RESUMO
The heat shock transcription factor 1 gene (HSF1) plays a key role in the heat stress response. We previously found a single nucleotide polymorphism (SNP) in the 3'-untranslated region (g.4693G>T) of HSF1 that was related to thermo tolerance in Chinese Holstein cattle through association analysis. However, it is not known whether other SNPs also affect thermo tolerance.In this study a novel SNP, g.1451G>T, was identified by DNA sequencing and genotyped using creating restriction site-polymerase chain reaction methodology. The g.1451G>T polymorphic site met Hardy-Weinberg equilibrium (P > 0.05). Association analysis demonstrated that this SNP had no effect on thermo tolerance traits in Holstein cattle. Findings of the study compared to the analysis of g.4693 G>T further indicated that g.4693 G>T may play an important role in thermo tolerance, although the mechanism is not clear. RNA hybrid and Targetscan prediction showed that the minimum free energy hybridization of bta-miR-484 with HSF1 3'-UTR was -31.9 kcal/mol and g.4693 G>T was in the seed sequence of bovine HSF1 that binds to bta-miR-484. Analysis by Luciferase assay indicated that HSF1 expression was directly targeted by bta-miR-484 in HEK 293T cells, and the Rluc/luc ratio of wildtype (GG) was lower than that of the mutant (TT) (P < 0.05). These results suggest that g.4693 G>T affects binding of HSF1 to bta-miR-484.
Assuntos
Proteínas de Choque Térmico/genética , MicroRNAs/metabolismo , Polimorfismo de Nucleotídeo Único/genética , Regiões 3' não Traduzidas/genética , Animais , Bovinos , MicroRNAs/genética , Ligação ProteicaRESUMO
We explored the molecular mechanism of the regulation of vacuolar-type-H+-ATPase B1 (VHAB1) in elvers in the response to salinity. The full-length cDNA of VHAB1 in Anguilla marmorata (designated as AmVHAB1), which was 1741 base pairs (bp) in length, was found to encompass a 1512-bp open reading frame encoding a polypeptide with 503 amino acids (55.9 kDa), an 83-bp 5'-untranslated region, and a 146-bp 3'-untranslated region. The mRNA and protein expression levels of AmVHAB1 in the gill were evaluated at different time points (0, 1, 3, 6, 12, 24, 48, 72, and 96 h, and 15 days) during the exposure to various salinity levels (0, 10, and 25). The results indicated that the expression levels of AmVHAB1 mRNA in the gill significantly increased and reached the highest level at 1 h exposure in the brackish water (BW, 10) group and at 6 h exposure in the seawater (SW, 25) group. The salinity level affected the relative expression level of AmVHAB1 mRNA in the gill, which was increased by approximately 44-fold in the SW group when compared with that in fresh water. Immunoblotting analysis showed that VHA expression was significantly higher in the BW and SW groups, with the highest expression level was detected at 96 h exposure. We found that the AmVHAB1 gene in elvers from A. marmorata plays an important role in the adaptation to seawater.