pMDC85 promoter contains 2x35s constitutive promoter and I've put in another tissue-specific promoter down-stream of 35s. Now I am thinking what tissues in plants will GFP label?
pMDC85 looks like a binary vector with both right border (RB) and left border (LB) of a T-DNA for plant genetic transformation [attached picture]. The 2x 35S promoter locates within the T-DNA. So, the 2x 35S promoter with your tissue-specific promoter and GFP will be co-transferred into the plant genome at a same locus if there is no truncation or re-arrangement occurs. The 2x 35S promoter carries 2 enhancers. It is well known that the enhancers can affect the nearby promoters of genes. So, I think you should probably still get GFP expression at the specific tissue, but the expression level may be altered.
Do let us know, when you get results.
Yuan-Yeu, The doubt came into my mind before I was gonna cut out the 2x35s promoter from pMDC85. I then searched online and couldn't find any relevant information. Now I think it might be a good idea to test it in parallel with the constructs I am working with. After the constructs are made, I will transform them into Arabidopsis. Do you think it worth a try?
I would if it won't take too much time. The best way to find out is to compare them parallel, using one with the 2x 35S promoter and one without, for transformation. But you need to factor in the 'position effect' factor when you compare those transgenic lines you obtain later. Position effect can also affect the expression of a transgene. Maybe you can land yourself with an extra paper for this, who knows :).
Hi Jiexi,
This is a piece of info related to what I posted earlier: 35S enhancers affect the expressions of neighboring genes.
Researchers had constructed a vector with four 35S enhancers (pPCVIcEn4HPT) and transformed it into plants for gene function study (see attached figure). Once the enhancers are integrated into a chromosomal spot near genes, it will activate the genes and produce 'phenotypes'. From checking on the 'phenotypes', researchers can go back to check the potential genes near the tagged cassette (with four 35S enhancers), and fish out the genes. This has been developed into a technology called "Activation Tagging" (see attached paper as one example). Although they used 4 enhancers and yours are only 2, but yours are very close to the tissue-specific promoter, so I think it should still have the effect.
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