ABSTRACT
The heat shock protein 90 (Hsp90) is a copious and ubiquitous molecular chaperone which plays an essential role in many cellular biological processes. The objective of this study was to identify single nucleotide polymorphisms (SNPs) in the Hsp90AA1 gene and to determine their association with heat stress traits in Chinese Holstein cattle breed. Direct sequencing was used to identify new SNPs. Luciferase reporter assay methods were used to assess g.- 87G > C and g.4172A > G loci in the promoter activity and 3'-UTR, respectively. Quantitative real-time PCR was utilized to quantify the gene expression profile. Five SNPs were identified in 130 multiparous lactating cows: one SNP in the promoter, three SNPs in the coding region, and one in 3'-UTR were novel and reported for the first time in this study. As a result of promoter assay using dual luciferase assay system, the genotype CC showed the highest transcription activity region (13.67 ± 0.578) compared to the wild-type GG (3.24 ± 0.103). On the other hand, the result revealed that one of the selected microRNAs (dme-miR-2279-5p) was found to interact with the Hsp90AA1 3'-UTR sequence and to suppress the reporter activity markedly in the presence of the allele G (2.480 ± 0.136). The expression of Hsp90AA1 in cow bearing mutant allele C was higher (4.18 ± 0.928) than cows bearing wild-type allele G (1.008 ± 0.0.129) in stress season. In summary, there was an association between genetic variations in the Hsp90AA1 and thermoresistance. This association could be used as a marker in genetic selection for heat tolerance in Chinese Holstein cattle breeds.
Subject(s)
Genetic Association Studies , HSP90 Heat-Shock Proteins/genetics , Polymorphism, Single Nucleotide/genetics , Thermotolerance/genetics , 3' Untranslated Regions/genetics , 5' Untranslated Regions/genetics , Animals , Base Sequence , Cattle , Codon/genetics , Diploidy , Gene Frequency/genetics , Genetic Loci , HSP90 Heat-Shock Proteins/metabolism , Haploidy , HeLa Cells , Humans , Linkage Disequilibrium/genetics , Luciferases/metabolism , MicroRNAs/metabolism , Promoter Regions, Genetic , RNA, Messenger/genetics , RNA, Messenger/metabolismABSTRACT
In the present study the melatonin receptor 1A gene (MTNR1A) was proposed to be a candidate gene for egg production in Yangzhou geese. A total of 210 goose blood samples were collected to investigate the association of the MTNR1A gene with the number of eggs produced. Using a direct sequencing method, a single nucleotide polymorphism (SNP; g.177G>C) was detected in the 5' regulatory region of the MTNR1A gene (Genbank ss1985399687). Two alleles (G and C) and three genotypes were identified. Association analysis results showed that the g.177G>C SNP significantly affected the level of egg production within a 34-week egg-laying period (P < 0.05). Furthermore, the geese with the GG genotype produced significantly more eggs compared to the geese with the CC genotype. Quantitative real-time PCR analysis showed that the MTNR1A gene was highly expressed in small intestine, granulosa cell and ovary compared to other examined tissues. In addition, the mRNA expression level of MTNR1A in ovary indicated that significantly higher expression levels were recorded for geese with the GG genotype compared to those with the CC genotype. Moreover, a luciferase reporter assay showed that the CC genotype had significantly lower promoter activity than did GG. These results suggest that the identified SNP in the MTNR1A gene may influence the number of eggs produced and mRNA expression levels in Yangzhou geese and could be considered as a useful molecular marker in goose selection and improvement, especially for egg production.
