Hi Jiexi,

You said, "GFP signal was ectopically detected in all tissues of transformed plants". Are you sure this was not due to 'auto-fluorescence' of the tissue? Did you use a 'wild-type' side-by-side with your transgenic plants?

Hi Yuan-Yeu, I'm pretty sure it is not auto-fluorescence. I did have wild type side-by-side.

The 35S promoter has the ability to act as a bi-directional enhancing element; If you can use the NOS promoter to drive HptII you might have a better result. Can you upload a cartoon of the structure of your construct (this would be helpful in more clearly answering your question).

Dear Jiexi,

The presence of a 35S promoter absolutely can influence the spatial and temporal specificity of other promoters. We have seen this in our own research, and thankfully this has been documented in several publications. With the exception of publicly available T-DNA lines (SALK, etc.), I always try to avoid 35S vector containing plant lines unless I am using them for a defined non-physiological purpose such as protein purification. One option is to use the 35S-free pORE vectors or use the pORE vectors as a source to modify your pMDC.  I have used (and modified) the pOREs for years and have been quite satisfied, and since multiple selection promoters are available you can be confident in your data.

Good luck!

The 35S promoter used in a selectable marker gene of a plant transformation vector affects the expression of the transgene.

Planta. 2005 Jun;221(4):523-30. Epub 2005 Jan 29.

The cauliflower mosaic virus (CaMV) 35S promoter sequence alters the level and patterns of activity of adjacent tissue- and organ-specific gene promoters.

Plant Cell Rep. 2007 Aug;26(8):1195-203. Epub 2007 Mar 6.

pORE: a modular binary vector series suited for both monocot and dicot plant transformation.

Transgenic Res. 2007 Dec;16(6):771-81. Epub 2007 Feb 2.

Timothy is absolutely correct about pORE, we have made extensive use of this vector in our work as well. There are others out there as well that don't use the 35S promoter. Good luck!

I have a question rather than an answer. How did you determine that you had cloned the full promoter without missing any sequences that critical for phloem parenchyma-tissue specificity?

@Zhifen: That's the $64000 question, isn't it? The truth is that we still don't really know what constitutes a "full" promoter. Bioinformatic methods probably still haven't yet reached a point of reliability for prediction, and suffer from only being able to predict those motifs that have been previously described. Promoter work is still hard, grind it out by promoter bashing and empirically testing. Then you can maybe perform pull-down assays to confirm that transcription factors are binding. There is still much to discover with regard to promoters.

I saw this question rather late. I agree with the above answers, the 35S promoter could indeed act as enhancer affecting the signal strength of the promoter used for the GFP cassette. Nevertheless, the promoter used should retain (at least most) of its tissue specificity, but at higher levels. This is basically the mechanism of activation tagging. To avoid this problem you could use a vector with other promoter driving the antibiotic resistance marker and/or using larger fragment of the promoter in the GFP cassette. You could also have been unlucky where you happen to work with a promoter that is also strongly affected by the chromatin state of its genomic region, which makes promoter:reporter analysis problematic.

Hi, many thanks to those who answered my question. Your answers are very helpful to me. sorry for late reply. i'd only received email notification today so that I know your guys replied. :( anyway.

@Ray Collier

please see attached map of my construct.

@Timothy E Gookin @Eric van der Graaff

The promoter is phloem parenchyma-specific used and confirmed by a paper published on Science.I know i can use a different vector but the problem is that the promoter is a very complicated 2.9kb fragment and i've tried lots of efforts to amplify it but always have trouble getting an error-free fragment.  And because they sent us this promoter in pDONR221 entry vector which contains recombination sites so I did a LR combination and ligated this promoter into pMDC110. Now here comes the problem! So could you please suggest me what's the best option at this stage, is it to use the pORE vectors as a source to modify my pMDC?

I think your best bet will be to try to move the cassette into a vector that doesn't use the 35S promoter. I don't think there is any easy way out here. Sorry....

@Zhifen Zhang: Ray Collier is correct. this promoter has been characterized previously so that's why im pretty confident on its specificity.

@Ray Collier: Do  you think it is doable if I amplify the At4g15440 promoter (for basta selection) from pORE and replace the 35S promoter in pMDC110 with it?

I am unfamiliar with that promoter, but the criterion for any promoter you choose for selection is that it should be constitutive (or at least driving expression in the tissues that will be exposed to the selective agent). If you are confident that the At4g15440 promoter is satisfying this requirement then yes, you should be able to use it. I guess you don't want to use the NOS promoter for some reason (I have used this previously for BAR selection and it has been suitable)?

Dear Jiexi,

I previously constructed several Gateway modified pORE vectors (e.g. mVenus, mTurquoise, GUS, etc) for promoter analysis Contact me directly if you are interested.

The mas promoter works well for selection, as well as driving ectopic expression fluoroproteins, etc.  Note that it has 1'-2' activity  and is differentially responsive depending on tissue type and hormone levels (for the common transgenic plant selection orientation see pCambia derived pFGC5941, etc.). 

Tim