| چکیده انگلیسی مقاله |
Extended Abstract
Introduction and Objective: 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. Also, there is little information about the genetics of secondary metabolism in medicinal plants, so 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 is 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 in better understanding the evolution of species. Also, 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 aim of the present study was 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.
Material and Methods: since the sequences of C4H, HCT, and C3'H genes were not identified in S. arvensis, their sequences were recovered from plants that were 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 and conserved regions were identified. 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 extraction from leaf samples was performed by CTAB method and amplification of the studied gene fragments in S. arvensis was performed 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 for one to two hours and after staining with ethidium bromide, the 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 of 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 to verify the accuracy of the sequenced fragments, alignment (BLAST) of the sequenced fragments against DNA, RNA and protein databases was performed. Then, phylogenetic relationships and the frequency of nucleotide mutations in these genes were examined and analyzed. MEGA5 and BLAST softwares were used to examine phylogenetic relationships (maximum likelihood method) and analyze nucleotide sequences (including frequency and substitution). Clustal omega software was also used to examine the alignment of nucleotide and protein sequences. Amino acid analysis (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 part of the sequence of the coding region of C4H, C3'H and HCT genes was identified in Sonchus arvensis and registered in GenBank with accession numbers ON014597.1, ON014595.1, 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. Also, 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 the nucleotide and protein levels showed a high similarity to the evolutionarily close plants such as L. sattiva.
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 indicates the 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 level showed the highest percentage of similarity (lowest genetic distance) with plants such as L. sativa and Cichorium intybus, which confirmed the accuracy of the sequence results. Also, phylogenetic analysis based on the sequencing of C4H, C3'H, and HCT genes showed a close phylogenetic relationship between S. arvensis and L. sativa.
|