ایجاد توتون تراریخت تولیدکننده لاکاز قارچی با استفاده از یک پروموتر دارای بیان ریشه-ویژه

نوع مقاله: علمی پژوهشی

نویسندگان

1 استادیار

2 دانشجوی دکتری فیزیولوژی گیاهی، دانشگاه پیام نور تهران شرق،ایران

3 گروه زیست شناسی، دانشگاه پیام نور تهران شرق، تهران، ایران

4 گروه زیست شناسی، دانشکده زیست شناسی، دانشگاه دامغان، دامغان، ایران

چکیده

لاکازها گروهی از گلیکوپروتئین‏ها با ویژگی پلیفنلاکسیدازی هستند که می‌توانند ترکیبات گوناگونی را اکسید نمایند. این گروه آنزیمی در گیاهان، قارچ‌ها، حشرات و باکتری‌ها بیان می‌شوند و دارای نقش‌های مهمی در فعالیت‌های زیستی و کاربردهای گوناگونی در صنایع غذایی، دارویی، نساجی و حتی نظامی می‌باشند. با توجه به ساختار این آنزیم و این‌که تشکیلات بیان پروتئین در گیاهان قادر به تولید کامل پروتئین‌ها ازجمله گلیکوپروتئین‏ها می‌باشند به جهت کاربرد در پالایش زیستی، سازه‌ای طراحی گردید که لاکاز II قارچی تحت کنترل پیشرانه باکتریایی ویژه بیان در ریشه مانوپین سنتاز و تشدیدکننده ترجمه Tobacco Etch Virus در گیاه توتون بیان شود. افزودن سیگنال پپتید اندوکیتیناز اسیدی Q توتون به ابتدای آنزیم باعث ترشح آن به فضای آپوپلاستی می‌شود. با استفاده از واکنش زنجیره‌ای پلیمراز، تراریختی گیاهان توتون توسط ژن لاکاز به‌وسیله آگروباکتریوم تأیید گردید. نتایج نیمهکمی بیان ترانسکریپتومیک لاکاز در ریشه و برگ تراریخت‏های مفروض نشانگر بیان متمایز آن در ریشه و برگ بود که ناشی از کارکرد متمایز پیشرانه باکتریایی مانوپین سنتاز می‌باشد. نتیجه وسترن بلاتینگ تولید پروتئین بالغ در ریشه را تأیید کرد. وجود این پروتئین به‌صورت تک سایز نشانه عملکرد صحیح سیگنال پپتید و ترشح آن به فضای آپوپلاستی در ریشه توتون می‌باشد. با توجه به کاربرد این گیاهان جهت استخراج آنزیم یا پالایش زیستی می‌توان تراریخت‏های موردنظر را بر اساس میزان پروتئین و فعالیت آنزیمی غربالگری و گزینش نمود.

کلیدواژه‌ها

موضوعات


عنوان مقاله [English]

Generation of fungal laccase producing transgenic tobacco plants using a root-specific expression promoter

نویسندگان [English]

  • Seyed Javad Davarpanah 1
  • Majid Dana 2
  • Gholamreza Bakhshi Khaniki 3
  • Amir Abbas Mokhtarieh 4
1 Research Assistant Professor
2 Department of Biology, Faculty of Science, Payam Nour University, Tehran, Iran
3 Department of Biology, Faculty of Science, Payam Nour University, Tehran, Iran
4 Department of Biology, School of Biology, Damghan University, Damghan, Iran
چکیده [English]

Laccases are a group of glycoproteins which can oxidize a wide range of compounds with various biological activities and industrial applications in food and beverage, pharmaceutical, textile, and even military-related industries. Regarding this enzyme structure and the ability of plant protein production machinery for protein glycosylation, a construct consisting of fungal laccaseII under control of root-specific mannopine synthase promoter and tobacco etch virus translation enhancer was designed for tobacco transformation to be used in phytoremediation. N-terminal addition of acidic tobacco endochitinase Q to Laccase directs its apoplastic secretion. Putative laccase agrobacterium-mediated transformants were confirmed using polymerase chain reaction. Semi-quantitative PCR of roots and leaves of putative transformants showed differential expression of the laccase gene at the transcriptomic level resulting from the differential function of bacterial mannopine synthase promoter. Western blotting results confirmed production of mature protein in roots which also confirms the correct function of signal peptide and secretion of this enzyme into the apoplastic space of roots. Regarding their application for protein production or phytoremediation transgenics of interest should be screened based on protein concentration and enzyme activity.

کلیدواژه‌ها [English]

  • Laccase
  • Mannopine synthase
  • Rhizosecretion
  • Tobacco
  • Transformation
Bailey MR, Woodard SL, Callaway E, Beifuss K, Magallanes-Lundback M, Lane JR, Horn ME, Mallubhotla H, Delaney DD, Ward M, Van Gastel F (2004) Improved recovery of active recombinant laccase from maize seed. Appl Microbiol. Biot. 63: 390-397.

