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从长势良好‘阿拉好’黑莓(Rubus spp.)的果实中提取并分离总RNA,反转录成c DNA,根据已登录的黑莓转录组数据,参考其它植物透明外种皮基因(TRANSPARENT TESTA 12,TT12)设计引物,通过RT-PCR扩增得到目的条带,命名为Ru TT12-1。序列分析发现:Ru TT12-1基因全长1 659 bp,具有一个1 464 bp的开放阅读框,编码486个氨基酸,蛋白质分子量为53.09 k D,等电点为5.416。同源性分析表明,其核苷酸序列与其它植物TT12同源基因的一致性为71%~89%。实验分析表明,Ru TT12-1蛋白含有两个MATE结构域,说明此基因属于MATE家族;亚细胞定位预测显示,此基因定位于细胞质膜。蛋白二级结构预测显示,Ru TT12-1蛋白有15个α-螺旋,19个β折叠区,21个β-转角,其大多数氨基酸为具有疏水性。本研究初步了解了该基因的生物学信息特征,有助于今后进一步了解其相关功能,进而揭示花青素苷和原花青素由细胞质转移到细胞中央大液泡的转运过程。
Total RNA was extracted and isolated from the fruits of well-grown ’Rubus spp.’ And reverse transcribed into c DNA. Refer to the transcriptomes of other plant transplants (TRANSPARENT TESTA 12 , TT12). The target band was amplified by RT-PCR and named as Ru TT12-1. Sequence analysis showed that the gene of Ru TT12-1 was 1 659 bp in length and had a 1 464 bp open reading frame encoding a protein of 486 amino acids with a molecular weight of 53.09 kD and an isoelectric point of 5.416. Homology analysis showed that its nucleotide sequence was consistent with TT12 homologous genes of other plants by 71% ~ 89%. Experimental analysis shows that the Ru TT12-1 protein contains two MATE domains, indicating that the gene belongs to the MATE family. Subcellular localization prediction indicates that the gene is localized on the plasma membrane. Protein secondary structure prediction showed that the Ru TT12-1 protein has 15 α-helices, 19 β-sheet regions, 21 β-turns, and most of its amino acids are hydrophobic. The preliminary understanding of the biological characteristics of the gene information in this study will help to further understand its related functions in the future, and then reveal the transfer of anthocyanin and proanthocyanidins from the cytoplasm to the cell central vacuole.