RNA silencing of single and multiple members in a gene family of rice

Abstract

RNA silencing with inverted repeat (IR) constructs has been used to suppress gene expression in various organisms. However, the transitive RNA-silencing effect described in plants may preclude the use of RNA silencing for a gene family. Here, we show that, in rice (Oryza sativa), transitive RNA silencing (spreading of double-stranded RNA along the target mRNA) occurred with the green fluorescent protein transgene but not with the endogenous phytoene desaturase gene. We fused IR copies of unique 3' untranslated regions derived from the rice OsRac gene family to a strong promoter and stably introduced them into rice. Each of the seven members of the OsRac gene family was specifically suppressed by its respective IR construct. We also examined IR constructs in which multiple 3' untranslated regions were fused and showed that three members of the OsRac gene family were effectively suppressed by a single construct. Using highly conserved regions of the two members of the OsRac gene family, we also suppressed the expression of all members of the gene family with variable efficiencies. These results suggest that RNA silencing is a useful method for the functional analysis of gene families in rice and other plants.

Figure 1.

RNA silencing of the 35S-gfp transgene. A, Schematic representation of the gfp gene and IR construct. The central 314 bp (shaded region, nt 201–514; also used as trigger probe) of the 720-bp gfp gene was used as an RNA silencing trigger. The black horizontal lines indicate the regions of the gfp gene used as probes for RNA blotting. The 5′ probe, 196 bp (nt 1–196); the 3′ probe, 200 bp (nt 521–720). Diagram of the gfp IR construct is shown. B, GFP fluorescence in rice calli. GFP fluorescence was observed in the original M65 line and in three independent transgenic lines (1, 3, and 4) carrying the gfp-targeted RNA silencing trigger transgene. C, RT-PCR analysis of gfp mRNA expression in the original M65 line and in five independent transgenic lines carrying the gfp-targeted RNA silencing trigger transgene. gus linker, mRNA derived from the gus linker region of the gfp-IR transgene; hpt, mRNA of the hygromycin resistance gene; bar, the bar gene used for the transformation of the gfp-IR transgene; ubq, mRNA of rice ubiquitin used as a control. D, siRNA analysis of gfp-silenced rice plants. Probes used are shown to the left of each section. Arrowheads indicate the positions of 23- (bottom) and 27-nt (top) DNA oligomers, which are likely to correspond to 21-nt- and 24-nt-size-class siRNA detected in other plants, respectively. The bottom section shows 5S rRNA as a loading control.

Figure 2.

RNA silencing of the endogenous PDS gene. A, Schematic representation of the PDS gene and IR construct. The central 470 bp (shaded region, nt 1,260–1,730; also used as trigger probe) of the 2,027-bp PDS cDNA was used as an RNA silencing trigger. The black horizontal lines indicate the regions of the PDS gene used as probes for RNA blotting. The 5′ probe, 475 bp (nt 692–1,166); the 3′ probe, 203 bp (nt 1,768–1,970). A diagram of the PDS IR construct is shown. B, RT-PCR analysis of PDS mRNA expression. Five independent transgenic rice plants were examined by RT-PCR using PDS-specific primers (top). The wild type (WT) is a nontransgenic control. gus linker, mRNA derived from the gus linker region of the PDS-IR transgene; hpt, mRNA of the hygromycin resistance gene; ubq, mRNA of rice ubiquitin used as control. C, Analysis of siRNAs. Probes used are shown to the left of each section. Arrowheads indicate the positions of 23-nt (bottom) and 27-nt (top) DNA oligomers, which are likely to correspond to 21-nt- and 24-nt-size-class siRNA detected in other plants, respectively. The bottom section shows 5S rRNA as a loading control.

Figure 3.

Structures of the rice OsRac gene family and constructs for RNA silencing. A, Phylogenetic tree of the OsRac gene family. The dendrogram was generated from the coding region of the nucleotide sequences of OsRac1 to 7 using ClustalW. B, Nucleotide identities between the highly conserved coding sequence of OsRac1 or OsRac5 and those of the other OsRac genes. C, Diagrams of the OsRac genes and regions used to make constructs for RNA silencing. The majority of the coding regions of the OsRac1 to 7 genes are highly conserved, but the 3′ ends of the coding sequences and the 3′ UTRs are highly divergent. The 3′ coding regions and the 3′ UTR gene-specific regions (shaded boxes) were used to generate IR constructs for gene-specific RNA silencing (OsRac1, 309 bp; OsRac2, 328 bp; OsRac3, 316 bp; OsRac4, 332 bp; OsRac5, 246 bp; OsRac6, 367 bp; OsRac7, 221 bp). The conserved regions of OsRac1 and OsRac5 were used to make IR constructs to suppress the other OsRac genes (518 bp, black boxes). The length of cDNA for each of the OsRac genes is shown to the right. D, OsRac gene-targeting RNA silencing IR constructs. These OsRac sequences were placed in the sense (S)-gus linker-antisense (AS) orientation (for the gene-specific OsRac3 construct) or the AS-gus linker-S orientation (for the other constructs). These IR constructs were driven by the maize Ubq1 promoter.

