Overwintering plants have the ability to survive throughout the winter, and many of them contain thermal hysteresis proteins (THPs), which lower the freezing point and melting point of water. These proteins are present in fish and insects as well, but the mechanism is likely to be different. Unlike insects and fish, plant THPs allow them to survive the freezing of their extracellular water, rather than preventing freezing altogether. Solanum dulcamara is an example of an overwintering plant, and is at the center of this article. The objective of the research was to isolate and characterize a specific THP within Solanum dulcamara that allows it to survive through the winter’s harsh temperature decline.
Researchers collected and purified samples of Solanum dulcamara RNA from leaves at two separate points in time; one sample in September and the other in November. This was done to compare the RNA present during warmer weather and colder weather; they assumed that the THP would be present in the RNA extracted from the November leaves. The extracted RNA was then used to generate a cDNA expression libraries for both samples. An antibody generated from a previously purified S. dulcamara THP was used to screen the two libraries for the presence of matching THPs. A total of 20,000 recombinants from both the September and November samples were put through the antibody screening. The November sample was found to have eight matches and like the expected, the September sample had zero. Two of these matches were then isolated and prepared for cloning. Using PCR, a clone was generated, duplicated, and integrated into E. coli as a plasmid. Finally, the protein was grown in culture and extracted.
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