Abstract：Abstract Raffinose is ubiquitously occurred in higher plant as an important soluble carbohydrate, which is known to be involved in various abiotic stresses. Raffinose synthase (RS, EC2.4.1.82) is the key enzyme that catalyses the reversible galactosylation, yielding raffinose and myo-inositol. In this study, the raffinose synthase gene TaRS of wheat (Triticum aestivum) was cloned by homologous alignment. The TaRS was assigned to wheat chromosome 3B. Sequence analysis revealed that TaRS had a whole open reading frame (ORF, 2 349 bp) and belonged to the glycoside hydrolase super family (GH-D) which contained 2 conservative motifs, KxD and RxxxD. In evolutionary relationship, TaRS shared the highest homology with the indicated RS from Aegilops tauschii (XM020298559.1). Southern blot assay showed that there existed at least 4 allelic copies of TaRS in Chinese spring wheat genome. Subcellular localization analysis revealed that TaRS protein was localized at the cell membrane in wheat protoplasts. Tissue specific analysis showed that TaRS expressed in root, stem, leaf and seed, but had the highest expression level in leaves. Heterologous expression was conducted in Escherichia coli, and the crude extract was able to utilize sucrose and galactinol as substrates to synthesis raffinose in vitro based on the result of high performance liquid chromatography (HPLC). Furthermore, TaRS displayed an optimum activity at about pH 8.0. Expression pattern analysis under multiple abiotic stresses indicated that the expression of TaRS was induced by dehydration, high temperature, salinity, and low temperatur, and reached the highest expression level at 12, 1, 1, 48 h, respectively. These results indicated that TaRS may play critical roles in wheat tolerance of abiotic stresses and provides theoretical basis for further studying in wheat abiotic stress tolerance breeding.
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