مقایسه بیان موقت پروتیین HDA19 در برگ‌های Nicotiana tabacum و Nicotiana bentamiana

نوع مقاله: علمی پژوهشی

نویسندگان

1 دانشجوی دکتری گروه اصلاح نباتات و بیوتکنولوژی، دانشگاه علوم کشاورزی و منابع طبیعی ساری، ساری، ایران

2 دانشیار گروه اصلاح‌نباتات و بیوتکنولوژی، دانشگاه علوم کشاورزی و منابع طبیعی ساری، ساری، ایران

3 موسسه بیوشیمی بیماری‌های گیاهی، مرکز هلم هولتز مونیخ، مرکز تحقیق آلمان برای سلامت محیطی، نوهربرگ، 85764، آلمان

4 استادیار گروه اصلاح نباتات و بیوتکنولوژی، دانشگاه علوم کشاورزی و منابع طبیعی ساری، ساری، ایران

چکیده

اخیراً بیان موقت ژن به‌منظور فراهم سازی روش‌های بسیار سریع ارزیابی بافت‌های گیاهی به‌عنوان یک منبع تولید پروتیین در مقایسه با روش وقت گیر و پرهزینه تراریختی پایدار گیاهان گسترش یافته است. مطالعه حاضر بیان موقت پروتیین HDA19 را در دو گونه از گیاه tobacco (Nicotiana tabacum وNicotiana bentamiana) نشان می‌دهد. برای این منظور پرایمرهای اختصاصی برای واکنش زنجیره ای پلی مراز طراحی و برای همسانه سازی ژن HDA19 در وکتور بیانی pB2GW7 به‌کار رفتند. سازه نوترکیب به Agrobacterium tumefaciens strain GV3101 انتقال یافت سپس برای تراریختی موقت گیاهان tobacco از طریق اگروباکتریوم استفاده شد. در نهایت وجود ژن هدف در لاین‌های تراریخت اگروباکتریوم از طریق کلونی PCR تأیید گردید. از طرفی بیان پروتیین در لاین‌های تراریخت توسط روش دات بلات و الایزا تأیید شد. اگرچه روش دات بلات و SDS-PAGE حاکی از تراریختی هر دو رقم بود ولی روش الایزا تولید پروتیین نوترکیب در گیاه تراریخت Nicotiana tabacum را تایید کرد که 400 میکروگرم در گرم وزن بافت تر برگ گیاه بود. با توجه به یافته‌های فوق این گیاه میزبان مناسبی برای تولید HDA19 نوترکیب، نماینده‌ای از پروتیین‌های هیستون داستیلاز، نسبت به Nicotiana bentamiana می‌باشد.

کلیدواژه‌ها

موضوعات


عنوان مقاله [English]

Comparison of Transient HDA19 Protein Expression in Leaves of Nicotiana tabacum and Nicotiana bentamiana

نویسندگان [English]

  • Maryam Jamshidnia 1
  • Sayed Kamal Kazemitabar 2
  • Christian Lindermayr 3
  • Hamid Najafi Zarini 4
1 Ph.D. Student, Department of Plant Breeding & Biotechnology, Sari Agricultural Sciences and Natural Resources University (SANRU), Sari, Iran
2 Associate Professor, Department of Plant Breeding & Biotechnology, Sari Agricultural Sciences and Natural Resources University (SANRU), Sari, Iran
3 Institute of Biochemical Plant Pathology, Helmholtz Zentrum München, German Research Center for Environmental Health, Neuherberg, 85764, Germany
4 Assistant Professor, Department of Plant Breeding & Biotechnology, Sari Agricultural Sciences and Natural Resources University (SANRU), Sari, Iran
چکیده [English]

