Volume 17, Issue 2 (5-2025)
J Crop Breed 2025, 17(2): 82-93 |
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Azizyan R, Abdollahi Mandoulakani B. (2025). Phylogeny Analysis of Some Genes Involved in the Chlorogenic Acid Biosynthesis Pathway in the Medicinal Plant Sonchus arvensis L.. J Crop Breed. 17(2), 82-93. doi:10.61882/jcb.2024.1578
URL: http://jcb.sanru.ac.ir/article-1-1578-en.html
URL: http://jcb.sanru.ac.ir/article-1-1578-en.html
1- Department of Plant Production and Genetics, Faculty of Agriculture, Urmia University, Urmia, Iran
2- Department of Agricultural Biotechnology, Institute of Biotechnology, Urmia University, Urmia, Iran
2- Department of Agricultural Biotechnology, Institute of Biotechnology, Urmia University, Urmia, Iran
Abstract: (732 Views)
Extended Abstract
Background: Sonchus arvensis L. is a medicinal plant from the Asteraceae family. The valuable medicinal properties of this plant are due to the presence of important and valuable phenolic acids, such as chlorogenic acid. Reducing the risk of various diseases, such as heart disease, diabetes, and cancer, following the consumption of chlorogenic acid has been proven in clinical and research studies. Therefore, more research in the field of medicine and pharmacy on this plant will be considered by researchers in the future. Moreover, there is little information about the genetics of secondary metabolism in medicinal plants. Thus, the genes of many secondary metabolite biosynthesis pathways have not been identified, and little information exists about their regulation and function. Therefore, identifying biosynthetic pathways, knowing how the genes involved in these pathways are regulated, and analyzing their phylogeny are particularly important. On the other hand, examining the phylogenetic relationships of genes in different plant species and determining the evolutionary relationships between them can help better understand the evolution of species. Furthermore, knowledge of the nucleotide sequence of genes and the frequency of different nucleotides is essential for estimating the divergence time of species and reconstructing the evolutionary relationships between different species. The present study aimed to sequence part of the coding region of some genes involved in the chlorogenic acid biosynthetic pathway, such as cinnamate 4-hydroxylase (C4H), P-coumarol-ester 3'-hydroxylase (C3'H), and hydroxycinnamoyl coashimate/quinate hydroxycinnamoyl transferase (HCT), and to investigate their evolutionary and phylogenetic relationships in S. arvensis.
Methods: Since the sequences of C4H, HCT, and C3'H genes were not identified in S. arvensis, their sequences were recovered from plants that are evolutionarily close to S. arvensis, such as Cynara cardunculus var. scolymus and Cichorium. intybus. First, the sequences of these genes in plants phylogenetically close to S. arvensis were collected from GenBank and aligned using Clustal Omega software, followed by identifying conserved regions. Then, specific primers were designed based on the conserved regions using Fast PCR 4.0 and Gen Runner 3.05 software. In the next step, DNA from leaf samples was extracted by the CTAB method, and the studied gene fragments in S. arvensis were amplified using the designed specific primers and polymerase chain reaction (PCR). Electrophoresis of the resulting products was performed using a 1.5% agarose gel at a voltage of 80 V for one to two hours. After staining with ethidium bromide, a photograph was taken using a Gel Doc device (INFINITY, France). Given the specificity of the amplified products, the PCR products were sent directly to Microsynth Company (Switzerland) for sequencing part of the coding regions of the C4H, C3'H, and HCT genes. To ensure reliability, all PCR products were sequenced in both forward and reverse directions, and alignment (BLAST) of the sequenced fragments against DNA, RNA, and protein databases was performed to verify the accuracy of the sequenced fragments. Then, phylogenetic relationships and the frequency of nucleotide mutations were examined and analyzed in these genes. MEGA5 and BLAST software were used to examine phylogenetic relationships (the maximum likelihood method) and analyze nucleotide sequences (including frequency and substitution). Clustal Omega software was used to examine the alignment of nucleotide and protein sequences. Amino acid analysis (the percentage and weight of amino acids and inferred proteins) was performed with Bio Edit and MEGA5 software, and the Neutrality test was performed with MEGA5 software.
Results: For the first time in this research, a portion of the coding sequence of the C4H, C3'H, and HCT genes was identified in Sonchus arvensis and registered in GenBank with accession numbers ON014597.1, ON014595.1, and ON014596.1, respectively. The results of the analysis of nucleotide sequences showed that nucleotide frequency for C4H, C3'H, and HCT genes were the highest for adenine (28.1), guanine (28.4), and thymine (1.1), respectively, and the lowest nucleotide frequency belonged to thymine (13.2), thymine (20.1), and cytosine (19.7), respectively. Moreover, the neutrality test results showed the directional selection of these genes during evolution. The transition substitution mutation was more frequent than the transversion substitution mutation. The results of phylogenetic analysis based on C4H, HCT, and C3'H gene sequences showed a close phylogenetic relationship or a low genetic distance between S. arvensis and lettuce (L. sativa). The results of the sequence blast at both the nucleotide and protein levels showed a high similarity to evolutionarily close plants, such as L. sativa.
