Skip to main content
SpringerLink
Log in

Designing a new marker-free and tissue-specific platform for molecular farming applications

  • Original Article
  • Published:
Journal of Plant Biochemistry and Biotechnology Aims and scope Submit manuscript

Abstract

Tissue-specific expression of recombinant proteins in transgenic plants is of particular importance. Production of tissue-specific transplastomic plants is challenging due to lack of tissue-specific promoter in the plastome. The objective of the present study was to produce a transgenic platform enhancing tissue-specific transgene expression in the green organs of Nicotiana tabacum Cv. Jafarabadi. A bacteria/plant hybrid sigma factor sequence (hsig) under control of the PEP Carboxylase promoter (PEPC) was designed and cloned in a plasmid construct without selectable marker gene and finally transformed into the nucleus genome by Agrobacterium. Transformation was confirmed by PCR, RT-PCR, southern blot and real-time PCR analysis. The resulted platform was re-transformed using a plastid construct pFNGi containing gfp (reporter gene) and neo genes (a kanamycin resistance selectable marker gene). Expression of GFP was assayed under Epifluorescence microscopy. The results showed that the constructed platform was functional, and the GFP was expressed. Finally, it can be concluded that application of the hybrid sigma factor (hsig) under control of a tissue-specific promoter could be a suitable platform for tissue-specific expression of plastome integrated transgenes, and also it is proposed to use other more safer marker genes for plastid transformation instead of the antibiotic resistance gene.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2

Similar content being viewed by others

Abbreviations

FR:

Flanking region

PEP:

Plastid-encoded RNA polymerase

PMF:

Plant molecular farming

Sig:

Sigma factor

References

  • Ahmed I, Islam M, Arshad W, Mannan A, Ahmad W, Mirza B (2009) High-quality plant DNA extraction for PCR: an easy approach. J Appl Genet 50:05–107

    Article  Google Scholar 

  • An G, Wastson BD, Chiang CC (1986) Transformation of tobacco, tomato, potato and Arabidopsis thaliana using a binary Ti vector system. Plant Physiol 81301–305

  • Badri MA, Rivard D, Coenen K, Michaud D (2009) Unintended molecular interactions in transgenic plants expressing clinically useful proteins: the case of bovine aprotinin traveling the potato leaf cell secretary pathway. Proteomics 9:746–756

    Article  CAS  PubMed  Google Scholar 

  • Breyer D, Goossens M, Herman P, Sneyers M (2009) Biosafety considerations associated with molecular farming in genetically modified plants. J Med Plants Res 3(11):825–838

    Google Scholar 

  • Breyer D, De Schrijver A, Goossens M, Pauwels K and Herman P (2012) Biosafety of Molecular Farming in Genetically Modified Plants, in Molecular Farming in Plants: Recent Advances and Future Prospects. Springer pp 259–274

  • Buhot L, Horvath E, Medgyesy P, Lerbs-Mache S (2006) Hybrid transcription system for controlled plastid transgene expression. Plant J 46:7000–7007

    Article  Google Scholar 

  • Cui L, Chen Y, Shen G, Zhao L, Tang K (2011) Expression of bioactive Thymosin alpha 1 (Tα1) in marker-free transgenic lettuce (lactuca sativa). Plant Mol Biol Report 29(2):466–472

    Article  CAS  Google Scholar 

  • Garcia-Almodovar RC, Petri C, Padilla IMG (2013) Combination of site-specific recombination and a conditional selective marker gene allows for the production of marker-free tobacco plants. Plant Cell Tissue Organ Cult. doi:10.1007/s11240-013-0396-x

    Google Scholar 

  • Ghareyazie B, Alinia F, Menguito CA, Rubia LG, Palma GM, Liwanag EA, Cohen MB, Khush GS, Bennett J (1997) Enhanced resistance to two stem borers in an aromatic rice containing a synthetic cryIA(b) Gene. Mol Breed 3:401–414

    Article  CAS  Google Scholar 

  • Grevich J, Daniell H (2005) Chloroplast genetic engineering: recent advances and future perspectives. CRC Crit Rev Plant Sci 24:83–107

    Article  CAS  Google Scholar 

  • Koop H, Herz S, Golds TJ, Nickelsen J (2007) The genetic transformation of plastids. Cell Mol Biol Plastids Curr Genet 19:457–510

    Article  CAS  Google Scholar 

  • Lutz KA, Azhagiri AK, Tungsuchat-Huang T, Maliga PA (2007) guide to choosing vectors for transformation of the plastid genome of higher plants. Plant Physiol 145:1201–1210

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Lysenko EA (2007) Plant sigma factors and their role in plastid transcription. Plant Cell Rep 26(2007):845–859

    Article  CAS  PubMed  Google Scholar 

  • Magee AM, Kavanagh TA (2002) Plastid genes transcribed by the nucleus-encoded plastid RNA polymerase show increased transcript accumulation in transgenic plants expressing a chloroplast-localized phage T7 RNA polymerase. J Exp Bot 53:2341–2349

    Article  CAS  PubMed  Google Scholar 

  • Magee AM, Coyne S, Murphy D, Horvath EM, Medgyesy P, Kavanagh TA (2004) T7 RNA polymerase-directed expression of an antibody fragment transgene in plastids causes a semi-lethal pale-green seedling phenotype. Transgenic Res 13:325–337

    Article  CAS  PubMed  Google Scholar 

  • Magee AM, MacLean D, Gray JC, Kavanagh TA (2007) Disruption of essential plastid gene expression caused by T7 RNA polymerase-mediated transcription of plastid transgenes during early seedling development. Transgenic Res 4:415–428

