Cloning of seed specific promoter of gamma-kafirin gene from sorghum

BRAJESH KUMAR SINGH¹ and V. K. KHANNA²

¹Department of Molecular Biology and Genetic Engineering,

College of Basic Sciences and Humanities ²,

Department of Genetics and Plant Breeding,

College of Agriculture,

G. B. Pant University of Agriculture and Technology,

Pantnagar-263145 (U. S. Nagar, Uttarakhand)

ABSTRACT

Seeds of sorghum variety M 35-1 (R-99) were taken for this study. This variety was selected on the basis of its high kafirin content as per the data available. The long-term goal of the present study is the production of transgenic sorghum, which has soft endosperm with HMW-gs gene of gluten protein of the wheat. This makes the sorghum flour more palatable for making roti and also increases the dough quality and lysine content. One of the approaches to achieve this goal is to transform the HMW-gs genes with the seed specific g-kafirin gene promoter. Present investigation records the results of our efforts on trying to clone the g-kafirin gene promoter. The DNA extracted from M 35-1 (R-99) variety of sorghum were used to amplify the promoters and cloned in the pGEM vector. Genomic DNA was extracted from fresh etiolated young leaves of M 35-1 (R-99) sorghum variety. DNA sample were purified. Forward and reverse primers pairs were designed based on the published gene sequence of g-kafirin protein gene. These genomic DNAs were used as template to amplify the promoters with designed primers. The combination of the primers was also used to amplify the promoter region of different sizes. The promoters were amplified on a large scale and purified. For vector preparation the pGEM vector were isolated from pre-transformed E. coli. The identification of pGEM plasmid was confirmed by restriction analysis. The vector were prepared in large quantity and treated with RNAase A and phenol: chloroform: isoamyl treatment to get purified plasmid DNA. The digested plasmid DNA was standardized with EcoR-1. Linearized plasmid and amplified promoter was then ligated using T₄ DNA ligase. Ligation mixtures were used to transform bacterial strains DH 5a host cells and plated on LB plates with ampicillin. The transformants are to be analysed for the presence of promoter inserts.