Genes conferring kanamycin resistance to plastids, such as neo and aph(3 0 )IIa, are selectable in tissue culture (Carrer et al 1993;Huang et al 2002;Lutz et al 2004). A mild form of an aurea phenotype was first discovered as a phenotype caused by a neo gene variant encoded in plasmid pHK33 (Kuroda and Maliga 2002).…”

Section: Discussionmentioning

confidence: 99%

“…Typically two cycles of such purifying regeneration are required to obtain homoplastomic plants. Marker genes available in plastids for selective enrichment confer resistance to spectinomycin and streptomycin (aadA) , kanamycin (neo or aph(3 0 )IIa) (Carrer et al 1993;Huang et al 2002) (Lutz et al 2004), chloramphenicol (Li et al 2011) or the amino acid analogues 4-methylindole (4MI) and 7-methyl-DL-tryptophan (7MT) (ASA2) (Barone et al 2009). Because the plants that are expressing marker genes have no visual phenotype, homoplastomic state can be verified only by DNA gel blot analyses.…”

Section: Introductionmentioning

confidence: 99%

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Visual spectinomycin resistance (aadA au ) gene for facile identification of transplastomic sectors in tobacco leaves

et al. 2010

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Identification of a genetically stable Nicotiana tabacum (tobacco) plant with a uniform population of transformed plastid genomes (ptDNA) takes two cycles of plant regeneration from chimeric leaves and analysis of multiple shoots by Southern probing in each cycle. Visual detection of transgenic sectors facilitates identification of transformed shoots in the greenhouse, complementing repeated cycles of blind purification in culture. In addition, it provides a tool to monitor the maintenance of transplastomic state. Our current visual marker system requires two genes: the aurea bar (bar(au)) gene that confers a golden leaf phenotype and a spectinomycin resistance (aadA) gene that is necessary for the introduction of the bar(au) gene in the plastid genome. We developed a novel aadA gene that fulfills both functions: it is a conventional selectable aadA gene in culture, and allows detection of transplastomic sectors in the greenhouse by leaf color. Common causes of pigment deficiency in leaves are mutations in photosynthetic genes, which affect chlorophyll accumulation. We use a different approach to achieve pigment deficiency: post-transcriptional interference with the expression of the clpP1 plastid gene by aurea aadA(au) transgene. This interference produces plants with reduced growth and a distinct color, but maintains a wild-type gene set and the capacity for photosynthesis. Importantly, when the aurea gene is removed, green pigmentation and normal growth rate are restored. Because the aurea plants are viable, the new aadA(au) genes are useful to query rare events in large populations and for in planta manipulation of the plastid genome.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Visual spectinomycin resistance (aadA au ) gene for facile identification of transplastomic sectors in tobacco leaves

et al. 2010

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“…Petit havana) were generated and transformed via the polyethylene glycol-mediated (PEG) method adapted from Huang et al, 2002; Koop et al, 1996; Negrutiu et al, 1987. The transformed protoplasts were further incubated overnight in the dark.…”

Section: Methodsmentioning

confidence: 99%

SLocX: predicting subcellular localization of Arabidopsis proteins leveraging gene expression data

et al. 2011

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Despite the growing volume of experimentally validated knowledge about the subcellular localization of plant proteins, a well performing in silico prediction tool is still a necessity. Existing tools, which employ information derived from protein sequence alone, offer limited accuracy and/or rely on full sequence availability. We explored whether gene expression profiling data can be harnessed to enhance prediction performance. To achieve this, we trained several support vector machines to predict the subcellular localization of Arabidopsis thaliana proteins using sequence derived information, expression behavior, or a combination of these data and compared their predictive performance through a cross-validation test. We show that gene expression carries information about the subcellular localization not available in sequence information, yielding dramatic benefits for plastid localization prediction, and some notable improvements for other compartments such as the mitochondrion, the Golgi, and the plasma membrane. Based on these results, we constructed a novel subcellular localization prediction engine, SLocX, combining gene expression profiling data with protein sequence-based information. We then validated the results of this engine using an independent test set of annotated proteins and a transient expression of GFP fusion proteins. Here, we present the prediction framework and a website of predicted localizations for Arabidopsis. The relatively good accuracy of our prediction engine, even in cases where only partial protein sequence is available (e.g., in sequences lacking the N-terminal region), offers a promising opportunity for similar application to non-sequenced or poorly annotated plant species. Although the prediction scope of our method is currently limited by the availability of expression information on the ATH1 array, we believe that the advances in measuring gene expression technology will make our method applicable for all Arabidopsis proteins.

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“…A few other SMGs have been reported to allow for selection of transplastomic cells in vitro (reviewed in Rosellini 2012): the spinach betaine aldehyde dehydrogenase (BADH) gene for betaine aldehyde resistance , the E. coli nptII and aphA-6 genes for kanamycin resistance (Carrer et al 1993;Huang et al 2002), a tobacco mutant anthranilate synthase a subunit gene (ASA) for 5-methyltryptophan resistance (Barone et al 2009), and the E. coli chloramphenicol acetyltransferase (cat) gene for chloramphenicol resistance (Li et al 2011). Recently, the use of Agrobacterium tumefaciens ipt, encoding the enzyme isopentenyl transferase, as a selectable marker for plastid transformation has been demonstrated (Dunne et al 2014).…”

Section: Introductionmentioning

confidence: 99%

A mutant Synechococcus gene encoding glutamate 1-semialdehyde aminotransferase confers gabaculine resistance when expressed in tobacco plastids

et al. 2015

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A mutant glutamate 1-semialdehyde aminotransferase gene from the Synechococcus , inserted into tobacco plastid DNA by means of particle bombardment and antibiotic selection, conferred gabaculine resistance allowing to attain homoplasmy. Many plant species are recalcitrant to plastid genome transformation. New selections systems may help to overcome this limitation and to extend the application of this technology. A mutant hemL gene from the photosynthetic cyanobacterium Synechococcus, encoding a gabaculine-insensitive glutamate 1-semialdehyde aminotransferase (GSA), is an efficient selectable marker gene for nuclear transformation of tobacco, alfalfa and durum wheat. Since GSA functions in the plastid, we introduced the mutant hemL gene into the tobacco plastid genome along with the conventional antibiotic resistance aadA gene, in the attempt to develop a new selection system for plastome transformation. Although we were unable to directly regenerate gabaculine resistant transplastomic plants, we demonstrated the functionality of hemL in tobacco plastids by using gabaculine selection in the second and third rounds of in vitro selection that permitted to obtain the homoplasmic state in transgenic plants. Thus, the mutant hemL gene functions as a secondary selection marker in tobacco plastids. Our results encourage further attempts to test gabaculine resistant GSA for plastome transformation of crop plants in which gabaculine has stronger regeneration-inhibiting effects with respect to tobacco.

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Efficient plastid transformation in tobacco using the aphA-6 gene and kanamycin selection

Cited by 88 publications

References 25 publications

Visual spectinomycin resistance (aadA au ) gene for facile identification of transplastomic sectors in tobacco leaves

Visual spectinomycin resistance (aadA au ) gene for facile identification of transplastomic sectors in tobacco leaves

SLocX: predicting subcellular localization of Arabidopsis proteins leveraging gene expression data

A mutant Synechococcus gene encoding glutamate 1-semialdehyde aminotransferase confers gabaculine resistance when expressed in tobacco plastids

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