Volume 18, Issue 1 (3-2026)
J Crop Breed 2026, 18(1): 149-162 |
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Aghili H A, Ghasemnezhad A, Moradi H, Ghasemi Omran V. (2026). Bioinformatics Analysis of Flavanone-3՛-hydroxylase (F3՜H) in Silybum marianum. J Crop Breed. 18(1), 149-162. doi:10.61882/jcb.2026.1582
URL: http://jcb.sanru.ac.ir/article-1-1582-en.html
URL: http://jcb.sanru.ac.ir/article-1-1582-en.html
1- Horticulture Science, Plant Production, Gorgan University of Agricultural Science and Natural Resources, Gorgan, Iran
2- Horticulture Science, Crop Science, Sari University of Agricultural Science and Natural Resources, Sari, Iran
3- Crop Science Genetic & Agricultureal Biotecnology Institute of Tabarestan, Sari University Agricultural Science and Natural Resources, Sari, Iran
2- Horticulture Science, Crop Science, Sari University of Agricultural Science and Natural Resources, Sari, Iran
3- Crop Science Genetic & Agricultureal Biotecnology Institute of Tabarestan, Sari University Agricultural Science and Natural Resources, Sari, Iran
Abstract: (191 Views)
Extended Abstract
Background: Medicinal plants play a crucial role in human nutrition and the treatment of numerous diseases due to their ability to produce secondary metabolites with therapeutic properties. Silybum marianum L., a medicinal plant belonging to the Asteraceae family, contains silymarin as its principal bioactive compound, which is widely used in the treatment of liver-related disorders. A comprehensive understanding of the molecular pathways and the genes involved in the biosynthesis of this compound is essential for its improvement and enhanced production. Accordingly, functional analysis of the genes and proteins participating in the silymarin biosynthetic pathway holds significant importance. Flavonoid 3'-hydroxylase (F3'H) is one of the key enzymes involved in this pathway. Among the various approaches available for gene and protein function analysis, bioinformatics methods offer a powerful and efficient means of investigation. Therefore, a bioinformatics analysis of the nucleotide and protein sequences of the flavonoid 3'-hydroxylase (F3'H) gene was conducted in the present study.
Methods: A previously published article reporting the nucleotide sequence of this gene was utilized for the bioinformatics investigation of the nucleotide sequence of the flavonoid3-hydroxylase (F3'H) gene in the milk thistle plant (S. marianum). The mRNA sequence was retrieved from the NCBI database with the accession number KP861882.1. The nucleotide sequence analysis was performed using the ppuigbo computational database. Additionally, the Plantcare website was employed to analyze the promoter of the gene of interest. The protein sequence of the gene of interest was obtained from the NCBI database under the accession number ALA39990.1, and information regarding the flavonoid3-hydroxylase protein from the milk thistle plant was examined through the Uniprot database with the accession number A0A0K2F2U. Furthermore, the protein domain was identified using the NCBI and InterPro databases. The physicochemical properties of the protein were analyzed using the ProtParam database. Motifs were identified from the meme database, and the functionality of these motifs was assessed through the elm database. The modeling of the secondary and tertiary structures of the F3'H protein was conducted using the phyre2 database, and the accessibility of the F3'H protein bases to solvents was predicted with the I-TASSER server. Finally, the quality of the stereochemical structure of the modeled F3'H protein was evaluated with the PROCHECK software using the Ramachandran plot.
Results: The investigations carried out in the Materials and Methods section indicate that this gene consists of 1557 nucleotides, with a C-G content of 51.87%. From the perspective of mRNA, it comprises1317 base pairs that encode a protein with an amino acid length of 349. In the promoter sequence of the F3'H gene, 19 regulatory elements were predicted, with the TATA-box and CAAT-box being the most prominent regulatory elements in the promoter region of this gene. Additionally, this gene contains several light-responsive regulatory elements. The physicochemical properties obtained for this protein, which has 349 amino acids and a molecular weight of 48.320 kDa, indicated an aliphatic index of 32.99 and a predicted isoelectric point of 7.87, with a GRAVY index of -0.059. It was determined that there are 49 negatively charged amino acids (aspartic acid and glutamic acid) and 50 positively charged amino acids (arginine and lysine). Furthermore, the extinction coefficient of this protein was found to be 80,330 at 280 nm in an aqueous environment, with an instability index of 34.65. Evaluations also revealed that this protein contains one domain from the P450 superfamily and four identifiable motifs. The structure of this protein consists of 45% alpha helices, 5% beta sheets, and 4% membrane helices, and the corresponding ligand-binding site was predicted. The analysis of the Ramachandran plot for the predicted model indicated that 87.5% of the amino acids fell within the favored region, 11.7% in the allowed region, 0.3% in the nearly allowed region, and 0.5% in the disallowed region. Moreover, the highest density of permissible points corresponds to right-handed alpha helices and beta sheets. The combined favored and allowed regions amounted to 99.2, thus rendering the model presented for the F3'H protein of milk thistle acceptable in this study.
