北美鹅掌楸LtuHMGS基因的克隆与生物信息学分析

Cloning and bioinformatics analysis of Ltu HMGS in Liriodendron tulipifera

【目的】研究北美鹅掌楸LtuHMGS基因的结构特性、亚细胞定位及表达模式,为从分子水平探究北美鹅掌楸萜类合成途径提供理论基础。【方法】从北美鹅掌楸转录组数据库中鉴定出一条HMGS基因序列,采用RACE技术克隆其全长cDNA,将其命名为LtuHMGS。对LtuHMGS基因的ORF进行生物信息学分析,探究其理化性质及与同源基因的系统进化关系;利用烟草瞬时转化系统对LtuHMGS基因编码蛋白进行亚细胞定位;采用RT-qPCR技术分析LtuHMGS基因在北美鹅掌楸的组织表达谱。【结果】LtuHMGS基因的ORF全长1425 bp,共编码474个氨基酸,蛋白质分子式为C_(2340)H_(3608)N_(614)O_(721)S_(24),分子量为52.65 kD,α-螺旋为其氨基酸序列的主要二级结构且该蛋白不含信号肽和跨膜区域,是一种非分泌型蛋白。对LtuHMGS蛋白的保守结构域预测分析发现其氨基酸序列含有羟甲基戊二酰辅酶A合酶保守结构域,亚细胞定位表明LtuHMGS蛋白可能更多地存在于细胞质膜中。同源序列比对和系统进化树分析表明,北美鹅掌楸LtuHMGS蛋白序列与其他物种的序列高度相似且与樟科的沉水樟和山鸡椒的HMGS蛋白的进化关系更近。RT-qPCR结果发现,LtuHMGS基因在北美鹅掌楸雌蕊中的表达量远高于其他组织。【结论】从北美鹅掌楸中克隆得到1条编码羟甲基戊二酰辅酶A合酶的候选基因(LtuHMGS),该基因可能参与北美鹅掌楸萜类物质的合成,是研究北美鹅掌楸萜类合成机制的关键酶基因之一。

【Objective】The aim of this work was to study the structural characteristics,subcellular localization and expression patterns of LtuHMGS in Liriodendron tulipifera,in order to provide a theoretical basis for exploring the synthesis pathway of terpenoids in L.tulipifera at the molecular level.【Method】A novel HMGS homolog was identified from the L.tulipifera transcriptome database and named Ltu HMGS.The full-length cDNA was then cloned using RACE technology.Subsequently,the research predicted its physicochemical properties and conducted phylogenetic analysis via bioinformatics approaches.Further,subcellular localization of the encoding protein of the candidate Ltu HMGS gene was analyzed using tobacco transient transformation system,and the expression profile was characterized by RT-q PCR.【Result】The full-length ORF of Ltu HMGS gene is 1425 bp,encoding 474 amino acids in total.The molecular formula of its encoding protein is C_(2340)H_(3608)N_(614)O_(721)S_(24),with a molecular weight of 52.65 kD.Theα-helix is the main secondary structure of its amino acid sequence.The novel LtuHMGS does not contain any signal peptide or transmembrane region,suggesting that it is a non-secreted protein.Prediction of the conserved domains of the novel LtuHMGS protein suggested a 3-hydroxy-3-methylglutary-CoA synthase conserved domain,and the subcellular localization indicated that most LtuHMGS proteins may exist in cytoplasmic membrane.Homologous alignment and phylogenetic tree analysis showed that the LtuHMGS protein was highly similar to the sequences of other species and had a closer evolutionary relationship with HMGS proteins of Cinnamomum micranthum and Litsea cubeba from Lauraceae.The results of RTq PCR indicated that the Ltu HMGS transcripts accumulate mostly in the pistil rather than other tissues.【Conclusion】A candidate gene encoding 3-hydroxy-3-methylglutary-CoA synthase(Ltu HMGS)was cloned from L.tulipifera.This gene may be involved in the synthesis of terpenoids,which is one of the key genes in the study of terpenoids synthesis mechanism in L.tulipifera.