Cloning and Functional Analysis of Genes Working in Oxidative Stress Defense in Unicellular Flagellated Microalgae Euglena gracilis

Abstract

Microalgae has been developed as one of the bioresource alternatives for bioenergy production. In comparison to corn and sugarcane, microalgae require the least land area and energy consumption, yet able to produce 80% oil from its dry biomass. Euglena gracilis is a photosynthetic flagellated microalgae that is capable to survive the dynamic conditions in nature. However, severe environmental stress conditions, such as intense light exposure, can induce the formation of ROS, leading to cellular oxidative stress. To conquer this condition, the antioxidant systems of E. gracilis has been functionally evolved. Glutathione reductase (GR) and trypanothione reductase (TRYR) are two hydrophilic enzymes involved in the AsA-GSH antioxidant cycle and thiol redox system in E. gracilis, respectively. Albeit the genes that encode these enzymes has been discovered, its physiological function remains to be explored. In order to study the physiological roles of GR and TRYR enzymes, GR and TRYR genes were silenced using RNA interference. The samples were grown mixotrophically using ethanol-supplemented CM media. Semi-quantitative RT-PCR confirmed that GR had been successfully silenced, but not TRYR. The knockdown of GR resulted in lower GR enzyme activity, but it did not affect the cell growth.