Assessment of RNAi-Induced Silencing in Banana (Musa spp.) using the β-Glucuronidase (GUS) Reporter Gene Model

Banana (Musa spp.) is one of the world’s most important crops which provides staple food for hundreds of millions of people, particularly in (sub)tropical regions. The ultimate goal of a genome project like the one for banana that recently released the first full banana (A) genome sequence is to decipher the biological function(s) of each gene in the genome, and then to use the agronomically important genes for genetic improvement of one or more traits by intragenesis. RNA interference (RNAi)-induced gene silencing is an efficient tool to downregulate a gene or subset of genes in a polyploid like banana viathe production of sequence specific double-stranded RNA. Functional characterization of the silenced gene(s) is achieved in the resulting knock-down lines. However, an in-depth report focused on gene silencing in banana is still lacking.

In this study, using a stable ß-glucuronidase (GUS) transformed banana suspension as a model system we investigated the silencing of the gusA^INT transgene by intron-containing self-complementary hairpin RNA constructs (ihpRNA) targeting different sites of the GUS mRNA. The results showed that the ihpRNA vector yielding a 299 bp RNA duplex aimed at the 5’end of the GUS mRNA caused in in vitro plants a 7- and 4.5-fold reduction in GUS mRNA level and enzyme activity, respectively. Furthermore, detection of GUS specific siRNA by Northern hybridization showed correlation between siRNA expression level and GUS enzyme activity. In conclusion, RNAi-induced silencing of a stably integrated transgene works in banana, and warrants further silencing studies in banana of agronomically interesting genes for functional characterization.