Retrotransposons are genomic parasites activated by stress conditions that can be seriously detrimental for their host. In this work I demonstrate that Tto1, a typical plant LTR retrotransposon with insertion preference into genes can be turned into a synthetic molecular tool for gene tagging in plants and can be used to predict models for its replication steps. Although retrotransposons have been already used in plant mutagenesis, such application always required establishing protocols for tissue cultures and regeneration in vitro. Here, I show that sequence engineering of Tto1 provides the possibility to obtain transposition in vivo, with a simple screening method based on PCR and with the advantage to skip all in vitro manipulations. An artificial -estradiol inducible promoter has been used to obtain transposition “on demand” in Arabidopsis plants, which generates stable unlinked insertions that follow mendelian segregation in the progeny.
Comparing serial deletions of 3’ LTR of the engineered inducible Tto1 (iTto1), I have mapped its two natural terminators and identified the “minimal” R (redundant) region required to achieve the complete reverse transcription of the genomic mRNA into a new cDNA copy. Interestingly, the transcripts ending at the major “early” terminator cannot support reverse transcription, suggesting a mechanism of natural control on the expression. Transcripts with a more extended termination point contain 100 essential nucleotides that define the active nucleus of the R region. This sequence promotes the formation of a stable hairpin structure that “kisses” a complementary identical hairpin on the cDNA and determines the formation of the characteristic cDNA/mRNA heteroduplex. Since the LTR is a repeated sequence the definition of a minimal redundant region has also the important implication to reduce the only possible target for sequence-based gene silencing, which should lead to an increase of the mutagenic efficiency of iTto1.
Additional investigations have been carried out in attempt to identify points of improvement of iTto1 performances. By sequence alignment I identified different versions of the integrase that might have influence on insertion efficiency. Furthermore I tested the pOp6/LhGR-N system that will provide higher expression levels in different host plants. The final goal of my work is to extend the application of iTto1 to crop mutagenesis, therefore a big part of my work has been spent to develop Tto1 constructs with activity in barley. Transgenic plants have been obtained, however the constructs still need further experimentation
