با همکاری مشترک دانشگاه پیام نور و انجمن بیوتکنولوژی جمهوری اسلامی ایران

نوع مقاله : علمی پژوهشی

نویسندگان

1 استادیار پژوهشگاه ملی مهندسی ژنتیک و زیست فناوری

2 دانشیار پژوهشگاه ملی مهندسی ژنتیک و زیست فناوری

چکیده

ناقلین دوتایی مرسوم مثل pBI121 که هنوز به طور گسترده ای در انتقال ژن به گیاه به واسطه آگروباکتریوم استفاده می شوند، عمدتاً تعداد محدودی جایگاه منحصر به فرد شناسایی آنزیم های برشی در جایگاه همسانه سازی خود دارند. همچنین این ناقل ها فاقد بسیاری از عناصر و توالی های مفید مثل توالی‌های مورد استفاده در خالص سازی پروتئین نوترکیب هستند. در این مقاله یک ناقل بیانی جدید گیاهی با جایگاه همسانه سازی توسعه یافته شامل توالی شناسایی 13 آنزیم برشی منحصر به فرد، توالی رمز کننده His-Tag با تکرار هشت اسید آمینه هیستیدین برای خالص سازی پروتئین نوترکیب، توالی مورد شناسایی آغازگر عمومی M13، به عنوان آغازگر معکوس توالی یابی معرفی می شود. پس از طراحی و ساخت این ناقل، صحت ساخت آن با روش های مولکولی مانند PCR، بررسی الگوی هضم آنزیمی و تعیین توالی تایید شد. برای بررسی کارآیی ناقل جدید، ژن گـزارشگر -galactosidase‌β در آن همسانه سازی شده و سازه حاصل و همچنین ناقل pBI121 به عنوان کنترل، به آگروباکتریوم تومی فاشینس سویه LBA4404 منتقل و در نهایت به گیاه توتون، رقم سامسون منتقل شدند. پس از انجام مراحل باززایی، انتقال ناحیه T-DNA این ناقل به گیاهچه های توتون با روش PCR بررسی و تایید شد. سنجش فعالیت GUS در گیاهان تراریخت حاکی از بیان موثر ژن گزارشگر و کارآیی ناقل جدید در تراریختی گیاه می باشد.

کلیدواژه‌ها

موضوعات

عنوان مقاله [English]

Construction and functional analysis of pBI105, a plant expression vector to facilitate cloning and recombinant protein purification

نویسندگان [English]

  • Kasra Esfahani 1
  • Ali Hatef Salmanian 2

1 Assistant Professor, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran.

2 Associated Professor, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran.

چکیده [English]

Binary vectors such as pBI121 are widely used for introducing genes of interest into plants via Agrobacteriun-mediated transformation method. These vectors usually have low number of unique recognition sites of restriction enzymes in their multiple cloning sites. They also lack elements such as sequences necessary for protein purification. In this article, a new plant expression vectorwith a developed multiple cloning site including recognition sites of 13 restriction enzymes, a sequence for coding 8xHis-Tag to purify recombinant proteins and the sequence of M13 reverse sequencing primer, is introduced. Construction of new vector was confirmed by PCR, digestion pattern analysis, and sequencing. The T-DNA regions of the new vector and pBI121 as a control sample were transformed to Nicotiana tabbacum L. cv. Samsun by using Agrobacterium tumefaciensstrain LBA4404. Integration of T-DNA of the vector to the genome of plantlets, which were regenerated on selective culture, was analyzed by PCR. GUS assay also confirmed expression of the transgene in transgenic seedlings. These evidences indicated that the new plant expression vector, pBI105, is efficient for plant transformation.

کلیدواژه‌ها [English]

  • Plant expression vector
  • Agrobacteriun-mediated transformation
  • Plant transformation
  • pBI105
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