اخیراً توجه روزافزونی به گیاهان دارویی و متابولیتهای ثانویه میشود. بومادران (Achillea millefolium L.) یک گیاه دارویی است که دارای ترکیبات مهمی از جمله پینن و لینالول میباشد. این دو ترکیب به سبب اهمیت بالایی که دارند، زمینهساز توجه محققان به خود شدند. در این تحقیق میزان بیان رونوشت دو ژن کدکننده پیننسینتاز و لینالولسینتاز را که در انتهای مسیر مونوترپنی MEP قرار دارند و سبب تولید پینن و لینالول میشوند، با اعمال برونزاد تیمار جیبرلیکاسید (GA3( با سه سطح غلظتی (صفر، 25، 50 میلیگرم) در زمانهای مختلف نمونهبرداری (24، 48 و 72 ساعت پس از اعمال تیمار)، سال 1396-1395 در گلخانه دانشکده کشاورزی مورد بررسی قرار گرفتند. نتایج واکنش زنجیره پلیمرزدر زمان واقعی نشان دادکه بیشترین میزان رونوشت در اعمال تیمار جیبرلیک اسید با غلظت 25 میلیگرم و زمان نمونهبرداری 48 ساعت پس از اعمال تیمار، مربوط به ژن پیننسینتاز بود. در بیشتر نمونهها، با افزایش میزان رونوشت پیننسینتاز، میزان رونوشت لینالولسینتاز کاهش مییافت. شواهد حاکی از آن است که، کاربرد برونزاد جیبرلیکاسید، باعث ایجاد فرآیند مولکولی شده که منتج به پاسخدهی گیاه با تغییر بیان ژنهای کدکننده پیننسنتاز و لینالولسینتاز می شود.
عنوان مقاله [English]
Investigating the Expression of Pinene Synthase and Linalool Genes in Response to Gibberellic Acid in Yarrow (Achillea millelium L.) Plant
Background and Objectives
Currently, there is an immense attention towared medicinal plant and secondary motabiltes. Yarrow (Achillea millefolium L.) is a medicinal plant which consists of two important compounds; including penene and linalool. Since these two compounds have the importance values, the researchers significantly focused on them. The rate of transcript expression of penene synthesis and Linalol synthesis induced at the end of 2-c-methyl-D-erythritol-4-phosphate (MEP) pathway that caused to produce Penene and Linalol. In order to evaluate the synthesis of Penene and Linalol, a research study was carried to exame the exogenous treatment (zero, 25 and 50 mg/l) of gibberellic acid (GA3) at different times (24, 48 and 74 hr.) on Yarrow at the greenhouse on 1395-1397. The results of R-PCR indicated that significant rate of the transcript expression was related to the Penene synthesis at 25 mg concentration of GA3 at the 48 hr after treatment. In the most samples as the rate of the Penene synthesis increased the rate of Linalol synthesis decreased. The evidences suggested that the exogenous treatment of GA3 induced a transduction signal in the plant. In which at the end, the plant molecular process respond to it and showed in overall that evaluation of GA3 implemented different encoding gene expression for theses compounds, and illustrated that when the Penene synthesis inceased in the treated plant the Linalol synthesis dcreased simontenously.
Materials and Methods
A greenhouse pot trial was conducted at the Experimental Research Station of College of Agriculture at Shahid Chamran University of Ahvaz. The plants used in this study were provided from the National Center for Genetic and Biological Resources of Iran, with access code P1000093. Two months after seed germination, the desired amounts of gibberellic acid were prepared with distilled water (25 mg/l and 50 mg/l), and then each potted plant was sprayed uniformly with 10cc of prepared solution. Sampling was carried out by collecting the leaves of treated plants at 24, 48 and 72 hours after the treatment. The phenol-chloroform method (Wang and Ghabrial, 2002) was conducted with some modifications to extract RNA of collected leaves at the two biological replications. Quantity and quality of extracted RNAs were measured using Nanodrop and electrophoresis of %1 agarose gel. Synthesis of cDNA was performed using the Takara cDNA synthetase kit according to its manufacturer's instructions. Partial sequences of pinene synthase and linalool synthase genes were obtained in an early experiment in our laboratory (data is not published). The coding sequence of the target genes in other plants within the same family aligned with our obtained sequences to identify conserved segments for each gene using CLUSTAL W program. The standard housekeeping actin for yarrow with accession number JX679606.1, which is available in the public GenBank database, used to normalize the expression of the genes of interest. The primers of all genes (pinene synthase, linalool synthase and actin) were designed from the conserved sequences by PrimerQuest Tool, and then verified with oligoanalyzer v.3.1. Relative expressions of target genes were determined using real-time PCR with SYBR green fluorescence detection. Relative differences in target gene expression were calculated using REST software.
The acquired result of real-time PCR revealed that in pinene synthase gene, the highest amount of transcription occurred at 48 hours after the treatment with the concentration of 25 mg of gibberellic acid and, interestingly, the lowest expression of the transcript for linalool synthase gene observed at the same level of concentration and time. In most of the samples, with the increase in pinene synthase transcriptase, the amount of linalool synthase transcript decreased.
Taking into account the fact that both pinene and linalool are synthesized from the common substrate geranyl diphosphate. Therefore, an increase in the expression one leads to reduction in the transcript expression of the other one in the same pathway. Our results also confirmed that the transcription level of pinene synthase gene has been increasingly altered in response to the applied concentrations of exogenous gibberellic acid, resulting in a reduction in the expression of linalool synthase gene. Evidence suggests that the application of gibberellic acid triggers a cascade of molecular events which ultimately constitutes the plant response to the elicitor.