Expression And Functional Analysis Of BnGOLS1Gene From Brassica Napus

Orthodox seeds lose most of their water in the maturation phase of development. Seeds acquire desiccation tolerance during this period to protect themselves against the harmful dehydration. The accumulation of raffinose family oligosaccharides (RFO) are often suggested to be essential for acquisition of desiccation tolerance, and galactinol synthase (GOLS), a key enzyme in RFO synthesis, significantly affects this process. However, the detailed mechanism has not yet been elucidated. In this work, the time course of Arabidopsis thaliana seeds desiccation tolerance acquisition obtained by seed desiccation tolerance test. At the same time, this study also puts forward some improvement made on the seed desiccation method. When recombinant BnGolS1was expressed in E. coli, GOLS activity was detected in the cultured supernantant after induced by isopropyl-(3-d-l-thiogalactopyranoside(IPTG). Then, the enzymatic properties were also tested after purified by Ni-NTA Agarose. The optimum temperature and pH were39℃and8.0, respectively for BnGOLS1activity. It contains higher activity at35-55℃. The Km of BnGOLS1was4.98mM for myo-inositol and0.401mM for UDP-Gal. And its activity can be enhanced by Mn2+; the optimal concentration of Mn2+was8mM for recombinant BnGOLS1. Furthermore, we used oil rape (Brassica napus. L) and Arabidopsis thaliana (Col) in the experiments. B. napus and A. thaliana were transformed with Agrobacterium strain GV3101containing pBI-35S-GS、pBI-NAP-GS or pBI-GS-GUS through Agrobacterium-mediated floral dip or via the pollen tube pathway transformation. PCR and Southern Blot confirmed the integration of the introduced BnGOLS1in transgenic A. thaliana plants. The results indicated that the transgenic plants had higher GOLS activity and RFO concentration than those in wild type plants. Moreover, under heat and drought conditions, the transgenic plants were better able to withstand stress than wild type plants. These data suggest that BnGOLS1gene results in enhancement of both heat and drought stress tolerance in plants.