Expression of Insect (Dendroides Canadensis) Antifreeze Proteins In Drosophila Melanogaster and Arabidopsis Thaliana

Antifreeze proteins (AFPs), in the presence of a seed ice crystal, lower the freezing point of water without affecting the melting point. This difference between melting point and freezing point has been termed thermal hysteresis (TH). In freeze-avoiding species (those that die if frozen and, therefore, must prevent freezing), AFPs typically promote supercooling by inhibiting inoculative freezing across the cuticle initiated by external ice and by masking internal ice nucleators. The freeze avoiding beetle Dendroides canadensis produces a family of antifreeze proteins (DAFPs) some of which interact with one another to cause a synergistic increase in TH. CDNAs of two self-enhancing synergistic DAFPs, dafp-1 and/or dafp-4, were transferred to Drosophila melanogaster via P-element-mediated transformation. Northern and Western blots showed expression of DAFP(s) at both transcript and protein levels. All transgenics had TH, but the highest TH of 6.78 Ìâå± 0.12Ìâå¡C was detected in 5-day adult flies containing two copies of both DAFP-1 and DAFP-4 genes. Moderate synergistic enhancement of TH was found between DAFP-1 and DAFP-4 in transgenic D. melanogaster containing both DAFPs. Supercooling points without ice in contact with the insects were lowered in all 5 transgenic lines (heterozygous DAFP-1-producing line, heterozygous DAFP-4-producing line, homozygous DAFP-1-producing line, homozygous DAFP-4-producing line, and a heterozygous line that produces both DAFP-1 and DAFP-4) compared to controls, however, when ice was in contact with the flies, supercooling points were lowered only in the heterozygous DAFP-1+DAFP-4 transgenic line. Transgenic D. melanogaster exhibited higher survivorship compared to controls when placed at low, but non-freezing temperatures (0Ìâå¡C and 4Ìâå¡C). The same two DAFP cDNAs, dafp-1 and dafp-4, were introduced into the genome of Arabidopsis thaliana via agrobacterium-mediated floral dip transformation. Southern blots demonstrated multiple insertions of transgenes in transgenic A. thaliana. Both DAFP-1 and/or DAFP-4 were expressed in transgenic A. thaliana, as shown by RT-PCR and Western blots. TH of apoplastic protein from T3 DAFP-1+DAFP-4-producing transgenic A. thaliana was 1.20 - 1.35Ìâå¡C, by capillary method. The freezing temperature of DAFP-1+DAFP-4-producing transgenic A. thaliana was lowered by 2 - 3Ìâå¡C compared to the wild type. DAFP-4 and DAFP-6 share similar sequences with only four different amino acids. To determine why DAFP-4 has TH while DAFP-6 does not, five mutated proteins with various combinations of the four differing amino acids were generated, expressed, purified, and their TH determined. The comparison of TH among all mutated proteins and DAFPs -4 and -6 revealed that each amino acid position that differs between DAFP-4 and DAFP-6 plays an important role in conferring TH, but positions Q9S and S32T are less important than D17N and T26V.