بررسی ارتباط بیان افزوده ژن p5cs و تغییرات برخی آنتی اکسیدان های غیرآنزیمی در گیاه تراریخت تنباکو تحت تنش شوری در شرایط کشت در شیشه

ژن p5cs (پرولین-5 کربوکسیلاتسنتتاز) کد کننده آنزیم کلیدی در مسیر سنتز پرولین است و به عنوان یک اسمولیت باعث افزایش مقاومت گیاه به شوری می‌شود. ابتدا گیاهان تراریخت حاوی ژن p5cs و غیرتراریخت انتخاب گردیدند. حضور ژن p5cs در گیاهان تراریخت شده از تکنیک pcr با پرایمر اختصاصی ژن nptii:p5cs استفاده شد. به‌منظور بررسی صفات فیزیولوژیکی گیاهان تراریخت و غیرتراریخت به محیط کشت ms خاوی غلظت های 0، 100، 150 و 200 میلی مولار نمک (nacl) منتقل گردیدند. پس از چهار هفته نتایج نشان داد که مقادیر وزن‌تر، وزن خشک و کلروفیل در گیاهان تراریخت در مقایسه با گیاهان غیرتراریخت در پاسخ به شوری کمتر کاهش یافت. همچنین گیاهان تراریخت سدیم کمتری را نسبت به گیاهان غیرتراریخت در برگ تجمع دادند. سطح ترکیبات فنلی، آنتوسیانین و فلاونوئید در گیاهان تراریخت کمتر از گیاهان غیرتراریخت مشاهده شد. بعلاوه در گیاهان تراریخت در تمام غلظت‌های نمک نسبت به غیرتراریخت افزایش معنی‌داری در میزان پرولین مشاهده شد. میزان اسکوربات در گیاهان تراریخت و غیر تراریخت تغییر معنی داری نشان ندارد ولی پراکسید هیدروژن در اثر تنش شوری در گیاهان غیر تراریخت نسبت به غیر تراریخت کاهش معنی داری نشان داد. بنابراین به نظر می رسد گیاهان تراریخت با افزایش پرولین و تغییر برخی از انتی اکسیدان های غیر انزیمی در گیاهان تراریخت مقاومت به شوری تنش شوری بیشتری نشان داد.

Evaluation of relationship between P5CS gene over expression and changes of some nonenzymatic antioxidant in transgenic tobacco under in vitro salt stress condition

Introduction The growth and production of crop plants are strongly affected by various environmental stress. Salinity stress affects all stages of plant development including germination, bud growth, vegetative growth, flowering, and fruiting. During the past few years, several genes that encoding various structural and regulatory proteins have been used to produce tolerant plants of abiotic stress. Tolerant plants have been selected by improving our knowledge about molecular mechanism of stress tolerance in plants. Plants increase organic osmolytes such as proline, glycine betaine, polyols, alcoholic sugars, and soluble sugars to modulate osmotic stress. The P5CS gene (proline5carboxylatesynthase) is a key enzyme in the pathway for proline synthesis and this amino acid increases resistance of plants to salinity. Although there are many reports about the key role of accumulation proline in mechanism of salinity tolerance, a little information is available about physiological and nonenzymatic antioxidant response of transgenic plants by P5Cs overexpression in salt stress. Therefore, the aim of this study was to evaluate physiological and nonenzymatic antioxidant responses of transgenic and nontransgenic tobacco to salinity under in vitro culture. Materials and methods In order to achieve this goal, transgenic and nontransgenic plants were selected by PCR experiment with NPTII: P5CS proprietary primers. Consequently, the expression of the P5CS gene in transgenic plants was significantly higher than nontransgenic plants. To investigate the mechanism of salt tolerance in tobacco, transgenic and nontransgenic tobacco plants were grown on MS medium containing 0, 100, 150, 200 mM NaCl. After 4 weeks of treatments, fresh and dry weight, photosynthetic pigments, sodium and potassium, proline, phenol, anthocyanin, flavonoid, ascorbate, and hydrogen peroxide were measured. Results and discussion Based on the results, dry and fresh weight as well as chlorophyll content in transgenic plants decreased less than nontransgenic plant under salt stress. For example, the fresh weight of nontransgenic plants in the medium with 100 and 200 mM NaCl decreased by 47% and 33% and their dry weight decreased by 23 and 33%, respectively compared with transgenic plants. Total chlorophyll content of transgenic plants in the medium with 100, 150 and 200 mM salt was improved by 25%, 22%, and 41% compared with nontransgenic plants, respectively. Also, the leaves of transgenic plants accumulated less sodium than nontransgenic plants in response to salinity stress. By adding 100, 150 and 200 mM salt to medium, the level of sodium in transgenic plants decreased by 50%, 17%, and 18% compared with nontransgenic plants respectively. Moreover, the level of phenolic, anthocyanin and flavonoid compounds in the transgenic plants were less than nontransgenic plants by adding salt to medium. The proline content of both transgenic and nontransgenic plants increased in response to salinity. In addition, there was a significant increase in proline content of transgenic plants compared to nontransgenic plant under salt stress. The ascorbate content in transgenic and nontransgenic plants did not change significantly in response to salinity. However, the hydrogen peroxide decreased significantly in transgenic plants as compared with nontransgenic plants in salt stress. The results showed that the accumulation of hydrogen peroxide in transgenic plants in the medium with 100, 150 and 200 mM salt was 83%, 41%, and 23% lower than nontransgenic plants, respectively. So, it seems that transgenic and nontransgenic tobacco plants were salt tolerant and salt sensitive respectively. It has been suggested that high proline content may lead to salt tolerance in plants. According to our experiment, overexpression of P5CS gene increased proline content in other plants and improved salinity resistance in transgenic plants.