title: Molecular cloning, expression analysis and subcellular localization of a Transparent Testa 12 ortholog in brown cotton (Gossypium hirsutum L.).
abstract: 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 PAs from the cytoplasmic matrix to the vacuole. Taken together, our data revealed that GhTT12 was functional as a PAs transmembrane transporter.
authors.affiliation: School of Life Sciences, Anhui Agricultural University, Hefei 230036, China.
authors.forename: Jun-Shan
authors.initials: JS
authors.lastname: Gao
authors.affiliation: School of Life Sciences, Anhui Agricultural University, Hefei 230036, China.
authors.forename: Nan
authors.initials: N
authors.lastname: Wu
authors.affiliation: School of Life Sciences, Anhui Agricultural University, Hefei 230036, China.
authors.forename: Zhi-Lin
authors.initials: ZL
authors.lastname: Shen
authors.affiliation: Institute of Agricultural Economy and Information, Anhui Academy of Agricultural Sciences, Hefei 230031, China.
authors.forename: Kai
authors.initials: K
authors.lastname: Lv
authors.affiliation: School of Life Sciences, Anhui Agricultural University, Hefei 230036, China.
authors.forename: Sen-He
authors.initials: SH
authors.lastname: Qian
authors.affiliation: School of Life Sciences, Anhui Agricultural University, Hefei 230036, China.
authors.forename: Ning
authors.initials: N
authors.lastname: Guo
authors.affiliation: School of Life Sciences, Anhui Agricultural University, Hefei 230036, China.
authors.forename: Xu
authors.initials: X
authors.lastname: Sun
authors.affiliation: School of Life Sciences, Anhui Agricultural University, Hefei 230036, China.
authors.forename: Yong-Ping
authors.initials: YP
authors.lastname: Cai
authors.affiliation: School of Life Sciences, Anhui Agricultural University, Hefei 230036, China. Electronic address: yjsc01@ahau.edu.cn.
authors.forename: Yi
authors.initials: Y
authors.lastname: Lin
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D032561:Cotton Fiber; D020869:Gene Expression Profiling; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant; D003368:Gossypium / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D010802:Phylogeny; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D044945:Proanthocyanidins / Q000378:metabolism; D021381:Protein Transport; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions / Q000378:metabolism
chemical_list: D010940:Plant Proteins; D044945:Proanthocyanidins; D049452:Green Fluorescent Proteins
keywords: Brown cotton; GhTT12; PAs; Quantitative RT-PCR; Subcellular localization