中药青天葵黄酮醇合成酶基因的克隆与生物信息学分析

Gene Cloning and Bioinformatics Analysis of Flavonol Synthase from Nervilia fordii

【目的】克隆中药青天葵黄酮化合物生物合成途径的关键调控酶—黄酮醇合成酶(flavonol synthase,FLS)基因并分析其序列特征及编码蛋白的功能。【方法】在前期青天葵转录组测序获得的FLS基因序列片段的基础上,通过cDNA末端快速扩增技术(RACE-PCR)克隆FLS基因的cDNA全长及其编码区;利用生物信息学方法对其编码蛋白进行同源性比对和功能分析。【结果】青天葵FLS基因cDNA全长1268 bp,其中包含993 bp的开放阅读框,编码331个氨基酸。编码蛋白的氨基酸序列预测显示,青天葵FLS蛋白为酸性、亲水性蛋白,分子量约为37.3 kDa;不含有信号肽、转运肽和跨膜结构域,可能定位于细胞质中;具有黄酮醇合成酶保守功能域,与铁皮石斛等同科植物FLS蛋白的同源性可达82%。【结论】成功克隆了青天葵黄酮醇合成酶基因,为后续的基因功能鉴定和生物合成调控,进而提高青天葵黄酮类药效成分积累奠定了基础。

【Objective】This paper aimed to clone the encoding gene of flavonol synthase from herbaceous Nervilia fordii,and the sequence feature and function of its coding protein were analyzed.【Method】Based on the FLS sequence fragment acquired from previous transcriptome sequencing,the full-length cDNA and coding region of FLS was cloned from N.fordii leaves using rapid amplification of cDNA ends technique,and then the polygenetic alignment and function analysis of the encoding protein were performed by bioinformatics approaches.【Result】The length of FLS gene cloned from N.fordii was 1268 bp,including 993 bp open reading frame,encoded 331 amino acids.Sequence analysis and prediction revealed that Nf FLS protein with a molecular weight of 37.3 kDa was acidic and hydrophilic,with none signal peptide,transit peptide and transmembrane region,which demonstrated the protein probably located in cytoplasm.Nf FLS protein contained a flavonol synthase conserved function domain and shared an identity of 82 % with the FLSs from Orchidaceae species such as Dendrobium catenatum.【Conclusion】A FLS gene was successfully cloned from N.fordii,which provided firm foundation for its functional identification of flavones biosynthesis regulation in this species.