ABSTRACT
Ppmar1 and Ppmar2 are two active mariner-like elements (MLEs) cloned from moso bamboo (Phyllostachys edulis (Carrière) J. Houz) genome possessing transposases that harbour nuclear export signal (NES) domain, but not any nuclear localization signal (NLS) domain. To understand the functions of NES in transposon activity, we have conducted two experiments, fluorescence and excision frequency assays in the yeast system. For this, by site-directed mutagenesis, three NES mutants were developed from each of the MLE. In the fluorescence assay, the mutants, NES-1, 2 and 3 along with the wild types (NES-0) were fused with fluorescent proteins, enhanced yellow fluorescent protein (EYFP) and enhanced cyan fluorescent protein (ECFP) were co-transformed into yeast system. To differentiate protein localisation under the NES influence, ECFP alone was fused to wild and mutant NES domains either on N- or C-terminal and not to EYFP. Fluorescence assay revealed that blue fluorescence of ECFP was more intense than the red fluorescence of the EYFP in the yeast cell matrix. Further, ECFP had a wider localisation in the cellular matrix, but EYFP was largely located in the nucleus. The NES-1 domain was related to the comparatively high spread of ECFP, while NES-2 and NES-3 indicated a low spread, implying that NES activity on nuclear export increased when the NES is made leucine-rich, while the signalling activity was reduced when the leucine content was lowered in the NES domain. In the transposon excision assay, the mutant and wild type NES of both the Ppmar elements were integrated into an Ade2 vector, and within the Ade2 gene. Co-transformation of the vector together with non-autonomous Ppmar transposons and NES-lacking transposases was used to assess the differential excision frequencies of the mutants NES domains. In both the MLEs, NES-1 had the highest excision suppression, which was less than half of the excision frequency of the wild type. NES-2 and NES-3 elements showed, up to three times increase in transposon excision than the wild types. The results suggested that NES is an important regulator of nuclear export of transposase in Ppmar elements and the mutation of the NES domains can either increase or decrease the export signalling. We speculate that in moso bamboo, NESs regulates the transposition activity of MLEs to maintain the genome integrity.
