霍山石斛黄烷酮合酶Ⅱ的克隆及功能分析

Cloning and functional analysis of flavanone synthase Ⅱ from Dendrobium huoshanense

黄酮碳苷是一类广泛参与植物防御反应且有着广泛药理活性的天然产物,同时也是霍山石斛中一类重要活性成分。黄烷酮合酶Ⅱ已被证实是植物黄酮碳苷合成途径的关键酶,其催化产物2-羟基黄烷酮是多数已报道黄酮碳苷合成的前体化合物。该研究基于已报道的黄烷酮合酶Ⅱ氨基酸序列,从构建的霍山石斛基因组本地化数据库中筛选并验证出一个黄烷酮合酶Ⅱ(DhuFNSⅡ)基因,验证其功能发现该酶可体外催化柚皮素和乔松素分别生成芹菜素和白杨素。DhuFNSⅡ的开放阅读框(ORF)为1 644 bp,编码547个氨基酸,亚细胞定位显示该蛋白定位在内质网上。RT-qPCR结果表明DhuFNSⅡ在茎中的表达量最高,其次是叶、根。霍山石斛经4种生长过程中常遇的非生物胁迫处理后,各组织中DhuFNSⅡ等目标基因的表达量均发生显著上调,但在各处理组中的上调程度却各不相同,其中干旱和冷胁迫对基因表达量影响更为显著。通过对DhuFNSⅡ的鉴定与功能分析,将有助于解析霍山石斛品质代谢物黄酮碳苷形成的分子机制,并为其品质形成及科学栽培提供参考。

Flavonoid C-glycosides are a class of natural products that are widely involved in plant defense responses and have diverse pharmacological activities. They are also important active ingredients of Dendrobium huoshanense. Flavanone synthase Ⅱ has been proven to be a key enzyme in the synthesis pathway of flavonoid C-glycosides in plants, and their catalytic product 2-hydroxyflavanone is the precursor compound for the synthesis of various reported flavonoid C-glycosides. In this study, based on the reported amino acid sequence of flavanone synthase Ⅱ, a flavanone synthase Ⅱ gene(DhuFNSⅡ) was screened and verified from the constructed D. huoshanense genome localization database. Functional validation of the enzyme showed that it could in vitro catalyze naringenin and pinocembrin to produce apigenin and chrysin, respectively. The open reading frame(ORF) of DhuFNSⅡ was 1 644 bp in length, encoding 547 amino acids. Subcellular localization showed that the protein was localized on the endoplasmic reticulum. RT-qPCR results showed that DhuFNS Ⅱ had the highest expression in stems, followed by leaves and roots. The expression levels of DhuFNS Ⅱ and other target genes in various tissues of D. huoshanense were significantly up-regulated after four kinds of abiotic stresses commonly encountered in the growth process, but the extent of up-regulation varied among treatment groups, with drought and cold stress having more significant effects on gene expression levels. Through the identification and functional analysis of DhuFNSⅡ, this study is expected to contribute to the elucidation of the molecular mechanism of the formation of quality metabolites of D. huoshanense, flavonoid C-glycosides, and provide a reference for its quality formation and scientific cultivation.