    Article  Google Scholar 

  • McBride KE, Schaaf DJ, Daley M, Stalker DM (1994) Controlled expression of plastid transgenes in plants based on a nuclear DNA-encoded and plastid-targeted T7 RNA polymerase. Proc Natl Acad Sci U S A 91:7301–7305

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Menand B, Marechal-Drouard L, Sakamoto W, Dietrich A, Wintz H (1998) A single gene of chloroplast origin codes for mitochondrial and chloroplastic methionyl-tRNA synthetase in Arabidopsis thaliana. Proc Natl Acad Sci U S A 95:11014–11019

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Murashige T, Skoog FA (1962) revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497

    Article  CAS  Google Scholar 

  • Obembe OO, Popoola JO, Leelavathi S, Reddy SV (2011) Advances in plant molecular farming. Biotechnol Adv 29:210–222

    Article  PubMed  Google Scholar 

  • Oikawa K, Fujiwara M, Nakazato E, Tanaka K, Takahashi H (2000) Characterization of two plastid sigma factor, SigA1 and SigA2, that mainly function in mature chloroplasts in Nicotiana tabacum. Gene 261:221–228

    Article  CAS  PubMed  Google Scholar 

  • Puchta H (2003) Marker-free transgenic plants. Plant Cell Tissue Organ Cult 74:123–134

    Article  CAS  Google Scholar 

  • Rosales-Mendoza S, Rubio-Infante N, Monreal-Escalante E, Govea-Alonso D, García-Hernández AL, Salazar-Gonzalez JA, González-Ortega O, Paz-Maldonado LMT, Moreno-Fierros L (2014) Chloroplast expression of an HIV envelop-derived multiepitope protein: towards a multivalent plant-based vaccine. Plant Cell Tissue Organ Cult 116(1):111–123

    Article  CAS  Google Scholar 

  • Salehi Jouzani G, Tohidfar M, (2013) Plant molecular farming: future prospects and biosafety challenges. biosafety, omicsgroup.org

  • Sambrook J, Russel DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, New York

    Google Scholar 

  • Sparrow P, Broer I, Hood EE, Eversole K, Hartung F, Schiemann J (2013) Risk assessment and regulation of molecular farming - a comparison between Europe and US. Curr Pharm Design 19

  • Spok A, Twyman RM, Fischer R, Ma JK, Sparrow PA (2008) Evolution of a regulatory framework for pharmaceuticals derived from genetically modified plants. Trends Biotechnol 26:506–517

    Article  PubMed  Google Scholar 

  • Svab Z, Maliga P (1993) High-frequency plastid transformation in tobacco by selection for a chimeric aadA gene. Proc Natl Acad Sci USA 90:913–917

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Verma D, Samson NP, Koya V, Daniell H (2008) A protocol for expression of foreign genes in chloroplasts. Nat Protoc 3:739–758

    Article  CAS  PubMed  Google Scholar 

  • Wohlleben W, Arnold W, Broer I, Hillemann D, Strauch E, Pühler A (1988) Nucleotide-sequence of the phosphinothricin N-acetyltransferase gene from Streptomyces viridochromogenes-Tu494 and its expression in Nicotiana tabacum. Gene 70:25–37

    Article  CAS  PubMed  Google Scholar 

  • Yuan JS, Burris J, Stewart N, Mentewab A, Stewart CN (2007) Statistical tools for transgene copy number estimation based on real-time PCR. BMC Bioinforma 8:S6

    Article  Google Scholar 

Download references

Acknowledgment

This project was financially supported by Agricultural Biotechnology Research Institute of Iran (ABRII). We would like to thank all our colleagues in the Tissue Culture and Gene Transformation Department of ABRII for their kind support and assistance during the course of this investigation.

Author information

Authors and Affiliations

  1. Tissue Culture and Gene Transformation Department, Agricultural Biotechnology Research Institute of Iran (ABRII), Mahdasht Road, 31535-1897, Karaj, Iran

    Motahhareh Mohsenpour & Masoud Tohidfar

  2. Sari Agricultural Sciences and Natural Resources University, km 9 Khazarabad road, Sari Mazandaran, Iran

    Nadali Babaeian Jelodar

  3. Microbial Biotechnology and Biosafety Department, Agricultural Biotechnology Research Institute of Iran (ABRII), Karaj, Iran

    Gholamreza Salehi Jouzani

Authors
  1. Motahhareh Mohsenpour
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Masoud Tohidfar
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Nadali Babaeian Jelodar
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Gholamreza Salehi Jouzani
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Masoud Tohidfar.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Fig. 1

(DOCX 41 kb)

Supplementary Fig. 2

(DOCX 486 kb)

Supplementary Fig. 3

(DOCX 176 kb)

Supplementary Fig. 4

(DOCX 359 kb)

Supplementary Fig. 5

(DOCX 107 kb)

Supplementary Fig. 6

(DOCX 614 kb)

Supplementary Fig. 7

(DOCX 40 kb)

Supplementary Fig. 8

(DOCX 85 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Mohsenpour, M., Tohidfar, M., Jelodar, N.B. et al. Designing a new marker-free and tissue-specific platform for molecular farming applications. J. Plant Biochem. Biotechnol. 24, 433–440 (2015). https://doi.org/10.1007/s13562-014-0294-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s13562-014-0294-2

Keyword

Search

Navigation