Part 1: Investigating Heat Stress in Acropora humilis using qPCR

For this assignment, I am investigating the gene expression response of the coral Acropora humilis during a heat stress experiment. The coral fragments will be subjected to elevated temperatures to induce thermal stress, simulating the conditions of marine heatwaves that lead to coral bleaching.

To understand the molecular mechanisms underlying this stress response, I have selected two target genes and one reference gene for qPCR analysis.

1. Target Gene 1: Heat Shock Protein 70 (HSP70)

• Biological Function: HSP70 is a highly conserved molecular chaperone. Its primary role is to assist in protein folding, prevent the aggregation of denatured proteins, and help refold proteins that have been damaged by cellular stress.

• Expected Observation: I expect to see a significant upregulation in HSP70 expression in the heat-treated coral compared to the control group.

• Relevance to Condition: Elevated temperatures disrupt protein homeostasis, causing cellular proteins to denature (lose their functional shape). Increased expression of HSP70 is a primary, direct cellular response to thermal stress to combat this structural damage and maintain protein functionality.

2. Target Gene 2: Manganese Superoxide Dismutase (MnSOD)

• Biological Function: MnSOD is an antioxidant enzyme located in the mitochondria. It acts as a first line of defense against oxidative stress by catalyzing the dismutation of highly toxic superoxide radicals into less toxic hydrogen peroxide and oxygen.

• Expected Observation: I anticipate a strong upregulation of MnSOD expression in the heat-stressed samples.

• Relevance to Condition: During a bleaching event, thermal stress damages the photosynthetic apparatus of the coral’s symbiotic algae (zooxanthellae). This breakdown leads to the overproduction of Reactive Oxygen Species (ROS), which leak into the host coral’s tissues. The coral must upregulate antioxidant defenses like MnSOD to neutralize these ROS and prevent severe cellular damage and apoptosis.

3. Reference (Housekeeping) Gene: Elongation Factor 1-alpha (EF1-alpha)

• Justification: EF1-alpha is a fundamental protein translation factor responsible for the enzymatic delivery of aminoacyl tRNAs to the ribosome. It is routinely demonstrated to have high stability across diverse conditions and is continuously expressed at relatively high levels in most cell types.

• Stability: Fundamental cell maintenance and basal protein synthesis must continue regardless of environmental stress, so EF1-alpha’s expression is largely unaffected by heat treatment. This makes it a reliable internal baseline to compare the target genes to.

4. Scientific Reasoning

• Hypothesis: The target genes (HSP70 and MnSOD) will be significantly upregulated in the heat stress treatment in comparison to the control treatment.
• Rationale: Extended heat stress will lead to the production of ROS and the denaturing of key proteins, so the coral will upregulate the production of antioxidants and chaperone proteins to perform damage control. HSP70 upregulation will produce more chaperone proteins, while MnSOD will produce antioxidants to handle the ROS.