ساخت و آنالیز بیان ناقل گیاهی pCaBGi

نوع مقاله: مقاله پژوهشی

نویسندگان

1 استادیار پژوهشکد ه علوم گیاهی، د انشگاه فردوسی مشهد،

2 د انش آموخته کارشناسی ارشد بیوتکنولوژی، دانشگاه بین المللی قزوین

3 د انش آموخته کارشناسی ارشد بیوتکنولوژی، دانشگاه فردوسی مشهد،

4 د انش آموخته کارشناسی ارشد زیست شناسی سلولی و مولکولی، پژوهشگاه ملی مهندسی ژنتیک و زیست فناوری

چکیده

بیان موقت مبتنی بر تزریق اگروباکتریوم در برگ گیاه روش نسبتاً سریع و قابل اعتمادی برای آنالیز توالی‌های تنظیمی است. به منظور بهره‌گیری از مزایای این تکنیک یک ناقل بیانی مناسب برای انجام آزمون بیان موقت عناصر تنظیمی مبتنی بر تزریق اگروباکتریوم مورد نیاز است. در این مطالعه بمنظور ساخت یک ناقل بیانی گیاهی، جایگاه‌های آنزیمی مناسب از وکتور pBGi به عنوان قطعه در نظر گرفته شد و وکتور pCAMBIA3301 حامل ژن گزارشگر GUS پس از حذف پرموتر CaMV 35S به عنوان توالی پایه استفاده شد. وکتور جدید به نام pCaBGi از طریق وارد نمودن قطعه BamHI/SnaBI از وکتور pBGi به جای قطعه BamHI/SnaBI در وکتور pCAMBIA3301 ساخته شد. تایید ناقل جدید با استفاده از روش کلنی PCR و توالی یابی بوسیله آغازگرهای PSh4-F2/R انجام شد. عدم بیان ژن گزارشگر در میزبان پروکاریوتی با سنجش فعالیت آنزیم بتاگلوکورونیداز در سویه GV3101 اگروباکتریوم به عنوان یک سیستم پروکاریوتی تایید شد. تزریق سلول‌های اگروباکتریوم حامل وکتور pCaBGi در برگ توتون بیان پایه بسیار جزئی ناشی از فعالیت توالی حداقل پروموتری مشاهده شد. توالی وکتور pCaBGi با استفاده از برنامه sequin با شماره بازیابی MG719235 در بانک ژن ثبت شد.

کلیدواژه‌ها

عنوان مقاله [English]

Construction and expression analysis of plant binary vector pCaBGi

نویسندگان [English]

  • Farhad Shokouhifar 1
  • Nahid Abbaspour 2
  • Sahba Toosi 3
  • Nayereh Sadat Ghafarinia 4

1 Assistant Professor Research Center for Plant Sciences, Ferdowsi University of Mashhad, Mashhad, Iran

2 MSc. in Biotechnology from the International University of Qazvin, Qazvin, Iran

3 MSc. in Biotechnology from Ferdowsi University of Mashhad, Mashhad, Iran

4 MSc. in molecular and cellular biology, National institute of genetic engineering and biotechnology, Tehran, Iran.

چکیده [English]

Transient expression in the plant leaf cells is a relatively fast technique to analyses regulatory sequences. To gain the approach advantages, a convenient expression vector would be needed for transient analysis of regulatory elements. In this study, to construct such a vector we used pBGi which provides the regulatory elements cloning site::minimal promoter as insert and pCAMBIA3301 that after removing the CaMV 35S promoter was used as backbone. The new vector, pCaBGi was constructed by replacing BamHI/SnaBI fragment (cis-acting cloning site::Minimal promoter) of pBGi with a BamHI/SnaBI fragment (CaMV 35S promoter) isolated from the pCAMBIA3301. The recombinant colonies were screened using colony PCR with and accuracy of the construction steps has been confirmed by sequencing using PSh4-F2/R specific primers. GUS assay showed that the intron containing GUS gene do not expressed agrobacterium strain GV3101 as a prokaryotic system. Injection of GV3101 harboring pCaBGi in tobacco leaves showed very low Basal expression of minimal promoter. The Sequence of pCaBGi has been submitted to gene bank using sequin software with the Accession number MG719235.

کلیدواژه‌ها [English]

  • Binary vector
  • vector construction
  • Transient expression
  • Agroinjection
  • synthetic promoter analysis

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