Molecular cloning, expression, sequence analysis and in silico comparative mapping of trehalose 6-phosphate gene from Egyptian durum wheat

Abstract

Trehalose is a non-reducing disaccharide which consists of two glucose units that functions as a compatible solute to stabilize the membrane structures under heat and desiccation stress. Trehalose-6-phosphate synthase (TPS) and trehalose-6- phosphate phosphatase (TPP) are the key enzymes for trehalose biosynthesize in the plant kingdom. On the basis of bioinformatics prediction, fragment containing an open reading frame of 945 bp was cloned from durum wheat. Sequence comparison and analysis of conserved domains revealed the presence of a TPP domain. Full length of the gene was isolated using gene race technology. Semi-quantitative RT-PCR and real time quantitative PCR indicated that the expression of this gene is up-regulated in response to drought stress. The biochemical assay of the trehalase activity showed that the enzyme's activity decreased under the dehydration stress. The obtained phylogenic tree showed that the isolated TPP protein forms a distinct clad close to the Oryza sativa trehalose-6- phosphate phosphatase. In silico and comparative mapping indicated that the isolated TPP gene is localized on rice chromosome 8, durum wheat chromosome 20, bread wheat chromosome 3B, oat linkage group E, sorghum chromosome 4 and barley 5H.\r\r\tKey words: Abiotic stress tolerance, trehalose-6- phosphate phosphatase (TPP), durum wheat, trehalose, real time PCR, cloning, full length gene, drought stress.