Recently, transient gene expression has been developed to provide a more rapid means of assessing plant tissues as a protein production platform without the labor-intensive and time-consuming process of generating stably transformed transgenic plants. This study reports the expression of HDA19 gene in two species of tobacco plants (Nicotiana tabacum and Nicotiana bentamiana) by means of transient transformation. Specific primers were designed and used for PCR amplification and cloning of HDA19 gene in the plant expression vector pB2GW7. The recombinant construct was transferred into Agrobacterium tumefaciens strain GV3101, and was used for Agrobacterium mediated transformation of tobacco plants. The presence of the desired gene in transgenic lines was confirmed through colony PCR. The expression of the protein in transgenic lines was confirmed by immune-dot blot assay and ELISA. Although the transformation of the two species was confirmed by immune-dot blot assay and SDS-PAGE, recombinant protein production in Nicotiana tabacum plants was confirmed by ELISA and it was estimated 400 µg per gram wet weight of tobacco leaves. According to the results, this species is the appropriate host for the production of recombinant HDA19, one of the histone deacetylases, rather than Nicotiana bentamiana.

کلیدواژه‌ها [English]

  • KEYWORDS: HDA19؛ Tobacco؛ Recombinant protein
  • Nicotiana tabacum
  • Nicotiana bentamiana
Ahangarzadeh S, Daneshvar MH, Rajabi-Memari H, Galehdari H, Alamisaied Kh (2012) Cloning, Transformation and Expression of Human Interfron α2b gene in tobacco (Nicotiana tabacum cv.xanthi). Jundishapur. J. Nat. Pharm. Prod. 7(3): 111-116.

Bergmann L (1960) Growth and division of single cells of higher plants in vitro. J. Gen. Physiol. 43(4):841-51.

Chen CY, Wu K, Schmidt W (2015) The histone deacetylase HDA19 controls root cell elongation and modulates a subset of phosphate starvation responses in Arabidopsis. Sci. Rep. 28(5)15708.

Choi SM, Song HR, Han SK, Han M, Kim CY, Park J, Lee YH, Jeon JS, Noh YS, Noh B (2012) HDA19 is required for the repression of salicylic acid biosynthesis and salicylic acid-mediated defense responses in Arabidopsis. Plant J. 71(1): 135–146.

De La Riva GA, González-Cabrera J, Vázquez-Padrón R, Ayra-Pardo C (1998) Agrobacterium tumefaciens: a natural tool for plant transformation. Electron. J. Biotechn. 1(3):24-25.

Fischer R, Vaquero C, Sack M, Drossard J, Emans N, Commandeur U (1999) Toward Molecular Farming in the future: transient protein expression in plants. Biotechnol. Appl. Biochem. 30:113-116.

Fischer R, Schillberg S (2004) Molecular farming: Plant-made pH pharmaceuticals and technical proteins. Weinheim: Wiley-VCH verlag GmbH & Co. KGaA.

Fong MP, Tian L, Chen ZJ (2006) Arabidopsis thaliana histone deacetylase 1 (AtHD1) is localized in euchromatic regions and demonstrates histone deacetylase activity in vitro. Cell Res. 16(5): 479-488.

Gil F, Brun A, Wigdorovitz A, Catala R, Martinez-Torrecuadrada JL, Casal I, Salinas J, Borca MV, Escribano JM (2001) High yield expression of a viral peptide vaccine in transgenic plants. FEBS Lett. 488:13-17.

Glozak MA, Seto E (2007) Histone deacetylases and cancer. Oncogene 26:5420–5432.

Habibi-Pirkoohi M, Malekzadeh-Shafaroudi S, Marashi H, Moshtaghi N, Nassiri M, Zibaee S (2014) Transient Expression of Foot and Mouth Disease Virus (FMDV) Coat Protein in Tobacco (Nicotiana tabacom) via Agroinfiltration. Iranian J. Biotech. 12(3): e1015.

Jamshidnia M, Kazemitabar SK, Lindermayr C, Najafi Zarini H (2016) Designing and construction pB2GW7 vector encoding HDA19 gene of Arabidopsis thaliana. In: Proceeding of the 2nd International and 14th Iranian Genetics Congress. Iran.

Kang TJ, Han SC, Jang MO, Kang KH, Jang YS, Yang MS (2004) Enhanced expression of B-subunit of Escherichia coli heat-labile enterotoxin in tobacco by optimization of coding sequence. Appl. Biochem. Biotechnol. 117(3): 175-187.

