بررسی الگوی بیانی برخی microrna های کاندید در اطلسی تحت تاثیر تنش خشکی

Microrna ها rna های کوچک غیر کدکننده ای هستند که نقش مهمی در پاسخ به تنش های زیستی و غیر زیستی دارند. گیاهان در مواجه با تنش های محیطی، به صورت افزایش و یا کاهش بیان microrna ها با این تنش ها مقابله می کنند. از جمله microrna هایی که تحت تاثیر تنش خشکی بیان متغیری از خود نشان دادند می توان به microrna 393، microrna 159، microrna 169 و microrna 319 اشاره کرد. در این پژوهش نقش این 4 microrna در مقابله با تنش خشکی در گیاه اطلسی (petunia hybrida) بررسی شد. ابتدا گیاه اطلسی در مرحله 4 تا 6 برگی تحت تاثیر تنش خشکی 50% قرار داده شد و rna از گیاه تحت تنش و گیاه شاهد استخراج شد. سنتز cdna با آغازگرهای  stem-loop انجام شد. با qrt-pcr میزان بیان microrna ها در دو حالت تحت تنش و شاهد بررسی شد. نتایج بدست آمده نشان دادند در طی تنش خشکی 50درصد، میزان بیان 319 microrna تغییری نداشته است، امّا کاهش بیان در 393 microrna  مشاهده شد و افزایش بیان 4 و 6.5 برابری به ترتیب در 169microrna و 159microrna مشاهده شد. بیشترین میزان بیان در شرایط تنش نسبت به شاهد در microrna159 مشاهده شد که این امر نشان دهنده نقش حیاتی این microrna درشرایط تنش خشکی در گیاه اطلسی است. همچنین جهت بررسی میزان فعالیت آنزیم آنتی اکسیدانی کاتالاز در شرایط تنش خشکی در برگ های گیاه اطلسی از نمونه برگ تازه اطلسی در بافر فسفات پتاسیم 0.1 مولار با 7 ph= استفاده شد که این نتایج نشان دهنده روند افزایشی میزان فعالیت آنزیم کاتالاز در برگ های گیاه اطلسی تحت تاثیر تنش خشکی است.

studying the expression of some candidate micrornas in petunia under drought stress

small non-coding rnas known as micrornas are essential for responding to biotic and abiotic stresses. a few micrornas that exhibited varied expressions in the face of drought stress are mir393, mir159, mir169, and mir319. this study examined how these four micrornas helped petunia deal with drought stress. a 50% drought stress was applied to the petunia plants in the 4 to 6-leave stage, and rna was isolated from both stressed and control plants. the expression level of micrornas was examined using the qrt-pcr method under two conditions: control and stress. the data obtained indicated that the expression level of mir319 remained unchanged, while mir393 showed a decrease in expression, and mir169 and mir159 showed increases of 4 and 6.5-fold, respectively. introductionthe world is getting warmer, there is less rainfall, and other factors have changed the weather, making it riskier for plants to grow and thrive. it is anticipated that drought will become a more pressing issue in the ensuing decades. drought is a significant issue that impacts the physiological, biochemical, and molecular processes of plants. in addition to negatively affecting stomatal motions, nutrient absorption, and the synthesis of photosynthetic chemicals, this stress negatively impacts the plant’s metabolism and ultimately results in a drop in yield. like other plant activities, a plant’s reaction to stress depends on the proper control of gene expression, which is accomplished by a variety of processes. the alteration of gene expression by post-transcriptional regulation at various developmental stages is one of the most significant of them. sometimes the activity of short rnas or micrornas changes the expression of a gene. these small rnas play a crucial role in the body’s reaction to biotic and abiotic stresses. certain mirnas are overexpressed or reduced in response to environmental stresses to help plants deal with the stress. among the micrornas that exhibited varied expressions under drought stress are mir393, mir159, mir169, and mir319. petunia hybrida, the scientific name for this plant, belongs to the solanaceae family, which is commonly utilized to create greenery in urban areas. because drought stress is one of the most significant abiotic stresses and green space is essential to human existence, this study intends to explore the function of micrornas 159, 319, 169, and 393 in response to drought stress in petunia.materials and methodsan iranian cultivar of petunia hybrida was employed in this experiment. three generations of the same seeds were self-pollinated. after being sanitized in a 1.5% sodium hypochlorite solution, the petunia plant’s seeds were cleaned with sterile distilled water. the seeds were cultured on a culture tray with cleaned peat moss following disinfection. it was moved to the culture chamber, which had a temperature of 25 °c, 16 h of light, and 8 h of darkness. the plant grew to the stage of 4-6 leaves about 4-6 weeks after planting. based on the field capacity (fc), a 50% stress treatment was applied during this stage for 48 h.  to extract total rna, leaf tissue samples from the stressed plant and the control plant were collected independently. for both the control and stressed samples, total rna extraction was carried out using the riboex rna extraction solution according to the manufacturer’s instructions. the methods of varkoni et al. (2007) and chen et al. (2005) were used to develop primers. following primer design, cdna was produced using addbio’s reverse transcription enzyme and stem-loop pcr. then, using biorad equipment, the real-time syber green pcr method was used to examine the expression level of micrornas under 50% drought stress and in two standard circumstances. using the cyp gene as an internal control gene and the specific primers for each microrna, the pcr reaction was carried out using the prepared cdnas as templates. using the 2-δδct method, the quantitative expression values of genes were determined. catalase enzyme activity was also tested. analysis of variance was performed using jmp statistical software version 8 and the mean of treatments was compared using lsd test at the level of 5%.results and discussionafter extracting the rna from two samples under both stress and control, an agarose gel and spectrophotometry tests revealed that the isolated rna was of good quality. consequently, it was evident that the two bands, which corresponded to the 28s and 18s ribosomal rna, were sharp. additionally, the extracted samples’ absorption numbers at the 280/260 wavelength ranged from 1.85-2, demonstrating the high quality of the extracted rnas. the isolated rnas were converted into cdna. pcr was used to confirm the synthesized cdnas. next, they underwent qrt-pcr analysis using primers that were specifically created for them. according to the study’s findings, the expression level of microrna 319 is 50% lower during drought stress than it is under normal condition, essentially remaining the same. reduced expression of microrna 393 was observed during drought stress. on the other hand, when the petunia plant is stressed by drought, the expression levels of micrornas 169 and 159 have increased. following a 48 h 50% drought stress treatment, the petunia plant had a nearly 4-fold rise in microrna 169 expression and a 6.5-fold increase in microrna 159 expression. the obtained data indicated that, of the four micrornas discussed, mir159 has the highest level of expression, compared to the control state. these findings support the critical and significant role of mir159 in the petunia plant during drought stress.conclusionthis study’s findings demonstrated that elevating microrna159 expression in petunia  plants improves the plant’s resistance to drought stress. the impact that this mir has on the myb101 and myb33 proteins is what causes this elevated tolerance. mybs involved in cell division, morphological regulation, disease resistance, stress response, and the secondary biosynthesis pathway. they most likely fulfill their function in response to stresses including cold, salinity, and drought by altering the hormones auxin and abscisic acid.