ساخت و بررسی کارآیی ناقل بیانی گیاهی pBI105، با هدف سهولت همسانه سازی و خالص سازی پروتئین نوترکیب

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

Akbarzadeh A, Kordbacheh F, et al. (2010) A Binary Vector for Agrobacterium Mediated Plant Transformation with New Glyphosate tolerant Gene as a Selectable Marker. Biharean Biologist 4(1): 19-29.##Angenon G, Van Montagu M. et al. (1990) Analysis of the stop codon context in plant nuclear genes. FEBS Letters 271(1-2): 144-146.##Bevan M (1984) Binary Agrobacterium vectors for plant transformation. Nucleic Acids Research 12(22): 8711-8721.##Chen PY, Wang CK, et al (2003) Complete sequence of the binary vector pBI121 and its application in cloning T-DNA insertion from transgenic plants. Molecular Breeding 11(4): 287-293.##Esfahani K (2012) pBI121+2upGUS: The new plant expression vector with two new recognition sites of restriction enzymes upstream of β-glucuronidase. 12th Iranian Genetics Congress, Tehran, Iran.##Esfahani K (2012) pBI121GUS-6: The new plant expression vector with several recognition sites of different restriction enzymes. 12th Iranian Genetics Congress, Tehran, Iran.##Esfahani K, Salmanian AH (2013) Designing and construction of new plant expression vectors with more recognition sites of restriction enzymes. 8th National Congress of Biotechnology, Tehran, Iran. ##Esfahani K, Salmanian AH (2014) Plant expression vectors containing developed multiple cloning site and sequences required for sequencing and purification of recombinant proteins. Iran patent No. 82179.##Gallois P, Marinho P (1996) Leaf Disk Transformation Using Agrobacterium tumefaciens-Expression of Heterologous Genes in Tobacco. Plant Gene Transfer and Expression Protocols, Methods in Molecular Biology™, ed Jones H (Springer New York), 49: 39-48.##Gelvin SB (2003) Agrobacterium-mediated plant transformation: The biology behind the "gene-jockeying" tool. Microbiology and Molecular Biology Reviews 67(1): 16-37.##Hellens R, Mullineaux P, et al (2000) A guide to Agrobacterium binary Ti vectors. Trends in Plant Science 5(10): 446-451.##Holsters M, De Waele D, et al. (1978) Transfection and transformation of Agrobacterium tumefaciens. Molecular and General Genetics MGG 163(2): 181-187.##Jefferson RA, Kavanagh TA, et al. (1987) GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO Journal 6(13): 3901-3907.##Karimi M, Inzé D, et al. (2002) GATEWAY™ vectors for Agrobacterium-mediated plant transformation. Trends in Plant Science 7(5): 193-195.##Karimi M, Inzé D, et al. (2013) Gateway vectors for transformation of cereals. Trends in Plant Science 18(1): 1-4.##Kawasaki S (2004) Method for producing a transformed plant with a high capacity binary shuttle vector. US Patent Office.##Kochetov AV, Volkova OA, et al. (2011) Tandem termination signal in plant mRNAs. Gene 481(1): 1-6.##Komori T, Imayama T, et al (2007). Current status of binary vectors and superbinary vectors. Plant Physiology 145(4): 1155-1160.##Kozak M (1991) Structural features in eukaryotic mRNAs that modulate the initiation of translation. Journal of Biological Chemistry 266(30):19867-19870.##Lee LY, Gelvin SB (2008) T-DNA binary vectors and systems. Plant Physiology 146(2): 325-332.##Lin CY, Ku HM, et al. (2011) Construction of the binary vector with bi-selectable markers for generating marker-free transgenic plants. Botanical Studies 52(3): 239-248.##Lütcke H, et al (1987) Selection of AUG initiation codons differs in plants and animals. The EMBO Journal 6(1):43-48.##Nakamura S, Mano S, et al. (2010) Gateway binary vectors with the bialaphos resistance gene, bar, as a selection marker for plant transformation. Bioscience, Biotechnology and Biochemistry 74(6): 1315-1319.##Sambrook J, Russell DW (2001) Molecular cloning. Cold Spring Harbor, New York.##Sripriya R, Sangeetha M, et al. (2011) "Improved Agrobacterium-mediated co-transformation and selectable marker elimination in transgenic rice by using a high copy number pBin19-derived binary vector. Plant Science 180(6): 766-774.##Sugio T, Matsuura A, et al. (2010) Effect of the sequence context of the AUG initiation codon on the rate of translation in dicotyledonous and monocotyledonous plant cells. Journal of Bioscience and Bioengineering 109, 170-173.##Tanaka Y, Nakamura S, et al. (2011) Development of a series of Gateway binary vectors possessing a tunicamycin resistance gene as a marker for the transformation of Arabidopsis thaliana. Bioscience, Biotechnology and Biochemistry 75(4): 804-807.##Terpe K (2003) Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems. Appl. Microbiol. Biotechnol.  60: 523–533##Tohidfar M, Ghoreyshi E, Fakheri B, Mohsenpour M (2014) Design and Construction of Specific Chloroplast Vectors for Rice Plants Containing Betaine Aldehyde Dehydrogenase (badh) and Flavodoxin (fld) Genes for Resistance to Abiotic Stress. Crop Biotech. 6: 47-59.##Zamani K, Malboobi MA, Lohrasebi T,  Esfahani K (2008) Expression vectors for production and purification of plant recombinant proteins". Iran patent No. 64775.##