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目的克隆狭叶松果菊Echinacea angustifolia 3-脱氢奎尼酸合成酶基因并考察其在各个组织中的表达情况。方法采用快速扩增cDNA末端技术,以狭叶松果菊组培苗cDNA为模板,克隆出狭叶松果菊3-脱氢奎尼酸合成酶基因全长序列并通过半定量RT-PCR分析其在不同器官中的表达模式。结果克隆到的基因(命名为EanaroB)全长为1 424 bp,编码一个442个氨基酸残基组成的多肽。其氨基酸序列与植物来源的3-脱氢奎尼酸合成酶同源性都在80%左右。将得到的序列提交Genbank,序列号为EU293857。半定量RT-PCR结果表明,狭叶松果菊EanaroB基因在狭叶松果菊的根、茎、叶、花中均有表达,但花和叶中的表达量较高,在根和茎中较少。结论采用RACE和PCR的方法从狭叶松果菊中克隆出了咖啡酸类化合物生物合成途径上的一个基因EanaroB,为进一步研究咖啡酸类衍生物生物合成代谢途径提供一定依据,为今后利用基因工程技术提高咖啡酸类衍生物,包括苯乙醇苷类物质松果菊苷的代谢工程打下一定基础。
Objective To clone Echinacea angustifolia 3-dehydroquinate synthase gene and study its expression in various tissues. Methods The full-length cDNA sequence of 3-dehydroquinate synthase gene in Echinacea purpurea was cloned by rapid amplification of cDNA ends and cDNA of Prorocentrum japonicum tissue culture seedlings as a template. Semi-quantitative RT-PCR Its expression patterns in different organs. Results The cloned gene (designated as EanaroB) was 1 424 bp in length and encoded a polypeptide of 442 amino acid residues. Its amino acid sequence and plant-derived 3-dehydroquinate synthetase homology is about 80%. The resulting sequence was submitted to Genbank with the serial number EU293857. The results of semi-quantitative RT-PCR showed that EanaroB gene was expressed in roots, stems, leaves and flowers of Echinacea angustifolia, but higher in flowers and leaves, less. Conclusion The gene EanaroB of caffeic acid biosynthesis pathway was cloned from Echinacea purpurea by RACE and PCR, which provided a basis for further study on the biosynthetic metabolic pathway of caffeic acid derivatives. Engineering to improve caffeic acid derivatives, including phenylethanoid glycosides echinacoside metabolic engineering laid the foundation.