I am interested to know that does the selectable marker gene in the T-DNA region under NOS promoter will work in both plant and Agrobacterium cells. If yes, Kindly provide some reference .
Thanks,
Best wishes.
Naveed Anjum
First, what do you want this 'selectable marker gene' for? For bacterial selection on medium or for selecting your transgenic plants?
Dear @yuan-yeu yau,
For the both selection i.e. Agrobacterium selection for plasmid and on tissue culture medium for transgenic plant.
@ Naveed Anjum, what binary vector you are using? What selection marker gene you are using?
this link is useful
https://www.wikigenes.org/e/gene/e/1142638.html
regards
Dear @yuan-yeu yau,
I am using pGA482 vector. Practically it is providing selection on both tetracycline (for bacterial selection LBA4404) and Kanamycin (under Nos promoter for transgenic selection) in bacterial cell (LBA4404 and E. coli).
The vector is described here:
Article Vectors carrying two separate T-DNAs for co-transformation o...
Unfortunately, the resistance gene in the backbone of pGA482 appears to be for conferring to Agrobacterium resistance to tetracycline, which is an unfortunate choice (I personally strongly dislike using tet selection). Apparently it is important that one chooses the small Agrobacterium colonies from solid culture media supplemented with tetracycline, as the larger colonies are likely tet resistant mutants (which apparently occurs quite readily in Agrobacterium). If you can find an alternative split T-DNA plasmid which carries either nptIII (conferring resistance to kanamycin) or aadA1a (conferring resistance to spectinomycin) your work might be a bit easier.
@ Ray Collier,
From the attached figure (pGA482), I do see the NPTII gene is driven by NOS promoter (for transgenic plant selection), and the backbone is with tetracycline-resistant gene for bacteria selection. Do you know what promoter drives this tetracycline-resistant gene?
I don't think it is driven by another NOS promoter. It looks like a very old binary vector (1988).....
Dear @yuan-yeu yau,
A 29bp E. coli promoter for tetracycline efflux protein gene.
@Naveed; and my apologies for not directly answering your original question. The NOS promoter is typically utilized for plant expression, not expression of genes in Agrobacterium. In almost all binary vectors the antibiotic resistance genes, used for selection of Agrobacterium which contain the plasmid, are located outside the T-DNA, not within. Also, disregard my earlier comment about split T-DNA (that is not applicable to pGA482, I confused myself with the graphic from the Plant J paper). There is a very good reason why you should not use pGA482, and that is that the selection marker is located at the T-DNA Right Border. Given that T-DNA transfer initiates at the RB, and terminates at the Left Border, it is advised that one locates the selection marker at the LB to select for plants containing full length T-DNA insertions. You could rely on GUS staining, but this is a more costly method (and most often lethal) method for identification of the transgenics.
@ Naveed Anjum,
Thanks for your response. Interesting, I've not seen that short promoter (29bp).
But, why did you ask the question? Did you want to remove the current promoter and put a NOS promoter there?
Why don't you look for a pCambia or other more popular binary vectors?
@ Ray,
Why using GUS screening is most often lethal method for identification of the transgenicss? I understood that it could be costly for some labs to use GUS staining.
@Yuan-Yeu: Although there is a vital GUS protocol, typically the staining of the plant tissue is lethal. Of course, you don't typically stain the whole plant, thus you still should obtain propagules. I more meant that I like to use fluorescent markers for non-destructive identification of transgenic tissues.
@ Ray,
ok. now I know what you meant. Yes, people don't have to use the whole plant for staining, but use a small tissue to do it. I usually test it using a small part of tissue. Once it stains blue, I keep it for growing bigger. When, I will re-test again using different tissues from different leaves of the same plant to make sure it is real. Sometimes, it is false-positive (from contamination of not-eliminated Agrobacterium).
Dear @yuan-yeu yau,
Actually I am amazed that, how NOS promoter work in Agrobacterium/E.coli ! I am Finding/searching any research paper/expert opinion that about working of NOS promoter in Agrobacterium/E.coli (weaker expression) because we know that it has eukaryotic elements and express in plants. As in pGA482 I have practical experience that NOS work in Agrobacterium/E.coli (due to kana resistance).
Secondally, A 29bp E. coli promoter for tetracycline efflux protein gene. For this please see attached pSoup vector (SnapGene format) detail. pKR2 (tetracycline resistant) is used during pSoup and pGA482 vector construction.
@ Naveed Anjum,
Interesting, so you are saying that after you transformed pGA480 into Agrobacterium/E.coli, and spread on solid medium + kanamycin, surviving colonies are observed? And this resistant is due to Pnos::nptII construct (originally designed for plant selection) in the T-DNA region of the pGA480?
Did you have a control when you did this experiment? Did you also spread empty competent cells on the solid medium + kanamycin?
Did you miniprep the 'surviving' colonies and to see whether they contain pGA480?
Dear @yuan-yeu yau, Yup. I also isolated plasmid (pGA482) and do cloning.
Dear Ray Collier, Thanks for your precious suggestion (nptII on LB).
I am into the same problem now , some people say that plant-directed promoter for kanamicin also provides some antibiotic resistance to Agro and can be used for screening of positive clones... I am going to find it out. Would be amusing finding, if it is true. What i have found for sure is that Bla gene , conferring ampicillin resistance in E.coli is not providing it in Agro. Just not at all. Really strange discovery.
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