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Suppression of Arabidopsis genes by terminator-less transgene constructs

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Abstract

Transgene-mediated gene silencing is an important biotechnological and research tool. There are several RNAi-mediated techniques available for silencing genes in plants. The basis of all these techniques is to generate double-stranded RNA precursors in the cell, which are recognized by the cellular surveillance system, and marked for degradation by the Dicer family RNases into siRNAs. Improperly terminated, unpolyadenylated RNA are precursors of double-stranded RNA, and, therefore, can serve as silencing triggers in plants. Such transcripts can easily be synthesized from transgene constructs lacking transcription-terminator signals (terminator-less constructs). The present study determined the silencing efficiency of terminator-less constructs on six different genes in Arabidopsis: Phytochrome A (PHYA), Brassinosteroid Insensitive 1 (BRI1), Variegated 2 (VAR2), Constans (CO), Apetala 1 (AP1) and Transparent Testa Glabra 1 (TTG1). Expression of terminator-less gene fragments of PHYA, AP1, and VAR2 resulted in a ~90 % decline, and those of BRI1 and CO resulted in a ~70 % decline, in the steady state level of the respective transcript in transgenic lines compared to the wild-type. This suppression was accompanied by phenotypic aberrations in selected transgenic lines. Thus, targeted gene suppression in plants can be initiated by the expression of a simple construct design consisting of a gene fragment lacking transcription terminator signals.

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Acknowledgments

This work was supported by USDA-NIFA Grant (Award no. 2010-38821-21540), and ABI-Arkansas Division of Agriculture grant. We thank Chase Purnell for technical assistance.

Conflict of interest

The authors declare no conflicts of interest.

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Author notes

  1. Scott J. Nicholson

    Present address: USDA-ARS, 1301 N. Western Rd, Stillwater, OK, 74075, USA

Authors and Affiliations

  1. Department of Crop, Soil and Environmental Sciences, University of Arkansas, Fayetteville, AR, 72701, USA

    M. Aydın Akbudak, Scott J. Nicholson & Vibha Srivastava

  2. Department of Horticulture, University of Arkansas, Fayetteville, AR, 72701, USA

    Vibha Srivastava

  3. Cell and Molecular Biology Program, University of Arkansas, Fayetteville, AR, 72701, USA

    Vibha Srivastava

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  1. M. Aydın Akbudak
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Correspondence to M. Aydın Akbudak or Scott J. Nicholson.

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Supplemental Fig. 1 Transgene constructs SJN02 and SJN04

Supplemental Fig. 2 a Seedlings grown in FRc and transferred to white light. PHYA suppression results in long hypocotyls. PHYA silencing was observed among T2 progeny of the SJN02 transgenic lines, exemplified by 02-2. Intermediate seedlings are also apparent within the heterozygous population. b Bushy plant growth due to BRI1 silencing in lines B9 and B10 c Anomalies in gynoecium development due to AP1 silencing. Two AP1 silenced lines, A1 and A4, are presented along with COL (WT). d Reduction in leaf number and early senescing due to CO silencing.

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Akbudak, M.A., Nicholson, S.J. & Srivastava, V. Suppression of Arabidopsis genes by terminator-less transgene constructs. Plant Biotechnol Rep 7, 415–424 (2013). https://doi.org/10.1007/s11816-013-0278-z

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  • DOI: https://doi.org/10.1007/s11816-013-0278-z

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