Identification of two suites of cyclotide precursor genes from metallophyte Viola baoshanensis: cDNA sequence variation, alternative RNA splicing and potential cyclotide diversity
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Abstract
Cyclotides are a novel family of plant-derived defense peptides that are biosynthetically
produced via the processing of cyclotide precursor (CP) proteins containing one, two
or three cyclotide domains. By screening a cDNA library of Viola baoshanensis roots
and using RACE and RT-PCR methods, 23 cDNA clones were identified and then used to
deduce full CP proteins containing one (VbCP1S-5), two (VbCP6S), or three (VbCP7S)
cyclotide domains. RT-PCR and sequence analyses suggested that VbCP6S were resulted
from the alternative splicing of VbCP7S RNA. The significance of VbCP7S RNA splicing
is that it provides a mechanism for increasing the diversity of cyclotide expression
via the recombination of N-terminal repeat (NTR) regions and cyclotide domains. After
analyzing the full endoplasmic reticulum (ER) signals of known and novel CPs associated
with RT-PCR tests, three primers encoding the conserved sequence ALVLIATFA, AAFALPA-LA
and AAFALPA-AFA were proposed to be more efficient in cloning CP genes than the well-applied
primer encoding AAFALPA. Cyclotide sequence analyses indicated that the cDNA clones
encoded a variety of Möbius and bracelet cyclotides, which were likely involved in
the known bioactivities of cyclotides, and also might play a previously unreported
role in mediating the metal tolerance of V. baoshanensis. Overall, this study shows
that CP genes are varied in V. baoshanensis and cyclotide expression is subject to
transcriptional and post-transcriptional regulation in this plant.