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Outline
Cinnamic acid 4-hydroxylase (C4H) is a key rate-limiting enzyme in the plant phenylpropanoid metabolic pathway, which regulates the biosynthesis of flavonoids, lignins and phenolic acids, and plays essential roles in plant growth, development and stress adaptation. Euphorbia maculata is a traditional medicinal plant abundant in bioactive secondary metabolites. To date, the molecular features and catalytic function of C4H genes in E. maculata remain poorly characterized, limiting the understanding of its secondary metabolic regulatory mechanism. Therefore, this study was performed to clone the EmC4H2 gene and its promoter sequence, analyze its structural characteristics and spatiotemporal expression profiles, and validate its in vitro catalytic activity, aiming to clarify the biological function of EmC4H2 in phenylpropanoid metabolism of E. maculata.
Based on transcriptome and genomic data, the ORF and promoter sequences of EmC4H2 were cloned and identified. Bioinformatic tools were applied to analyze the protein's physicochemical features, conserved domains and phylogenetic relationships, and further conducted three-dimensional structure modeling and molecular docking against the substrate. The spatiotemporal expression pattern of EmC4H2 in different tissues and developmental stages of E. maculata was detected via RT-qPCR. Recombinant expression of EmC4H2 protein was induced by Escherichia coli, and the enzyme activity was detected in vitro.
The ORF and promoter sequences of EmC4H2 were 1 518 bp and 1 696 bp in length, respectively. The EmC4H2 protein contains 505 amino acids and is subcellular localized in the cytoplasm. It contains a conserved domain of the cytochrome P450 (CYP450) family and shares the highest similarity with the C4H protein from Euphorbia lathyris, with a similarity of 91.29%. The molecular docking of EmC4H2 with the substrate trans-cinnamic acid shows high affinity, with a binding energy (ΔG) of −5.98 kcal·mol−1. The expression of EmC4H2 had spatiotemporal specificity, and the expressed recombinant protein has a molecular weight of about 75 kDa. It has the activity of catalyzing the generation of 4-coumaric acid from trans-cinnamic acid in vitro, with an enzyme activity of 327 U·g−1.
This work systematically clarifies the sequence traits, phylogenetic topology, tissue expression patterns and catalytic properties of EmC4H2. Combined data confirm that EmC4H2 acts as an active catalytic enzyme involved in the phenylpropanoid metabolism of E. maculata. The study constructs a robust theoretical basis for exploring the molecular regulatory network of secondary metabolite synthesis and supplies excellent genetic resources for quality breeding of medicinal E. maculata.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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