Designing of polyoleosin-proinsulin fusion gene and its transformation to rapeseed (Brassica napus L.)

Document Type : Research Paper

Authors

1 M.Sc. Student, Department of Plant Production and Genetics, Agricultural Sciences and Natural Resources University of Khouzestan, Khouzestan, Iran.

2 Assistant Professors, Department of Plant Production and Genetics, Agricultural Sciences and Natural Resources University of Khouzestan, Khouzestan, Iran.

Abstract

Objective
The use of plant Oleosin gene is a recommended method for large-scale, easier and cheaper production of recombinant protein. Attachment of oleosin gene to target gene cause to accumulate recombinant protein on seed oil bodies surface. Recent researches indicate that application of tandem Oleosin Genes (polyoleosin) fused to the target gene to facilitate recombinant protein purification is more efficient than single Oleosin.
 
Materials and methods
In present research, we designed a synthetic fusion fragment containing 5´- Kozak sequence - His-tags, three Oleosin Genes, a proteolytic site for a peptidase enzyme, Proinsulin protein, and a tetranucleotide stop codon. This synthetic sequence was cloned in binary vector pBI121by enzymatic digestion and ligation method. Then for transformation of cotyledon and hypocotyl explants of Canola, confirmed recombinant construct is transformed to Agrobacterium LBA4404 strain. Probable transgenic shoots were regenerated on selective medium containing kanamycin after 4-6 weeks.
 
Results
The analysis of transgenic shoots at DNA level was performed by PCR and amplification of a 120-bp fragment indicate integration of fusion gene in transgenic plant genome. Finally, the Proinsulin gene transcription was also confirmed by RT- PCR.

