Thesis

Gene expression of proteins associated with lipid bodies in Emiliania huxleyi

Emiliania huxleyi is a marine haptophyte alga known for its ability to generate large volumes of calcite coccoliths. It also synthesizes a unique suite of neutral lipid (PolyUnsaturated Long Chain Alkenes, Alkenones and Alkenoates, or PULCA), whose biosynthetic pathways are currently unknown. Like energy-storage triacylglycerides, which they replace, these lipids are packaged into lipid bodies (LBs), and a prior study used a proteomics screen of isolated LBs and endomembranes from E. huxleyi CCMP 1516 to generate a list of candidate proteins possibly associated with PULCA biosynthesis, mobilization, and catabolism. The goal of this thesis was to study gene expression these proteins under conditions of neutral lipid accumulation and degradation, including batch growth with addition of sodium bicarbonate, and during light-dark manipulations. I designed quantitative PCR (qPCR) primers for almost 120 genes identified by the proteomics screen, and optimized mRNA extraction, reverse transcription to cDNA, and qPCR. I confirmed neutral lipid accumulation during nutrient (phosphate) limitation in the light after sodium bicarbonate dosing, and catabolism during continuous darkness. I was able to quantify gene expression changes of many LB-associated genes, most with low expression values, as well as a number of control genes and other genes used in other studies. I observed several striking patterns of gene expression in the experiments. In the growth curve + bicarbonate experiment, many LB-associated genes likely related to acyl lipid biosynthesis showed 10-100-fold expression increases as neutral lipids accumulated. However, the timing of increases varied: some increased before bicarbonate addition, while others only after. In contrast, several genes for prenyl and sphingolipid pathways did not show such striking increases, and a few genes possibly related to acyl lipid catabolism decreased. Most genes associated with photosynthesis, structural proteins, and trafficking did not show significant changes, but several genes for carbohydrate metabolism also increased sharply, highlighting the likely connection between carbohydrate and lipid metabolism. In contrast, the light-dark experiment showed few large changes in expression, and several genes showed strong down-regulation, although patterns were not consistent. But because of the large numbers of time points and treatments in this experiment, I was only able to survey a few genes by qPCR, and these results are very tentative. In both experiments, I was able to quantify only a fraction of the LB-associated genes, due to the large effort necessary to screen genes by qPCR, and also due to degradation of mRNA stored at -80 oC. Although my study examined far more genes and time points than most qPCR studies, and I confirmed the likely role of acyl lipid biosynthesis pathways in PULCA production, an important outcome of this study is the need in future to use more comprehensive genome-wide techniques, such as RNA-seq, to evaluate the large numbers of genes likely involved in the pathways of neutral lipid biosynthesis.

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