Novel high-throughput RNAi vectors for plant biotechnology

Abstract

With 2 figures and 1 table Abstract The knockdown of genes through RNA interference (RNAi) inducing vectors with one promoter regulating a sense and antisense sequence of the target gene separated by a spacer/intron region has been rampant in plant kingdom. Those constructs are difficult to clone because of long homologies. This study is the first to successfully use RNAi constructs with two promoters in inverse orientation flanking a DNA sequence of the target gene in plants. Different double-promoter constructs containing a β-d-glucuronidase gene–targeting sequence with constitutive 35S promoters or inducible heat shock promoters GmHSP17.5-E (Glycine max) were tested. We were able to knock down the β-d-glucuronidase expression in transgenic Nicotiana tabacum after transformation with these new RNAi constructs. The change in gene expression was tested with molecular, histochemical and fluorimetric methods. The results showed a very high and reliable knockdown rate for constitutive promoters. Owing to their consistent knockdown and the uncomplicated cloning, these new RNAi vectors are of outstanding importance for genomics and plant biotechnology.