[Intellectual Contribution][Advances in Technology]

Development of a precise marker excision system in plants

Ayako Nishizawa-Yokoi, Masaki Endo, Hiroaki Saika, Seiichi Toki
Plant Genome Engineering Research Unit
[Abstract]
The use of selectable marker genes such as antibiotic resistant genes, is necessary to isolate transformed cells from among non-transformed cells. Therefore, a suite of strategies has been developed to remove selectable marker genes from plant genomes after selection of transformed cells and plants. In this study, we revealed that animal-derived piggyBac transposon can be transposed without leaving footprints at the excised sites and can be utilized for precise marker excision in plants.
[Keywords]
Keywords: rice, transgenic plants, piggyBac transposon, marker excision

[Background]

In order to generate and utilize transgenic plants, the selectable marker genes are needed to eliminate following the establishment of transgenic cells. To date, it has been reported that several methods, such as transposition and site-specific recombination, were employed successfully to remove selectable marker genes from transgenic plants. However, marker excision using these methods leaves dispensable sequences such as the residual footprint and recombinase recognition sequences at the excised site. Meanwhile, animal-derived piggyBac transposon excises without leaving a footprint at the excised site and has been used for precise marker excision in animal cells. In this study, we investigated whether piggyBac can also be transposed accurately in rice cells.
[Results and Discussion]
  1. We designed an assay system that allows visualization of transposase-mediated transposition of piggyBac as luminescence derived from reconstituted luciferase (LUC) expression cassettes (Fig. 1A). The reporter construct carrying a LUC expression cassette containing piggyBac transposon was introduced into rice calli.
  2. Transgenic rice calli harboring reporter constructs were infected with Agrobacterium to introduce the control vector or expression vector of transposase PBase. After antibiotic selection, LUC luminescence was detected on PBase-expressing rice calli but not on control calli (Fig. 1B). Furthermore, sequence analysis showed that piggyBac was transposed accurately from the reporter construct (Fig. 1C).
  3. Regenerated plants were obtained from LUC-positive calli and were subjected to piggyBac-excision and re-integration assay. PCR analysis revealed that piggyBac transposon was excised from the reporter construct in more than 70% of PBase-expressing plants (Table 1). In addition, the piggyBac transposon was lost from the reporter construct in 30% of regenerated plants without concomitant re-integration of the transposon (Fig. 1A and Table 1).
  4. Segregation after crossing resulted in T1 progeny without the selectable marker gene as well as PBase expression.
[Future prospects]
  1. The piggyBac transposon has been shown to transpose efficiently not only in many animal species but also in rice. It is expected that piggyBac would also be functional in other plant species and could therefore be widely used as an efficient and precise marker excision system for plant genomes.
  2. We are currently developing a reversible transgenesis system in plants in which the delivery of the transgene from T-DNA to genome and sequential excision of the transgene from the genome are both mediated by piggyBac transposon.

Fig.1. Analysis of piggyBac transposition from reporter constructs in rice calli. (A) Schematic representation of excision assay to detect the transposition of piggyBac transposon as luciferase luminescence in rice calli. (B) LUC luminescence images of control (left) and PBase-expressing calli (right) after 4-weeks of selection. The luminescence intensity is shown with the false color scale. (C) Nucleotide sequences of the original reporter construct (top) and piggyBac excision site in the LUC reporter cassette (bottom) in PBase transgenic rice calli.


Table.1. PCR analysis of piggyBac excision and re-integration events in PBase- expressing T0 plants

 

[Collaborator]
Keishi Osakabe (University of Tokushima)

[Reference]

  1. Nishizawa-Yokoi A, Endo M, Osakabe K, Saika H, Toki S (2014) Precise marker excision system using an animal-derived piggyBac transposon in plants The Plant Journal 77(3):454-463
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