斑地锦3-脱氢奎尼酸脱水酶基因(EmDHQD)及其启动子克隆与表达分析OA

Objective:To clone the 3-dehydroquinate dehydratase(DHQD)gene EmDHQD and its promoter of Euphorbia maculata and analyze the expression patterns of this gene in different tissue samples at various growth stages.Methods:PCR was employed to clone EmDHQD and its promoter from E.maculata,followed by bioinformatics analysis.The expression patterns of EmDHQD in different tissue samples at different growth stages were examined by real-time quantitative PCR.Results:The open reading frame(ORF)of EmDHQD(GenBank accession No.PX984471)was amplified,with a length of 1 596 bp,encoding 531 amino acid residues.The predicted molecular mass of the protein was 57.34 kDa.The protein was predicted to localized to the mitochondria and belonged to the PLN02520 family.It was hypothesized that EmDHQD might be a soluble enzyme protein stably present in the cytoplasm.The amino acid sequence of EmDHQD shared 79.32%-93.22%similarity with those of DHQD proteins from 21 other plant species,with the highest similarity to that of the DHQD protein of Euphorbia lathyris.A promoter fragment of the EmDHQD gene,approximately 1 700 bp in length,was cloned.In addition to the core eukaryotic promoter elements(TATA-box and CAAT-box),the promoter harbored cis-acting elements responsive to plant hormones,defenses,stress,and low temperatures.EmDHQD was expressed in both stems and leaves during the reproductive growth stage.The relative expression level of EmDHQD in stems was higher than that in leaves on days 1,30,45,and 60 after the first flower opened(P<0.01),while on day 15 after the first flower opened,the relative expression in stems was lower than that in leaves(P<0.01).These results indicated that EmDHQD expression exhibited temporal and tissue specificity.Conclusion:The EmDHQD gene and its promoter are successfully cloned and preliminarily characterized,which provides a theoretical reference for subsequent research on the regulation of gallic acid biosynthesis by EmDHQD in E.maculata.