Biotechnology Bulletin ›› 2024, Vol. 40 ›› Issue (8): 174-185.doi: 10.13560/j.cnki.biotech.bull.1985.2024-0149

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Cloning and Functional Analysis of bHLH Gene Related to Anthocyanin Synthesis in Paeonia qiui

LI Yu-qing(), WU Nan, LUO Jian-rang()   

  1. College of Landscape Architecture and Arts, Northwest A & F University, Yangling 712100
  • Received:2024-02-07 Online:2024-08-26 Published:2024-06-26
  • Contact: LUO Jian-rang E-mail:liyuqing@nwafu.edu.cn;luojianrang@nwafu.edu.cn

Abstract:

【Objective】Using Paeonia qiui as the experimental material, the regulatory mechanism of PqbHLH1 gene on anthocyanin synthesis during leaf development was studied.【Method】Based on the transcriptome data obtained in the early stage, primers were designed and the bHLH1 gene was cloned from the leaves of Paeonia qiui using PCR technology. Bioinformatics methods were used to analyze the physicochemical properties, hydrophobicity, theoretical isoelectric points, protein secondary structure, and evolutionary relationships of the PqbHLH1 gene. Fluorescence quantitative PCR technology was used to detect its expressions in different tissues and stages. Agrobacterium mediated method was employed to overexpress it in Arabidopsis thaliana and detect the effect of overexpressing PqbHLH1 on the anthocyanins of peony leaves.【Result】The total length of the coding region of PqbHLH1 gene was 1 920 bp, encoding 639 amino acids. The PqbHLH1 protein was hydrophilic and does not contain signal peptides or transmembrane structures, and was localized in the nucleus. Phylogenetic analysis showed that the PqbHLH1 protein in peony ovale had the closest genetic relationship with VvMYCA1 in grapes. Fluorescence quantitative PCR analysis showed that the expression of PqbHLH1 gene in peony leaves had a trend of first decreasing and then increasing, and the expression was significant in the S6 stage of leaves. The expressions of anthocyanin synthesis structural genes PqCHS, PqF3'H, PqDFR, and PqANS showed a trend of first increasing and then decreasing with leaf development. In A. thaliana strains overexpressing the PqbHLH1 gene, the expressions of structural genes related to anthocyanin synthesis was significantly higher than that of wild-type Arabidopsis, and the expression trend of anthocyanin content was consistent with that of structural genes AtCHI, AtF3'H, and AtUFGT, while the expression trend of flavonol content was consistent with that of structural gene AtFLS.【Conclusion】The PqbHLH1 gene positively regulates the synthesis of anthocyanins and flavonols in peony leaves by regulating the expressions of genes related to anthocyanin synthesis.

Key words: Paeonia qiui, bHLH transcription factor, anthocyanin, expression analysis