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Abstract BackgroundDNA transposable elements are mobilized by a “cut and paste” mechanism catalyzed by the binding of one or more transposase proteins to terminal inverted repeats (TIRs) to form a transpositional complex. Study of the rice genome indicates that themPingelement has experienced a recent burst in transposition compared to the closely relatedPingandPongelements. A previously developed yeast transposition assay allowed us to probe the role of both internal and terminal sequences in the mobilization of these elements. ResultsWe observed thatmPingand a syntheticmPongelement have significantly higher transposition efficiency than the related autonomousPingandPongelements. Systematic mutation of the internal sequences of bothmPingandmPongidentified multiple regions that promote or inhibit transposition. Simultaneous alteration of single bases on bothmPingTIRs resulted in a significant reduction in transposition frequency, indicating that each base plays a role in efficient transposase binding. Testing chimericmPingandmPongelements verified the important role of both the TIRs and internal regulatory regions.Previous experiments showed that the G at position 16, adjacent to the 5′ TIR, allows mPingto have higher mobility. Alteration of the 16th and 17th base frommPing’s3′ end or replacement of the 3′ end withPong3′ sequences significantly increased transposition frequency. ConclusionsAs the transposase proteins were consistent throughout this study, we conclude that the observed transposition differences are due to the element sequences. The presence of sub-optimal internal regions and TIR bases supports a model in which transposable elements self-limit their activity to prevent host damage and detection by host regulatory mechanisms. Knowing the role of the TIRs, adjacent sub-TIRs, and internal regulatory sequences allows for the creation of hyperactive elements.
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Development of mPing ‐based activation tags for crop insertional mutagenesis
Abstract Modern plant breeding increasingly relies on genomic information to guide crop improvement. Although some genes are characterized, additional tools are needed to effectively identify and characterize genes associated with crop traits. To address this need, themPingelement from rice was modified to serve as an activation tag to induce expression of nearby genes. Embedding promoter sequences inmPingresulted in a decrease in overall transposition rate; however, this effect was negated by using a hyperactive version ofmPingcalledmmPing20. Transgenic soybean events carryingmPing‐based activation tags and the appropriate transposase expression cassettes showed evidence of transposition. Expression analysis of a line that contained a heritable insertion of themmPing20Factivation tag indicated that the activation tag induced overexpression of the nearby soybean genes. This represents a significant advance in gene discovery technology as activation tags have the potential to induce more phenotypes than the originalmPingelement, improving the overall effectiveness of the mutagenesis system.
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- Award ID(s):
- 1444581
- PAR ID:
- 10454593
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Plant Direct
- Volume:
- 5
- Issue:
- 1
- ISSN:
- 2475-4455
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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