The specific protein in this legume crop is also not identified yet, so it should be isolated and amino acid sequence should be identified too. My idea is first to identify the protein and then design DNA probes complimentary to that protein. On the other hand I'm planning to extract mRNA from the plant and then produce cDNA, and clone it into expression vector and host cell respectively.
Hi Malitha
I never used this kind of methods since we're always used in going from DNA to proteins, but you can use some bioinformatics tools as SMS (http://www.bioinformatics.org/sms2/rev_trans.html). because of degeneration code, if your protein is large, you'll have a lot of results you must blast against the plant genome at NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome). at this step, just choose the species/genome.
you'll have a lot of work, I advice you ta take only a part of the protein, and see after blasting if the remaining sequences are right to your protein.
all the best
fred
You can use the benchling tool back translate and select a related organism (this tool include codon optimization considering the frequency of some codons on an specific organism) . However, Once you have the AA sequence you can also search for homologous proteins with anotated genes. This, you will have an idea to nominate some candidates based on homology.
Frederic Lepretre Thank you so much for the idea. I agree with you, finding out the AA sequence of protein may not sounds easy as I mentioned above since proteins are very complex structures. If you were me, what steps would you follow to find this gene of interest.
Miguel Fernández-Niño Thank you so much for take time to answer my question. I have never use this benchling tool before, and thank you for the information. What steps would you suggest me to follow to discover the gene of interest? Your opinion would be highly appreciated as an expert in the field.
Hi Malitha,
I would take a sequence of 20 AA for which you don't have doubt, reverse them into RNA and blast all reversed transcripts to see were they preferentially blast. I would afterward see the highest result and see if the genomic sequence defines a potential gene that could give you the rest of your protein.
it can represent lot of work, it depends mainly on what is known on your targeted plant, but sometimes simple methods lead to more reliable results.
fred
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