جداسازی ژن‌های بتا1 و3- گلوکاناز از یونجه یکساله، بیان آنها در باکتری BL21 E.coli و بررسی خاصیت ضد قارچی پروتئین های بیان شده
محورهای موضوعی : یافته های نوین کشاورزینیره نوابی 1 , بهروز نجمی 2 , محمد رسولی 3
1 - کارشناس ارشد بیوتکنولوژی کشاورزی
2 - کارشناس ارشد زراعت و اصلاح نباتات
3 - دانشجوی دکتری اصلاح نباتات واحد علوم و تحقیقات تهران
کلید واژه: یونجه یکساله, بتا1و3- گلوکاناز, پروتئین های PR,
چکیده مقاله :
پروتئین های مرتبط با بیماریزایی (Pathogenesis-related protein) آن دسته از ترکیبات پروتئینی هستند که توسط گیاه میزبان در پاسخ به حمله عوامل بیماری‌زا یا تنش‌های محیطی تولید می شوند. تاکنون بتا1 و3-گلوکانازهای زیادی در گیاهان مختلف شناسایی شده است که خاصیت ضد قارچ آنها در شرایط آزمایشگاه بررسی شده است. هدف این تحقیق جداسازی ژن های بتا1و3- گلوکاناز یونجه یکساله، کلون ژن‌ها، بیان فراوانی و بررسی فعالیت های فیزیولوژیک و خاصیت ضد قارچ پروتئین های به دست آمده در شرایط آزمایشگاه می باشد. در این تحقیق با همردیف سازی ژن های بتا1 و3-گلوکاناز یونجه چند ساله و نخود روی ژنوم یونجه یکساله سه چارچوب خواندن با شباهت بالای 50% و دارا بودن رمز شروع و پایان، شناسایی و جداسازی شده و قطعات به ناقل PET21c منتقل و در باکتری BL21 E.coli بیان شد. با تکنیک SDS-PAGE مشخص شد که این پروتئین ها به صورت اجسام نا محلول تولید شده اند. خاصیت ضد قارچی این پروتئین ها پس از تا شدن و به دست آوردن ساختار فضایی صحیح روی قارچ های Alternaria alternata و Fusarium graminearum آزمایش شد. نتایج آزمون بررسی خاصیت ضد قارچ پروتئین ها پس از تجزیه و تحلیل آماری با نرم افزار SAS در سطح 5% معنی دار بود.
Pathogenesis related proteins are compounds that produced by response to pathogens or environmental stress. Up to now numerous kind of β-1 and 3-Glucanase are identified in various plants and thein fungicide effect were tested. The aim of this research was isolation, over-expression and analysis of physiological activities of β-1 and 3-Glucanase (PR-2) from Medicago truncatula. In this case by Medicago sativa and Pisum sativum β-1 and 3-Glucanase gene alignment on Medicago truncatula genome three open reading frame with %50 similarity by having start and stop codon were identified and isolated that fragments were cloned in PET21c vector and expressed in E.coli BL21. SDS PAGE technique reveal that proteins were produce as an inclusion bodies. We also test their fungicide effect after folding them to achieve their natural structure on Alternaria alternaria and Fusarium graminearum. The result were significant ( P< 0.05) by SAS system analysis.
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