Function And Transcriptional Regulation Of Lin28B Gene In Dolang Sheep During Puberty Initiation

Puberty is the critical period when female animals first appear in estrus and ovulate.It plays a vital role in the life cycle of animals and is an important symbol of female animals’ reproductive ability.The time of puberty directly affects the reproductive ability of animals.In the early stage,the research group preliminarily explored the relationship between the Lin28 B gene and the onset of puberty in Dolang sheep.So this study continues to use Lin28 B as a candidate gene to explore the relationship between Lin28 B gene and puberty initiation.In this study,on the basis of the previous research group’s experiments,the overexpression vector was constructed through the obtained CDS region sequence of Dolang sheep and transfected into ovine granulosa cells,and the expression of Lin28 B gene was detected for its related pathway genes.In order to preliminarily analyze the function of Lin28 B gene in sheep ovary granulosa cells.Secondly,this study cloned the sequence of the promoter region of Lin28 B gene and constructed the promoter deletion vector of Lin28 B gene through bioinformatics analysis to understand the structure and function of the promoter.Finally,this study further clarified the transcriptional regulation of Lin28 B gene by analyzing the methylation status of hypothalamus and transcription factors SP1 and Egr1 in Dolang sheep.The main findings of this study are as follows:In this study,by constructing the vector of the C DS region sequence of Lin28 B gene of Dolang sheep,and transfecting it into ovine granulosa cells,it was found that overexpression of Lin28 B inhibited the expression of its pathway genes Let7-b and Let7-g,and promoted Estradiol secretion and proliferation of ovine granulosa cells.Lin28B gene promoter region in Dolang sheep was obtained by cloning and through bioinformatics analysis,it was found that there was one Cp G island in the sequence,four transcription initiation sites,and multiple transcriptional regulation related to puberty.Associated transcription factors.In this study,a promoter deletion fragment vector was also constructed.Through double luciferase detection,it was found that the core promoter region of Lin28 B gene is located at-837 ～-338 bp.Associated transcription factors.By bisulfite sequencing(BSP),this study detected the methylation of the Cp G island in the promoter region of the Lin28 B gene in the hypothalamus of Dolang sheep.The expression of Lin28 B in the hypothalamus of Dolang sheep was detected by real-time quantitative PCR method before puberty,puberty and post puberty.The results showed that from prepuberty to postpuberty,the methylation level increased,while the expression level of Lin28 B decreased,indicating that the expression of Lin28 B was negatively correlated with the methylation level of the promoter.Analysis of variance showed that there were significant differences in the methylation status of Cp G 5,Cp G 7 and Cp G9 from before puberty to after puberty(P <0.05).The data of this experiment showed that the expression of Lin28 B was increased by the demethylation of promoter Cp G islands,among which Cp G5,Cp G7 and Cp G9 were considered as key regulatory sites.SP1 and Egr1 point mutation vectors and Egr1 overexpression vectors were constructed.Through dualluciferase detection,it was found that the transcriptional activity of Lin28 B promoter was significantly reduced after the deletion of SP1 or Egr1 single transcription factor,and the transcriptional activity decreased even more after Egr1 mutation.Significant,by overexpressing the Egr1 vector,it can be seen that the activity of the Lin28 B gene promoter is significantly increased.Therefore,it is preliminarily deduced that the transcription factors SP1 and Egr1 have an effect on Lin28 B.The transcriptional regulation of genes plays an important role.Taken together,this study demonstrated that Lin28 B overexpression of the gene in sheep ovary granulosa cells has certain effects on Let7-b,Let7-g and estradiol.And this study also shows that the methylation status and the pair of transcription factors SP1 and Egr1 can directly affect the transcription of Lin28 B gene.Therefore,this study clarified the regulatory mechanism of methylation status and transcription factors SP1 and Egr1 involved in regulating the transcription of Lin28 B gene,thus providing a theoretical basis for the regulation mechanism of sheep Lin28 B gene on puberty initiation.