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采用RT-PCR和RACE技术相结合的方法,从石蒜花瓣中克隆到1个黄烷酮3–羟化酶(F3H)基因的cDNA全长序列,全长1293bp,包含1098bp的完整开放阅读框,编码365个氨基酸;氨基酸序列比对显示该序列编码的氨基酸与水仙的F3H具有高达91%的同源性,将其命名为LrF3H。二级结构预测表明,随机卷曲是LrF3H蛋白最大量的结构元件,而α–螺旋和延伸链散布于整个蛋白中。保守结构域预测表明该基因编码的蛋白具有典型的F3H蛋白功能结构域,其保守结构域中含有铁离子及2–O–酮戊二酸结合位点,属于2OG-FeⅡ_Oxy双加氧酶超家族。实时荧光定量PCR分析表明,LrF3H在整个花发育过程中均表达,而且从初蕾期到盛开期随着花瓣着色加深表达量逐渐增加,盛开期表达量最高,之后随着花朵萎蔫表达量下降;在不同器官中,LrF3H的表达量在花瓣和花葶中最高,而在根、鳞茎和叶片中都很低,推测该基因在石蒜花色形成过程中起着关键作用。
The full-length cDNA sequence of F3H gene was cloned from the petals of Lycoris radiata using RT-PCR and RACE techniques. The full-length cDNA of F3H was 1293bp and contained a complete open reading frame of 1098bp , Encoding a protein of 365 amino acids. The amino acid sequence alignment showed that the amino acid sequence encoded by this sequence shared as high as 91% homology with that of Narcissus, and named it as LrF3H. Secondary structure prediction shows that random curl is the largest number of LrF3H structural elements, while the α-helix and the extended stretch are scattered throughout the protein. Conserved domain prediction showed that the protein encoded by this gene has a typical functional domain of F3H protein. Its conserved domain contains iron ions and 2-O-ketoglutaric acid binding sites, belonging to the 2OG-FeⅡ_Oxy dioxygenase superfamily . Real-time PCR analysis showed that LrF3H was expressed throughout the flower development, and gradually increased from the early bud stage to the full bloom stage with the deepening of flower petal coloring, the highest expression in full blossom stage, and then decreased with the floral wilt; In different organs, the expression of LrF3H was highest in petals and scallops, but lower in roots, bulbs and leaves, suggesting that LrF3H plays a key role in the floral formation of Lycoris radiata.