Cloning Trehalose Synthase Gene And Its Expression In Arabidopsis thaliana

Abstract

Trehalose is a well known as biological stabilizer functioning as stress protecting agent. It is ubiquitously found in bacteria, fungi, invertebrates, and certain desert-adapted resurrection plants such as Selaginella. In most flowering plants, trehalose is synthesized in amounts nearing the detection limit. Gene(s) encoding trehalose-metabolizing enzymes from E.coli, trehalose synthase and trehalose phosphate phosphatase (OtsA and or OtsB) and the same gene from yeast (TPS1 and or TPP) have been introduced to plants and increased stresses resistance, yet resulted in phenotypic defect, due to accumulation of its intermediate product, trehalose-6-phosphate (T6P). T6P increases along with the increase of trehalose level. This research was aimed to find out if introducing trehalose synthase gene (TreS) which does not produce T6P which prevent phenotypic defect resulted from trehalose accumulation, and whether the presence of TreS in plant improves plant resistance to stresses. The use of TreS as an alternative among non toxic selectable markers was also assessed. For this reason, genes encoding trehalose synthase originated from Thermobifida fusca (TfTreS) and originated from Mycobacterium tuberculosis (MtTreS) had been cloned into plant expression vector (pBIN1935S). TfTreS was isolated from T. fusca using PCR and was inserted to pGemT for sequencing. DNA sequent analysis of the 2 clones, TfTreS within pGemT were identical to the published data yet several silent point mutations occured. TfTreS from these clones were inserted into pBIN19 with CaMV35S promoter. It has been successfully transferred to Arabidopsis thaliana via Agrobacterium mediated transformation. MtTreS readily available in pBlunt TOPO II, was exiced from the plasmid and was inserted to pBin1935S, and transferred to Arabidopsis in the same manner, but it has not been successfully transferred to the plant.