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林业科学 ›› 2009, Vol. 12 ›› Issue (5): 11-19.doi: 10.11707/j.1001-7488.20090502

• 论文 • 上一篇    下一篇

金桂芳樟醇合成酶基因的克隆与序列分析

唐丽1 唐芳2 段经华3 刘友全1 钟秋平1,4   

  1. (1.中南林业科技大学林学院 长沙 410004;2.中国林业科学研究院林业研究所 北京100091; 3.中国林业科学研究院 经济林研究开发中心 郑州 450003; 4.中国林业科学研究院亚热带林业实验中心 分宜 336600)
  • 收稿日期:2009-01-22 修回日期:1900-01-01 出版日期:2009-05-25 发布日期:2009-05-25

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

Tang Li1,Tang Fang2,Duan Jinghua3,Liu Youquan1,Zhong Qiuping 1.4   

  1. (1.College of Forestry,Central South University of Forestry and Technology Changsha 410004;2.Research Institute of Forestry,CAF Beijing 100091; 3. Non-Timber Forestry Research and Development Center, CAF Zhengzhou 450003;4.The Experimental Centre of Subtropical Forestry,CAF Fenyi 336600)
  • Received:2009-01-22 Revised:1900-01-01 Online:2009-05-25 Published:2009-05-25

摘要:

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 fragransvar. thunbergii linalool synthase,GenBank登录号FJ645727)。OfLis基因全长cDNA长度为2 003 bp,包含1个1 731bp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67.29 ku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。

关键词: 金桂, 单萜烯合成酶, 芳樟醇合成酶, 逆转录PCR, 快速扩增cDNA末端

Abstract:

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 cDNA Ends (RACE) technology,and Blastn analysis. The gene was named as OfLis (Osmanthus fragrans var. thunbergiilinalool synthase) and deposited under GenBank accession No. FJ645727. The OfLis full length cDNA was 2 003 bp and contained a complete open reading frame (ORF) of 1 731bp encoding 576 amino acids. Sequence analysis showed that OfLis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)8W which is essential for the enzymatic activity of many monoterpene synthases. The molecular weight and isoeletric point of OfLiswere predicted to be 67.29 ku and 5.26,respectively. Most of the amino acid sequences of OfLiswere hydrophilic regions. At the amino acid sequence level,OfLis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 OfLis 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.

Key words: Osmanthus fragransvar. thunbergii, monoterpene synthase, linalool synthase, reverse transcription-PCR, Rapid Amplification of cDNA Ends