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Title: Suppression of Arabidopsis genes by terminator-less transgene constructs

Author
item AKBUDAK, AYDIN - University Of Arkansas
item Nicholson, Scott
item SRIVASTAVA, VIBHA - University Of Arkansas

Submitted to: Plant Biotechnology Reports
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/24/2013
Publication Date: 10/1/2013
Publication URL: http://handle.nal.usda.gov/10113/58590
Citation: Akbudak, A.M., Nicholson, S.J., Srivastava, V. 2013. Suppression of Arabidopsis genes by terminator-less transgene constructs. Plant Biotechnology Reports. 7(4):415-424.

Interpretive Summary: Genes may be silenced, or suppressed, by a number of methods that end in the generation of a specific double-stranded RNA molecule that is either produced within, or introduced into, an organism. The plant silencing system contains enzymes called RNA-Dependent RNA Polymerases, that are not found in many other organisms. This enzyme, in association with other members of the plant silencing pathway, may associate with unusual RNA molecules, such as messenger RNA molecules that, due to the absence of a termination sequence, lack the typical poly-A tail found on normal messenger RNA molecules. These transcripts may be used to invoke silencing of specific sequences within the plant. This study details the use of unterminated transcripts to silence native genes in the model plant Arabidopsis, and reports on the efficiency of such an approach. Of six genes targeted, four were reduced by approximately 90%, and the two remaining genes displayed less significant reductions. The technique described in this study may be used to efficiently suppress specific targets in plants using a simpler construct design than is currently utilized.

Technical 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, BRI1, CO, AP1, and VAR2 resulted in approximately 90% 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.