Designing Primers with a Plant Signal Peptide to Enhance the Expression of GBA1 in Transgenic Soybean Plants
Main Article Content
Abstract
Transgenic plants offer advantages for the manufacture of recombinant proteins with terminal
mannose residues on their glycan chains. So plants are chosen as source of pharmaceutical products and for
the development of alternative expression systems to produce recombinant lysosomal enzymes. In the
present study the sequence of the natural cDNA encoding for the human lysosomal enzyme
glucocerebrosidase (GCD) was modified to enhance its expression in soybean plants. The glucocerebrosidase
gene signal peptide was substituted with that signal peptide for the Arabidopsis thaliana basic endochitinase
gene to support the co-translational translocation into the endoplasmic reticulum (ER), and the storage
vacuole. So, targeting signal from tobacco chitinase A, to facilitate GCD trafficking from the ER to the
storage vacuole, appropriate primers were designed containing both an ER and vacuolar targeting signals,
(VTS). Those primers were used for PCR amplification of the human GBA gene (Hu-GBA) gene from
constructed PGEM-GBA plasmid which was cloned in the plant expression vector pCAMBIA1304. The
resulted construct was transported in Agrobacterium tumefaciens strain LBA4404 and was used for
transformation of cotyledon explants. After 5-day of seedling, cotyledons were cut and used as explants.
After infection and co-cultivation, hygromycin B was added in selection media as a selective agent for the
transformants cotyledons. The presence of the Hu-GBA transgene in the genomes of transgenic plants was
determined by polymerase chain reaction PCR as a band of size1587 bp. The GBA mRNA expression in
modified soybean was detected by qRT-PCR compared with control GBA mRNA.
Received 13/11/2019
Accepted 5/1/2020
Published Online First 30/4/2021
Article Details
This work is licensed under a Creative Commons Attribution 4.0 International License.
How to Cite
References
Vojta L, Ljuma-Skupnjak L, Budimir A, Vukičević, S, Fulgosi H. Rapid transient expression of human granulocyte-macrophage colony-stimulating factor in two industrial cultivars of tobacco (Nicotiana tabacum L.) by agroinfiltration. Biotechnol Rep. 2015; (7): 81-86.
Kachoie E A, Behjatnia S A, Kharazmi S. Expression of Human Immunodeficiency Virus type 1 (HIV-1) coat protein genes in plants using cotton leaf curl Multan betasatellite-based vector. PLoS One. 2018Jan; 13(1): e0190403.
Owczarek B, Gerszberg A, Hnatuszko-Konka K. A Brief Reminder of Systems of Production and Chromatography-Based Recovery of Recombinant Protein Biopharmaceuticals. Biomed Res. Int. 2019 Jan; 2019:13. doi: 10.1155/2019/4216060.
Jennifer S, Richard S. Plant glyco-biotechnology. Semin. Cell Dev. Biol. 2018 August; 80: 133-141. https://doi.org/10.1016/j.semcdb.2017.07.005
Lim CY, Lee K J, Oh D B, Ko K. Effect of the developmental stage and tissue position on the expression and glycosylation of recombinant glycoprotein GA733-FcK in transgenic plants. Front. Plant Sci. 2015 Jan; 5: 778-788.
Abdul-Hammed M, Breiden B, Schwarzmann G, Sandhoff K. Lipids regulate the hydrolysis of membrane bound glucosylceramide by lysosomal β-glucocerebrosidase. J Lipid Res 2017 Mar; 58(3): 563-577. doi: 10.1194/jlr.M073510.
Santos D, Tiscornia G. Induced pluripotent stem cell modeling of Gaucher’s disease: what have we learned? Int J Mol Sci. 2017 Apr; 18(4): 888-905.
Migdalska‐Richards A, Schapira A H. The relationship between glucocerebrosidase mutations and Parkinson disease. J Neurochem. 2016 Oct; 139(1): 77–90. doi: 10.1111/jnc.13385
Berg-Fussman A, Grace M E, Ioannou Y, Grabowski G A. Human acid beta-glucosidase. N-glycosylation site occupancy and the effect of glycosylation on enzymatic activity. J. Biol. Chem. 1993Jul; 268(20): 14861-66.
Cox T M. Gaucher disease: clinical profile and therapeutic developments. Biologics: targets & therapy. 2010 Dec ; 4: 299-313.
Serratrice C, Carballo S, Serratrice J, & Stirnemann J. Imiglucerase in the management of Gaucher disease type 1: an evidence-based review of its place in therapy. Core evidence. 2016 Oct 11:37-47.