Behnam M, Davarpanah SJ, Karimian R (2016) Designing and cloning a masp1-based synthetic gene in binary vector for transient expression in cotton fiber. NBR. 3: 238-248.

Chhabra M, Mishra S, Sreekrishnan TR (2015) Immobilized laccase mediated dye decolorization and transformation pathway of azo dye acid red 27. J. Environ. Health. Sci. 13:38.

Davarpanah SJ, Ahn JW, Ko SM, Jung SH, Park YI, Liu JR, Jeong WJ (2012) Stable expression of a fungal laccase protein using transplastomic tobacco. Plant Biotechnol. Rep. 6: 305-12.

Davarpanah SJ, Jung SH, Kim YJ, Park YI, Min SR, Liu JR, Jeong WJ (2009) Stable plastid transformation in Nicotiana benthamiana. J. Plant Biol. 52: 244-50.

de Wilde C, Uzan E, Zhou Z, Kruus K, Andberg M, Buchert J, Record E, Asther M, Lomascolo A (2008) Transgenic rice as a novel production system for Melanocarpus and Pycnoporus laccases. Transgenic. Res. 17: 515.

Doyle JJ (1990) Isolation of plant DNA from fresh tissue. Focus. 12:13-5.

Drake PM, Barbi T, Sexton A, McGowan E, Stadlmann J, Navarre C, Paul MJ, Ma JK (2009) Development of rhizosecretion as a production system for recombinant proteins from hydroponic cultivated tobacco. FASEB. J. 23: 3581-9.

Drake PM, Chargelegue DM, Vine ND, van Dolleweerd CJ, Obregon P, Ma JK (2003) Rhizosecretion of a monoclonal antibody protein complex from transgenic tobacco roots. Plant. Mol. Biol. 52: 233-41.

Feltkamp D, Baumann E, Schmalenbach W, Masterson R, Rosahl S (1995) Expression of the mannopine synthase promoter in roots is dependent on the mas elements and correlates with high transcript levels of mas-binding factor. Plant. Sci. 109: 57-65.

Foo CW, Kaplan DL (2002) Genetic engineering of fibrous proteins: spider dragline silk and collagen. Adv. Drug. Deliver. Rev. 54:1131-43.

Fujihiro S, Higuchi R, Hisamatsu S, Sonoki S (2009) Metabolism of hydroxylated PCB congeners by cloned laccase isoforms. Appl. Microbiol. Biot. 82: 853.

Gaume A, Komarnytsky S, Borisjuk N, Raskin I (2003) Rhizosecretion of recombinant proteins from plant hairy roots. Plant. Cell. Rep. 21: 1188-93.

Gleba D, Borisjuk NV, Borisjuk LG, Kneer R, Poulev A, Skarzhinskaya M, Dushenkov S, Logendra S, Gleba YY, Raskin I (1999) Use of plant roots for phytoremediation and molecular farming. P. Natl. Acad. Sci. USA. 96: 5973-7.

Guevara-Garcia A, Lopez-Bucio J, Herrera-Estrella L (1999) The mannopine synthase promoter contains vectorial cis-regulatory elements that act as enhancers and silencers. Mol. Gen. Genet. 262: 608-17.

Gupta G, Rajendran V, Atanassov P (2003) Laccase biosensor on monolayer‐modified gold electrode. Electroanal. 15:1577-83.

Gutiérrez SP, Saberianfar R, Kohalmi SE, Menassa R (2013) Protein body formation in stable transgenic tobacco expressing elastin-like polypeptide and hydrophobin fusion proteins. BMC Biotechnol. 13: 40.

Hirai H, Kashima Y, Hayashi K, Sugiura T, Yamagishi K, Kawagishi H, Nishida T (2008) Efficient expression of laccase gene from white-rot fungus Schizophyllum commune in a transgenic tobacco plant. FEMS. Microbiol. lett. 286: 130-5.

Hood EE, Bailey MR, Beifuss K, Magallanes‐Lundback M, Horn ME, Callaway E, Drees C, Delaney DE, Clough R, Howard JA (2003) Criteria for high‐level expression of a fungal laccase gene in transgenic maize. Plant. Biotechnol. J. 1:129-40.

Kiiskinen LL (2005) Characterization and heterologous production of a novel laccase from Melanocarpus albomyces. VTT Technical Research Centre of Finland.

Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 227: 680.

Lee LY, Kononov ME, Bassuner B, Frame BR, Wang K, Gelvin SB (2007) Novel plant transformation vectors containing the superpromoter. Plant. Physiol. 145: 1294-300.

Leuzinger K, Dent M, Hurtado J, Stahnke J, Lai H, Zhou X, Chen Q (2013) Efficient agroinfiltration of plants for high-level transient expression of recombinant proteins. J. Vis. Exp: JoVE. 77.

Li J, Yuan H, Yang J (2009) Bacteria and lignin degradation. Frontiers of biology in China. 4: 29-38.

