| ATP-binding cassette(ABC) transporters are one of the largest families of transmembrane proteins, which are widely exist in prokaryotes and eukaryotes, Most of the ABC transporters are able to combine with ATP, and regulation transmembrane transportation by the release energy that hydrolysis of ATP, transportation of substrates include: amino acids, polysaccharides, peptides, metal ions, alkaloids, phenols, terpenoids, waxes and drugs, et al., ABC transporters play a very important role in the process of accumulation and transportation of secondary metabolites. According to ABC transporters conservative sequence homology and the composition types of structural domain, ABC transporters can be divided into nine sub-family(ABCA – ABCI). The WBC(white brown complex) type transporters is a member of ABCG sub-family, In order to understand transport mechanism of secondary metabolites accumulation and transportation of WBC transporters in the molecular level, this experiment with blueberry(Vaccinium SPP.) "Northland"(V. corymbosum L.) roots, stems, leaf buds, leaves, blossoms, blossom buds, green fruits, pink fruits, blue fruits, seeds of blue fruit, peel of blue fruit and pulps of blue fruit as experimental materials, real-time fluorescence quantitative PCR(qRT-PCR) was performed on seven significantly up-regulated Unigenes and one of down-regulated highly expressed in peel of Unigene10823_All to examine the expression pattern during blueberry development stage and several different organs, and successful cloned the Unigene10823_All full-length sequence, named VcWBC3. The main experimental analysis results were as follows:(1) 21 Unigenes encoding WBC transporter were screened out from blueberry fruit transcriptome, total 10 Unigenes were up-regulated expression(the expression of peel highly than pulp), of which seven Unigenes were significantly up-regulated; and 11 genes were down-regulated expression(the expression of pulp highly than peel).(2) VcWBC genes expressed in all tested tissues and organs of blueberry, but had different expression levels. Unigene14517_All and Unigene29820_All highly expressed in stems and blue-fruits, respectively; Unigene5067_All and Unigene27766_All highly expressed in roots; Unigene10823_All and Unigene27928_All highly expressed in blossoms; furthermore, Unigene34381_All and Unigene34438_All showed an increasing expression pattern from green-fruits, pink-fruits to blue-fruits.(3) The full-length sequence of VcWBC3 was contained a 2157 bp ORF(open reading frame), eneoding718 amino acids, the protein molecular weight was 78.71 kDa, isoelectric point was 7.54,and it was a unstable protein; VcWBC3 protein secondary structure consisted of 267 alpha-helix(37.19%), 264 random coil(36.77%), 125 extended strand(17.41%) and 62 beta turn(8.64%); VcWBC3 had a signal peptide and three transmembrane domains, and mainly located in plasma membrane, and belong to hydrophilic protein; VcWBC3 and other plant amino acids showed that the amino acid sequences were highly conserved in the motifs common to all ABC transporters of “Walker A”, “Walker B” and “ABC signature”, the amino acid sequence homology of VcWBC3 with Vitis vinifera(VvABCG7), Malus domestica(MdABCG7) and Arabidopsis thaliana(AtABCG7) was 81.62%, 77.30% and 75.77%, respectively, VcWBC3 was closely related to Arabidopsis thaliana of AtABCG26(AtWBC27) and AtABCG25(AtWBC26); VcWBC3 protein gene if want give play to its transfer function that it must be form dimers with itself or other half molecules G family ABC transporters of blueberry.This will provide the molecular basis and theoretical basis to understand the functional diversity and evolutionary status of the WBC transporters in blueberry. These results demonstrate favorable clues to study that the regulation of the secondary metabolites of WBC transporters in blueberry. In addition, it also provides a reference for regulation of the secondary metabolites of WBC transporters in other plants in the future. |
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