Cloning and Characterization of a Tissue Specific Promoter GluB-1 from Nipponbare by Transformation in Rice

Abstract

Tools of plant biotechnology can be applied as a routine procedure for improving rice grain quality or production of desired recombinant proteins in the rice seeds. For this purpose, endosperm specific promoter is a useful choice as desired gene expression would take place only in rice seed and not in root or shoot. Also, rice seed has become an attractive bioreactor for the production of recombinant proteins compared to other cereals. Glutelins are the most abundant storage proteins in rice grain which constitute up to 80% of the total protein content. The promoter region of GluB-1, one of the glutelin genes in rice, has been used as a model to study the regulation of seed-storage protein accumulation. In this study the upstream region (~2.4 kb) of the GluB-1 gene was amplified from the genomic DNA of the Nipponbare cultivar of Oryza sativa (japonica group) and then cloned successively into an entry and promoter‐characterization binary destination vector having the reporter gene β‐glucuronidase (GUS) by applying Gateway Technology. Three plants generated by Agrobacterium mediated tissue culture were confirmed by PCR with GluB-1 specific primer. In the transformed plants, histochemical GUS assay showed no expression in the root and shoot but was prominent in the endosperm of the T1 seeds. Therefore this 2.4 kb promoter from Nipponbare rice can be successfully used to improve rice grain quality or express stable recombinant proteins in rice seeds for therapeutic purposes.