| چکیده انگلیسی مقاله |
Extended Abstract
Introduction and Objective: Artemisia absinthium, commonly known as Afsantin, is an important perennial medicinal herb native to Asia, the Middle East, Europe, and North Africa. During the evolutionary process, plants have developed mechanisms that not only allow them to survive under the most severe radiation doses but also respond effectively to small changes in radiation intensity through physiological adjustments. One of the plant defense mechanisms against radiation is evolutionary changes in gene expression levels. Among these genes, farnesyl diphosphate synthase (FDS) is a key enzyme in the terpenoid metabolic pathway that catalyzes the synthesis of sesquiterpenoid compounds from the farnesyl pyrophosphate (FPP) precursor and plays an important role in regulating plant growth and development. The present study investigated the effects of radiation from radioactive waste in an abandoned uranium mine on the expression changes of the farnesyl diphosphate synthase (FDS) and squalene synthase (SQS) genes in the A. absinthium, which had grown for many years in the radioactive sampling site environment.
Material and Methods: Plant material sampling was carried out from the altitudes of Kojanaq village located 18 km northwest of Meshginshahr city with the geographical coordinates 38°29'17.7"N, 47°30'15.1"E. All calculations related to geographical position were performed using a Garmin satellite positioning device (Oregon model, 650). For complete access to radiation contamination information in the study area, a point-by-point radon radiation map was prepared at 10 points using a radiation meter over two consecutive years with a Victoreen 451 radiation meter (Fluke Biomedical Company, USA). These measurements included points at the same altitude on two opposite mountains (mountain with radioactive material points A and non-radioactive B). Sampling of A. absinthium shoots was carried out systematically and in three different biological replicates. Total RNA was extracted from the sampled plant leaves, and the first cDNA strand was synthesized. The Primer 3 Plus online software was used to design qPCR primers, which were then analyzed using the Oligo Analyzer tool and the NCBI/Primer-BLAST plugin in the NCBI genetic database. Initially, using the synthesized cDNA for all control and non-control samples as templates, the gene regions controlling the farnesyl diphosphate and squalene synthase enzymes were amplified to ensure the accuracy of the designed primers. Quantitative analysis of the target gene expression was performed by the Real-Time PCR method using the Corbett Real-Time PCR (3000) and the CyberGreen kit (Sinaclon Company). To measure the gene expression pattern changes in all synthesized cDNAs from the studied samples, the Amplicon SYBR Green High ROX master mix was used.
Results: Statistical analysis using GraphPad Prism 10 software showed that the expression level of both studied genes was higher in samples collected from radioactive points compared to non-radioactive points. The highest relative increase in gene expression was observed in the squalene synthase-controlling gene, which is one of the key genes in the biosynthetic pathway of the isoprenoid metabolite group. Statistical comparison of the relative expression pattern of the squalene synthase (SQS) gene in non-radioactive (Bsqs) and radioactive (Asqs) samples showed a significant difference in squalene synthase gene expression levels between samples collected from radioactive and non-radioactive areas at a 99% probability level. Furthermore, statistical analysis of the relative expression pattern of the farnesyl diphosphate synthase (FDS) gene in non-radioactive (Bfds) and radioactive (Afds) samples revealed no significant difference in farnesyl diphosphate synthase gene expression levels between samples collected from radioactive and non-radioactive areas at a 99% probability level. Additionally, the P-value for relative expression of the squalene synthase gene in radioactive and non-radioactive area samples was calculated as 0.0077, and for the farnesyl diphosphate synthase gene, it was 0.6039.
Conclusion: Considering that the expression level of the squalene synthase gene was highest at an altitude of 860-900 meters with an average radiation intensity of 0.567 mSv, and at an altitude of 910-930 meters with the highest radiation level (1.25 mSv), the squalene gene expression level showed a severe decrease. It can be inferred that the optimal radiation intensity for inducing squalene synthase gene expression is around 0.5 mSv, and an increase in radiation intensity leads to a significant reduction in the expression of this gene. Presumably, with an increase in radiation intensity and ionizing rays, the plant intelligently modulates the biosynthetic pathway of squalene and directs the biosynthetic pathway towards the synthesis of sesquiterpenoids, which compete with the squalene synthase enzyme for the common precursor farnesyl diphosphate. Since the mutagenic effect of radioactive elements has been proven, it can be concluded that long-term exposure of perennial plants to radioactive radiation leads to genetic changes. These mutations, by altering the structure and behavior of enzymes involved in the biosynthetic pathways of secondary metabolites, likely represent a genetic-biochemical protective response of the plant to combat the damage caused by free radicals generated by radioactive materials.
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