The design and application of a qPCR assay for the study of light regime as a driver of rhodopsin expression in high Arctic phytoplankton.

• Main Author: Nicholls, Emilie Kavanagh
• Format: Master Thesis
• Language: English
• Published: 2023
• Subjects: qPCR assay; polar night; phytoplankton; time series; rhodopsins; Arctic; Phytoplankton
• Online Access: http://hdl.handle.net/10852/108188

Several months of darkness during polar night impose unique challenges on polar phototrophic organisms, whereby solar energy capture via plastid-based photosystems remains unattainable for an extended period of time. Nevertheless, energy acquisition via the contribution of retinal- based phototrophy from microbial rhodopsins in high Arctic phytoplankton remains entirely unexplored. Light regime at the subsurface, via the investigation of spectral composition, intensity and duration, was studied throughout this time series spanning the transition from polar night to the spring equinox to explore this as a potential driver of rhodopsin expression. Blue- and green-light was observed as the dominant light type, especially during field campaigns associated with the lowest solar declination angles, suggesting that light-harvesting activity from blue- and green-light absorbing rhodopsins could be sustained given spectral composition and intensity. A quantitative PCR assay based on the use of SYBR Green was developed to investigate rhodopsin gene abundance and expression in two strains of high Arctic phytoplankton as the light climate evolves. Preliminary testing via PCR was performed whilst continuously optimizing reaction conditions (primer concentrations, temperature trials, genetic material type and quantity). With the addition of a standard curve to standardize amplification, the assay was applied to DNA and RNA extracts from environmental samples collected bimonthly throughout the time series. Stochasticity and primer entropy was observed, likely due to their use on environmental samples containing very little genetic material. The assay did provide consistent results and demonstrated that stronger deductions can be made for rhodopsin gene abundance and expression in high Arctic phytoplankton should a higher yield of target DNA/RNA be present in the samples. Ambient environmental conditions at the sampling site (temperature, salinity, nutrients, and fluorescence) displayed values expected for a prebloom phase and ...