Abstract: To elucidate the AFPⅢ multimerization molecular mechanisms on cold tolerance, we cloned tetramer (LD4) of the type Ⅲ AFP from a multimer type Ⅲ antifreeze protein gene LD12 cDNA which was characterized from the Antarctic eelpout (Lycodichthys dearborni). The eukaryotic expression plasmid Tol2-actin-LD4-2A-EGFP was constructed and transfected into zebrafish cells (ZF4). It was found that LD4 was abundantly expressed in ZF4 cell line and reduced mortality of zebrafish cells under low temperature stress. Transcriptome sequencing and gene expression analysis were carried out at WT, EGFP and LD4 cells under different processing temperatures (28 ℃, 18 ℃ for 2 days, 18 ℃ for 2 days and 10 ℃ for 2 days), the results indicated 26 differentially expressed transcription factors, in which I3MB13 and ZNF687b were up-regulated and JUN and Cremb were down-regulated. The expression patterns of 26 selected genes were verified by qRT-PCR (quantitative real-time PCR). Based on the KEGG pathway analysis, the transcription factors were suggested to be involved in apoptosis, cell cycle and proliferation regulation. So, AnnexinV-PE/7-AAD double staining was used to detect the effect of LD4 on apoptosis. Unfortunately, apoptosis rate was not significantly decreased in LD4 cells compared with the control group (EGFP and WT). That is, LD4 might be involved in another channel rather than inhibiting apoptosis to resist low temperature, which provides a certain theoretical basis for further research on the mechanism of LD4.

Key words: multimer type Ⅲ, antifreeze protein, zebrafish cell line, transcriptome sequencing, cell apoptosis