| In order to investigate the function of the tumor necrosis factor receptor-associated factor (TRAF1) gene of black seabream under low temperature stress, the full-length cDNA sequence of the TRAF1 gene in black seabream was obtained using RACE cloning technology. The gene structure was analyzed using bioinformatics tools, and the expression levels in various tissues of black seabream and the gene expression patterns under low temperature stress were analyzed by qPCR. SNP sites of the TRAF1 gene related to low temperature tolerance in black seabream were screened by direct sequencing. The results showed that the full-length cDNA of the TRAF1 gene in black seabream was 3528bp, including a 5'UTR of 93bp, an ORF of 1566bp, and a 3'UTR of 1869bp, encoding a total of 521 amino acids, including one ring finger domain and one MATH domain. The TRAF1 gene expression of black seabream was detected in various tissues including liver, brain, muscle, gills, kidney, heart, and intestine, with the highest expression level in the tissue of kidney, significantly higher than other tissues (P<0.05); Under low-temperature stress, compared to the control group, the TRAF1 gene showed an increased expression level in the low-temperature tolerant group and a decreased expression level in the low-temperature sensitive group in the tissues of heart, spleen, muscle, brain, and liver. Comparing the full-length DNA sequences of the TRAF1 gene between the tolerant and sensitive groups of black seabream, a total of 16 SNP loci were identified through screening. Among them, 2 SNP loci were located in the exon region and 14 were located in the 3'UTR region. The SNP g.5862 locus in the exon region and the SNP g.7827 locus in the 3'UTR region were significantly correlated with the low temperature tolerance trait of black seabream (P<0.05); Real-time fluorescence quantitative detection revealed that among the three SNP loci of the black sea bream TRAF1 gene, the GG genotype at each locus exhibited significantly higher gene expression levels compared to the other two genotypes (P< 0.05), and can be used as candidate markers for molecular marker assisted breeding of low temperature tolerance trait in black seabream. |
