همسانه سازی و بیان پپتید ضدمیکروبی امیگانان پنتاهیدروکلراید (MBI226) در توتون (Nicotiana tabacum) به منظور ایجاد مقاومت به باکتری‌های بیماری‌زا

نوع مقاله: علمی پژوهشی

نویسندگان

1 دانشجوی دکتری بیوتکنولوژی کشاورزی، دانشکده کشاورزی، دانشگاه لرستان، خرم آباد، ایران

2 استاد گروه زراعت و اصلاح‌نباتات، دانشکده کشاورزی، دانشگاه لرستان، خرم آباد، ایران

3 دانشیار گروه زراعت و اصلاح‌نباتات، دانشکده کشاورزی، دانشگاه لرستان، خرم آباد، ایران

4 استادیار گروه بیوتکنولوژی، دانشکده کشاورزی، دانشگاه بوعلی‌سینا، همدان، ایران

چکیده

پپتیدهای ضدمیکروبی، مولکول‌های قدیمی و حفاظت شده‌ هستند که در مکانیسم‌های دفاعی موجودات زنده مانند باکتری‌ها، جانوران و گیاهان دیده می‌شوند. شناسایی و معرفی پپتیدهای ضدمیکروبی جدید، روشی مقرون به صرفه برای مقابله با میکروب‌های بیماری‌زا و همچنین بهبود مقاومت گونه‌های گیاهان زراعی با استفاده از تکنولوژی DNA نوترکیب است. به این منظور یک سازه ژنی حاوی توالی ژن کد کننده پپتید ضدمیکروبی امیگانان (Omiganan) نوتروفیل گاو پس از همسانه‌سازی به کمک اگروباکتریویوم تومفاشینز به دیسک‌های برگی توتون انتقال داده شد. با روش PCR حضور ژن کد کننده پپتید ضدمیکروبی در ژنوم گیاهان تراریخت اثبات و تعداد 6 گیاه تراریخت به همراه شاهد انتخاب شدند. پروتئین کل استخراج و از آن برای کنترل رشد برخی باکتری‌های مهم انسانی مثل؛ E.coli ، Staphylococcus aureus و Bacillus cereus و همچنین برخی باکتری‌های گیاهی مانند Xanthomonas campestris و Pseudomonas aeruginosa به روش دیسک و تشکیل هاله مهار کننده استفاده شد. نتایج نشان داد که پروتئین کل گیاهان تراریخت در مقایسه با گیاه غیرتراریخت، به‌طور معنی‌داری (05/0P

کلیدواژه‌ها

موضوعات


عنوان مقاله [English]

Cloning and expression of an omiganan pentahydrochloride (MBI226) antimicrobial peptide in tobacco (Nicotiana tabacum) confers resist to bacterial pathogens

نویسندگان [English]

  • Azam Badr Hadad 1
  • Farhad Nazarian Firouzabadi 2
  • Ahmad Ismaili 3
  • Hedayat Bagheri 4
1 Ph.D. Student, Department of Biotechnology, Agricultural Faculty, Lorestan University, Khorramabad, Iran
2 Professor, Department of Agronomy and Plant Breeding, Faculty of Agriculture, Lorestan University, Khorramabad, Iran
3 Associate Professor, Department of Agronomy and Plant Breeding, Faculty of Agriculture, Lorestan University, Khorramabad, Iran
4 Assistant Professor, Department of Biotechnology Bu-Ali Sina University, Hamedan, Iran
چکیده [English]

Antimicrobial peptides are ancient and conserved molecules which are found in defense mechanisms of almost all living organisms from bacteria to animal and plant species. Identification and introduction of novel antimicrobial peptides, is a cost-effective way to improve crop plants resistance to pathogens by using recombinant DNA technology. Therefore, an expression construct containing omiganan antimicrobial encoding gene from the cytoplasmic granules of bovine neutrophils, was cloned and transferred to the tobacco leaf disk using Agrobacterium tumefaciens mediated-transformation. The presence of the antimicrobial peptide encoding gene in the genome of transgenic plants was confirmed by PCR analysis. Six putative transgenic lines and a non-transgenic control plant were selected for further molecular analysis. Total protein was extract from transgenic and non-transgenic control plants and used for antimicrobial activity assay against some human; E. coli, Salmonella, Staphylococcus, Bacillus, and plant: Xanthomonas and Pseudomonas pathogens by disc diffusion method. Results of this experiment showed that total protein extract from transgenic lines, as compared to non-transgenic plant, was significantly (P

کلیدواژه‌ها [English]

  • Antimicrobial peptides
  • Agrobacterium tumefaciens
  • pathogen
  • recombinant protein
  • Tobacco
Alan A, Blowers A, Earle E (2004) Expression of a magainin-type antimicrobial peptide gene (MSI-99) in tomato enhances resistance to bacterial speck disease. Plant Cell Reports. 22: 388-396.

Bauer A (1966) Antibiotic susceptibility testing by a standardized single disk method. Am. J. Clin. Pathol. 45: 493-496.

Burt S (2004) Essential oils: their antibacterial properties and potential applications in foods-a review. International Journal of Food Microbiology. 94: 223-253.

Chmara H, Milewski S, Andruszkiewicz R, Mignini F, Borowski E (1998) Antibacterial action of dipeptides containing an inhibitor of glucosamine-6-phosphate isomerase. Microbiology. 144: 1349-1358.

Diaz AH, Kovacs I, Lindermayr C (2016) Inducible Expression of the De-Novo Designed Antimicrobial Peptide SP1-1 in tomato confers resistance to Xanthomonas campestris pv. vesicatoria. PLoS One 11: e0164097.

