مطالعه بیان عوامل رونویسی ap2-domain، hd-zip، wrky و myb در آفتابگردان روغنی (.helianthus annuus l) تحت تنش خشکی

به‌منظور بررسی تاثیر تنش خشکی بر بیان عوامل رونویسی ap2-domain، hd-zip، wrky و myb در آفتابگردان روغنی، دو لاین با واکنش متفاوت انتخاب و در قالب طرح کاملاً تصادفی با 3 تکرار در گلخانه در سال زراعی 1395-1396 کشت شدند. گیاهان تا مرحله 8 برگی از لحاظ وضعیت آبی در 100 درصد ظرفیت زراعی نگهداری شدند. بعد از این مرحله تعدادی از گلدان‌ها در همان ظرفیت زراعی نگهداری شدند، اما به بقیه تنش خشکی 80، 60 و 40 درصد ظرفیت زراعی اعمال شد. نمونه برداری از گیاهان یک و سه هفته بعد از اعمال تنش انجام گرفت. مطالعه بیان ژن‌های مذکور با استفاده از تکنیک واکنش زنجیره ای پلی مراز در زمان واقعی انجام شد. مقایسات میانگین بیان عوامل رونویسی ap2-domain، wrky و myb در دو لاین ensat254 (متحمل) و lc1064c(حساس) نشان داد که بیان ژن‌ها یک هفته بعد از اعمال تنش در شدتْ تنش‌های مختلف محسوس نبوده اما در هفته سوم بعد از اعمال تنش در شدت تنش 40 درصد میزان بیان ژن‌ها خصوصا در لاین ensat254 افزایش یافته است. در رابطه با عامل رونویسی hd-zip میزان بیان یک هفته بعد از اعمال تنش در شدت تنش 40 درصد در لاین ensat254خیلی بیشتر از لاین lc1064c بود. میزان بیان در هفته سوم بعد از اعمال تنش در هر دو لاین در شدت تنش 40 درصد افزایش یافت، هر چند افزایش بیان در لاین lc1064c اندکی بیشتر بود؛ بنابراین، به نظر می‌رسد بیان زود هنگام عامل رونویسی hd-zip در افزایش مقاومت ژنوتیپ به تنش خشکی دخیل باشد. یافته های این تحقیق می‌تواند در برنامه‌های اصلاحی آفتابگردان برای تولید و توسعه ارقام متحمل به تنش خشکی مفید و فایده واقع شود.

Study on expression of transcription factors AP2-Domain, HD-ZIP, WRKY and MYB in oily sunflower (Helianthus annuus L.) under drought stress

IntroductionSunflower (Helianthuse annuus L.) is an annual plant from Composite with a chromosome number of 2n = 2x = 34 which is widely cultivated for supplying edible oil. Drought is one of the most important environmental stresses that limits the growth and distribution of plant more than other factors. This plant is classified as semitolerant to drought stress; however, its performance is negatively affected by drought. Transcription factors are molecules that play an important role in the understanding and transmission of stress messages as well as many physiological processes. One of the most effective ways to deal with stress is to produce resistant hybrids. Investigation and study of expression of genes post stress application and identification of genes involved in resistance and especially regulatory genes such as transcription factors is vital and necessary for molecular breeding programs. Materials and methodsIn order to investigate the effect of drought stress on the expression of transcription factors: AP2Domain, HDZIP, WRKY and MYB in oilseed sunflower, two lines with different susceptibility to drought stress were selected and cultivated in a completely randomized design with three replications in greenhouse. The seeds were planted in 3 cm depth of 30 × 25 cm pots containing farm soil and sand mixture in the ratio of 2:1. The plants were grown in controlled conditions at 25 ± 3 °C, 65% relative humidity and 12 h darklight photoperiod and were irrigated regularly at 100% of field capacity up to 8leaf stage. After this stage, a number of pots were kept at the same field capacity however, some other were exposed to 80, 60 and 40% of field capacity. Samplings were done in two times, one and three weeks after drought stress application. The study of the expression of genes was performed using real time PCR by SYBR Green method. RNA extraction kit RNXplusTM (Sinoclon Co., Iran) and complementary DNA (cDNA) synthesis Kit (Fermentas LIFE SCIENCE # K1621) were used according to the manufacturer’s protocols. Quantitative reverse transcriptionPCR (qRTPCR) was performed in triplet using 6.25 μl of Maxima SYBR Green/ Fluorescein qPCR Master Mix (2X) (Thermo Fisher Scientific, Germany), 5 pM of forward and reverse primers and 50 ng of cDNA for each reaction in a final volume of 12.5μl. Relative gene expression was analyzed by comparative Ct method, 2−ΔΔC. Target gene was normalized by the reference gene, ACTIN and calibrated for each sample against the control. Results and discussionThe results of statistical analyzes showed that the expression of the genes in the susceptible and resistant lines of sunflower is different. Mean comparisons of expression of AP2Domain, WRKY and MYB transcription factors in the two genotypes ENSAT254 (tolerant) and LC1064C (susceptible) showed that the expression level was not tangible in the first week after drought stress application, but the expression of genes was increased in 40% of field capacity in the third week post drought stress application especially in ENSAT254 genotype. In relation to HDZIP transcription factor, the expression was much higher in ENSAT254 genotype than LC1064C genotype in the first week of sampling at 40% stress intensity. In the third week of sampling, the expression level of both genotypes increased in 40% of field capacity, although the expression was slightly higher in LC1064C genotype. ConclusionsEarly expression of HDZIP transcription factor appears to be involved in increasing genotype resistance to drought stress. The results of the present study can be useful in sunflower improvements programs for producing and developing drought tolerant cultivars.