Kim KC, Lai Z, Fan B, Chen Z (2008) Arabidopsis WRKY38 and WRKY62 transcription factors interact with histone deacetylase 19 in basal defense. Plant Cell. 20: 2357-2371.

Krenek P, Samajova O, Luptovciak I, Doskocilova A, Komis G, Samaj J (2015) Transient plant transformation mediated by Agrobacterium tumefaciens: Principles, methods and applications. Biotechnol. Adv. 33 (6): 1024-1042.

Kumar G, Ganapathi T, Bapat V (2007) Production of hepatitis B surface antigen in recombinant plant systems: an update. Biotechnol. Prog. 23(3):532-539.

Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 227: 680-685.

Lauren B, Curtis R.A, Curtis R.W (2005) Comparison of transient protein expression in tobacco leaves and plant suspension culture. Biotechnol. Prog. 21: 946-952.

Leelavathi S, Reddy VS (2003) Chloroplast expression of His-tagged GUS-fusions: a general strategy to overproduce and purify foreign proteins using transplastomic plants as bioreactors. Mol. Breeding. 11(1): 49-58.

Lund P, Dunsmuir P (1992) A plant signal sequence enhances the secretion of bacterial ChiA in transgenic tobacco. Plant Mol. Biol. 18(1): 47-53.

Ma L, Lukasik E, Gawhnes F, Takken FLW (2012) The use of Agroinfiltration for transient expression of plant resistance and fungal effector proteins in Nicotiana benthamiana leaves. Melvin D, Bolton and Bart P.H.J. Thomma (eds.) Plant Fungal Pathogens: Methods and Protocols, Methods in Molecular Biology, vol. 835, Springer Science+Business Media.

Mohammadzadeh S, Khabiri A, Roohvand F, Memarnejadian A, Salmanian AH, Ajdary S, Ehsani P (2014) Enhanced-Transient Expression of Hepatitis C Virus Core Protein in Nicotiana tabacum, a Protein With Potential Clinical Applications. Hepat. Mon. 14(11): e20524.

Rajabi Memari H, Ramanan RN, Ariff AB (2010) Comparison of expression systems for the production of human interferon-α2b. Cent. Eur. J. Biol. 5(4): 446-455.

Sala F, Rigano M, Barbante A, Basso B, Walmsley AM, Castiglione S (2000) Vaccine antigen production in transgenic plants: strategies, gene constructs and perspectives. Vaccine. 21:803-808.

Sambrook J, Russell DW (2006) SDS-Polyacrylamide Gel Electrophoresis of Proteins. CSH Protoc.

Soltanmohammadi B, Jalali-Javaran M, Rajabi-Memari H, Mohebodini M (2014) Cloning, Transformation and Expression of Proinsulin Gene in Tomato (Lycopersicum esculentum Mill.). Jundishapur. J. Nat. Pharm. Prod. 9(1): 9-15.

Steffany A, Bennett L, Roberts DCS (2003) Analysis of protein expression in brain tissue by ELISA. Series Methods in Molecular Medicine. Vol. 79, Drugs of Abuse: Neurological Reviews and Protocols. Wang JQ, (eds.) Humana Press Inc., Totowa, NJ, pp 283-295.

Streatfield SJ, Jilka JM, Hood EE, Turner DD, Bailey MR, Mayor JM, Woodard SL, Beifuss KK, Horn ME, Delaney DE, Tizard IR, Howard JA (2001) Plant-based vaccines: unique advantages. Vaccine. 19(17-19): 2742-2748.

Wang S, Guo AY, Zheng WJ, Zhang Y, Qiao H, Kennedy IR (2007) Development of ELISA for the determination of transgenic Bt-cottons antibodies against Cry1Ac protein from Bacillus thuringiensis HD-73. Eng. Life Sci. 7(2): 1-7.

Zhou C, Zhang L, Duan J, Miki B, Wu K (2005) histone deacetylase 19 is involved in jasmonic acid and ethylene signaling of pathogen response in Arabidopsis. Plant Cell. 17: 1196–1204.