Silencing of the aleurone-specific Ltp2-gus gene in transgenic rice is reversed by transgene rearrangements and loss of aberrant transcripts

Abstract

An Ltp2 promoter was isolated from barley as an aleurone-specific promoter, and its tissue specificity was maintained in transgenic rice. Expression of the Ltp2-gus gene in rice could be detected by X-gluc staining of the seeds. Previously, we reported the isolation of silenced plants in the R2 generation and the involvement of antisense gus transcripts in aleurone-specific Ltp2-gus gene silencing in transgenic rice, the L3.3 line. In the current study, we analyzed the L0.1 line, a sibling of the L3.3 line, and the partially revertant line from the L0.1 line accompanied by a transgene structural change. Strong silencing of the Ltp2-gus gene was detected over five generations in the L0.1 line. DNA and RNA analysis revealed that there were incomplete transgenes and that several aberrant RNAs that carried an antisense gus region were expressed in the L0.1 line. Determination of the transgene structure in the L0.1 line revealed that the partial antisense gus gene and the partial Ltp2 promoter region fused to the Ltp2 promoter were lacking in the revertants. RNA analysis demonstrated that the antisense gus and the promoter transcripts were produced and found in the poly(A)- fraction. Results of these analyses suggested that the observed aberrant transcripts, including antisense and promoter transcripts, were associated with Ltp2-gus silencing in the L0.1 line.