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Cloning multiple genes in plant vector
#1
This is post no. 1 under the main topic.
I need your help with following information and it would be great if you could help with this as soon as possible. I am basically trying to clone two genes in a plant-based vector and express it in either tobacco/ Arabidopsis. Old method of doing this was cloning two separate genes in two different plant vectors and go for sequential transformation. Its important to ensure that two vectors are compatible for the experiment, as in they should have different promoters and selection markers. In recent times TA cloning and gateway cloning vectors have been designed by various labs, so cloning multiple genes in single plant based vector is possible but these are not commercially available so it becomes essential to contact various labs.

If have any information on the same then please share and I would also like information on how to obtain these vectors. I would ideally like to have a single vector in which multiple genes can be cloned and expressed in plants. If not then I would like to have some more information on what criteria I should use in selecting two different vectors and choose which plant for transgenic expression.

I hope I have explained myself well. If you have any questions please ask me.
Thanks!
 
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#2
This is post no. 2 under the main topic.
Dear member,

I saw your question just now. Well, you can clone multiple genes in same vector. As it is Eukaryota system, it is important to have following things for expression.

1- Suitable promoter for your system (Plant specific promoters)
2- Ribosome binding sites (for initiation of translation)
3- Transcription termination (Termination of transcription)

If you want to added multiple gene in same vector and you have to duplicate theses expression elements then vector size will be very big and may cause difficulties for cloning and further use in expression.

There are several IRES (Internal Ribosome entry sites) known for plant system and you can add any of these sites between your two genes. As there will be translation termination codon for your both the genes they will be transcribed with same promoter and transcription terminator but two different proteins will be synthesized.

Please read these two articles, it will help you

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC157974/
http://www.ncbi.nlm.nih.gov/pubmed/12848831

For more advancement in your system, you may use tissue specific promoters to your vector.

Here below i have given a link for the list of vectors which you can use for your system.

http://www.arabidopsis.org/abrc/catalog/vector_1.html

Hope you will be able to find solution from my answer, if you have any further question, please write it here. I will try
to attend that.

good luck

Brijesh
 
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