Studies on the expression pattern of seed-specific napin promoter (BcNAI) in transgenic (Nicotiana tabacum L.) tobacco seeds.

Abstract

BcNA1 gene from Brassica napus, is highly expressed in developing seeds. The promoter of this gene, referred to as napin promoter, has been isolated and is shown to confer seed specific expression pattern in heterologous systems and thus has been used in many genetic engineering experiments aimed at expressing transgenes, especially those involved in fatty acid or TAG biosynthesis, in developing seeds. We were interested in studying the expression pattern of seed specific, napin promoter, in the developing seeds of tobacco. Therefore we were interested in isolating the napin promoter and validate the same in tobacco seeds. Promoter specific primers for napin were synthesized using the sequences available in the database and were used for amplifying the promoter using the genomic DNA isolated from Brassica napus. The obtained amplicon of the expected size (1752 bp) was cloned in T/A cloning vector and confirmed by restriction analysis as well as sequencing. The sequence data was subjected to BLAST analysis, which confirmed the cloning of napin gene. The isolated napin promoter was cloned in binary vector pCAMBIA1391Z upstream of uidA gene encoding β- glucuronidase and the confirmed clone was mobilized into Agrobacterium strain LBA4404. Putative transgenic tobacco shoots obtained using this vector were confirmed through PCR and standard GUS assay in the developing seeds of tobacco.