A Preliminary Study On The Functions Of A Multimer Type â…¢ Antifreeze Protein In Low Temperature Tolerance

Fish antifreeze proteins are produced by fishes inhabiting the polar marine environments, serving as biological antifreezes that lower the freezing point of the blood and body fluids. Antifreeze proteins function by a mechanism termed as‘absorption-inhibition’, by which the antifreze molecules bind with ice and inhibit ice-growth. For a long time, people viwed the antifreeze proteins as a type of proteins function only to physically bind with ice crystals, without any other biochemical functions. However, in recent years, accumulating evidence indicated that some antifreeze proteins could confer cold protection functions to transgenic plants or animals even in temperatures above the freezing point when no ice could exist. In this laboratory, we identified a gene encoding a new type Ⅲ antifreeze proteins, termed LD4, from an Antarctic eelpout, which contained 4 domains of ice binding domain and showed stronger cold protection activities than other fish antifreeze proteins. In this study, we attempt to elucidate the mechanisms underlying the cold protection function of LD4.We first cloned the tetramer(LD4) of the AFPⅢfrom a cDNA characterized from the Antarctic Eel pout(L. dearborni) into an eukaryotic expression plasmid, and produced the Tol2-actin-LD4-2A-EGFP plasmid. The construct was transfected into the zebrafish embryo fibroblast cells(ZF4). LD4 was abundantly expressed in ZF4 cell line and reduce significantly reduced the mortality of the cell at low temperatures. We then conducted transcriptome sequencing of the transfected cells and compared to the gene expression patterns of the untransfected cells under the same cultural conditions. 26 differentially expressed transcription factors were identified. We found that factors like I3MB13 and ZNF687 b were up-regulated, while JUN and Cremb were down-regulated in the LD4 transfected cells. The expression patterns of the 26 selected genes were verified by RT-qPCR. KEGG pathway analysis suggested that the transcription factors were involved in apoptosis, cell cycle and cell proliferation regulation. We then performed annexinV-PE / 7-AAD double staining to examine the effects of LD4 on apoptosis. We found that the apoptosis rates in the cells were not significantly affected in the LD4 cells compared with the control groups(EGFP and WT), suggested that LD4 might function through other pathways rather than inhibiting apoptosis to confer higher cell survival rate. This result provided an angel for discovering the mechanisms of LD4 in cold-resistance.