| China is a traditional sheep-raising country with the largest number of sheep in the world.However,most of the local sheep are seasonal-estrous breeds,seasonal reproduction is an important evolutionary result of sheep’s adaptation to survival,but it reduces production efficiency in actual production,which is one of the main limiting factors for the low efficiency of sheep production in the country and the world.Related research found that the successful reproduction of mammals was composed of a series of hormone-dependent behaviors and physiological activities regulated by the circadian clock.The PER gene is the core molecule of the circadian clock and can restart the rhythm oscillator,but there are few studies on PER gene in Chinese local sheep breeds currently.In this study,we first detected the expression of PER in different tissues of year-round estrus and seasonal estrus in sheep,then Sequenom Mass ARRAY? SNP assay was applied to genotype the single nucleotide polymorphism sites（SNPs）of PER,gene and genotype frequencies of those sites were analyzed in sheep with different breeding traits.And the association between PER gene polymorphism and litter size were also analyzed in Small Tail Han sheep（STH）.In addition,our previous study found that the expression of miR-30a-5p was significant difference between year-round and seasonal estrous sheep breeds,and some study in mice found that miR-30a-5p could regulate the expression of PER2 gene.The relationship between miR-30a-5p and PER2 gene was verified in sheep,which will preliminary clarify the molecular mechanism of miR-30a-5p regulating seasonal estrus in sheep.The main results were as follows:（1）qPCR showed that PER（including PER1、PER2 and PER3）gene expressed in all tissues selected.The expression levels of PER1 in Sunite sheep（SNT）was higher than Small Tail Han sheep（STH）in all tested tissues,and reached significant differences in pineal gland and ovary,and uterus（P<0.05）.There were three genotypes in both year-round estrous breeds and seasonal estrous breeds.The allele frequencies of the PER1g.27342699G>A was significantly different between year-round estrous and seasonal estrous sheep（P<0.05）.Association analysis indicated that 2 SNPs of PER1 gene had no significant correlation with the litter size of each parity in STH.（2）The results showed that the expression of PER2 in SNT was significantly higher than that in STH of ovary and uterus,but it was conversely expressed in oviduct（P<0.05）.There were three genotypes in both year-round estrous and seasonal estrous sheep breeds,and the genotype frequencies and allele frequencies of the PER2 g.2852655T>C locus were significantly different between year-round and seasonal estrous sheep（P<0.05）,however,there was no significant correlation between this locus and litter size of each parity in STH.（3）The results showed that the expression of PER3 in SNT was higher than that in STH except oviduct,and reached extremely significant differences in pineal gland and uterus（P<0.01）.From genotyping,this study found the genotype and allele frequencies of5 SNPs（including g.43657503A>G,g.43670012T>C,g.43670807A>G,g.43677259T>C and g.43707227G>A）were significantly different between year-round and seasonal estrous sheep（P<0.05）,but 12 SNPs of PER3 had no significant correlation with the litter size of each parity in STH.（4）3’RACE and PCR were used to amplify the 3’UTR and CDS region of PER2 gene in sheep,then we found that there was only one binding site between PER2 and miR-30a-5p in the CDS region of PER2 gene,and the 3’UTR binding site has mutated though the prediction of targetscan bioinformatics software.（5）To verify the target interaction between miR-30a-5p and PER2 gene:the wild-type and mutant sequences of CDS region and 3’UTR of PER2 gene were constructed into the dual fluorescent vector,miR-30a-5p was constructed into the vector with green fluorescence,and then co-transfected into 293T cells.The results showed that the relative luciferase activity of miR-30 and PER2-CDS group was significantly reduced compared with the control group（P<0.01）,and there was no significant change in miR-30and PER2-3’UTR group.In summary,the clock gene PER gene expressed in all tissues selected.The expression of PER gene in SNT was higher than that in STH,indicating that higher levels of PER gene may be involved in seasonal estrus regulation.The genotype frequencies and allele frequencies of 6 SNPs（including PER2 gene g.2852655T>C and PER3 gene g.43657503A>G,g.43670012T>C,g.43670807A>G,g.43677259T>C,g.43707227G>A）were significantly different between seasonal estrous and year-round estruos sheep breeds,and they might be the key sites for seasonal estrus regulation.In addition,there was no significant between the polymorphisms of PER gene and the litter size of each parity in STH,indicating that the clock gene PER could not directly affect the litter size of STH.Furthermore,this experiment verified that there was only a binding site in the CDS region of PER2 gene between PER2 and miR-30a-5p in sheep. |
