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.

RNA interference for wheat functional gene analysis

“…1a, Yan et al 2004). Results from the previous two studies indicate that in order to trigger RNAi the two sequences need to share a continuous stretch of identical nucleotides longer than 18-nt, which is consistent with the ''21-nt rule'' that indicates that at least 21-nt of continuous perfect identity are required to trigger RNAi (Miki et al 2005;McGinnis et al 2007).…”

“…During the RNAi response in plants, cleavage of dsRNA produces siRNAs of 21-26 nt (Hamilton et al 2002;Llave et al 2002;Tang et al 2003;Qi et al 2005). Therefore, the presence of a continuous stretch of at least 21 identical nucleotides between the trigger and the target gene is required, although it is not always sufficient to produce efficient silencing (Miki et al 2005;Yue et al 2007;McGinnis et al 2007).…”

“…However, if the objective is to silence all members of a particular multigene family, the conserved region would be preferred for the RNAi trigger in order to target multiple members of the family simultaneously. This was recently accomplished for seven members of the rice OsRac gene family (Miki et al 2005) whose transcript levels were all simultaneously down-regulated using a single RNAi construct targeting a conserved region. These types of constructs are still difficult to design in wheat because its complete genome sequence is still not available.…”

“…The determination of the upper limit for RNAi trigger size may be also related to the number of targets that can be effectively included in a single RNAi construct to knockdown multiple target transcripts. For example, Miki et al (2005) reported a progressive decrease in silencing efficiency in rice when increasing the size of a chimeric construct from 553 (two targets) to 1,089-bp (four targets).…”

Genome sequencing reveals agronomically important loci in rice using MutMap

“…pCAMBIA-empty was used as control in this study. For RNAi of the OsCAO1 gene, a 337-bp OsCAO1 partial fragment was amplified from wild-type Hitomebore genomic DNA by PCR and subcloned into binary vector pANDA 21 to yield pANDA-CAO1. All binary vectors were introduced into Agrobacterium tumefaciens strain EHA105 for rice transformation.…”