山葡萄无色花色素双加氧酶基因（LDOX）cDNA的克隆与表达

为获得山葡萄LDOX基因的全长序列,采用RT-PCR与SMART RACE技术克隆LDOX基因,并对该基因进行生物信息学分析。结果显示,山葡萄LDOX基因全长1 353bp,其中开放阅读框(ORF)为1 068bp,编码355个氨基酸,氨基酸序列的分子质量为40.19ku,等电点为5.61;VAmLDOX基因(GenBank登陆号:FJ645769)属于双加氧酶基因家族,不含信号肽,VAmLDOX蛋白属于不稳定亲水蛋白,二级结构中随机卷曲含量最高;山葡萄VAmLDOX氨基酸序列与欧亚种葡萄、苹果、大豆、三花龙胆和紫苏等的同源性系数分别为99%、81%、80%、77%和75%;半定量RT-PCR分析显示,在山葡萄果实着色过程中,VAmLDOX在不同时期的果皮中均有表达,在转色期的叶片、茎、果肉中也均有表达,且表达量相近。

Cloning and Analysis of Leucoanthocyanidin Dioxygenase (LDOX) in Vitis amurensis Rupr

To obtain the full length cDNA sequences and analyze the bioinformatics of LDOX gene in the skins of Vitis amurensis, the technology of RT PCR and SMART RACE were used. The results showed that the full length of LDOX cDNA was 1 353 bp with open reading frame of 1 068 bp, which encoded 355 amino acids. The product of LDOX gene expression was unstable protein with the molecular mass of 40.19 ku, isoelectric point of 5.61. The VAmLDOX gene landing GenBank No. was FJ645769, which belongs to the leucoanthocyanidin dioxygenase supergene family. In the secondary structure, the levels of random coil was the highest and the LDOX gene was unstable hydrophilic protein and which was not containing a signal peptide. The amino sequence of LDOX in Vitis amurensis had the homology with LDOX of Vitis vinifera (EF192468), Malus domesstica (FJ817488), Glycine max (AF382828), Gentiana triflora (ABI93310) and Perilla frutescens (AB003779), and the likeness coefficient was 99%, 81%, 80%, 77% and 75% respectively. The results of semi quantitative RT PCR showed that:in the fruit coloring process, the expression of VAmLDOX happened in various period of the shins and also expressed in stems, pulb and skins, and the expression quantity was similar.