Molecular analysis of activation tagged aspen-Populus variants II
Final Report Abstract
Based on the Ac/Ds two element transposition system from maize an activation tagging approach was suggested for the hybrid aspen (Populus tremula x P. tremuloides) line ‘Esch5’. 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. Here, an inducible two element Ac/ATDs element system was suggested to induce activation tagged variants following two independent transformation steps. The transposase gene is under control of a heat shock promoter, and the ATDs element is only active and jumps into new genomic positions when Ac-transposase is induced. In combination with a 35S enhancer tetramer and outward facing two CaMV 35S promoter located near both ends of the ATDs element, expression of genes can be elevated which are located adjacent to the new integration site of the ATDs element. As recognition marker for ATDs transposition, the phenotypic selective marker gene rolC from A. rhizogenes and the positive selection marker gene (tms2) from A. tumefaciens are considered. Molecular analyses of already available different putative ATDs-rolC variants yielded in the amplification and sequencing of ATDs flanking genomic regions in 26 putative variants. Obtained sequences were blasted against P. trichocarpa in the Phytozome database (https://phytozome.jgi.doe.gov/pz/portal.html#!info?alias=Org_Ptrichocarpa). To produce new HS-transposase/ATDs-tms2 double transgenic lines, already available lines just carrying the transposase gene were super-transformed with ATDs-tms2. In total, we have produced 21 additional (new) double transgenic lines. The new double transgenic lines were tested in PCR and in Southern Blot analyses. In total, 17 lines were transgenic for both gene constructs and revealed presence of one or two copies of the ATDs construct. From these, only 9 lines revealed at least one time ATDs transposition following heat shock treatments. The remaining 9 new lines and one already available HS- transposase/ATDs-tms2 lines were subjected to 3 heat shock experiments. In total, about 1.4 Mio poplar micro explants were heat treated and 2,090 putative new activation tagged variants were regenerated. Surprisingly, when analysing 425 putative variants from 10 different independent original HS-transposase/ATDs-tms2 double transgenic lines for presence of empty donor site following ATDs transposition, just 66 were tested positive for empty donor cassette (15.5%). The ratio of ATDs transposition varied between the different double transgenic lines from 0 to 100%. From the 66 ATDs positive transposed putative variants, ATDs flanking sequences could be amplified in 12 variants. Blast results of obtained sequences revealed 7 putative tagged poplar genes.
Publications
-
(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, CRC Press, pp. 469-482
Fladung M
-
(2015) Transposon activation tagging in plants for gene function discovery. In: Vettori C, Vendramin GG, Paffetti D, Travaglini D (Eds) Proc of the IUFRO Tree Biotechnology 2015 Conference: “Forests: the importance to the planet and society”; pp 248-249
Fladung M
-
(2016) Genetic Engineering Contribution to Forest Tree Breeding Efforts. In: Vettori C et al (Eds), Biosafety of Forest Transgenic Trees. Springer Science+Business Media Dordrecht, Forestry Sciences 82, pp. 11-29. ISSN 0924-5480
Häggman H, Sutela S, Fladung M
-
(2016) Transposon activation tagging in plants for gene function discovery. In: Lüttge U, Cánovas FM, Matyssek R (Eds) Progress in Botany 77, Springer International Publishing, Switzerland, 265-289
Fladung M
