Abstract
The 5′ regulatory sequences of seed-storage protein genes, arcelin 5-I (Arc), phaseolin (Phas) of common bean (Phaseolus vulgaris L.) and legumin (Leg) gene of Cicer arietinum (L.) were evaluated for seed-specific expression of β-glucuronidase (uidA) and recombinant human α1-proteinase inhibitor (α1-PI) in transgenic chickpea. Histochemical assay of transformed plants developed with seed-specific promoters, showed GUS expression in seeds and not in other plant tissues. Fluorometric assay of β-glucuronidase revealed that phaseolin promoter with arcelin 5′ UTR (pPAG) resulted into maximum GUS activity in seeds, followed by somatic tissues and minimal expression in leaves. The expression profile of GUS driven by different seed-specific promoters in chickpea, are in order phaseolin > arcelin > legumin respectively. RT-PCR analysis confirmed that transcripts of β-glucuronidase and α1-PI genes, driven by the phaseolin promoter are stable in chickpea and their accumulation is confined into the seed tissues. Analysis of α1-PI expression in seeds of transgenic chickpea was performed by direct antigen coating-enzyme linked immunosorbent assay (DAC-ELISA), residual porcine pancreatic elastase activity assay and Western immunoblotting. Results of DAC-ELISA showed that modified α1-PI gene driven by the phaseolin promoter with arcelin 5′ UTR showed maximum accumulation of α1-PI up to 1.95 μg mg−1 of fresh weight in seeds. Biological activity of recombinant α1-PI, confirmed by elastase inhibition assays exhibiting 0.264 μg mg−1 of active protein. Western immunoblot analysis confirmed the molecular mass of plant-expressed recombinant α1-PI of molecular mass ~50 kDa in the seeds of transgenic chickpea.
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Abbreviations
- α1-PI:
-
α1-Proteinase inhibitor
- DAC-ELISA:
-
Direct antigen coating-enzyme linked immunosorbent assay
- PPE:
-
Porcine pancreatic elastase
- TSP:
-
Total soluble protein
- UTR:
-
Untranslated region
- GUS:
-
β-Glucuronidase
- MUG:
-
4-Methyl umbelliferyl glucuronide
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Acknowledgments
Authors express their grateful thanks to Dr. C. S. Nautiyal, Director, CSIR-National Botanical Research Institute for providing infrastructural support at CSIR-NBRI, Lucknow. We thank Dr. T. C. Hall, Institute of Developmental and Molecular Biology and Department of Biology, Texas A&M University for providing plasmid −1470 +34 PHAS–GUS-3′ +pUC and Dr. Ann Depicker, Department of Applied Biological Sciences, Laboratory of Plant Genetics, Universiteit Brussels, Belgium, for providing plasmid pbeta-phas-G4 and pParc5I-G4, Dr. S. V. Sawant, Scientist, Plant Molecular Biology Lab, CSIR-NBRI, Lucknow, for providing the gene construct with legumin promoter. We thankfully acknowledge Council of Scientific and Industrial Research, New Delhi for providing research fellowships to SM and SJ and University Grants Commission, New Delhi to RS. We appreciate the generous supply of chickpea seeds by Dr. Dheer Singh, Scientist, GBPUAT, Pantnagar. This work was carried out under the CSIR-NBRI In-house project OLP 0031.
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Mishra, S., Jha, S., Singh, R. et al. Transgenic chickpea expressing a recombinant human α1-proteinase inhibitor (α1-PI) driven by a seed-specific promoters from the common bean Phaseolus vulgaris (L.). Plant Cell Tiss Organ Cult 115, 23–33 (2013). https://doi.org/10.1007/s11240-013-0336-9
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DOI: https://doi.org/10.1007/s11240-013-0336-9