In our lab also we are working on the similar lines, but with a minimal promoter of a large regulatory region of the ubi gene from Porteresia. We are trying to make a synthetic tissue specific promoter (root specific) We have added some enhancer elements like MAT and scaffolding elements. Also few repeats of the tissue specific elements. We have transformed it in tobacco using gus as the reporter gene. We compared with the constitutive promoter. In roots its expression is better than the one transformed with the constitutive promoter.

Hi Suramonian,

Thanks for the reply. I want to add that we have been using the strategy it for two different tissues one is for root and the other is pollen. To add to the question i would like to know how many such repeats of motifs be required for the activity? and what was the reason behind choosing large region of minimal promoter?

If your work is published can you please provide me the link?

Thanks

Hi Amita

Sorry for the delayed response. Let me explain little more in detail. Earlier we have isolated a ubi promoter from Porteresia (2.3 Kb) and this was giving excellent expression in rice and sugarcane and we are using this promoter for sugarcane transformations. From this we have taken 83 bp core promoter region containing TATA box etc. Our objective was to make synthetic promoters for root and shoot specific expression. We designed and synthesized two promoters each for stem and root specific expression. One was with addition of 2 copies of root motif/stem motifs along with single copy of other essential motifs (stress related elements, pathogen related, auxin responsive, root meristem and MART box) in the upstream of the core promoter region and another one with both in the upstream and down stream of the core promoter region. In the case of root specific promoter, where the elements are added both in the upstream & down stream of the core promoter, we were able get high expression of gus, though there was some leaky expression in the areal parts. This may be because of the light induceable elements in the core region. However, the "Shoot specific promoter" is behaving like constitutive promoter.

Thank you very much for your response.

Hi Amita,

Distance between the core promoter and cis-elements is important to allow proper interaction of regulatory proteins (proteins that bind DNA directly or through other proteins). You may want to keep it close to what is seen in native promoters where these motifs are present and active.

Subbaiah

Dear Subbaiah,

Thanks for your reply.