Biotechnology Bulletin ›› 2023, Vol. 39 ›› Issue (4): 148-156.doi: 10.13560/j.cnki.biotech.bull.1985.2022-1130

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Cloning and Analysis of Chalcone Synthase Gene and Its Promoter from Euphorbia maculata

GUO San-bao1(), SONG Mei-ling2, LI Ling-xin3, YAO Zi-zhao3, GUI Ming-ming2, HUANG Sheng-he1()   

  1. 1. Department of Pharmacy, Jiangxi College of Traditional Chinese Medicine, Fuzhou 344000
    2. Department of Basic Medicine, Jiangxi College of Traditional Chinese Medicine, Fuzhou 344000
    3. Fuzhou Medical College, Nanchang University, Fuzhou 344000
  • Received:2022-09-15 Online:2023-04-26 Published:2023-05-16

Abstract:

Quercetin is one of the main medicinal ingredients of Euphorbia maculata, and chalcone synthase(CHS)is one of key enzymes in the biosynthesis of quercetin. To explore the mechanism of quercetin biosynthesis in E. maculata, a CHS gene named EmCHS(GenBank access number: ON652865)was cloned from E. maculatabased on transcriptome sequencing results, combined with technologies of 3' RACE and Tail-PCR. The amino acid sequence was aligned, and physicochemical properties, transmembrane region, subcellular localization and phylogenetic relationships of EmCHS protein were analyzed. And the expressions of EmCHS in different tissues during different growth stages were detected with RT-qPCR. The results showed that the ORF of EmCHS gene was 1 194 bp, encoding 397 amino acids. The EmCHS protein was located in the cytoplasm. The theoretical isoelectric point of the protein was 5.96 and the theoretical molecular weight was 43.48 kD, and it did not contain a transmembrane region and was a hydrophilic protein with stable structure. The results of amino acid sequence alignment and phylogenetic relationships analysis revealed that EmCHS had the highest similarity(91.92%)with the amino acid sequence of Manihot esculenta in Euphorbiaceae family, which was consistent with the characteristics of plant taxonomy. The analysis of RT-qPCR confirmed that EmCHS gene was expressed in different tissues during different growth stages, and there were significant differences. The expression of EmCHS gene was the lowest in the leaf at the reproduction stage, while the highest in the root at the reproduction stage. Besides, the promoter(GenBank access number: OP626754)length of EmCHS gene cloned was 971 bp, including TATA-box, CAAT-box, MYB and MYC transcription factor binding sites, multiple light response and hormone response cis-acting elements, and so on. These results will provide a foundation for further research on gene function and expression regulation of EmCHS gene.

Key words: Euphorbia maculata, CHS, promoter, cloning, tail-PCR, bioinformatics, gene expression