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

【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.