Furbish F S, Steer C J, Krett N L, Barranger J A. Uptake and distribution of placental glucocerebrosidase in rat hepatic cells and effects of sequential deglycosylation. Biochim Biophys Acta. 1981 Apr ;673(4):425-34.
Hudson L C, Garg R, Bost K L, Piller K J Soybean seeds: a practical host for the production of functional subunit vaccines Biomed Res. Int., 2014 Apr; 2014: 13. doi: 10.1155/2014/340804.
Lambirth K C, Whaley A M, Blakley I C, Schlueter J A, Bost K L, Loraine A E, et al. A comparison of transgenic and wild type soybean seeds: analysis of transcriptome profiles using RNA-Seq. BMC Biotechnol. 2015 Oct 1;15:89. doi: 10.1186/s12896-015-0207-z.
Lambirth K C, Whaley A M, Blakley I C, Schlueter J A, Bost K L, Loraine A E, et al. A comparison of transgenic and wild type soybean seeds: analysis of transcriptome profiles using RNA-Seq. BMC biotechnology.2015 Oct.15(1):89-105.
Hudson L C, Bost K L, Piller K J. Optimizing recombinant protein expression in soybean. In Soybean-Molecular Aspects of Breeding. IntechOpen. 2011 Apr. doi: 10.5772/15111.
Hudson L C, Garg R, Bost K L, Piller K J. Soybean seeds: a practical host for the production of functional subunit vaccines. BioMed research international. 2014. Apr (2014):13.
Jia Y, Yao X, Zhao M, Zhao Q, Du Y, Yu C, et al. Comparison of soybean transformation efficiency and plant factors affecting transformation during the Agrobacterium infection process. Int J Mol Sci. 2015 Aug 7;16(8):18522-43. doi: 10.3390/ijms160818522.
Kumari S, Krishnan V, Dahuja A, Vinutha T, Jolly M, Sachdev A A. Rapid method for optimization of Agrobacterium-mediated transformation of Indian soybean genotypes IJBB. 2016 Dec; 53: 218-226.
Rojas A, Lopez-Pazos S, Chaparro-Giraldo A. Screening of Colombian soybean genotypes for Agrobacterium mediated genetic transformation conferring tolerance to Glyphosate. Agron. Colomb. 2018Mar; 36(1): 24-34.
Güler-Gane G, Kidd S, Sridharan S, Vaughan T J, Wilkinson T C. Tigue N J .Overcoming the refractory expression of secreted recombinant proteins in mammalian cells through modification of the signal peptide and adjacent amino acids. PLoS One. 2016 May 19;11(5):e0155340.
Tannous A, Pisoni G B, Hebert D N, Molinari M. N-linked sugar-regulated protein folding and quality control in the ER. In Seminars in cell & developmental biology. Semin Cell Dev Biol. 2015 May;41:79-89.
Chen L, Cai Y, Liu X, Yao W, Guo C, Sun S, et al. Improvement of soybean Agrobacterium-mediated transformation efficiency by adding glutamine and asparagine into the culture media. Int J Mol Sci. 2018 Oct; 19(10): 3039-3055. doi: 10.3390/ijms19103039.
Jafari M, Daneshvar M H, Lotfi A. In vitro shoot proliferation of Passiflora caerulea L. via cotyledonary node and shoot tip explants. BTa. JBCBB. 2017 June ; 98(2):113-119. doi: 10.5114/bta.2017.68309.
Norkunas K, Harding R, Dale J, Dugdale B. Improving agroinfiltration-based transient gene expression in Nicotiana benthamiana. Plant Methods. 2018 Aug; 25(14):71-84.10.1186/s13007-018-0343-2.
Bhowmik D, Shekhar S, Cheng A Y, Long H, Tan G Z , Hoang T M , et al. Robust Genetic Transformation System to Obtain Non-chimeric Transgenic Chickpea. Front Plant Sci. 2019 Apr; 26(10): 524-537.
Guan P. Dancing with hormones: A current perspective of nitrate signaling and regulation in Arabidopsis. Front. Plant Sci. 2017 Sep; 8: 1697-1717. https://doi.org/10.3389/fpls.2017.01697
Ying L, Lei S, Ling Y, Rui G, Zheng q, Xiao h, et al. Hippocampal gene expression profiling in a rat model of functional constipation reveals abnormal expression genes associated with cognitive function. Neurosci. Lett., 2018 May; 675(14): 103-109. https://doi.org/10.1016/j.neulet.2018.03.023