Molecular Dissections of the DNA Transposon Tam3 Responded to Low-Temperature Stresses in Antirrhinum

Introduction. Several active transposons have been identified in Antirrhinum, among which Tam3 is a class-II transposon belonging to the hAT (hobo, Ac, Tam3) superfamily, whose members are widely distributed in multicellular organisms, including plants, animals and fungi. Unlike the majority of other DNA transposons, Tam3 exhibits an unusual and remarkable feature of low-temperature dependent transposition (LTDT): activation at low growth temperatures (15°C), but inhibition at high temperatures (above 25°C). The low temperature allows a portion of TPase (about 20%) to transfer into nuclei resulting in the transposition of Tam3, but high temperature rigorously inhibits this nuclear import. In this study, we identified a cis-motif in TAM3 TPase (T3TP) which is involved in LTDT of Tam3. Furtherly, we did the yeast-two-hybrid screening to isolate the T3TP-interacting factors. And for those isolated T3TP-interacting factors, we would do some selections to identify the true host factor(s) involved LTDT, and finally, to clarify the mechanism of LTDT of Tam3.

Materials and Methods. Antirrhinum HAM22 (palida^rec::Tam3), showing variegation in flowers under low temperature due to the transposition of Tam3, were grown under both 15°C and 25°C. And then the mesophyll protoplasts were used for identifying the subcellular localization of Tam3 TPase and a series of its truncated constructs. The flowers from HAM22 growing under 25°C were also applied for the subcellular location experiment. The gene gun was utilized to transform these constructs into petal cells. cDNA library for yeast-two-hybrid was constructed by using the leaves and flowers of HAM22 growing under 25°C. And then y2h screening was carried on to isolate the Tam3 TPase-interacting proteins.

Results/Discussion.  We examined GFP constructs carrying a series of truncated Tam3 TPase gene to observe their subcellular localization. Most of the constructs showed that GFP localized on the membrane at the high temperature. However, the constructs lacking normal BED-zinc finger domain exhibited nuclear localization of GFP signal at 25°C. The BED-zinc finger domain is present at amino acid positions181to 234 from N terminal. When the cis-regulatory element (BED-zinc finger domain) is intact, the location of Tam3 TPase showed remarkable feature of low-temperature dependent nuclear import. But disruption of this cis-domain resulted in the nuclear import of Tam3 TPase even under high temperature. It suggests that the cis-domain is necessary for the LTDT of Tam3. To confirm this result, the same constructs were transformed into the petal cells of HAM22 under 25°C by particle-bombardment. And similar result was acquired, the host suppresses the nuclear import of Tam3 TPase through the zinc-finger domain, serving as a protein binding motif here, which also have been identified as a DNA binding domain to initiate the transposition of Tam3. Thus, the motif has biofunctional activities. As the domain is involved in the regulation from the host, it provides us some clues to isolate the host factor(s) which regulate the activity of Tam3 at the post-translational level. Till now, through yeast-two-hybrid screening, we have got some genes, interacting with Tam3 TPase. Next, obtain the candidate gene(s) involved in LTDT are impendency for carrying on the subsequent work.