I am trying to transform a gene to a plant. So I am designing a primer for the gene of interest. But the softwares are not giving primers for the full length gene. So, shall I go with manually designing the primers for the full length gene or the primers that are obtained from the software for truncated gene?
If your purpose is to introduce a functional gene, of course you should design the primers such as to amplify the full gne. I don’t see the problem for this. You can use software provided by most DNA polymerase suppliers to check the length your primer needed to amplify the full gene should have to possess a Tm adequate for PCR amplification.
Likhith R K Sure, you need a complete gene sequence + promoter and terminator. It is important to know the source of your gene of interest - virus, bacteria, or plant. Plant genes can be too long for one-step PCR and include introns. Bacterial genes may have different codon usage and must be re-designed for optimal expression in plants.
Alex Ignatov Thanks for the response. I am trying to insert viral gene into the plant
Likhith R K I think it will be useful to follow the listed below publications:
da Câmara Machado, M.L., da Câmara Machado, A., Hanzer, V. et al. Regeneration of transgenic plants of Prunus armeniaca containing the coat protein gene of Plum Pox Virus. Plant Cell Reports 11, 25–29 (1992). https://doi.org/10.1007/BF00231834
Expression of alfalfa mosaic virus coat protein gene confers cross‐protection in transgenic tobacco and tomato plants | The EMBO Journal (embopress.org)
Di, R., Purcell, V., Collins, G.B. et al. Production of transgenic soybean lines expressing the bean pod mottle virus coat protein precursor gene. Plant Cell Reports 15, 746–750 (1996). https://doi.org/10.1007/BF00232220
Likhith R K
You mentioned "truncated gene". That probably is a cDNA. You can use a cDNA or the full-length gene (introns+exons) for plant transgene expression. But the full-length gene is usually in long length, not as simple in cloning manipulation (compared to shorter cDNA). If you want to obtain cDNA of the gene, you should perform RT-PCR, starting from the gene's mRNA. But, some genes only express (producing mRNA) at a certain stage of plant development. This you need to take into consideration.
Alex Ignatov
Thanks for reminding that to me, I didn't notice it is a viral gene.
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