Cloning of the LkF3H2 Gene in Larix kaempferi and Its Function in Regulating Flavonoid Metabolism

doi:10.13560/j.cnki.biotech.bull.1985.2022-1508

Abstract

Abstract:

【Objective】 The Larix kaempferi flavanone 3-hydroxylase 2（LkF3H2）gene in Larix kaempferi plays a crucial role in flavonoid biosynthesis. This study is aimed to investigate the function of the LkF3H2 in L. kaempferi and plant flavonoid biometabolism. 【Method】 The LkF3H2 gene of L. kaempferi was cloned based on preliminary transcriptome data, and bioinformatics analysis and tissue expression analysis were performed. The gene was then stably transformed into tobacco to examine the relationship between gene expression and flavonoid content using tissue expression analysis and flavonoid content determination. 【Result】 The cDNA sequence length of LkF3H2 gene is 1 074 bp, and its protein encodes 358 amino acids with a molecular formula of C₁₇₈₅H₂₈₀₇N₄₈₁O₅₄₁S₁₈ and a molecular weight of 40.24 kD, indicating that it is an unstable hydrophilic protein lacking a signal peptide. Moreover, phylogenetic analysis revealed that LkF3H2 is closely related to the F3H genes of Pinus taeda, Pinus radiata, Picea abies, and Picea glauca. In addition, the LkF3H2 gene had the highest expression in the leaves of one-month-old L. kaempferi seedlings and the stems of one-year L. kaempferi seedlings, as determined by tissue expression analysis. Moreover, it had the highest expression in the transgenic tobacco leaves; notably, transgenic tobacco leaves had the highest flavonoid content, followed by stems and roots, indicating a positive relationship between flavonoid content and LkF3H2 gene expression in transgenic tobacco. 【Conclusion】 The LkF3H2 gene of L. kaempfer belongs to the 2-oxoglutarate-dependent dioxygenase family and is pivotal in flavonoid biosynthesis.

Key words: flavanone 3-hydroxylase gene, transgenic tobacco, tissue expression, flavonoids

LI Can, JIANG Xiang-ning, GAI Ying. Cloning of the LkF3H2 Gene in Larix kaempferi and Its Function in Regulating Flavonoid Metabolism[J]. Biotechnology Bulletin, 2024, 40(2): 245-252.

Figures/Tables 7

Fig. 1 Cloning of LkF3H2 gene（Marker: DL2000）

Fig. 2 Amino acid sequence alignment of LkF3H2 protein

Fig. 3 LkF3H2 protein secondary and tertiary structure prediction, conserved domain prediction A: Secondary structure prediction. B and C: Tertiary structure prediction. D: Conserved domain prediction

Fig. 4 Construction of phylogenetic tree

Fig. 5 Expressions of LkF3H2 gene in different tissues in Larix kaempferi Lk-1: One-month seedling. Lk-2: One-year seedling. The data are analyzed with Tukey’s multiple comparisons test and shown as the mean ± SE of three independent replicates. * and ** indicate significant differences at 0.05 and 0.01 levels

Fig. 6 Growth and development stages of transgenic tobacco A: Pre-culture. B: Regeneration culture. C: Rooting culture. D: Transgenic tobacco. E: PCR identification result

Fig. 7 Expression of the LkF3H2 gene and flavonoid content in transgenic tobacco A: Expression of the LkF3H2 gene. B: Flavonoid content of transgenic tobacco. C: Relationship between the expression of the LkF3H2 gene and flavonoid content. The data are analyzed with Tukey’s multiple comparisons test and shown as the mean ± SE of three independent replicates. * and ** indicate significant differences at 0.05 and 0.01 levels

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