as far as I know your response is "NO". you have not said about the construct or where you have inserted the construct in chloroplast genome but normally a eukaryotic promoter should not be able to express a gene in chloroplast protein synthesis machinery.

I am wondering how you have been made sure that the construct inserted in the chloroplast genome or is being expressed?

Thank you for your attention.

We used a carrier which tranfer our construct to chloroplast.

Thanks again

1. You did not use 'gene bombardment' to deliver the construct into chloroplast genome, through homologous recombination?

2. You should consider to include a 'positive' control in your experiment: ex. MPI::gus (or GFP)**. Deliver this construct side-by-side with your MPI::GOI (gene of interest) in your experiment. Construct MPI::gus should tell you whether or not the MPI promoter works in the chloroplast.

**MPI::gus should be tested before it is delivered into plastids, to make sure that the construct is correct and has no problem expressing GUS. You can simply test it with a transient assay [ex. bombard on onion (monocot) skin]. 

By the way, although I'm not sure whether promoter from nucleus is active in the chloroplasts, the other way around, researchers have found that some chloroplast promoters are active in nucleus.

Here are two examples, chloroplast psbA promoter (1989) and chloroplast promoter, 16S rrn (2014). Both promoters could drive genes in the nucleus genome. However, psbA promoter showed 'weakly' active in nucleus (see attachment).

So, probably you need to conduct an experiment to find out.

Thank you so much, Yuan-Yeu.

I will try MPI:GUs.

Thanks again

Zahra,

Salam. I think Yuan has offered you a nice solution. 

Salam.

Yes, I will try this.

Thanks 

@ Zahra,

I also found this paper. A review paper about 'chloroplast transformation' (see attachment, yellow highlights). It summarized several important things for some genetic elements used in building a chloroplast transformation vector, including promoter....etc.

In the 'promoter' section (p. 2-3), it points out:

"Plastid transcription is accomplished by the combined actions of two RNA polymerases recognizing different promoters, a T7-like single-subunit nuclear-encoded polymerase (NEP) and a bacterium-like α2ββ’ plastid-encoded polymerase (PEP). Transcription of transgenes inserted into the plastid genome is typically driven by plastid promoters included in the plastid transformation vector, usually the 16S rRNA promoter (Prrn16) or the psbA promoter (PpsbA)."

I have quickly looked through the internet, but I haven't found papers using nucleus promoter to drive gene in chloroplasts. Look like 'plastid promoters' are typically used for chloroplast transformation so far. If so, you probably need a plan B, just in case the promoter doesn't work in chloroplasts.

Thank you so muchYuan-Yue,

We used Mesoporous silica nanoparticles to transfer our construct.

As I study more, it can go to nucleus ,as well. I transferred 35:Gus  construct using MSNPs and the result of Gus assay was positive. As I know 35Spromoter cannot express in chloroplast like MPI promoter. 

Thank you for your attention.

as I mentioned above there is no evidence that a eukaryotic promoter can express a gene in chloroplast. when we try chloroplast transformation, the construct enters the nucleus,too. so in your case expression of GOI under control of MPI does not mean gene expression in chloroplast. if you like to do more experiments you can study GFP expression under control of MPI promoter and visualize it under fuorescent microscope. also you can isolate gDNA from your putative transplastomic plants!(containing MPI promoters+gfp) then transform  E.coli. if you see any GFP fluorescence in E.coli colonies then you might assume the MPI promoter can express GOI in chloroplast because both are prokaryotic systems.

Good Luck

Thank you for yor reply,

The purpose of  our study is to have expression using MSNPs. This is not important that the expression occure in chloroplast or nucleus. I thought MSNPs just enter in chloroplast, But they can enter to nucleus as well.

Thanks

Hi Zahra,

I am a little confused. So, you are saying:

(1). You used MSNP to deliver construct 35S::gus to nucleus, it worked and GUS staining turned blue.

(2). You said: "35S promoter cannot express in chloroplast like MPI promoter"

(3). What is above (1) to do with (2)? I am confused. I thought that you are delivering construct MPI::GOI to chloroplasts?

Hi Yuan-Yeu,

Let me explain more, I used MSNPs to transferred my construct to tomato. According to the papers, MSNPs commonly entered the chloroplast. But now I know that  when the nano particle size is less than 70nm, it can pass through nuclear pore and enter nucleus.

