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利用简并PCR和RACE技术从银杏叶片中分离到黄酮3’羟化酶基因Gb F3’H的c DNA全长序列(Gen Bank No.:KP056747)。其全长为2 144 bp,含有一个1 671 bp的开放式阅读框(ORF),编码556个氨基酸序列,预测氨基酸大小为63.47 k D,等电点为7.71。蛋白质同源序列分析表明,Gb F3’H与其他物种F3’H同源性较低,而与菊苣(Cichorium intybus)同源性最高,为56.3%。同源建模分析显示Gb F3’H与P450家族蛋白的三维结构及活性位点高度相似,最终定位于微粒体膜上。进化树分析结果显示Gb F3’H与其他植物分化较早,序列差异较大。组织表达分析显示,Gb F3’H在银杏不同组织中都有表达,其中雄蕊表达水平最高,其次为成熟叶。诱导表达分析显示,Gb F3’H转录水平受UV-B、6-BA、SA、ABA和IAA影响诱导上调,而伤害处理对其表达量无明显影响。Gb F3’H诱导表达模式与调控银杏黄酮含量变化呈正相关,其上游调控序列存在与黄酮调控相关的顺式元件,意味着Gb F3’H可能参与了银杏黄酮类物质的合成代谢,并与黄酮类物质向花青素合成分流有关。
The full-length cDNA sequence of flavone 3 ’hydroxylase gene Gb F3’H was isolated from Ginkgo biloba leaves using degenerate PCR and RACE (Gen Bank No.:KP056747). The full-length cDNA was 2 144 bp in length and contained a 1 671 bp open reading frame (ORF) encoding a 556 amino acid sequence with a predicted amino acid size of 63.47 kD and an isoelectric point of 7.71. Homologous sequence analysis showed that Gb F3’H had lower homology with F3’H of other species and highest homology with Cichorium intybus, which was 56.3%. Homology modeling analysis showed that Gb F3’H was highly similar to the three-dimensional structure and active site of the P450 family of proteins and was eventually located on the microsomal membrane. Phylogenetic tree analysis showed that Gb F3’H was earlier differentiated from other plants and had a large difference in sequence. Tissue expression analysis showed that Gb F3’H was expressed in different tissues of Ginkgo biloba, with the highest expression level of stamens, followed by mature leaves. Inducible expression analysis showed that the transcription level of Gb F3’H was up-regulated by UV-B, 6-BA, SA, ABA and IAA, while injury treatment had no significant effect on the expression of Gb F3’H. Gb F3’H induced expression pattern is positively correlated with the regulation of ginkgo flavone content, and its upstream regulatory sequence contains flavonoid-related cis-elements, which means Gb F3’H may be involved in the synthesis and metabolism of flavonoids, and flavonoids Anthocyanin-like substances to the diversion of related.