ORIGINAL PAPER Transgenic chickpea expressing a recombinant human a 1 -proteinase inhibitor (a 1 -PI) driven by a seed-specific promoters from the common bean Phaseolus vulgaris (L.) Smrati Mishra • Shweta Jha • Rani Singh • Sonam Chaudhary • Indraneel Sanyal • Devindra Vijay Amla Received: 14 February 2013 / Accepted: 25 May 2013 Ó Springer Science+Business Media Dordrecht 2013 Abstract The 5 0 regulatory sequences of seed-storage pro- tein 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 b-glucuronidase (uidA) and recombinant human a 1 -protein- ase inhibitor (a 1 -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 b-glucuronidase revealed that phaseolin promoter with arcelin 5 0 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 b-glucuronidase and a 1 -PI genes, driven by the phaseolin promoter are stable in chickpea and their accumulation is confined into the seed tissues. Analysis of a 1 -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 Wes- tern immunoblotting. Results of DAC-ELISA showed that modified a 1 -PI gene driven by the phaseolin promoter with arcelin 5 0 UTR showed maximum accumulation of a 1 -PI up to 1.95 lg mg -1 of fresh weight in seeds. Biological activity of recombinant a 1 -PI, confirmed by elastase inhibition assays exhibiting 0.264 lg mg -1 of active protein. Western immu- noblot analysis confirmed the molecular mass of plant- expressed recombinant a 1 -PI of molecular mass *50 kDa in the seeds of transgenic chickpea. Keywords a 1 -Proteinase inhibitor Á Phaseolin promoter Á Chickpea Á KDEL sequence Á Seed-specific expression Á uidA Abbreviations a 1 -PI a 1 -Proteinase inhibitor DAC-ELISA Direct antigen coating-enzyme linked immunosorbent assay PPE Porcine pancreatic elastase TSP Total soluble protein UTR Untranslated region GUS b-Glucuronidase MUG 4-Methyl umbelliferyl glucuronide Introduction Chickpea is an important pulse crop of the Indo-Mexican, Australian and Mediterranean regions. However, chickpea is regarded as an orphan legume crop, although grown in 24 % acreage of total legume cultivation around the globe (Hire- math et al. 2011). Chickpea alone constitutes about 20–26 % of the total global consumption of grain legumes as food and feed. Seeds, especially those of legumes, besides being a source of dietary protein can also be used as a reservoir for heterologous proteins. Many different platforms for the pro- duction of recombinant proteins in plants have been described S. Mishra Á S. Jha Á R. Singh Á S. Chaudhary Á I. Sanyal Á D. V. Amla (&) Plant Transgenic Lab, CSIR-National Botanical Research Institute, Rana Pratap Marg, P.O. Box 436, Lucknow 226 001, India e-mail: dvamla@rediffmail.com; dvamla@nbri.res.in Present Address: S. Jha Department of Botany, Jai Narain Vyas University, Jodhpur 342 001, India 123 Plant Cell Tiss Organ Cult DOI 10.1007/s11240-013-0336-9