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Subcloning and expression of blf1 gene isolated from Burkholderia pseudomallei in hairy roots of tobacco plants | ||
| Iranian Journal of Genetics and Plant Breeding | ||
| دوره 10، شماره 2 - شماره پیاپی 20، دی 2021، صفحه 71-81 اصل مقاله (990.87 K) | ||
| نوع مقاله: Research paper | ||
| شناسه دیجیتال (DOI): 10.30479/ijgpb.2023.17386.1317 | ||
| نویسندگان | ||
| Zahra Moshiri1؛ Raheem Haddad* 1؛ Hossein Honari2 | ||
| 1Department of Biotechnology, Faculty of Agriculture and Natural Resource, Imam Khomeini International University, Qazvin, Iran. | ||
| 2Biology Science and Technology Center, Faculty of Basic Sciences, Imam Hossein University, Tehran, Iran. | ||
| تاریخ دریافت: 24 خرداد 1401، تاریخ بازنگری: 28 اردیبهشت 1402، تاریخ پذیرش: 28 اردیبهشت 1402 | ||
| چکیده | ||
| Melioidosis is a common disease between human and animals caused by Burkholderia pseudomallei. There is currently no effective vaccine for it. The aim of this research was to express blf1 gene in tobacco plants. By transforming the pBI121 vector into Agrobacterium tumefaciens, the blf1 gene was transferred to the tobacco plants and the primary transgenic plants were obtained. The explants obtained from the transgenic plants were used to induce hairy roots. The presence of the blf1 gene was investigated in the obtained hairy roots by PCR and Western blot analysis. The titer of antigen production was measured by ELISA technique. The insertion of the blf1 gene construct into pBI121 vector containing the ctxB gene was confirmed by PCR. The tobacco explants inoculated with A. tumefaciens were cultured on the MS medium containing benzyl-aminopurine (BAP) and 1-naphthalene acetic acid (1 -NAA). After callus formation and seedling regeneration, the seedlings with active meristems were transferred onto the hormone-free medium for rooting and transgenic tobacco plants containing the blf1 gene were produced. By preparing an explant of transgenic plants by A. rhizogenes, inoculum and hairy roots were obtained. In hairy roots, the presence of blf1 gene was confirmed using PCR and its expression was confirmed by Western blotting. By using the ELISA technique, the titer of BLF1 antigen production in the total soluble protein of hairy roots was determined to be 0.56%. | ||
| کلیدواژه‌ها | ||
| Gene transfer؛ Tobacco؛ Melioidosis؛ Cloning | ||
| عنوان مقاله [English] | ||
| زیرهمسانه سازی و بیان ژن blf1 بورخولدریا سودومالئی در ریشه های مویین گیاه تنباکو | ||
| نویسندگان [English] | ||
| زهرا مشیری1؛ رحیم حداد1؛ حسین هنری2 | ||
| 1گروه بیوتکنولوژی، دانشکده کشاورزی و منابع طبیعی، دانشگاه بین‌المللی امام خمینی (ره)، قزوین، ایران. | ||
| 2مرکز علوم و فناوری زیست شناسی، دانشکده علوم پایه، دانشگاه امام حسین، تهران، ایران. | ||
| چکیده [English] | ||
| Melioidosis یک بیماری مشترک بین انسان و حیوان است که توسط Burkholderia Pseudomallei ایجاد می شود. در حال حاضر هیچ واکسن موثری برای آن وجود ندارد. هدف از این تحقیق بیان ژن blf1 در گیاه تنباکو می باشد. با انتقال وکتور pBI121 به Agrobacterium tumefaciens، ژن blf1 به گیاه تنباکو منتقل شد و گیاه تراریخته کامل بدست آمد. از ریزنمونه گیاه تراریخته توسط Agrobacterium rhizogenes برای تهیه ریشه های مویین استفاده شد. وجود ژن blf1 در ریشه های مویین به دست آمده با روش PCR و وسترن بلات بررسی شد. میزان تولید آنتی ژن با روش الایزا اندازه گیری شد. نتایج آزمایش ساخت سازه ژن با درج blf1 در وکتور pBI121 حاوی ژن ctxB با استفاده از پرایمرهای عمومی با تکنیک PCR تایید شد. ریزنمونه تنباکو پس از تلقیح با Agrobacterium tumefaciens، روی محیط کشت MS حاوی هورمون‌های بنزیل آمینوپورین (BAP) و 1-نفتالین استیک اسید (1-NAA) قرار داده شد. پس از تشکیل کالوس و باززایی گیاهچه، نهال هایی با مریستم فعال جهت ریشه زایی به محیط بدون هورمون منتقل و بوته های تنباکوی تراریخته با ژن blf1 تولید و از طریق تست PCRصحت انتقال ژن تایید شد. با تهیه ریزنمونه ای از گیاهان تراریخته توسط آگروباکتریوم رایزوژنز، تلقیح و ریشه های مویین به دست آمد. در ریشه های مویین، ژن blf1 با استفاده از PCR و بیان آن با تکنیک وسترن بلات تایید شد. با استفاده از تکنیک الایزا، تولید آنتی ژن نوترکیب BLF1 در ریشه های مویین، 0.56 درصد پروتئین محلول کل تعیین گردید. | ||
| کلیدواژه‌ها [English] | ||
| انتقال ژن, توتون, میلوئیدوزیس, همسانه سازی | ||
| مراجع | ||
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