Organism: Brassica rapa ssp. pekinensis
We have obtained the sequences of BrGGP2 (Bra035554; ortholog to AT5G55120) and BrGPP (Bra040607; ortholog to AT3G02870) from the Brassica database (http://brassicadb.org/brad/). Upon multiple sequence alignment and further database search, we found that the sequences of Bra035554 and Bra040607 are experienced adjacent gene fusions. Bra035554 gained one domain (PF04885.8 – stigma specific domain; function unknown) coded by two exons at the N-terminus and Bra040607 gained one domain (PF12874.2 – zinc finger (C2H2 type) domain) coded by seven exons at the C-terminus. In order to validate these gene fusions by experiments, we performed PCR with different primer pairs to clone the full-length of Bra035554 and Bra040607 using cDNAs of different tissues. If the gene fusion is true, then we must get the amplification of full-length of Bra035554 and Bra040607 by using B. rapa cDNA samples. But, we did not get any amplification with any cDNAs. Intriguingly, when I ran PCR with N-terminus and C-terminus specific primers, we got amplification (see the attached figure). Meanwhile, I also searched NCBI-EST database and found no sequence to support gene fusion. Now, I can conclude either (i) the sequences of Bra035554 and Bra040607 provided in Brassica database may be wrong? (Brassica is a primary database for Brassica species, so the sequences must be genuine), or (ii) further research is required for the transcriptional regulation of Bra035554 and Bra040607. What else I should do to confirm whether the gene fusions in Bra035554 and Bra040607 are true?
Hi Panneerselvam
It is always difficult to prove something is not the case, eg it is harder to prove that there is no fusion thatn showing that there is a fusion. Instead of going for cloning the full-length cDNA you could try to do shorter PCRs linking the Nterminal and Cterm halves. Especially show that the same primers do work with other primers in their respcetive half but not when they bridge.
Also try to blast the bridging area (not the whole full-length just 50 bp of each half and the bridge) to EST databases to get a clue if there is such a fused mRNA, in Brapa, Bnapus or other plants.
It looks like the introns between the halves are very long....
ANd about BrassicaDB (and any sequence deposit), the sequence may be genuine but the annotation may be faulty, especially now that more and more sequence is just compiled by computers and not annotated by "real" biologists. Annoation might get better with incorporation of more long RNAseq reads in annoation pipelines.
If you can show by (semi)qPCR that the 2 halves are independently regulated I would argue against fusion......
Dear Dr. Henk-Jan,
Thank you very much for your suggestion. I already did semi-quantitative RT-PCR with N- and C-termini specific primers (as shown in the above-attached figure) which suggest that the expression of N- and C- termini of both of my target genes were not dependent each other.
Following your suggestion, I will try couple of more PCRs with adjoining primers and let's see.
Dear Andrew,
Thank you very much for your suggestion.
No experimental evidence available to-date. I retrieved the sequence from the Brassica Genome Sequence database.
I consider your thought about the length of cDNA products. I designed few primer pairs to validate the gene fusions. I will update the results once i get.
After trying several primer combination, private whole genome sequencing data and NCBI ref seq data to check whether the above mentioned gene fusion is genuine, we concluded that it is (most probably) not true. Thank you :)
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