Keywords


References
Alam S, Khaleda L, Mohammad Al-Forkan S (2013) Establishment of regeneration protocol for Canola (Brassica napus L.). Global J Sci Frontier Res 13, 7-11.
Bhat WW, Lattoo SK, Rana S et al. (2012) Efficient plant regeneration via direct organogenesis and Agrobacterium tumefaciens-mediated genetic transformation of Picrorhiza kurroa: an endangered medicinal herb of the alpine Himalayas. In Vitro Cell Develop Biol Plant 48, 295-303.
Banilas G, Daras G, Rigas S, Moloney MM (2011) Oleosin di-or tri-meric fusions with GFP undergo correct targeting and provide advantages for recombinant protein production. Plant Physiol Biochem 49, 216-222.
Bhalla PL, Singh MB (2008) Agrobacterium-mediated transformation of based vectors. Plant Cell Rep 6, 321–325
Bhatla SC, Kaushik V, Yadav MK (2010) Use of oil bodies and oleosins in recombinant protein production and other biotechnological applications. Biotechnol Adv 28, 293-300.‏
Boothe J, Nykiforuk C, Shen Y et al. (2010) Seed‐based expression systems for plant molecular farming. Plant Biotechnol J 8, 588-606.‏
Boothe JG, Saponja JA, Parmenter DL (1997) Molecular farming in plants: oilseeds as vehicles for the production of pharmaceutical proteins. Drug Develop Res 42, 172-181.
Brown CM, Stockwell PA, Trotman CN, Tate WP (1990) The signal for the termination of protein synthesis in procaryotes. Nucleic Acids Res 18, 2079-2086
Brown TA (1990) Gene cloning and DNA analysis an introduction. Wiley-blackwell. P. 246.
Chaudhary S, Parmenter DL, and Moloney MM (1998). Transgenic Brassica carinata as vehicle for the production of recombinant proteins in seeds. Plant Cell Rep 17, 195–200.
Chen MX, Zheng SX, Yang YN et al. (2014) Strong seed-specific protein expression from the Vigna radiata storage protein 8SGα promoter in transgenic Arabidopsis seeds. J Biotechnol 174,.49-56.
Chen R, Zhang C, Yao B et al. (2013) Corn seeds as bioreactors for the production of phytase in the feed industry. J Biotechnol 165, 120-126.
Conley AJ, Joensuu JJ, Richman A, Menassa R (2011) Protein body-inducing fusions for high-level production and purification of recombinant proteins in plants. Plant Biotechnol J 9, 419–433.
Feng L, Chan WW, Roderick SL, Cohen DE (2000) High-level expression and mutagenesis of recombinant human phosphatidylcholine transfer protein using a synthetic gene: evidence for a C-terminal membrane binding domain. Biochem 39, 15399-15409.
Gustafsson C, Govindarajan S, Minshull J (2004) Codon bias and heterologous protein expression. Trends Biotechnol 22, 346-353.
Hiwasa-Tanase K, Nyarubona M, Hirai T et al. (2011) High-level accumulation of recombinant miraculin protein in transgenic tomatoes expressing a synthetic miraculin gene with optimized codon usage terminated by the native miraculin terminator. Plant Cell Reports 30, 113-124.
Holsters M, De Waele D, Depicker A et al. (1978) Transfection and transformation of Agrobacterium tumefaciens. Mol General Genet MGG 163, 181-187.
Joshi CP, Zhou H, Huang X, Chiang VL (1997) Context sequences of translation initiation codon in plants. Plant Mol Biol 35, 993-1001.
Kozak M (1999) Initiation of translation in prokaryotes and eukaryotes. Gene 234, 187-208.
Ling H (2007) Oleosin fusion expression systems for the production of recombinant proteins. Biologia 62, 119-123.
Nykiforuk CL, Boothe JG, Murray EW et al. (2006) Transgenic expression and recovery of biologically active recombinant human insulin from Arabidopsis thaliana seeds. Plant Biotechnol J 4, 77-85.
Ohta S, Mita S, Hattori T, Nakamura K (1990) Construction and expression in tobacco of a β-glucuronidase (GUS) reporter gene containing an intron within the coding sequence. Plant Cell Physiol 31, 805-813.
Ólafsdóttir G, Svansson V, Ingvarsson S et al. (2008) In vitro analysis of expression vectors for DNA vaccination of horses: the effect of a Kozak sequence. Acta Veterinaria Scandinavica 50, 1-7.
Parmenter DL, Boothe JG, Van Rooijen GJH et al. (1995) Production of biologically active hirudin in plant seeds using oleosin partitioning. Plant Mol Biol 29, 1167-1180.
Perlak FJ, Fuchs RL, Dean DA et al. (1991) Modification of the coding sequence enhances plant expression of insect control protein genes. Proceedings of the National Academy of Sciences 88, 3324-3328.
Richards E, Reichardt M, Rogers S (1994) Preparation of genomic DNA from plant tissue. Current Protocols Mol Biol 27, 2-3.
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning, Vol. 2: 14-9.
Schnell JA, Han S, Miki BL, Johnson DA (2010) Soybean peroxidase propeptides are functional signal peptides and increase the yield of a foreign protein. Plant Cell Reports 29, 987-996.
Scott RW, Winichayakul S, Roldan M et al. (2010) Elevation of oil body integrity and emulsion stability by polyoleosins, multiple oleosin units joined in tandem head‐to‐tail fusions. Plant Biotechnol J 8, 912-927.‏
Scott RW, Arcus VL, Roberts NJ (2012) Agriculture Victoria Services Pty Ltd and Agresearch Limited. Polyoleosins. U.S. Patent 8, 309-794.
Van Rooijen GJ, Moloney MM (1995) Plant seed oil-bodies as carriers for foreign proteins. Nature Biotechnol 13, 72-77.‏
Winichayakul S, Pernthaner A, Livingston S et al. (2012). Production of active single-chain antibodies in seeds using trimeric polyoleosin fusion. J Biotechnol 161, 407– 413.
Wu Y, Zhao D, Song L, Xu W (2009) Heterologous expression of synthetic chicken IFN-γ in transgenic tobacco plants. Biologia 64, 1115-1122.