Hi. I would like to check the differential expression of two versions of this gene, one of this containing an insertion in the upstream. I planned to transform Arabidopsis with the whole gene (not only the coding sequence), but don't know whether there is a better strategy or not? Thank you very much.
It is a good strategy - better full-lenth gene than only CDS.
Then you need to check many independent transgnoc lines, or you can pool several, lines to avoid the positional effect of the transgene insertion.
Yes. The CDS does not contain the motifs I want to test. Also, any recommendation in which vector should I use? Thank you.
I would insert the upstream sequence (including the promoter) fused to a reporter gene, in that case you can compare if the upstream sequence with the insertion is a functional promoter). Maybe you can fuse it with a GFP so it is easy to screen.
Thank you Ana! This is a good suggestion. It will be one of my experiments.
If you are still looking for a vector, I like the pKGWFS7 from Ugent: https://gateway.psb.ugent.be/search
It has a Gateway site upstream of a GFP fused to a GUS, so you can assess both GFP and GUS expression. This one carries a Kanamycin resistance in plants; there are also versions which confer Basta or Hygromycin resistance if you prefer (pBGWFS7 or pHGWFS7, respectively).
Hi Joanne,
Thank you for this suggestions. We have the pGWB533 in the lab, but if we need to order a new one, I would take your advice.
Regards,
Jose
Hi Jose
It is always a good idea to do a phylogenetic shadowing to find conserved regulatory sequences in the promoter region among closely related species. Maybe by this quick analysis you can already find highly conserved motifs. To do that you align your upstream sequence (round about 6 kb upstream of the ATG) with the upstream regions of species orthologues. It is recommendable to have at least 4 orthologues sequences.
Use either mVista
http://genome.lbl.gov/vista/mvista/submit.shtml
Or the JBROWSE in Araport
https://www.araport.org/
Have fun
Mara
Hi Mara,
Thank you. The point is that we did this comparison among several species and hybrids (around 40), so we found some non-conserved putative regulatory motifs. This sequences are the ones we are interested in. Also, thank you for the links!
Jose
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