Abstract
BackgroundIn plants, RNA- based gene silencing mediated by small RNAs functions at the transcriptional or post-transcriptional level to negatively regulate target genes, repetitive sequences, viral RNAs and/or transposon elements. Post-transcriptional gene silencing (PTGS) or the RNA interference (RNAi) approach has been achieved in a wide range of plant species for inhibiting the expression of target genes by generating double-stranded RNA (dsRNA). However, to our knowledge, successful RNAi-application to knock-down endogenous genes has not been reported in the important staple food crop banana.ResultsUsing embryogenic cell suspension (ECS) transformed with ß-glucuronidase (GUS) as a model system, we assessed silencing of gusAINT using three intron-spliced hairpin RNA (ihpRNA) constructs containing gusAINT sequences of 299-nt, 26-nt and 19-nt, respectively. Their silencing potential was analysed in 2 different experimental set-ups. In the first, Agrobacterium-mediated co-transformation of banana ECS with a gusAINT containing vector and an ihpRNA construct resulted in a significantly reduced GUS enzyme activity 6–8 days after co-cultivation with either the 299-nt and 19-nt ihpRNA vectors. In the second approach, these ihpRNA constructs were transferred to stable GUS-expressing ECS and their silencing potential was evaluated in the regenerated in vitro plants. In comparison to control plants, transgenic plants transformed with the 299-nt gusAINT targeting sequence showed a 4.5 fold down-regulated gusA mRNA expression level, while GUS enzyme activity was reduced by 9 fold. Histochemical staining of plant tissues confirmed these findings. Northern blotting used to detect the expression of siRNA in the 299-nt ihpRNA vector transgenic in vitro plants revealed a negative relationship between siRNA expression and GUS enzyme activity. In contrast, no reduction in GUS activity or GUS mRNA expression occurred in the regenerated lines transformed with either of the two gusAINT oligo target sequences (26-nt and 19-nt).ConclusionsRNAi-induced silencing was achieved in banana, both at transient and stable level, resulting in significant reduction of gene expression and enzyme activity. The success of silencing was dependent on the targeted region of the target gene. The successful generation of transgenic ECS for second transformation with (an)other construct(s) can be of value for functional genomics research in banana.Electronic supplementary materialThe online version of this article (doi:10.1186/1756-0500-7-655) contains supplementary material, which is available to authorized users.
Highlights
In plants, RNA- based gene silencing mediated by small RNAs functions at the transcriptional or post-transcriptional level to negatively regulate target genes, repetitive sequences, viral RNAs and/or transposon elements
Insertional mutagenesis is hampered by complex T-DNA insertions which might cause DNA rearrangement, lethal knockouts, or the necessity of making homozygous lines which is impossible for edible bananas due to their sterility
The transient GUS silencing system yields promising short interference RNAs (siRNAs)-mediated silencing results intron-spliced hairpin RNA (ihpRNA) vectors construction To assess RNA interference (RNAi)-induced silencing of gusAINT at the transient level, we constructed three ihpRNA constructs pIMHKUL3, pIMHKUL4 and pIMHKUL5 containing 299-nt, 26-nt, and 19-nt of the gusA genomic sequence, respectively, and targeting different sites of the gusA mRNA
Summary
RNA- based gene silencing mediated by small RNAs functions at the transcriptional or post-transcriptional level to negatively regulate target genes, repetitive sequences, viral RNAs and/or transposon elements. Insertional mutagenesis is hampered by complex T-DNA insertions which might cause DNA rearrangement, lethal knockouts, or the necessity of making homozygous lines which is impossible for edible bananas due to their sterility This is an inherently random approach limited by (i) its non-sequence-specific gene silencing activity, (ii) gene redundancy often present in gene families, and (iii) polyploidy in the case of for example, wheat and banana. The RNAi-induced gene silencing approach can overcome these disadvantages since it mediates PTGS of the target gene(s), resulting in inhibition of gene expression in a sequence-specific manner via the formation of double-stranded RNA (dsRNA) [15,16]. It allows silencing of single or multiple members of a gene family or, of homologous gene copies in polyploids by targeting unique sequences shared by these gene members [17,18,19]
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