斑地锦黄酮醇合成酶基因(EmFLS)及其启动子克隆及表达分析

Cloning and expression analysis of EmFLS gene and its promoter in Euphorbia maculata

  • 摘要: 【目的】克隆斑地锦黄酮醇合成酶基因(EmFLS)及其启动子序列,并检测EmFLS基因在斑地锦不同生长期不同组织中的表达模式,为后续研究该基因对黄酮醇化合物生物合成的调控机制提供理论参考。【方法】以斑地锦为材料,采用cDNA末端快速扩增(RACE)技术和热不对称交互式PCR(Tail-PCR)技术克隆EmFLS基因及启动子序列,对其进行生物信息学分析,并通过实时荧光定量PCR检测其在不同生长期不同组织中的表达模式。【结果】扩增获得EmFLS基因开放阅读框(ORF)(GenBank登录号ON821211.1),长度为1002 bp,编码333个氨基酸残基,蛋白相对分子质量为37.62 kD,定位于细胞质,为不稳定的亲水性蛋白,属于2OG-Fe (II)加氧酶超家族。EmFLS蛋白与其他13种植物FLS蛋白的氨基酸序列相似性为63.55%~70.51%,其中与木薯FLS蛋白的氨基酸序列相似性最高,且在系统发育进化树上处于一个独立的分支。EmFLS基因启动子约1800 bp,除含有真核生物启动子核心元件TATA-box和CAAT-box外,还含激素响应、胁迫响应和光响应等顺式作用元件。EmFLS基因在营养生长期和生殖生长期的根、茎、叶和果中均有表达,但在根和茎中的相对表达量显著高于叶和果(P<0.05,下同),斑地锦从营养生长期到生殖生长期EmFLS基因在根和茎中的相对表达量显著下降,而在叶中无显著变化(P>0.05)。【结论】 EmFLS基因表达具有组织特异性,且在根和茎中具有发育阶段特异性,推测EmFLS基因主要参与调控斑地锦根和茎中黄酮醇化合物的生物合成。

     

    Abstract: 【Objective】 In order to provide a theoretical reference for future research on the regulation of flavonol biosynthesis by the flavonol synthase gene in Euphorbia maculata(EmFLS),the EmFLS gene and promoter of E. maculata were cloned,and the expression patterns of the EmFLS gene in different tissues during different growth periods were detected.【Method】 Using E. maculata as the material,the EmFLS gene and promoter were cloned by rapid amplification of cDNA ends(RACE) technology and thermal asymmetric interlaced PCR(Tail-PCR) technology. The EmFLS gene was analyzed by bioinformatics analysis,and the expression patterns of EmFLS gene in different tissues during different growth periods were detected by real-time fluorescence quantitative PCR.【Result】 The open reading frame(ORF) of EmFLS gene(GenBank accession number ON821211.1) obtained by amplification was 1002 bp,encoding 333 amino acid residues with a protein relative molecular weight of 37.62 kD. EmFLS protein was located in the cytoplasm and was a hydrophilic protein with an unstable structure,belonging to the 2OG-Fe(II) oxygenase superfamily. The amino acid sequence similarity of EmFLS protein and 13 other plant FLS proteins was 63.55%-70.51%. EmFLS protein had the highest similarity with the amino acid sequence of Manihot esculenta. Phylogenetic tree analysis showed that it was a relatively independent branch. The EmFLS promoter was about 1800 bp,which had the core elements of eukaryotic promoter TATA-box and CAAT-box,and also contained cis acting elements related to hormone,stress,and light response. EmFLS gene was expressed in the root,stem,leaf and fruit during vegetative growth stage and reproductive growth stage,but the relative expression in the root and stem was significantly higher than that in leaf and fruit(P<0.05,the same below). And the relative expression of EmFLS gene in the root and stem decreased significantly from the vegetative growth stage to the reproductive growth stage,but there was no significant difference in the leaf(P>0.05).【Conclusion】 The expression of EmFLS gene has tissue specificity,and has developmental stage specificity in the root and stem. It is speculated that EmFLS is mainly involved in regulating the biosynthesis of flavonols in the root and stem of E. maculata.

     

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