Linthorst HJ, Van Loon LC, van Rossum CM, Mayer A, Bol JF, van Roekel JS, Meulenhoff EJ, Cornelissen BJ (1990) Analysis of acidic and basic chitinases from tobacco and petunia and their constitutive expression in transgenic tobacco. Mol. Plant-Microbe. Interact. 3:252-8.

Madhavi V, Lele SS (2009) Laccase: properties and applications. BioResources. 4: 1694-717.

Maestre-Reyna M, Liu WC, Jeng WY, Lee CC, Hsu CA, Wen TN, Wang AH, Shyur LF (2015) Structural and functional roles of glycosylation in fungal laccase from Lentinus sp. PLOS One. 10: e0120601.

Min SR, Davarpanah SJ, Jung SH, Park YI, Liu JR, Jeong WJ (2015) An episomal vector system for plastid transformation in higher plants. Plant. Biotechnol. Rep. 9: 443-9.

Montazer M, Sadeghian A (2008) Application of laccases with cellulases on denim for clean effluent and repeatable biowashing. J. Appl. Polym. Sci. 110: 3121-9.

Ñopo L, Woffenden BJ, Reed DG, Buswell S, Zhang C, Medina-Bolivar F (2012) Super-promoter: TEV, a powerful gene expression system for tobacco hairy roots. In Recombinant Gene Expression (pp. 501-526). Humana Press, Totowa, NJ.

Pannu JS, and Kapoor RK (2014) Microbial laccase: a mini review on their production, purification and applications, Int. J. Pharm. Arch. 3, 528- 536.

Parand M, Ranaei SS, Yamchi A (2015) cloning and extracellular expression of laccase enzyme from bacillus of Iranian hot spring into yeast cell Pichia pastoris. Modern Genet. J.1-10.

Passarini MR, Ottoni CA, Santos C, Lima N, Sette LD (2015) Induction, expression and characterisation of laccase genes from the marine-derived fungal strains Nigrospora sp. CBMAI 1328 and Arthopyrenia sp. CBMAI 1330. AMB Express. 5: 19.

Payne G, Ahl P, Moyer M, Harper A, Beck J, Meins F, Ryals J (1990) Isolation of complementary DNA clones encoding pathogenesis-related proteins P and Q, two acidic chitinases from tobacco. P. Natl. A. Sci. USA. 87: 98-102.

Petersen TN, Brunak S, von Heijne G, Nielsen H (2011) SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat. Methods. 8: 785.

Restrepo MA, Freed DD, Carrington JC (1990) Nuclear transport of plant potyviral proteins. Plant. Cell. 2: 987-98.

Rezaei S, Tahmasbi H, Mogharabi M, Ameri A, Forootanfar H, Khoshayand MR, Faramarzi MA (2015) Laccase-catalyzed decolorization and detoxification of Acid Blue 92: statistical optimization, microtoxicity, kinetics, and energetics. J. Environ. Health. Sci. 13: 31.

Sadeghian A, Montazer M (2009) The effect of cellulase and laccase enzymes on denim color. J. Color. Sci. Technol. 3: 53-64.

Scheller J, Gührs KH, Grosse F, Conrad U (2001) Production of spider silk proteins in tobacco and potato. Nat. Biotechnol. 19: 573.

Shraddha, Shekher R, Sehgal S, Kamthania M, Kumar A (2011) Laccase: microbial sources, production, purification, and potential biotechnological applications. Enzyme. Res. 2011.

Soden DM, O’callaghan J, Dobson AD (2002) Molecular cloning of a laccase isozyme gene from Pleurotus sajor-caju and expression in the heterologous Pichia pastoris host. Microbiol. 148: 4003-14.

Sonoki T, Kajita S, Ikeda S, Uesugi M, Tatsumi K, Katayama Y, Iimura Y (2005) Transgenic tobacco expressing fungal laccase promotes the detoxification of environmental pollutants. Appl. Microbiol. Biotechnol. 67: 138-42.

Stokes KD, McAndrew RS, Figueroa R, Vitha S, Osteryoung KW (2000) Chloroplast division and morphology are differentially affected by overexpression of FtsZ1 and FtsZ2 genes in Arabidopsis. Plant. physiol. 24: 1668-77.

Umbeck P, Johnson G, Barton K, Swain W (1987) Genetically transformed cotton (Gossypium hirsutum L.) plants. Nat. Biotechnol. 5: 263.

UniProt Consortium (2018) UniProt: the universal protein knowledgebase. Nucleic acids Res. 46: 2699.

Virk AP, Sharma P, Capalash N (2012) Use of laccase in pulp and paper industry. Biotechnol. Prog. 28:21-32.

Xu L, Zhu M, Chen X, Wang H, Zhang G (2015) A novel laccase from fresh fruiting bodies of the wild medicinal mushroom Tricholoma matsutake. Acta Biochim. Pol. 62.

Yoshida, H (1883). Chemistry of Lacquer (Urbz] part 1. J. Chem. Sol. 43: 472-486.