日本落叶松LkF3H2基因克隆及调控类黄酮代谢功能研究

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

摘要/Abstract

摘要：

【目的】日本落叶松黄烷酮3-羟化酶（flavanone 3-hydroxylase 2，LkF3H2）基因在类黄酮生物合成过程中发挥着重要的调控功能。探究LkF3H2基因在日本落叶松中发挥的具体功能和植物类黄酮生物代谢过程。【方法】根据实验室前期转录组数据克隆日本落叶松LkF3H2基因并进行生物信息学分析及组织表达分析。随后将该基因转化烟草进行转基因烟草组织表达分析及类黄酮含量测定，以探究基因表达量与类黄酮含量之间的关系。【结果】LkF3H2基因cDNA序列长度为1 074 bp，其蛋白编码358个氨基酸，分子式C₁₇₈₅H₂₈₀₇N₄₈₁O₅₄₁S₁₈，分子量为40.24 kD，该蛋白是不稳定的亲水性蛋白且不含信号肽；同时系统进化分析得出日本落叶松LkF3H2与火炬松、辐射松、白云杉和欧洲云杉F3H亲缘关系较近并且该蛋白含有保守结构域2OG-Fe（II）oxygenase superfamily，属于2-酮戊二酸依赖性双加氧家族。另外组织表达分析显示LkF3H2基因在一月龄日本落叶松幼苗叶中表达量最高，在一年生日本落叶松幼苗茎中表达量最高，并且在转基因烟草叶中也显示了最高的表达量。值得一提的是，转基因烟草叶中的类黄酮含量也是最高的，其次为茎和根，转基因烟草中类黄酮含量与LkF3H2基因表达量呈正相关关系。【结论】日本落叶松LkF3H2基因属于2-酮戊二酸依赖性双加氧家族并且在类黄酮生物合成过程中发挥着重要功能。

关键词: 黄烷酮3-羟化酶基因, 转基因烟草, 组织表达, 类黄酮

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

李灿, 蒋湘宁, 盖颖. 日本落叶松LkF3H2基因克隆及调控类黄酮代谢功能研究[J]. 生物技术通报, 2024, 40(2): 245-252.

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.

图/表 7

图1 LkF3H2基因克隆（Marker: DL2000）

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

图2 LkF3H2蛋白氨基酸序列比对

Fig. 2 Amino acid sequence alignment of LkF3H2 protein

图3 LkF3H2蛋白二三级结构预测、保守结构域预测 A：二级结构预测；B和C：三级结构预测；D：保守结构域预测

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

图4 系统进化树构建

Fig. 4 Construction of phylogenetic tree

图5 LkF3H2基因在日本落叶松中不同组织部位表达量变化 Lk-1：一月龄幼苗；Lk-2：一年生幼苗。图中数据采用Tukey多重比较检验进行分析，并以3个独立重复的±SE表示。* 表示在0.05水平上差异显著，** 表示在0.01水平上差异显著，*** 表示在0.001水平上差异显著

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

图6 转基因烟草生长发育各阶段 A：预培养；B：再生培养；C：生根培养；D：转基因烟草；E：PCR鉴定结果

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

图7 转基因烟草中LkF3H2基因表达量与类黄酮含量 A：LkF3H2基因表达量；B：转基因烟草类黄酮含量；C：LkF3H2基因表达量与类黄酮含量之间关系。数据采用Tukey多重比较检验进行分析，并以3个独立重复的±SE表示。* 表示在0.05水平上差异显著，** 表示在0.01水平上差异显著，*** 表示在0.001水平上差异显著

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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