Transgenic chickpea expressing a recombinant human α1-proteinase inhibitor (α1-PI) driven by a seed-specific promoters from the common bean Phaseolus vulgaris (L.)

摘要

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.