Identification Of Cis-regulatory Element From Zebrafish Transcriptomic Under Cold Stress And Cloning Of ATP6V0C From Antarctic Notothenioid Fish Dissostichus Mawsoni And Identification Of Its Function Of Cold Tolerance In HeLa Cell

The transcriptional programs of ectothermic teleosts are directly influenced by water temperature. However, the systematic molecular networks governing the temperature responses in teleosts are still unknown. We profiled the transcriptional responses in eight tissues of zebrafish exposed to graded cold temperatures, ranging from normal(28°C) to mild(18°C) and severe(10°C) cold, using RNA-seq. Fuzzy k-means clustering revealed 34 gene clusters of distinct expression patterns, demonstrating diverse tissue-specific responses in conjunction with multiple aspects of ubiquitous cross-tissue responses to cold. Thirty-one GO terms were over-represented which are involved in basic cellular processes, such as RNA splicing and proton transport, as well tissue-specific processes, such as ‘negative regulation of endopeptidase’ in the kidney. Genes exhibiting similar expression patterns are likely to be co-regulated at the level of transcription through a programmed interaction between the cis-regulatory elements and the trans-factors(e.g., transcription factors). We screened for enriched nucleotide motifs(potential cis-regulatory elements) within 1 kb of the promoter region of the clustered genes using the motif-finding algorithm DREME. The p values for the identified motifs were calculated based on the hypergeometric distribution, and those with values lower than 1x10-4 were regarded as significantly over-represented motifs of the cluster; thus, these motifs may participate in the regulatory network that orchestrates the similar expression patterns among the motif-bearing genes upon cold challenge. The over-represented motifs were compared with known motifs in the JASPAR database using the tool TOMTOM. We identified 17 motifs from 7 clusters(clusters 0, 1, 5, 7, 16, 19 and 21). Of these, 10 were identical in sequence to known transcription factor binding sites in the JASPAR database; thus, these motifs were regarded as known motifs. The percentage of the genes within a cluster represented by a specific motif varied; the highest representation(WCACCTGW in cluster 7) reached 48%, suggesting that approximately half of the co-expressed genes in this cluster might be influenced by this motif. A novel AGMAACCA motif and the AP-1 motif were experimentally validated to control of gene expression under cold stresses. These enriched motifs and accordingly transcription factors would largely contribute to orchestrating the cold-responsive pathways.To investigate the ATPase responsive for the cold adaptation in Antarctic fish, we cloned the Antaratic notothenioid Dissostichus mawsoni ATP6 VOC from the Dissostichus mawsoni c DNA library, and then transfected the dm ATP6 VOC gene into the He La cell to detect cell viability under cold stress(10℃) by flow cytometry. The complete coding sequence with 462 bp of dm ATP6V0 C gene was cloned and sequenced. We inserted the dm ATP6V0 C coding sequence into the pc DNA3.1(-) vector by Xho I and Bam HI restriction sites for gene expression in He La cell. The result shows that compared with the control, over-expression of dm ATP6V0 C gene in He La cell reduce the cell death rate from 19.23±1.87% to 8.13%±0.04%(P<0.05) under cold stress. These results indicated that the expression level of ATP6 VOC highly related to cell viability under cold stress and ATP6 VOC could be considered as a candidate for breeding cold resistant fish.