بررسي بيان ژن 3- هيدروكسي 3- متيل گلوتاريل كوآنزيم A ردوكتاز (HMGR)و گاماترپينن سينتاز (TPS2) تحت تنش كم‌آبي در آويشن باغي (Thymus vulgaris L)

The Study on Gene Expression of 3-Hydroxy-3-Methylglutaryl-Coa Reductase (HMGR) and γ-Terpinene Synthesis in Thymus Vulgaris L. Under Water Deficit Stress

اين آزمايش با هدف بررسي بيان ژن‌هاي دخيل در مسير بيوسنتز مونوترپن‌ها و برخي صفات مورفولوژي (شامل وزن تر ريشه و اندام هوايي، ارتفاع بوته، طول ريشه و تعداد ساقه جانبي) در قالب طرح بلوك‌هاي كامل تصادفي با 4 تيمار شامل شرايط 100 درصد (T1، شاهد)، 70 درصد (T2، تنش ملايم)، 40 درصد (T3، تنش متوسط) و 20 درصد (T4، تنش شديد) ظرفيت مزرعه‌اي در گلخانه تحقيقاتي پژوهشكده گياهان دارويي جهاد دانشگاهي كرج در سال 1394 اجرا گرديد. نتايج حاصل از صفات مورفولوژي نشان داد كه با افزايش تنش كم‌آبي وزن تر ريشه، وزن تر اندام هوايي، ارتفاع بوته، طول ريشه و تعداد ساقه‌هاي جانبي كاهش يافت. نتايج بررسي بيان ژن‌ها نشان داد كه بيشترين ميزان بيان ژن‌هاي TPS2 و HMGR و بيشترين ميزان تيمول و كارواكرول در تيمار T2 حاصل شد. روند تغييرات بيان ژن‌هاي TPS2 و HMGR با روند تغييرات ميزان مونوترپن تيمول و كارواكرول در همه تيمارها مشابه بود؛ به‌طوري‌كه بيشترين ميزان مونوترپن‌ها و بيان ژن‌هاي TPS2 و HMGR در تيمار T2 و سپس در تيمار T1 مشاهده شد و كمترين نيز در تيمارهاي T3 و T4 حاصل شد. در مجموع با توجه به نتايج حاصل از اين پژوهش، تنش ملايم بر افزايش بيان ژن‌هاي TPS2 و HMGR مؤثر بود و منجر به توليد بيشتر مونوترپن‌ها گرديد.

Background and Objectives Drought is a major abiotic stress that limits agricultural crop production. Thyme (Thymus vulgaris L.) is an aromatic and medicinal plant which is very important for the herbal industry. This study was aimed to investigate the variation some of the secondary metabolites and morphological traits as well as changes in expression of genes involved in the biosynthesis of thymol (including HMGR and TPS2) by real-time PCR. One of the main goals of the present study was to find out the relationship between the transcript of these genes and related metabolites in thyme. Materials and Methods This study was conducted in the greenhouse of Iranian Institute of Medicinal Plants of Jahad Daneshgahi (Karaj, Iran) in 2016. The experimental design was a randomized complete block design with 5 replication and 4 water deficit stress treatments, including 100% (T1, control), 70% (T2, mild stress), 40% (T3, moderate stress), and 20% (T4, severe stress) of field capacity (FC). Total RNA was extracted from leaves using TRIzol reagent following the manufacturer’s instructions. First strand synthesis of cDNA was performed immediately using 1 µg of total RNA with simultaneous use of oligo-dT (50 µM) primers and random hexamer and reverse transcriptase enzyme following the manufacturer protocol. In order to examine gene expression by real-time PCR, the method of fluorescence dye SYBR green (Fermentase, USA) was used. The amount of thymol and carvacrol were measured by HPLC. Results Results of morphological traits showed that increasing the water deficit stress caused a decrease in root biomass, shoot fresh weight, plant height, root length, and the number of side branches. Results of gene expression profiling showed that the highest expression of TPS2 and HMGR genes and the highest amount of thymol and carvacrol was obtained in treatment T2 (70% FC). Discussion TPS2 and HMGR gene expression changes were similar to the changes of monoterpenes thymol and carvacrol in all treatments and the highest number of monoterpenes and gene expression were obtained in mild water deficit stress treatment in comparison with control, moderate, and severe stresses. Altogether, according to the results of this study, mild stress had a significant effect on increasing the expression of HMGR and TPS2 and led to a higher production of monoterpenes.