Activation tagging in poplar
Isolation of tree-specific genes using a transposon-tagging approach
Trees are characterized by a number of specific characteristics which are under genetic control, like longevity and wood formation. It is expected that several genes are involved in the mentioned processes. In this project, an inducible two element Ac/ATDs system is suggested to establish an activation tagging system in hybrid aspen (Populus tremula x tremuloides).
Background and Objective
The aim of this task is the isolation and characterization of regulatory and other different gene sequences which are specific for trees.
In this proposal the confirmation of the mobility of the ATDs activation construct via PCR and Southern Blot analyses is planned. The ATDs integration sites will be characterized and flanking genomic regions sequenced. For the activation tagging experiments using the GIP- as well as the heat-shock systems more than 2.000 small pieces of transgenic regenerating callus from each at least ten different double transgenic lines will be treated for excision of ATDs. Finally, novel integration sites of the ATDs element will be molecularly characterized.
The proposed approach is based on results obtained from our earlier work on the genetic transfer of the maize transposable element Ac and its functional analysis in hybrid and pure aspen lines. All constructs described in the first proposal were established (carrying the transposase gene under two inducible promoters [GIP or heat-shock], and the activation construct (ATDs) with either rolC or tms). Transgenic poplar plants were obtained carrying either one or two of the constructs. Following induction of the GIP promoter a transposase signal could be detected in RT-PCR experiments. A strong transposase signal could be detected in RT-PCR experiments following induction of the heat-shock promoter. One activation tagging experiment was initiated using eight different double transgenic lines.
The strategy is based on results of earlier work. Here it was shown for the first time that the genetic transformation of poplar using the transposon Ac from maize is feasible. The results show that Ac excises from its original position in the construct and re-integrates somewhere in the genome (transposition). Further it was demonstrated, that transposition of Ac still occurs in six-years old transgenic plants.
Fladung M (2014) Prospects of using a modified Ac/Ds transposon system from maize for activation tagging in the tree species Populus. In: Ramawat KG, Mérillon J-M, Ahuja MR (eds) Tree biotechnology. Boca Raton: CRC Press ; Taylor & Francis, pp 469-482
Fladung M, Polak O (2012) Ac/Ds-transposon activation tagging in poplar: a powerful tool for gene discovery. BMC Genomics 13(61), DOI:10.1186/1471-2164-13-61
Fladung M, Polak O (2011) Activation tagging in poplar by using an inducible Ac/Ds transposon system. BMC Proc 5(Suppl. 7):O 51, DOI:10.1186/1753-6561-5-S7-O51