Molecular Cloning And Expression Analysis Of Procyanidins Transporter GhTT12 Gene From The Fiber Of Brown Cotton (Gossypium Hirsutum L.)

Brown cotton belongs to Gossypium hirsutum L. witha naturally colorful fiber, so it dose not need to print and dye in post processing, which is more friendlier to environment and lower production cost than a traditional white cotton. Nevertheless such problems as being less color, uneven coloring, low color fastness and low color saturation restrict it’s application and development to a great extent. Because cotton fiber is a kind of specialized cells from seed coat and the pigment of seed coat comes from the oxidation of procyanidins (PAs), we can inference that procyanidins may be a component of the fiber pigment in brown cotton. Present research shows that Transparent Testa 12(TT12) is as a membrane protein of the multidrug and toxic efflux transporter (MATE) family in procyanidins metabolic pathway, and TT12 may transport a glucoside of epicatechin, which is the precusor substance of procyanidins, into a vacuole by a reverse way of transportation, and discharge the H+ from the vacuole at the same time. So we research the function of TT12 gene in brown cotton fiberto provid a reference for genetic improvement of brown cotton.In this study, we designed the primers by the conserved domains of TT12 homologous genes from different species and isolated a TT12 gene from brown cotton fiber using RACE techniques, and performed corresponding bioinformatics analysis. In addition, we studied the relationship between the expression level of GhTT12 gene andthe accumulation of procyanidins at different developmental stages of brown cotton fiber. A prokaryotic expression vector was constructed and used to induce the expression of GhTT12 in Escherichia coli. A eukaryote expression vector was constructed and used to observe the subcellular localization of GhTT12 in Nicotiana tabacum. The results were as follows:(1) A TT12 gene was cloned from the fiber of brown cotton using homology-based cloning methods, named GhTT12 (GenBank accession No. KF240564). The full-length GhTT12 cDNA comprised 1733bp with an OFR of 1503 bp, which encoded a putative protein containing 500 amino acid residues with a typical MATE conservative domain. GhTT12 had a Mw of 54 kDa and a PI of 6.99, and its molecular formula was C2507H3929N615O668S21.(2) Homology comparison indicated that the amino acid sequence of GhTT12 was highly homologous to the sequences of amino acids eccoded by other TT12 genes, which suggested that TT12 gene was conservative in the process of evolution. Phylogenetic tree indicated that the GhTT12 protein had the highest identity (85.17%) to TcTT12, which mean that the function of GhTT12 might be closely related to TcTT12.(3) The contents of the soluble PAs and the insoluble PAs in brown cotton fiber increased firstly and then decreased, and it reached the maximum at 15 days post anthesis (DPA). The content of PAs in brown cotton fiber were far higher than that in white cotton fiber at all developmental stages. Semi-quantitative RT-PCR and real-time quantitative PCR showed that the expression of GhTT12 in brown fibers were much more than that in white fibers at different developmental stages, and especially the maximum was 6 folds at 5 and 10 DPA, but the expression of GhTT12 was lower in both brown and white fibers at 15 DPA and hereafter. Consequently, the results indicated that GhTT12 gene might participate in the pigment synthesis, and the procyanidins might be a component of the pigment in brown cotton fiber.(4) The prokaryotic expression vector pET-32a(+)-GhTT12 was constructed and was transformed into E. coli to induce the expression of GhTT12 with different IPTG concentrations, induction time and induction temperature. But there were no an interest protein induced, which was maybe because the hydrophobic structures of GhTT12 were too many to express in E. Coli. The plant expression vector pCambial304-GhTT12-GFP was constructed, and the GhTT12 protein was observed to localize in a vacuolar membrane by a laser scanning confocal microscope. The recombinant plasmid pCambia1304-GhTT12-GFP was transformed into Arabidopsis tt12 mutant by Agrobacterium-mediated floral dip method, and screened 1 resistant plant on MS medium by antibiotics.