Fritsche TR, Rhomberg PR, Sader HS, Jones RN (2008) Antimicrobial activity of omiganan pentahydrochloride tested against contemporary bacterial pathogens commonly responsible for catheter-associated infections. Journal of Antimicrobial Chemotherapy. 52(3): 1187-1189.

Furman N, Kobayashi K, Zanek M. C, Calcagno J, Garcia M. L, Mentaberry A (2013) Transgenic sweet orange plants expressing a dermaseptin coding sequence show reduced symptoms of citrus canker disease. Journal of Biotechnology 167(4): 412-419.

Guaní-Guerra E, Santos-Mendoza T, Lugo-Reyes SO, Terán LM (2010) Antimicrobial peptides: general overview and clinical implications in human health and disease. Clinical Immunology 135: 1-11.

Hancock RE, Lehrer R (1998) Cationic peptides: a new source of antibiotics. Trends in Biotechnology. 16: 82-88.

Horsch RB, Fry J, Hoffmann N, Neidermeyer J, Rogers SG, Fraley RT. 1989. Leaf disc transformation. In Plant molecular biology manual, pp. 63-71. Springer.

Jan P-S, Huang H-Y, Chen H-M (2010) Expression of a synthesized gene encoding cationic peptide cecropin B in transgenic tomato plants protects against bacterial diseases. Applied and Environmental Microbiology. 76: 769-775.

Jaynes JM, Nagpala P, Destéfano-Beltrán L, Hong Huang J, Kim J, Denny T, Cetiner S (1993) Expression of a Cecropin B lytic peptide analog in transgenic tobacco confers enhanced resistance to bacterial wilt caused by Pseudomonas solanacearum. Plant Science. 89: 43-53.

Mangena T, Muyima N (1999) Comparative evaluation of the antimicrobial activities of essential oils of Artemisia afra, Pteronia incana and Rosmarinus officinalis on selected bacteria and yeast strains. Letters in Applied Microbiology. 28: 291-296.

Melo MN, Castanho MA (2007) Omiganan interaction with bacterial membranes and cell wall models. Assigning a biological role to saturation. Biochimica et Biophysica Acta (BBA)-Biomembranes 1768: 1277-1290.

Melo MN, Dugourd D, Castanho MA (2006) Omiganan pentahydrochloride in the front line of clinical applications of antimicrobial peptides. Recent patents on anti-infective drug discovery. 1: 201-207.

Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiologia Plantarum. 15: 473-497.

Nawrocki KL, Crispell EK, McBride SM (2014) Antimicrobial peptide resistance mechanisms of gram-positive bacteria. Antibiotics. 3: 461-492.

Niidome T, Anzai S, Sonoda J, Tokunaga Y, Nakahara M, Hatakeyama T , Aoyagi H (1999) Effect of amino acid substitution in amphiphilic α‐helical peptides on peptide–phospholipid membrane interaction. Journal of Peptide Science 5(7): 298-305.

Oard S, Enright F (2006) Expression of the antimicrobial peptides in plants to control phytopathogenic bacteria and fungi. Plant Cell Reports. 25: 561-572.

Osusky M, Osuska L, Hancock RE, Kay WW, Misra S (2004) Transgenic potatoes expressing a novel cationic peptide are resistant to late blight and pink rot. Transgenic Research. 13: 181-190.

Osusky M, Zhou G, Osuska L, Hancock RE, Kay WW, Misra S (2000) Transgenic plants expressing cationic peptide chimeras exhibit broad-spectrum resistance to phytopathogens. Nature Biotechnology. 18: 1162-1166.

Richards E, Reichardt M, Rogers S (1994) Preparation of genomic DNA from plant tissue. Current Protocols In Molecular Biology. 2.3. 1-2.3. 7.

Sabat A, Kosowska K, Poulsen K, Kasprowicz A, Sekowska A, van den Burg B, Travis J, Potempa J (2000) Two allelic forms of the aureolysin gene (aur) within Staphylococcus aureus. Infection and Immunity 68: 973-976.

Sader HS, Fedler KA, Rennie RP, Stevens S, Jones RN (2004) Omiganan pentahydrochloride (MBI 226), a topical 12-amino-acid cationic peptide: spectrum of antimicrobial activity and measurements of bactericidal activity. Antimicrobial Agents And Chemotherapy. 48: 3112-3118.

Scorzoni L, Benaducci T, Almeida A, Silva DHS, Bolzani VdS, Mendes-Giannini MJS (2007) Comparative study of disk diffusion and microdilution methods for evaluation of antifungal activity of natural compounds against medical yeasts Candida spp and Cryptococcus sp. Revista de Ciências Farmacêuticas Básica e Aplicada 28(1): 25-34.

Selsted ME, Novotny MJ, Morris WL, Tang Y-Q, Smith W, Cullor JS (1992) Indolicidin, a novel bactericidal tridecapeptide amide from neutrophils. Journal of Biological Chemistry. 267: 4292-4295.

Sindambiwe J, Calomme M, Cos P, Totte J, Pieters L, Vlietinck A, Berghe DV (1999) Screening of seven selected Rwandan medicinal plants for antimicrobial and antiviral activities. Journal of Ethnopharmacology. 65: 71-77.

Subbalakshmi C, Sitaram N (1998) Mechanism of antimicrobial action of indolicidin. FEMS microbiology letters. 160: 91-96.

Subczynski WK, Wisniewska A (2000) Physical properties of lipid bilayer membranes: relevance to membrane biological functions. Acta Biochimica Polonica. 47: 613-626.

Vanden B, Vlietinck A (1991) Screening methods for antibacterial and antiviral agents from higher plants. In methods in plant biochemistry ed. Dey PM and Harborne JB. London academic press.

Zasloff M (2002) Antimicrobial peptides of multicellular organisms. Nature 415: 389-395.