Figure 4.

Gene-specific RNA silencing of the OsRac gene family in transgenic rice. A, RT-PCR analysis of OsRac1 to 7 mRNA expression. The wild type (WT) is a nontransgenic control. gus linker, mRNA derived from the gus linker region; hpt, mRNA of the hygromycin resistance gene; ubq, mRNA of rice ubiquitin used as control. B, Analysis of siRNAs. Probes used for hybridization are as follows: top, the highly conserved region (Fig. 3C, black boxes); middle, the gene-specific region (Fig. 3C, shaded boxes); bottom, 5S rRNA. Arrowheads indicate the positions of 23-nt (bottom) and 27-nt (top) DNA oligomers, which are likely to correspond to 21-nt- and 24-nt-size-class siRNA detected in other plants, respectively. C, Quantitative analysis on mRNA levels of all the OsRac genes measured by real-time PCR. All of the OsRac1 to 7 values are normalized by the ubq (ubiquitin) control and then normalized to OsRac1 to 7 values in the wild type. Error bars represent the sd calculated from five independent transgenic lines. Bar shadings are identified to the right.

Figure 5.

Gene-specific RNA silencing of multiple genes in the OsRac gene family by a single IR construct. A, Chimeric OsRac sequences used in RNA silencing. Gene-specific regions of OsRac1, 3, 5, and 7 (Fig. 3) were used to generate chimeric RNA silencing constructs. 1/5, OsRac1 and OsRac5 gene-specific regions; 1/5/3, OsRac1, OsRac5, and OsRac3 gene-specific regions; 1/5/3/7, OsRac1, OsRac5, OsRac3, and OsRac7 gene-specific regions. B, RT-PCR analysis of OsRac1 to 7 mRNA expression in five independent transgenic lines. The wild type (WT) is a nontransgenic control. gus linker, mRNA derived from the gus linker region; hpt, mRNA of the hygromycin resistance gene; ubq, mRNA of rice ubiquitin used as a control. C, Analysis of siRNAs. Probes used for hybridization are as follows: top, the highly conserved region of OsRac1 (Fig. 3C, hatched box); middle, regions corresponding to the chimeric RNA silencing trigger sequences in A; bottom, 5S rRNA. Arrowheads indicate the positions of 23-nt (bottom) and 27-nt (top) DNA oligomers, which are likely to correspond to 21-nt- and 24-nt-size-class siRNA detected in other plants, respectively. D, Quantitative analysis of OsRac mRNA expression using real-time PCR. Error bars represent the sd calculated from five independent transgenic lines. Bar shadings are identified to the right.

Figure 6.

RNA silencing of the OsRac gene family by the highly conserved sequences of OsRac1 or OsRac5. A and B, RT-PCR analysis. Wild type (WT) is a nontransgenic control. Five independent transgenic rice plants expressing the highly conserved region of OsRac1 (A) or OsRac5 (B) were analyzed. C and D, RNA gel-blot analysis to detect siRNAs. Probes used are the conserved regions of OsRac1 (top) or OsRac5 (middle) and the gene-specific region of OsRac1 (bottom; Fig. 3C). Arrowheads indicate the positions of 23-nt (bottom) and 27-nt (top) DNA oligomers. The bottom section shows 5S rRNA as a loading control. E, Expression levels of all the OsRac genes, measured by real-time PCR analysis. Relative expression of each OsRac gene in transgenic rice is shown with the expression of their corresponding wild-type genes taken as 100%. White bars, Transgenic plants expressing dsRNA of the OsRac1 conserved region; black bars, transgenic plants expressing dsRNA of the OsRac5 conserved region. Error bars represent the sd calculated from 11 (OsRac1) or 10 (OsRac5) independent transgenic lines.