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以金桂的花器官为试材,设计芳樟醇合成酶基因简并引物,通过逆转录PCR、快速扩增cDNA末端技术和Blastn分析等,获得金桂芳樟醇合成酶基因的全长cDNA序列,命名为OfLis(Osmanthus fragransvar.thunbergii linalool synthase,GenBank登录号FJ645727)。OfLis基因全长cDNA长度为2003bp,包含1个1731bp的开放阅读框,编码576个氨基酸。序列分析表明:OfLis具有单萜烯类合成酶基因典型的保守结构域,即DDXXD和(N,D)D(L,I,V)X(S,T)XXXE;具有多数单萜烯类合成酶活性必需的RR(X)8W功能基团。OfLis推导氨基酸序列的预测分子质量和等电点分别为67.29ku和5.26;疏水性分析结果表明其大部分氨基酸区域为亲水结构。氨基酸水平上,OfLis与薰衣草芳樟醇合成酶基因的相似性最高,为63.6%,与山字草芳樟醇合成酶基因的相似性最低,为19.0%。逆转录PCR结果表明:整个花期内OfLis基因在花瓣、雌蕊和雄蕊中均有表达,而在萼片和叶片中不表达。研究结果为香味植物的育种、品种改良及香味成分的调控提供理论基础。
The floral organ of Jingui was used as experimental material to design the degenerate primer of linalool synthase gene. The full length cDNA of lignin synthase gene was obtained by reverse transcription PCR, rapid amplification of cDNA ends and Blastn analysis, It was named OfLis (Osmanthus fragrans var. Thunbergii linalool synthase, GenBank accession number FJ645727). The full-length cDNA of OfLis is 2003bp in length and contains a 1731bp open reading frame encoding 576 amino acids. Sequence analysis showed that OfLis has the typical conserved domains of monoterpene synthase genes, namely DDXXD and (N, D) D (L, I, V) X (S, T) RR (X) 8W functional groups necessary for enzyme activity. The predicted molecular mass and isoelectric point of the deduced amino acid sequence of OfLis were 67.29ku and 5.26, respectively. Hydrophobicity analysis indicated that most of the amino acid regions were hydrophilic. At amino acid level, the similarity between OfLis and lavender linalool synthase gene was the highest (63.6%), with the lowest similarity (19.0%) to the Linalool synthase gene. The results of RT-PCR showed that OfLis gene was expressed in petals, pistils and stamens throughout the flowering period but not in sepals and leaves. The results provide the theoretical basis for the breeding of the fragrant plants, the improvement of varieties and the regulation of the flavor components.