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.)
Oriented cloning of genes from pUC based vectors into binaries can be done with one restriction enzyme. The unique 8 base Sfil restriction site at the border of the MCS makes it possible to switch easily between different vectors with different plant selection markers.
Cloning of the genes can be easily done in pUC-based vectors with different orientation of the MCS and blue-white selection
The selection marker is located at the left border of the binary.
The 35S promoter, which drives the selection marker is located close to the left border. Promoter-fusions with detectable markers (GUS,GFP) cloned into the MCS at the right border will have reduced enhancement of expression from the 35S-enhancer compared to other vectors (Cambia).
Beside nptll (pLH9000+pLH9500) there are also binaries available with bar (pLH7000+pLH7500) and hpt (pLH6000) as a selection marker. So a second and third transformation of the transgenic plant is possible with a different selection marker.
The most exceptional feature of these vectors is the pVS1-ori which makes the binary more stable in Agrobacteria. This leds to a higher transformation rate compared to the pBin19 vector.
For selection of plasmids in bacteria Streptomycin/Spectinomycin can be used.
The ColE1-ori allows elevated copy numbers in bacteria.