Suppression of Arabidopsis genes by terminator-less transgene constructs

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.