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کلونینگ و بیان ژن رمزگذار آنزیم تجزیه‌کننده ترکیبات آلی فسفره (opd) در باکتری Escherichia coli

نوع مقاله : مقاله پژوهشی

نویسندگان

1 دانشجوی دکتری، گروه گیاهپزشکی، پردیس کشاورزی و منابع طبیعی دانشگاه تهران، کرج

2 استاد، گروه گیاهپزشکی، پردیس کشاورزی و منابع طبیعی دانشگاه تهران، کرج

3 پژوهشگر پسا دکتری، گروه زیست شناسی، دانشگاه مک مستر، همیلتون کانادا

چکیده

کاربرد گستردۀ ترکیب‌های آلی فسفرۀ منجر به بروز اثر زیان‌آور زیست‌محیطی بسیاری شده است. استفاده از میکروارگانیسم­ها در پالایش و اندازه‌گیری این ترکیب‌های ناگوار به‌عنوان یک رویکرد دوستدار محیط­زیست و مناسب شناخته شده است. آنزیم ارگانوفسفروس هیدرولاز از آنزیم‌های هیدرولیزکنندۀ فسفوتری­استری است که در برخی میکروارگانیسم­های خاک به‌ویژه Flavobacterium sp.شناسایی شده و قادر به هیدرولیز تعدادی از ترکیب‌های فسفرۀ آلی است. در این پژوهش بهینه‌سازی کدون‌های توالی رمزگذار این پروتئین برای بیان در باکتری Escherichia coli انجام شد و سپس همراه با پروموتر lac، در وکتور پروموتر-پروب pTH1705 به‌صورت ادغام الگوبرداری کلون شد و ترانسفورماسیون‌ در دو سویۀ‌ باکتری E. coli انجام گرفت. محیط کشت معدنی M9 حاوی غلظت 10و 50 میلی­گرم در لیتر دیازینون، برای سنجش عملکرد باکتری تراریخت بیان‌کنندۀ ژن opd (به‌صورت in trans) استفاده شد. بررسی منحنی رشد استرین E. coli V103 نشان داد، رشد باکتری در غلظت 50 میلی­گرم در لیتر دیازینون تا حد زیادی تحت تأثیر (وجود آفت­کش) قرار می­گیرد درحالی‌که در غلظت 10 میلی‌گرم در لیتر دیازینون این تأثیر کمتر است. نتایج حاصل از کروماتوگرافی مایع با کارایی بالا به‌عنوان یک روش استاندارد نشان داد، غلظت دیازینون در محیط کشت باکتری، در چهار استرین V100، V101، V102 و V103 پس از 24 ساعت از 10 میلی­گرم در لیتر به ترتیب به 38/6، 19/7، 09/7 و 74/5 میلی­گرم در لیتر کاهش یافت. این نتایج بیانگر انتقال موفق و نیز بیان ژن مورد نظر در باکتری است و این باکتری نوترکیب مهندسی‌شده توانایی تجزیۀ ترکیب‌های فسفره را دارد.

کلیدواژه‌ها


عنوان مقاله [English]

Cloning and studing the function of opd gene encoding organophosphorus hydrolase in Escherichia coli

نویسندگان [English]

  • Seyedeh Minoo Sajadian 1
  • Vahid Hosseini naveh 2
  • Khalil Talebi 2
  • Maryam Zamani 3
1 Ph. D. Candidate, Department of Plant Protection, University College of Agriculture & Natural Resoucrs, University of Tehran, Karaj, Iran
2 Professor, Department of Plant Protection, University College of Agriculture & Natural Resoucrs, University of Tehran, Karaj, Iran
3 Postdoc Fellowship, Department of Biology, McMaster University, Hamilton, Canada
چکیده [English]

Organophosphorus compounds are widely used, and their presence in different components of the ecosystem has led to harmful effects on the environment. The use of microorganisms in detoxification of xenobiotic compounds and measurement of pesticide residues is considered as an environmental-friendly and appropriate method. Organophosphorus hydrolase (opd), is a phosphotriester hydrolase enzyme which is discovered in some soil microorganisms such as Flavobacterium sp. and has a wide range of substrates and is able to hydrolyze many organophosphorus compounds. In the present study, codon optimization was done to express the protein in Escherichia coli. Following omission of the signal peptide sequence and substitution of serine by methionine as the start codon, this fragment was cloned under the control of lac promoter in a promoter-probe vector (pTH1705) as a transcriptional fusion and was used for transformation in two Escherichia coli strains DH5α and XL1-blue. To measure the performance of the transgenic bacteria expressing opd gene (as in trans), they were cultured in M9 mineral medium containing 10 and 50 mg/L diazinon and appropriate available carbon and nitrogen sources. Studying the growth curve of V103 strain revealed that bacterial growth in the presence of 50 mg/L diazinon had been affected while this effect in the presence of 10 mg/L diazinon was minor. Results obtained from high-performance liquid chromatography as a standard method to compare with the performance of obtained transgenic strain showed that after 24 hours, diazinon concentration (initially 10mg/L ) in the bacterial culture medium of V100, V101, V102 and V103 strains reached to 6.38, 7.19, 7.09 and 5.74, respectively. These results reflected the successful transfer and expression of target gene in the bacterium and this genetically engineered bacterium was able to degrade organophosphorus compounds directly.

کلیدواژه‌ها [English]

  • degradation
  • HPLC
  • organophosphorus compounds
  • transgenic bacterium
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