Molecular cloning, expression analysis and subcellular localization of a Transparent Testa 12 ortholog in brown cotton (Gossypium hirsutum L.)

Transparent Testa 12 (TT12) is a kind of transmembrane transporter of proanthocyanidins (PAs), which belongs to a membrane-localized multidrug and toxin efflux (MATE) family, but the molecular basis of PAs transport is still poorly understood. Here, we cloned a full-length TT12 cDNA from the fiber of brown cotton (Gossypium hirsutum), named GhTT12 (GenBank accession No. KF240564), which comprised 1733 bp with an open reading frame (ORF) of 1503 bp and encoded a putative protein containing 500 amino acid residues with a typical MATE conserved domain. The GhTT12 gene had 96.8% similarity to AA genome in Gossypium arboretum. Quantitative RT-PCR analysis denoted that the relative expression of GhTT12 in brown cotton was 1-5 folds higher than that in white cotton. The mRNA level was the highest at 5 days post anthesis (DPA) and reduced gradually during the fiber development Expressing GhTT12-fused green fluorescent protein (GFP) in Nicotiana tabacum showed that GhTT12-GFP was localized in the vacuole membrane. The content of PAs increased firstly and decreased afterwards, and reached the maximum at 15 DPA in brown cotton. But for white cotton, the content of PAs remained at a low level during the fiber development. We speculate that GhTT12 may participate in the transportation of PM from the cytoplasmic matrix to the vacuole. Taken together, our data revealed that GhTT12 was functional as a PAs transmembrane transporter. (C) 2015 Elsevier B.V. All rights reserved.