Conclusion: The results of this research positively evaluated the process of natural selection during evolution for the studied C4H, C3'H, and HCT genes, which indicates the effects of genetic drift or the balancing effects of population evolution throughout history, and a low difference between the frequencies of the polymorphisms. The BLAST results of sequenced C4H, HCT, and C3'H gene fragments in S. arvensis at the nucleotide and protein levels showed the highest percentage of similarity (lowest genetic distance) to plants such as L. sativa and Cichorium intybus, which confirmed the accuracy of the sequence results. Additionally, phylogenetic analysis based on the sequencing of C4H, C3'H, and HCT genes showed a close phylogenetic relationship between S. arvensis and L. sativa.
Background: Sonchus arvensis L. is a medicinal plant from the Asteraceae family. The valuable medicinal properties of this plant are due to the presence of important and valuable phenolic acids, such as chlorogenic acid. Reducing the risk of various diseases, such as heart disease, diabetes, and cancer, following the consumption of chlorogenic acid has been proven in clinical and research studies. Therefore, more research in the field of medicine and pharmacy on this plant will be considered by researchers in the future. Moreover, there is little information about the genetics of secondary metabolism in medicinal plants. Thus, the genes of many secondary metabolite biosynthesis pathways have not been identified, and little information exists about their regulation and function. Therefore, identifying biosynthetic pathways, knowing how the genes involved in these pathways are regulated, and analyzing their phylogeny are particularly important. On the other hand, examining the phylogenetic relationships of genes in different plant species and determining the evolutionary relationships between them can help better understand the evolution of species. Furthermore, knowledge of the nucleotide sequence of genes and the frequency of different nucleotides is essential for estimating the divergence time of species and reconstructing the evolutionary relationships between different species. The present study aimed to sequence part of the coding region of some genes involved in the chlorogenic acid biosynthetic pathway, such as cinnamate 4-hydroxylase (C4H), P-coumarol-ester 3'-hydroxylase (C3'H), and hydroxycinnamoyl coashimate/quinate hydroxycinnamoyl transferase (HCT), and to investigate their evolutionary and phylogenetic relationships in S. arvensis.
Methods: Since the sequences of C4H, HCT, and C3'H genes were not identified in S. arvensis, their sequences were recovered from plants that are evolutionarily close to S. arvensis, such as Cynara cardunculus var. scolymus and Cichorium. intybus. First, the sequences of these genes in plants phylogenetically close to S. arvensis were collected from GenBank and aligned using Clustal Omega software, followed by identifying conserved regions. Then, specific primers were designed based on the conserved regions using Fast PCR 4.0 and Gen Runner 3.05 software. In the next step, DNA from leaf samples was extracted by the CTAB method, and the studied gene fragments in S. arvensis were amplified using the designed specific primers and polymerase chain reaction (PCR). Electrophoresis of the resulting products was performed using a 1.5% agarose gel at a voltage of 80 V for one to two hours. After staining with ethidium bromide, a photograph was taken using a Gel Doc device (INFINITY, France). Given the specificity of the amplified products, the PCR products were sent directly to Microsynth Company (Switzerland) for sequencing part of the coding regions of the C4H, C3'H, and HCT genes. To ensure reliability, all PCR products were sequenced in both forward and reverse directions, and alignment (BLAST) of the sequenced fragments against DNA, RNA, and protein databases was performed to verify the accuracy of the sequenced fragments. Then, phylogenetic relationships and the frequency of nucleotide mutations were examined and analyzed in these genes. MEGA5 and BLAST software were used to examine phylogenetic relationships (the maximum likelihood method) and analyze nucleotide sequences (including frequency and substitution). Clustal Omega software was used to examine the alignment of nucleotide and protein sequences. Amino acid analysis (the percentage and weight of amino acids and inferred proteins) was performed with Bio Edit and MEGA5 software, and the Neutrality test was performed with MEGA5 software.
Results: For the first time in this research, a portion of the coding sequence of the C4H, C3'H, and HCT genes was identified in Sonchus arvensis and registered in GenBank with accession numbers ON014597.1, ON014595.1, and ON014596.1, respectively. The results of the analysis of nucleotide sequences showed that nucleotide frequency for C4H, C3'H, and HCT genes were the highest for adenine (28.1), guanine (28.4), and thymine (1.1), respectively, and the lowest nucleotide frequency belonged to thymine (13.2), thymine (20.1), and cytosine (19.7), respectively. Moreover, the neutrality test results showed the directional selection of these genes during evolution. The transition substitution mutation was more frequent than the transversion substitution mutation. The results of phylogenetic analysis based on C4H, HCT, and C3'H gene sequences showed a close phylogenetic relationship or a low genetic distance between S. arvensis and lettuce (L. sativa). The results of the sequence blast at both the nucleotide and protein levels showed a high similarity to evolutionarily close plants, such as L. sativa.
Conclusion: The results of this research positively evaluated the process of natural selection during evolution for the studied C4H, C3'H, and HCT genes, which indicates the effects of genetic drift or the balancing effects of population evolution throughout history, and a low difference between the frequencies of the polymorphisms. The BLAST results of sequenced C4H, HCT, and C3'H gene fragments in S. arvensis at the nucleotide and protein levels showed the highest percentage of similarity (lowest genetic distance) to plants such as L. sativa and Cichorium intybus, which confirmed the accuracy of the sequence results. Additionally, phylogenetic analysis based on the sequencing of C4H, C3'H, and HCT genes showed a close phylogenetic relationship between S. arvensis and L. sativa.
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