Conclusion: In the bioinformatics study of the flavonoid3-hydroxylase gene, regulatory elements related to light response, abscisic acid response, cold temperature response, zein metabolism, and flavonoid biosynthesis pathways were identified in the promoter of this gene. Additionally, the bioinformatics analysis of the flavonoid3-hydroxylase protein demonstrates that this protein is positioned among relatively stable proteins. Furthermore, a domain of the P450 superfamily was identified in this protein, and the predicted model of the protein, with 45% alpha helices, 5% beta sheets, 4% membrane helices, and a corresponding ligand-binding site, also exhibits a favorable conformation. Considering the importance of silymarin in the treatment of liver diseases, improving and enhancing the production of this vital bioactive compound can lead to the advancement of pharmaceutical product quality and increased efficiency in medicinal plant agriculture. Therefore, the findings of this study have significant practical applications in biotechnology and the genetic improvement of S. marianum aimed at increasing silymarin content.
Background: Medicinal plants play a crucial role in human nutrition and the treatment of numerous diseases due to their ability to produce secondary metabolites with therapeutic properties. Silybum marianum L., a medicinal plant belonging to the Asteraceae family, contains silymarin as its principal bioactive compound, which is widely used in the treatment of liver-related disorders. A comprehensive understanding of the molecular pathways and the genes involved in the biosynthesis of this compound is essential for its improvement and enhanced production. Accordingly, functional analysis of the genes and proteins participating in the silymarin biosynthetic pathway holds significant importance. Flavonoid 3'-hydroxylase (F3'H) is one of the key enzymes involved in this pathway. Among the various approaches available for gene and protein function analysis, bioinformatics methods offer a powerful and efficient means of investigation. Therefore, a bioinformatics analysis of the nucleotide and protein sequences of the flavonoid 3'-hydroxylase (F3'H) gene was conducted in the present study.
Methods: A previously published article reporting the nucleotide sequence of this gene was utilized for the bioinformatics investigation of the nucleotide sequence of the flavonoid3-hydroxylase (F3'H) gene in the milk thistle plant (S. marianum). The mRNA sequence was retrieved from the NCBI database with the accession number KP861882.1. The nucleotide sequence analysis was performed using the ppuigbo computational database. Additionally, the Plantcare website was employed to analyze the promoter of the gene of interest. The protein sequence of the gene of interest was obtained from the NCBI database under the accession number ALA39990.1, and information regarding the flavonoid3-hydroxylase protein from the milk thistle plant was examined through the Uniprot database with the accession number A0A0K2F2U. Furthermore, the protein domain was identified using the NCBI and InterPro databases. The physicochemical properties of the protein were analyzed using the ProtParam database. Motifs were identified from the meme database, and the functionality of these motifs was assessed through the elm database. The modeling of the secondary and tertiary structures of the F3'H protein was conducted using the phyre2 database, and the accessibility of the F3'H protein bases to solvents was predicted with the I-TASSER server. Finally, the quality of the stereochemical structure of the modeled F3'H protein was evaluated with the PROCHECK software using the Ramachandran plot.
Results: The investigations carried out in the Materials and Methods section indicate that this gene consists of 1557 nucleotides, with a C-G content of 51.87%. From the perspective of mRNA, it comprises1317 base pairs that encode a protein with an amino acid length of 349. In the promoter sequence of the F3'H gene, 19 regulatory elements were predicted, with the TATA-box and CAAT-box being the most prominent regulatory elements in the promoter region of this gene. Additionally, this gene contains several light-responsive regulatory elements. The physicochemical properties obtained for this protein, which has 349 amino acids and a molecular weight of 48.320 kDa, indicated an aliphatic index of 32.99 and a predicted isoelectric point of 7.87, with a GRAVY index of -0.059. It was determined that there are 49 negatively charged amino acids (aspartic acid and glutamic acid) and 50 positively charged amino acids (arginine and lysine). Furthermore, the extinction coefficient of this protein was found to be 80,330 at 280 nm in an aqueous environment, with an instability index of 34.65. Evaluations also revealed that this protein contains one domain from the P450 superfamily and four identifiable motifs. The structure of this protein consists of 45% alpha helices, 5% beta sheets, and 4% membrane helices, and the corresponding ligand-binding site was predicted. The analysis of the Ramachandran plot for the predicted model indicated that 87.5% of the amino acids fell within the favored region, 11.7% in the allowed region, 0.3% in the nearly allowed region, and 0.5% in the disallowed region. Moreover, the highest density of permissible points corresponds to right-handed alpha helices and beta sheets. The combined favored and allowed regions amounted to 99.2, thus rendering the model presented for the F3'H protein of milk thistle acceptable in this study.
Conclusion: In the bioinformatics study of the flavonoid3-hydroxylase gene, regulatory elements related to light response, abscisic acid response, cold temperature response, zein metabolism, and flavonoid biosynthesis pathways were identified in the promoter of this gene. Additionally, the bioinformatics analysis of the flavonoid3-hydroxylase protein demonstrates that this protein is positioned among relatively stable proteins. Furthermore, a domain of the P450 superfamily was identified in this protein, and the predicted model of the protein, with 45% alpha helices, 5% beta sheets, 4% membrane helices, and a corresponding ligand-binding site, also exhibits a favorable conformation. Considering the importance of silymarin in the treatment of liver diseases, improving and enhancing the production of this vital bioactive compound can lead to the advancement of pharmaceutical product quality and increased efficiency in medicinal plant agriculture. Therefore, the findings of this study have significant practical applications in biotechnology and the genetic improvement of S. marianum aimed at increasing silymarin content.
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