Our particles size is around 50 nm. Therefore, we used 35S:GUS  as well to verify the above Hypothesis. The MPI and 35S promoters can express in nucleus. So, when we detected blue color after GUS assay, we concluded that MSNPs can enter to the nucleus. So, the concerns about lack of expression of the MPI promoter in chloroplast were resolved, because our MSNPS can enter to the nucleus and release the construct there. 

I hope the above information is clear. 

We need to have expression of the MPI:GOI construct in tomato, and this is not important  where it will be release in plant cell. We want examine the gene transferring approach using MSNPs and the efficiency of the MPI promoter .

Hi Zahra,

1. Thanks for your response.

2. I think that those you mentioned above (if all correct) only prove: (1) MSNPs can go into nucleus or reach chloroplasts, (2) 35S::gus works and MPI promoter can drive a gene (which gene you did not mention: gus or GOI) in nucleus. But, you still don't have direct evidence that whether or not MPI promoter is active in chloroplasts.

3. How did you use MSNPs to transferred your construct ? Did you use bombardment? It has been reported that researchers use MSNP to deliver 'stuff' (such as protein, enzymes...) into plant cells. Such as the attached paper, where the authors used MSNPs to deliver Cre protein into plant cells to perform site-specific recombination. I can understand that protein is carried with MSNP into plant cells for 'transient' activities, so no 'foreign' gene/DNA would be integrated into plant genome. But if you deliver your 'DNA construct' with MSNPs into plant cells, why didn't you just bombard the naked DNA construct directly into plant cells? What is the advantage to wrap your construct inside the MSNPs first, then shoot them into the plant cells and release the constructs inside the cells?

4. If the MSNPs can deliver your constructs into the plant cells, once they go into the plant cells, would you like them to be integrated into the plant genome (either nucleus or chloroplast) permanently?

5. Your MPI promoter was from corn (monocot), will it work in tomato??

Hi Yuan-Yeu,

We tested the MPI promoter efficiency in tomato using agroinfilteration. We used AGL1 strain containing MPI:GUS construct and transferred it into tomato leaves by agroinfilteration. The results of GUS assay were positive and we concluded that the MPI promoter can be express in dicot ( tomato). 

We used some different approach to deliver the MSNPs into plant cell. We did not use bombardment. We want to introduce some cheaper, easier and faster approach (to deliver MSNPs into plant cell) than bombardment.

The main and first purpose of our project  is transient expression. but in countinue we want to use local recombination to integrate our construct to plant genome as well.

Thank you so much for your kind reply.

Hi Zahra,

1. Thanks for your response. Now, it makes perfect sense. I was guessing that you are trying to develop a way (not bombardment) to deliver your construct to chloroplasts through using MSNPs. When they reach chloroplasts, the DNA constructs are released, and through homology-depend recombination they can be integrated into chloroplast genome.

2. For this, I have a question: which method will be cheaper to deliver plasmids? Bombardment with gold-coated plasmids (gold particles can be expensive), or manufacture MSNPs to contain your plasmids?

synthesis of MSNP which contain plasmid is more cheaper and easier than bombardment. We want to introduce a method which can done  without expensive  equipment.

@ Zahra,

1. Your project is interesting. Majority of genetic transformations are done in nucleus by Agrobacterium-mediated approach, even for some important monocots. However, the most successful method used so far for chloroplast transformation is using gene gun (bombardment). However, Biolistic Particle Delivery Systems for bombardment can be expensive and not affordable for some institutes or individuals, and hinder the development of chloroplast transformation for them. Therefore, to develop a easier, cheaper and efficient way for delivering plasmids into plastid genome will benefit a lot of researchers worldwide.

2. When you used MSNPs to successfully deliver the 35S::gus construct into nucleus, have you found any construct integrating into the nucleus genome? Or, it was a transient expression?

Hi Yuan-Yeu,

We  used 35:GUS for transient expression. We just confirmed this transformation using PCR, RT-PCR and GUS assay.

Beside this project, we used MPI:GOI  for assessment of MSNPs stable transformation potentially. The next generation of these tomatoes were examined using antibiotic selection and PCR of GOI. The results were positive. We guess the local recombination has been occurred between our construct and chromosome 11 of tomato (about 680 bp) and the transformation construct has been integrated in the nuclear genome.  In continue we want to test these plants more.