北美鹅掌楸LtuFPPS1基因克隆与组织表达分析

Cloning and tissue expression analysis of LtuFPPS1 gene in Liriodendron tulipifera

关键词:  北美鹅掌楸, LtuFPPS1基因, 基因克隆, 生物信息学分析, 组织表达

Key words:  Liriodendron tulipifera, LtuFPPS1 gene, gene cloning, bioinformatics analysis, tissue expression

摘要:

法尼基焦磷酸合酶(farnesyl diphosphate synthase, FPPS)是植物萜类物质合成前体法尼基焦磷酸(farnesyl pyrophosphate,FPP)的关键合成酶。为探究北美鹅掌楸(Liriodendron tulipifera)萜类合成途径的分子机制,该文利用北美鹅掌楸转录组数据,通过设计特异性引物,采用RACE技术克隆得到LtuFPPS1基因的全长序列并进行生物信息学分析。结果表明:(1)LtuFPPS1基因全长1 460 bp,开放阅读框(ORF)长1 056 bp,编码351个氨基酸,蛋白质分子量为40 589.45 D,等电点为5.19,不稳定系数为43.50,归类为不稳定蛋白; LtuFPPS1基因所编码的蛋白不含信号肽,定位于线粒体,是一种不稳定亲水性蛋白,具有类异戊二烯类化合物合酶的特征结构域,α-螺旋为其氨基酸序列的主要二级结构。(2)进化树和同源序列分析表明,北美鹅掌楸LtuFPPS1蛋白与乐昌含笑(Michelia chapensis)的FPPS蛋白的亲缘关系更近。(3)组织表达分析结果发现,LtuFPPS1基因在北美鹅掌楸雌蕊中的表达量最高,其高低顺序为雌蕊>花芽>茎>雄蕊>萼片>叶片>花瓣>根,据此推测萜类代谢物在花器官中的合成相对较多。综上所述,LtuFPPS1基因为萜类合成酶基因,可为从分子生物学层面探究北美鹅掌楸萜类物质的合成提供一定的理论帮助。

Abstract:

Farnesyl diphosphate synthase(FPPS)is a key synthase for the synthesis of farnesyl pyrophosphate(FPP), a precursor of plant terpenoids. In order to investigate the molecular mechanism of terpenoid synthesis in Liriodendron tulipifera, the full-length sequence of the LtuFPPS1 gene was cloned by RACE technology and analyzed by bioinformatics using the transcriptome data of L. tulipifera, by designing specific primers. The results were as follows:(1)The length of LtuFPPS1 gene was 1 460 bp and ORF was 1 056 bp, coding 351 amino acids. The protein molecular weight was 40 589.45 D, the isoelectric point was 5.19, and the instability coefficient was 43.50, which was classified as unstable protein. The protein encoded by the LtuFPPS1 gene was an unstable hydrophilic protein that was localized to mitochondria and contains no signal peptide. It had a characteristic domain of isoprenoid compound synthase, and the secondary structure of amino acid sequence was mainly α-helix.(2)The phylogenetic tree and homologous sequence analysis showed that the LtuFPPS1 protein of L. tulipifera was more closely related to the FPPS protein of Michelia chapensis.(3)The results of tissue expression analysis revealed that the LtuFPPS1 gene was the most highly expressed in the pistil of L. tulipifera, and its order was pistil > flower bud > stem > stamen > sepal > leaf > petal > root. Based on this, it is speculated that terpenoid metabolites are relatively more synthesized in flower organs. In summary, the LtuFPPS1 gene is a terpene synthase gene, which can provide some theoretical help for exploring the synthesis of terpenoids in L. tulipifera from the molecular level.