Reporter gene silencine by production of hairpin RNA in Tobacco Nicotiana tabacum L.

Abstract

RNA interference (RNAi) is a post-transcriptional gene-silencing phenomenon induced by double-stranded RNA (dsRNA) molecules that contain nucleotide sequences complementary to target mRNA sequences causing degradation of the latter. It is an effective approach in inhibiting expression of a target gene for gene function analysis. Here, transgenic tobacco plants in which a β-glucuronidase (gus) gene was silenced were generated by introducing a gus hairpin (hpGUS) sequence into a transgenic tobacco (Nicotiana tabacum L. cv. Virginia Coker) harboring a gus gene by Agrobacterium-mediated transformation. Then, transgenic tobaccos were selected on MS media containing kanamycin. All putative independent lines were confirmed by PCR amplification for the presence of the hpGUS construct using their genomic DNA as template. Three independent lines of the hpGUS-expressing double transfomants were obtained from the regeneration of kanamycin-resistant cells and confirmed by Southern blot analysis. Northern blot analysis showed silencing of the gus transcript in all independent lines as compared with the control plants. The reduction of gus mRNA level was confirmed by histochemical and spectrophotometric GUS assay. GUS activity of the hpGUS-expressing double transfomants was lower than the control double tansformants indicating that hpGUS can silence expression of the gus gene at the protein level.