金桂芳樟醇合成酶基因的克隆与序列分析(英文)

Cloning and Sequence Analysis of a Homologous Linalool Synthase Gene Involved in Floral Scents in Osmanthus fragrans var. thunbergii

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。

In this study, a full length cDNA encoding linalool synthase was successfully cloned from floral organs of Osmanthus fragrans var. thunbergii with newly designed degenerate primers by reverse transcription （RT）-PCR, Rapid Amplification of eDNA Ends （RACE） technology, and Blastn analysis. The gene was named as OfLis （ Osrnanthus fragrans var. thunbergii linalool synthase） and deposited under GenBank accession No. FJ645727. The Of Lis full length eDNA was 2 003 bp and contained a complete open reading frame （ORF） of 1 731 bp encoding 576 amino acids. Sequence analysis showed that Of Lis presented two typical conserved motifs of terpene synthases, i. e DDXXD and （ N, D ） D （ L, I, V ） X （ S, T） XXXE, which are essential for substrate binding and ionization. There was a N-terminal peptide sequence RR（ X）sW which is essential for the enzymatic activity of many monoterpene synthases. The molecular weight and isoeletric point of Of-Lis were predicted to be 67.29 ku and 5.26, respectively. Most of the amino acid sequences of Of Lis were hydrophilic regions. At the amino acid sequence level, Of Lis exhibited the highest similarity （63.6%） with Lavandula angustifolia linalool synthase, and the lowest similarity （19.0%） with Clarkia concinna linalool synthase gene. RT-PCR analysis revealed that Of Lis was specifically expressed in petals, pistils and stamens, but not in sepals and leaves in the whole bloom stage. This study lays a scientific basis for breeding, improvement and regulation and control of flavor profile of fragrant plant varieties.