Seasonal Differences In Methylation Of KiSS-1 Promoter Region In Black Wire Hamster

Cricetulus barabensis is commonly known as small gills mouse, hamster dorsal striatum,raddei hamster. It is the dominant species of rodents in Chinese northern farmland.It is widely distributed and has strong productive ability and great harm. Its breeding activity has seasonal rhythms, it belongs to seasonal breeding animals, spring and autumn are breeding seasons every year. Studying the seasonal breeding mechanism of Cricetulus barabensis can help to understand how the external environment affects the internal breeding signal, can explore the rodent eradication methods from a biological point. Ki SS-1/GPR54 system is an extremely important part in the system that regulating the seasonal breeding activity. Found that in Cricetulus barabensis: the m RNA expression Ki SS-1and kisspeptin content is present seasonal differences,their expression in winter are the lowest, the expression in summer is relatively higher; Ki SS-1m RNA expression proportional to the length of the light; Melatonin inhibits Ki SS-1 expression.Study on Mechanism of Ki SS-1 expression in the present experiment starting from the promoter DNA methylation, to explain the specific mechanisms that Ki SS-1 gene is involved in the regulation of seasonal breeding activity.First we use the liver tissue of Cricetulus barabensis, amplified by PCR, cloned and sequenced to obtain Ki SS-1 gene promoter sequence. Bioinformatic analysis this sequence,identify its core promoter and lay the foundation for checking the methylation status. The full-length of this sequence is 2012 bp, Gen Bank accession number is:KU965557,compared with coding sequence, find the translation initiation codon, there is a sequence of 1997 bp before the initiation codon, the back 12 bp sequence belongs to Ki SS-1 gene coding sequence. The GC content of this sequence is 52.3%,it is higher than AT content. It found that the transcription start site located upstream 500 bp of the start codon. And found that the core promoter region TATA box, located upstream of the transcription start site at-22 bp, and found that cis-acting elements CAAT box in the upstream of the TATA box, located upstream of the transcription start site at-227 bp. Predicted Cp G islands are combined with the promoter sequence and the coding sequence, full-length 420 bp, GC content of 58.6%, much higher than the AT content, containing11 Cp G sites.Each season we selected three Cricetulus barabensis to obtain hypothalamus tissue, extract genomic DNA, then detects Ki SS-1 gene promoter Cp G island methylation status using bisulfite sequencing method, screened 10 clones for each individual.Using biq 2.0 Analysis of sequencing results to make a point-like diagram, by analyzing the dot diagram can be found: only the second sites in 11 Cp G sites are all unmethylated state;only the fifth Cp G site of summer is unmethylated, the other seasons are all methylated. We can speculate that the second site can regulate Ki SS-1 gene expression; the fifth site can result Ki SS-1 gene expressing highest in summer. The result shows that the methylation status of Cricetulus barabensis in four seasons are : 81.82%, 76.67%, 84.85%, 89.70, the methylation status in winter is highest, the methylation status in spring and autumn are significantly lower than that of winter, the methylation status in summer is the lowest. The correlation analysis of the Ki SS-1 m RNA expression, with the Ki SS-1 promoter methylation of hypothalamus of Cricetulus barabensis in four seasons showed that the correlation coefficient r =-0.781, p = 0.003. Description barabensis Ki SS-1 m RNA expression with Ki SS-1 promoter methylation status was moderately negative correlation, ie, the higher the degree of methylation, the lower the amount of m RNA expression,can be speculated that promoter methylation of suppressor genes expression. Compare the methylation status of Ki SS-1 promoter with photoperiod of four seasons. Found that light-time length and Ki SS-1 promoter methylation were negtively correlated: the longer the exposure time,the lower the degree of methylation. The intrinsic signal optical signal is melation, and melatonin also inhibites the expression of Ki SS-1 gene, so speculate that optical signal affects Ki SS-1 by that melatonin affects Ki SS-1 promoter methylation. So speculate that: increased exposure time will inhibit the secretion of melatonin, melatonin decreased result in reduced Ki SS-1 promoter methylation through a certain way, enhance Ki SS-1 expression, promote the synthesis of kisspeptin, promoting the expression of GPR54, GPR54 promotes the synthesis and release of Gn RH, promoting the release of LH and FSH, promoting reproduction.In summary, this study combined Nature Seasons sampling with bisulfite sequencing studying the effect of Ki SS-1 promoter methylation in Ki SS-1 / GPR54 system, further revealing the role of Ki SS-1 / GPR54 system in regulation of seasonal breeding, providing a scientific theory for the further study of seasonal breeding and biological regulatory mechanism Rodents.