橡胶草法尼基焦磷酸合酶基因的克隆与功能分析

doi:10.3864/j.issn.0578-1752.2016.06.002

摘要/Abstract

摘要： 【目的】法尼基焦磷酸合酶（farnesyl diphosphate synthase，FPS）是天然橡胶生物合成的甲羟戊酸途径中的关键酶，获得橡胶草FPS基因并转化野生橡胶草，为研究FPS基因在橡胶草橡胶生物合成过程中的调控作用奠定基础。【方法】采用同源克隆的方法获得橡胶草FPS基因全长cDNA序列，并对该基因进行生物信息学分析。对橡胶草和其他18种不同高等植物的FPS氨基酸序列进行多序列比对及进化树分析。提取橡胶草不同组织：根、叶柄、叶、花梗、花和种子RNA，对FPS基因进行组织特异性表达分析，并对不同生长时期的橡胶草FPS表达量进行比较，分析橡胶草生长过程中FPS的调节作用。将该基因在野生型橡胶草中过表达，对得到的转基因和野生型植株FPS相对表达水平进行分析，比较转基因和野生型植株橡胶含量的变化。碱煮法提取转基因和野生型橡胶草根部的橡胶，测定橡胶含量。【结果】获得橡胶草FPS基因全长cDNA序列，命名为TKFPS（GenBank登录号 KJ558350.1）。序列分析表明该基因包含1个1 029 bp的完整开放阅读框，编码342个氨基酸，编码的蛋白质相对分子量为39.35 kD，理论等电点（pI）为5.41，平均疏水性为-0.242。多序列比对结果表明，橡胶草FPS氨基酸序列含有高等植物FPS基因的5个保守结构域，系统发育分析结果显示新疆野生橡胶草与蒲公英处于同一分支，其亲缘关系最近。TMHMM和TargetP亚细胞定位分析得知，TKFPS 蛋白无跨膜区域，可能定位于细胞质中发挥功能。组织特异性分析结果表明TKFPS在橡胶草的根、叶柄、叶、花梗、花、种子中均有表达，其中，在叶中表达量最高，根次之，在花中表达量最低。重组子pCAMBIA2300-35S-TKFPS经农杆菌介导法转化野生橡胶草叶盘，得到6株转基因植株。实时荧光定量PCR检测发现，与野生型相比，转基因株系FPS的相对表达量水平明显升高，6株转基因株系TKFPS表达量平均约是野生型的2.8倍。橡胶含量测定结果表明6株转基因株系的根中橡胶质量分数平均比野生型增加了3.92%。【结论】成功从橡胶草中克隆获得TKFPS，并转化野生橡胶草得到转TKFPS的高产橡胶草株系，FPS在橡胶草产胶过程中发挥重要功能。

关键词: 橡胶草, 法尼基焦磷酸, 生物信息学, Real-time PCR

Abstract: 【Objective】 The Farnesyl pyrophosphate synthase (FPS) is a key enzyme in the mevalonate biosynthesis pathway of the crude rubber. The FPS was cloned from Taraxacum kok-saghyz Rodinand transformedinto the wild types, this study will lay a foundation for the research of regulating effects of FPS gene on T.kok-saghyz biosynthesis. 【Method】 Homologous cloning was used to clone the full-length cDNA of this gene, bioinformatic analysis was conducted. Multiple sequence alignment and phylogenetic tree analysis were performed using typical sequences of T.kok-saghyz and other 18 plants FPS proteins. The total RNA was extracted from root, petiole, leaf, peduncle, flower and seed to analysis tissue-specific expression of FPS gene in T.kok-saghyz. The comparation of relative expression levels of the FPS from different growth stages of T.kok-saghyz was processed to research the regulating role of TKFPS in different developing periods of T.kok-saghyz. And this gene was overexpressed in T.kok-saghyz, the expression level of TKFPS and rubber content changes were analyzed after the FPS overexpressed. The rubber was extracted from roots of the wild-type and transgenic strains with Alkali cooking method for determining rubber content. 【Result】 The full-length cDNA of the FPS from T.kok-saghyz was obtained (TkFPS, KJ558350.1). The ORF of 1029 bp encodes 342 amino acid residues with the predicted molecular weight of 39.35 kD, isoeletric point value of 5.41 and average hydrophobicity value of -0.242. Amino acid sequence analysis showed that it contains five conserved domains of FPS gene in higher plants. Phylogenetic tree analysis showed that the T.kok-saghyz had the closest evolutionary relationship with Herba Taraxaci. Subcellular localization analysis of TMHMM and TargetP indicated that it might be targeted to the cytoplasm without transmembrane region. TKFPS gene was expressed in the root, petiole, leaf, peduncle, flower and seed of T.kok-saghyz by the tissue-specificity expression. The expressive content of TKFPS gene in different tissues of T.kok-saghyz was different: the highest was leaf, the next was root, and the lowest level was in flower. Six transgenic strains were obtained after recombinant pCAMBIA2300-35S-TkFPS was transformed to wild T.kok-saghyz by Agrobacterium-mediated transformation. The fluorescent quantitative RT-PCR showed that the relative expression levels of the FPS in transgenic lines were significantly higher, with the increases up to 2.8 times over the wild types. On average, rubber assay results showed that rubber content in roots of six transgenic lines was increased by about 3.92% compared to the wild types. 【Conclusion】 The FPS was successfully cloned from T.kok-saghyz and transformed into the wild types. The obtained transgenic lines had much more rubber than the wilds, this result indicated that TKFPS does play an important role in the biosynthetic pathway of the rubber in T.kok-saghyz.

Key words: Taraxacum kok-saghyz Rodin;farnesyl pyrophosphate synthase; bioinformatics, real-time PCR

曹新文，王秀珍，李永梅，赵李婧，马海霞，闫洁. 橡胶草法尼基焦磷酸合酶基因的克隆与功能分析[J]. 中国农业科学, 2016, 49(6): 1034-1046.

CAO Xin-wen, WANG Xiu-zhen, LI Yong-mei, ZHAO Li-jing, MA Hai-xia, YAN Jie. Molecular Cloning and Functional Analysis of Farnesyl Pyrophosphate Synthase Genes from Taraxacum kok-saghyz Rodin[J]. Scientia Agricultura Sinica, 2016, 49(6): 1034-1046.

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