Molecular cloning and characterization of twoBrassica napus TTG1 genes reveal genus-specific nucleotide preference, extreme protein-level conservation and fast divergence of organ-specificity

Encoding a WD40 protein,Arabidopsis thaliana TRANSPARENT TESTA GLABRA1 (AtTTG1) regulates trichome and root hair differentiation as well as flavonoids and seed mucilage deposition in plants. Here, twoBrassica napus TTG1 (BnTTG1) genes were isolated, and Southern hybridization also generated only two bands. The 1511-bpBnTTG1-1 and the 1555-bpBnTTG1-2 both have one intron, and show alternative sites for transcription start, polyadenylation and intron right border splicing. EST and GSS tags suggested thatBnTTG1-1 was derived from B. rapa, whileBnTTG1-2 from B. oleracea. Evidence implies that TTG1 was possibly triplicated in Brassiceae, but some triplicated members were lost soon, which might involve fragmental rearrangements.BnTTG1-1 shares 88.7% genomic and 95.7% mRNA identities withBnTTG1-2. Deduced BnTTG1-1 and BnTTG1-2 proteins both are 337 aa, differed only by substitution of a similar residue. They resemble AtTTG1 in WD40 domain and all conserved motifs. TTG1 / AN11-type WD40 proteins are extremely conserved even across kingdoms. Homological and structural characterizations identifiedBnTTG1-1 andBnTTG1-2 to be orthologs of AtTTG1. Several non-coding motifs are conserved between AtTTG1 and BnTTG1. BnTTG1 coding regions tend to evolve high GC contents through T/A→C/G substitutions especially T→C transition, butAtTTG1 shows opposite base preference.BnTTG1 genes also evolve a GA-stretch in the leader sequence. RT-PCR detected complementation in organ-specificity betweenBnTTG1-1 andBnTTG1-2.BnTTG1-2 is more like AtTTG1 and is expressed in all major organs.BnTTG1-1 is more organ-specific with lower expression in seed and root, possibly withdrawing from regulating seed coat pigment /mucilage deposition and root hair formation.