1- Gorgan University Agricultural Science and Natural Resources
2- Sari University Agricultural Science and Natural Resources
2- Sari University Agricultural Science and Natural Resources
Abstract: (6 Views)
Extended Abstract:
Introduction and Objective: Medicinal plants play a significant role in human nutrition and health due to the production of secondary metabolites with pharmacological effects. In many parts of the world, medicinal plants are widely used not only for curation but also for the prevention of diseases in the form of functional foods. Silybin and silymarin are valuable antioxidant compounds based on polyphenols, abundantly found in the seeds of the milk thistle plant (Silybum marianum L.). This plant belongs to the Asteraceae family and is well-known in medical and therapeutic circles for its importance in preparing liver-nutrition medicines. Considering the importance of secondary metabolites, particularly those that cannot be artificially produced in laboratories, understanding the biosynthetic pathways and factors involved in their biosynthesis is crucial for the potential artificial production of these compounds. Therefore, a precise understanding of the molecular pathways and genes involved in the production of these compounds holds particular significance. Consequently, analyzing the function of genes and proteins is of great importance. Flavonoid3-hydroxylase (F3'H) is one of the enzymes involved in the biosynthetic pathway of silymarin. One of the methods for analyzing the function of genes and proteins is through bioinformatics studies. This research focuses on the bioinformatics analysis of the nucleotide and protein sequences of the flavonoid3-hydroxylase (F3'H) gene.
Materials and Methods: For the bioinformatics investigation of the nucleotide sequence of the flavonoid3-hydroxylase (F3'H) gene in the milk thistle plant (S. marianum), a previously published article reporting the nucleotide sequence of this gene was utilized. 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, to analyze the promoter of the gene of interest, the Plantcare website was employed. The protein sequence of the gene of interest was obtained from the NCBI database under accession number ALA39990.1, and information regarding the flavonoid3-hydroxylase protein from the milk thistle plant was examined through the Uniprot database with 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.
Findings: The investigations carried out in the Materials and Methods section indicated that this gene consists of1557 nucleotides, with a C-G content of51.87%. From the perspective of mRNA, it comprises1317 base pairs that encode a protein with an amino acid length of349. 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 has349 amino acids and a molecular weight of48.320 kDa, indicated an aliphatic index of32.99 and a predicted isoelectric point of7.87, with a GRAVY index of -0.059. It was determined that there are49 negatively charged amino acids (aspartic acid and glutamic acid) and50 positively charged amino acids (arginine and lysine). Furthermore, the extinction coefficient of this protein was found to be80,330 at280 nm in an aqueous environment, with an instability index of34.65. Evaluations also revealed that this protein contains one domain from the P450 superfamily and four identifiable motifs. The structure of this protein consists of45% alpha helices,5% beta sheets, and4% membrane helices, and the corresponding ligand-binding site was predicted. The analysis of the Ramachandran plot for the predicted model indicated that87.5% of the amino acids fell within the favored region,11.7% in the allowed region,0.3% in the nearly allowed region, and0.5% 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 to99.2, 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 demonstrated 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, with45% alpha helices,5% beta sheets,4% membrane helices, and a corresponding ligand-binding site, also exhibits a favorable conformation.
Introduction and Objective: Medicinal plants play a significant role in human nutrition and health due to the production of secondary metabolites with pharmacological effects. In many parts of the world, medicinal plants are widely used not only for curation but also for the prevention of diseases in the form of functional foods. Silybin and silymarin are valuable antioxidant compounds based on polyphenols, abundantly found in the seeds of the milk thistle plant (Silybum marianum L.). This plant belongs to the Asteraceae family and is well-known in medical and therapeutic circles for its importance in preparing liver-nutrition medicines. Considering the importance of secondary metabolites, particularly those that cannot be artificially produced in laboratories, understanding the biosynthetic pathways and factors involved in their biosynthesis is crucial for the potential artificial production of these compounds. Therefore, a precise understanding of the molecular pathways and genes involved in the production of these compounds holds particular significance. Consequently, analyzing the function of genes and proteins is of great importance. Flavonoid3-hydroxylase (F3'H) is one of the enzymes involved in the biosynthetic pathway of silymarin. One of the methods for analyzing the function of genes and proteins is through bioinformatics studies. This research focuses on the bioinformatics analysis of the nucleotide and protein sequences of the flavonoid3-hydroxylase (F3'H) gene.
Materials and Methods: For the bioinformatics investigation of the nucleotide sequence of the flavonoid3-hydroxylase (F3'H) gene in the milk thistle plant (S. marianum), a previously published article reporting the nucleotide sequence of this gene was utilized. 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, to analyze the promoter of the gene of interest, the Plantcare website was employed. The protein sequence of the gene of interest was obtained from the NCBI database under accession number ALA39990.1, and information regarding the flavonoid3-hydroxylase protein from the milk thistle plant was examined through the Uniprot database with 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.
Findings: The investigations carried out in the Materials and Methods section indicated that this gene consists of1557 nucleotides, with a C-G content of51.87%. From the perspective of mRNA, it comprises1317 base pairs that encode a protein with an amino acid length of349. 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 has349 amino acids and a molecular weight of48.320 kDa, indicated an aliphatic index of32.99 and a predicted isoelectric point of7.87, with a GRAVY index of -0.059. It was determined that there are49 negatively charged amino acids (aspartic acid and glutamic acid) and50 positively charged amino acids (arginine and lysine). Furthermore, the extinction coefficient of this protein was found to be80,330 at280 nm in an aqueous environment, with an instability index of34.65. Evaluations also revealed that this protein contains one domain from the P450 superfamily and four identifiable motifs. The structure of this protein consists of45% alpha helices,5% beta sheets, and4% membrane helices, and the corresponding ligand-binding site was predicted. The analysis of the Ramachandran plot for the predicted model indicated that87.5% of the amino acids fell within the favored region,11.7% in the allowed region,0.3% in the nearly allowed region, and0.5% 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 to99.2, 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 demonstrated 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, with45% alpha helices,5% beta sheets,4% membrane helices, and a corresponding ligand-binding site, also exhibits a favorable conformation.
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