Tnt1 is a retrotransposon isolated from tobacco. The structure of Tnt1 is typical of a retrotransposon, with a long open reading frame (ORF) in the central domain encoding the various enzymatic functions for replication and transposition. This region is flanked by 610 bp-long terminal repeats (LTRs) in the same orientation. Tnt1 has been shown to transpose in Arabidopsis during in vitro culture, and becomes stable in mature plants. Tnt1 shows similar behavior in the model legume M. truncatula. In both cases the Tnt1 was able to reach relatively high copy number (>10) and inserts preferentially into gene rich regions.
The goals for our work on Tnt1 include:
- Regenerating 200 plants through tissue culture each year
- Map these insertions using a modified Illumina library protocol initially developed for mPing populations
- Screen Tnt1 lines in the field for maturity and seed quantity and composition traits
- Ultimately Tnt1 insertions are expected in 30,000 soybean genes
Transposition of Tnt1 in soybean upon repeated regeneration.
P. Original parental line arising from cot-node A. tumefaciens-mediated transformation. Note that this line has 4 insertions based on Southern blotting.
R1. Southern blot using DNA from a progeny line obtained by micropropagation of the original parental line. Note that micropropagation resulted in the appearance of two additional Tnt1 insertions (compare to P).
R2. Two sibling progeny arising from micropropagation of the R1 line. Note that in this case at least two additional bands appear, while others appear to have been lost.
Previous work has led to the creation of twenty-seven independent transgenic lines carrying Tnt1 insertions. Southern blot anlaysis revealed that the copy number of transposed Tnt1 elements ranged from 4 to 19 with an average of 8 copies per line. These insertions showed Mendelian segregation and did not transpose under normal conditions. Analysis of 99 Tnt1 flanking sequences revealed the element preferentially inserts into genes (62% of insertions were in genes). Tnt1 insertions were found in all 20 soybean chromosomes, indicating that Tnt1 transposed throughout the soybean genome. FISH (fluorescence in situ hybridization) experiments were conducted to validate the presence of Tnt1 insertions in multiple chromosomes. Passage of transgenic lines through two different tissue culture treatments resulted in Tnt1 tranposition, signficantly increasing the number of insertions per line. We have reported that the Tnt1 retrotransposon can be used for effective large-scale insertional mutagenesis in soybean (Cui et al. 2013)1.