Molecular Cloning and Partial Characterization of Granule-Bound Starch Synthase (GBSS) Gene Promotors from Potato Cultivars

Abstract

Though each and every cell of a multicellular organism, whether plant or animal, contains the same set of genes, yet every cell is differentiated to perform a specific function, which leads to division of labour in multicellular organisms. There are many different cell types in a multicellular organism. These different cell types arise due to differential gene expression and regulation of gene expression by various regulatory regions present upstream to these genes. One of these regulatory regions is known as the promoter. Eukaryotic promoters are short DNA segments, which are responsible for regulation of gene expression. The present thesis work deals with molecular cloning studies on the granule-bound starch synthase (GBSS) gene promoter in Indian potato cultivars. Four Indian potato cultivars, viz., Kufri Chipsona-1, Kufri Chipsona-2, Kufri Jyoti and Kufri Chandramukhi, were used for this study. Total DNA was isolated from young and tender micropropagated plantlets using a rapid and simple method and its quality was checked. Two forward primers and one reverse primer were designed based on the GBSS gene sequence and used in two combinations for PCR, keeping the reverse primer common. In case of first primer pair, it was able to amplify one DNA fragment (size ~1.3 kb) from Kufri Chipsona-1 and two DNA fragments from Kufri Chipsona-2, Kufri Jyoti and Kufri Chandramukhi (size ~1.3 kb and 1.4 kb). It is likely that the primer has amplified two different isoforms of GBSS in these cultivars. In case of second primer pair, only one DNA fragment was amplified in all the four cultivars (size ~0.9 kb). It implies that the region amplified by second primer pair is fairly conserved. The PCR-amplified DNA fragments using first and second primer pair in Kufri Chipsona-1 and Kufri Chandramukhi were polished by Klenow enzyme treatment and cloned into the SmaI site of the pUC19 vector. The recombinant clones were checked and characterised using PCR and restriction analyses. These data strongly suggest that the clones generated are GBSS-specific, corresponding to the 5'-region of the gene. The cloned promoters need to be further characterised functionally for determining their strength and identifying the regulatory regions present in their sequences. The cloned and characterised promoters can be used for driving heterologous gene expression as well as for plastid targeting of recombinant proteins.