香猪类固醇激素合成关键基因差异表达的分子机制研究

香猪类固醇激素合成关键基因差异表达

的分子机制研究

中文摘要

香猪是主产于贵州的地方猪种资源，也是世界上优秀的猪种资源之一，具有较高的营养价值和经济效益，广受关注。本文以香猪高产仔和低产仔群体为研究对象，利用Illumina HiSeq2000转录组测序技术，比较两个群体发情期卵巢基因表达差异，选择与类固醇激素合成相关，且转录组测序结果差异显著的8个基因，采用qRT-PCR，BSP-PCR技术等方法，进行基因表达定量的验证。启动子结构差异和甲基化修饰表观遗传学研究；目的在于了解影响香猪类固醇激素合成基因表达差异的分子机制，以期对香猪进行合理开发和科学保护提供分子理论基础。通过研究获得了下列结果：

1.从香猪卵巢组织cDNA文库中通过荧光定量PCR扩增出8个差异表达的基因，高、低产香猪卵巢8个基因的表达量存在差异表达，高产群体表达量显著高于低产群体，实时荧光定量PCR结果与转录组测序一致，1-3月卵巢8个基因的表达量逐渐增加。

2.从香猪卵巢基因组中克隆差异表达基因的启动子区域，经生物信息学分析，存在TATA box，CAAT box，CG box，Octamer特征结构。SCARB1基因转录起始点上游1200bp处有C/G多态性，LDLR基因转录起始点上游376bp处存在G/T多态性，CYP11A1基因转录起始点上游69bp处存在C/T多态性，CYP17A1基因转录起始点上游130bp处存在T/C多态性，StAR基因转录起始点上游554bp处存在A/G多态性、155bp处存在C/T多态性，AKR1C1基因转录起始点上游261bp处存在T/C多态性，HSD3B1基因转录起始点上游300bp处存在T/C多态性。以上这些SNP位点位于基因启动子转录起始关键位置，影响转录因子的结合，可能影响基因转录效率，可能是基因表达量差异的原因之一。

3.高、低产香猪卵巢HSD3B1基因转录起始点上游78bp处存在CpG2甲基化位点，该位点为转录起始的核心启动子CAAT box区域，CpG2位点的甲基化频率与表达

量之间的相关系数为0.724，中等程度相关。检测到低产香猪甲基化频率高于高产香猪，可能影响基因转录效率，影响HSD3B1基因表达。

综上所述，高、低产香猪卵巢类固醇激素合成7个基因的表达差异可能与基因启动子转录起始关键位置的SNP多态性有关，及与转录因子结合元件的结构差异相关；HSD3B1基因转录起始点上游78bp处甲基化频率可能影响转录效率，可能影响基因表达，进一步引起香猪产仔数的变化。

关键词：香猪；卵巢类固醇激素；差异表达；实时荧光定量PCR；启动子；甲基化

Differential Expression Mechanism of Key Genes Related to Steroid Hormone Synthesis in Xiang Pigs

Abstract

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 HiSeq 2000transcriptome sequencing technology.Then,8genes which significant differences expression in two group were selected to vertify by Real-time PCR.The8genes were closely associated with the steroid hormone synthesis process.Further,the promoter regions of the8 genes were cloned to analyze promoter region structure polymorphism;BSP-PCR and was performed to analysis the HSD3B1genes,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.The8genes which associated with the steroid hormone synthesis process and1 control 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 the8genes by real-time quantitative PCR were coinside with the results of transcriptome sequencing.All of the8 genes were higher expression in high litter size group when compared with the low litter size group.Moreover,The expression of the8genes in Xiang pig showed a progressive increase in the first three developmental stage.

2.The promoter regions of the8genes 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 the8genes were characterized by TATA box,CAAT box, CG box,and Octamer.There was a C/G polymorphism site at the1200bp in the upstream of the SCARB1gene transcription start site.There was a G/T polymorphism site at the376bp in the upstream of LDLR gene transcription start site.There was a C/T polymorphism at the69 bp in the upstream of CYP11A1gene transcription start site.There was a T/C polymorphism