广藿香法呢基焦磷酸合成酶基因(FPS)的克隆及生物信息学分析

Cloning and Bioinformatics Analysis of Farnesyl Diphosphate Synthase(FPS) Gene in Pogostemon cablin

为进一步阐明广藿香(Pogostemon cablin (Blanco) Benth.)中百秋李醇(Patchouli alcohol)生物合成的分子调控机制,本研究采用RT-PCR技术从广藿香叶片中克隆得到法呢基焦磷酸合成酶(farnesyl diphosphate synthase) FPS基因,并通过qRT-PCR技术分析FPS基因在广藿香幼苗期(扦插后60 d)与成熟期(扦插后240 d)叶片中的相对表达量。对克隆得到的广藿香FPS基因进行生物信息学分析,序列片段长为1 177 bp,其中包含1个长度为1 050 bp的开放阅读框,编码由349个氨基酸残基构成的蛋白质,与其它已知FPS氨基酸序列的唇形科植物一致性高达93.10%,与已知的米团花(Leucosceptrum canum Smith) FPS蛋白亲缘关系最近。预测FPS蛋白质分子质量约为40.09 kD,理论等电点(pI)为5.34,推测其为亲水性蛋白,无跨膜区且不含信号肽,属于类异戊二烯生物合成家族。qRT-PCR结果表明FPS基因在成熟期叶片中表达量高于幼苗期,与转录组数据分析结果一致。本研究结果为进一步研究FPS基因在广藿香中的应用,探究广藿香中百秋李醇生物合成途径中关键酶的表达模式及通过调控百秋李醇生物合成提高广藿香挥发油中有效成分含量提供了理论依据。

In order to further understand the molecular mechanism of patchouli alcohol biosynthetic pathway in Pogostemon cablin. In this study, a famesyl diphosphate synthase (FPS) gene was cloned from Pogostemon cablin leaves by RT-PCR technique. The Real-time PCR was applied to analyze the relative quantitative expression of seedling stage (about 60 days after cutting) and maturation stage (about 240 days after cutting). Bioinformatics analysis was carried on to FPS, The sequence of FPS obtained by the PCR amplification technique was 1 177 bp, this sequence contain a length of 1 050 bp open reading frame (ORF), the enzyme consists of 349 amino acid, the similarity is 93.10% compared with other plants (Lamiaceae), it performed closest relationship with Leucosceptrum canum Smith FPS protein. FPS protein was predicted that the molecular mass was about 40.09 kD, the pl was about 5.34. This enzyme was hydrophilic, which also contained no transmembrane and signal peptide, it was belong to isoprenoid Biosynthesis Cl superfamily. The qRT-PCR results showed that the relative quantitative expres sion in maturation stage was higher than seedling stage of P. cablin leaves, this was consistent with the result of transcriptomic analysis. This study provides a theoretical basis to further study the application of FPS gene in Pogostemon cablin, to explore the expression pattern of key enzymes in the biosynthesis pathway of patchouli alcohol in Pogostemon cablin and to improve the effective content of ingredient in essential oil in Pogostemon cablin by regualting the biosynthesis of patchouli alcohol.