Isolation and Characterization of Medicago truncatula U6 Promoters for the Construction of Small Hairpin RNA-Mediated Gene Silencing Vectors

RNA silencing using vector-based double-stranded RNA is an attractive strategy to suppress gene expression in plants. Especially, a short hairpin RNA (shRNA)-mediated approach is potent for specific target gene silencing. Here, shRNA expression vectors were constructed based on U6 small nuclear RNA (snRNA) gene promoters in Medicago truncatula . Ten U6 snRNA gene loci identified showed highly homologous sequences with those of other eukaryotes. Their RNA polymerase III-specific promoters had a conserved upstream sequence element and TATA box that were located approximately three helical DNA turns apart. The U6 promoters were also insensitive to α-amanitin, actively guiding transcription of fused GUS reporter gene fragments. When the U6 fusion constructs were introduced into M . truncatula via Agrobacterium rhizogenes -mediated transformation, GUS fragment transcripts were detected in nearly every transformed root albeit in varying amounts. Consistently, under the control of U6 promoters, constructs of 21-nucleotide-stem shRNAs directed silencing of GUS expression in transformed roots. Moreover, a phytoene desaturase gene ( MtPDS3 )-targeting 27-nucleotide-stem U6-shRNA constructs, when introduced into hairy roots, decreased the MtPDS3 transcript levels by approximately 80 % as a result of endogenous gene silencing, as verified by the presence of shPDS-derived siRNAs detected by stem-loop PCR. The U6 promoter-directed shRNA expression plasmids constructed herein can be a useful tool for functional gene analyses in this model legume.