Visual spectinomycin resistance (aadA au ) gene for facile identification of transplastomic sectors in tobacco leaves
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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.
KeywordsAurea aadA aadAau Leaf variegation Plastid transformation Spectinomycin resistance Nicotiana tabacum
This work was supported by grants from the USDA National Institute of Food and Agriculture Biotechnology Risk Assessment Research Grant Program Award No. 2005-33120-16524 and 2008-03012. Kristina Marie Slivinski was supported by a fellowship from the Disabled American Veterans.
- Baena-Gonzales E, Allahverdieva Y, Svab Z, Maliga P, Josse EM, Kuntz M, Mäenpää P, Aro EM (2003) Deletion of the tobacco plastid psbA gene triggers post-transcriptional up-regulation of thylakoid-associated terminal oxidase (PTOX) and the NAD(P)H complex. Plant J 35:704–716. doi: 10.1046/j.1365-313X.2003.01842.x CrossRefGoogle Scholar
- Li W, Ruf S, Bock R (2011) Chloramphenicol acetyltransferase as selectable marker for plastid transformation. Plant Mol Biol. doi: 10.1007/s11103-010-9678-4
- Sharwood RE, von Caemmerer S, Maliga P, Whitney SM (2008) The catalytic properties of hybrid rubisco comprising tobacco small and sunflower large subunits mirror the kinetically equivalent source Rubiscos and can support tobacco growth. Plant Physiol 146:83–96. doi: 10.1104/pp.107.109058 PubMedCrossRefGoogle Scholar
- Tungsuchat-Huang T, Sinagawa-Garcia SR, Paredes-Lopez O, Maliga P (2010) 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. Plant Physiol 153:252–259. doi: 10.1104/pp.109.152892 PubMedCrossRefGoogle Scholar