Differential Expression Molecular Mechanism Of Key Genes Related With Steroid Hormone Synthesis In Xiang Pigs

Xiang pig is a local breed mainly produced in Guizhou Province.It is as one of the world's outstanding pig breeds with high nutritional value and economic benefit.Xiang pig is famous in all of the world for its excellent characters.In this study,Xiang pig was separated two groups,high litter size group(number>12)and low litter size group(number<7).The gene expression profile of ovary were compared between the two groups using the Illumina HiSeq2000 transcriptome sequencing technology.Then,8 genes which significant differences expression in two group were selected to vertify by Real-time PCR.The 8 genes were closely associated with the steroid hormone synthesis process.Further,the promoter regions of the 8genes were cloned to analyze promoter region structure polymorphism;BSP-PCR and was performed to analysis the HSD3B1 genes,promoter region methylation level.The purpose of the study is to understand the difference in steroidogenic between two Xiang pig groups and provide molecular theoretical basis for protection and developing the Xiang pig breed.The following results are obtained through the study:1.The 8 genes which associated with the steroid hormone synthesis process and 1control gene were amplified by the Real-time PCR from the Xiang pig ovary cDNA library.All of the amplification curves were S types and the melting curves were single peak which indicated the PCR primer were specifically.The expression pattern of the 8 genes by real-time quantitative PCR were coinside with the results of transcriptome sequencing.All of the 8genes were higher expression in high litter size group when compared with the low litter size group.Moreover,The expression of the 8 genes in Xiang pig showed a progressive increase in the first three developmental stage.2.The promoter regions of the 8 genes were cloned and sequenced from the genomic DNA of Xiang pig ovary with high and low litter sizes.After analysis with the bioinformatics software,the promoter regions of the 8 genes were characterized by TATA box,CAAT box,CG box,and Octamer.There was a C/G polymorphism site at the 1200 bp in the upstream of the SCARB1 gene transcription start site.There was a G/T polymorphism site at the 376 bp in the upstream of LDLR gene transcription start site.There was a C/T polymorphism at the 69 bp in the upstream of CYP11A1 gene transcription start site.There was a T/C polymorphismsite at the 130 bp in the upstream of the CYP17A1 gene transcription start site.There were a A/G polymorphism site at 554 bp and a C/T polymorphism site at 155 bp in the upstream of the StAR gene transcription start site.There was a C/T polymorphism site at 261 bp in the upstream of the AKR1C1 gene transcription start site.There was a T/C polymorphism site at300 bp in the upstream of the HSD3B1 gene transcription start site.All of the these polymorphism sites were located at the critical region of the promoter,which play an impotant role to affect the gene transcription efficiency and further affect molecular regulatory steroid hormone synthesis,ultimately regulated the litter size of Xiang pig.3.There was a methylation site at the 78 bp in the upstream of the HSD3B1 gene transcription start site in both high and low litter sizes Xiang pigs ovary.This site located at the CAAT box promoter core region.The methylation frequency of CpG2 locus was significantly correlated with the expression level of Spearman(P < 0.01),with a correlation coefficient of 0.724 moderate correlation.The methylation frequency was higher in the low litter sizes group when compared to the high litter sizes group in Xiang pig.Methylation level may affect the HSD3B1 gene transcription efficiency and further affect the synthetic steroid hormone molecular regulatory,ultimately regulate the little sizes of Xiang pig.In summary,the difference in the expression of 7 genes ovarian steroid hormone in high and low yield Xiang pig groups may be resulted from the polymorphisms in the key position of gene promoter and the diverse of transcription factor binding elements.The methylation frequency at 78 bp upstream of the transcription start point of HSD3B1 gene may affect gene transcription efficiency.