Revolution in plant RNAi
inverted promoter instead of inverted DNA-fragments
Double stranded RNA is triggering a RNAi effect which can result in gene silencing in eucaryotic organisms. In these vectors the sense and antisense strand are produced constitutively, developmental specific or tissue specific by two inverted promoters. All double promoter constructs can be inserted into binary vectors with SfiI.
To proof the concept, afragment of 549 bp from the gus gene was inserted between two 35S promoters. To terminate the RNA formation twotermination signals from the 35S gene were used. One of them was slightly modified to create sequencing primers for the inserts. This double promoter construct was transferred into a binary and tobacco plants, containing a strongly expressed 35S-gus gene, were transformed with Agrobacteria,. The regenerated plants were assayed with a histochemical GUS test and with the fluorometic Mu/MUG assay (Schmidt et al. 2009 submitted). Knockout plants showed a reduction of more than 99% in the fluorometic MU/MUG assay.
Binaries with 35s double promoter and Ubi10 promoter
To increase stability of binary vectors with 35S double promoters, we used the Ubq10 promoter from Arabidopsisto drive the selection marker in these constructs. Binary vectors with different selection markers are available (bar, nptII and hpt). According to Sun CW, Callis J. (Plant J. 1997 May;11(5):1017-27) Ubq10 shows the most constant mRNA levels in all organs compared to other ubiquitine genes. Strength of expression in different organs should be comparable with a single 35S promoter (Wally et. Al Plant Cell Rep 2008, 27:279-87.)