牛朊蛋白基因（PRNP）23bp和12bp插入/缺失不同单倍型的分子调控网络解析及其新标记发掘

Recently independent studies from our team and foreign scientists have revealed that they are dramatic effects of four different haplotypes consisting by the 23 bp insertion/deletion (I/D) within the promoter and the 12 bp I/D within the intron 1 of the prion protein gene (PRNP) on the PRNP gene mRNA expression within Bos animals. Therefore, how do they affect or adjust the relative genes expression and furthermore have association with the resistance to bovine spongiform encephalopathy (BSE) after conducting their biological functions in vivo?. In the present study, firstly, we will genotype the polymorphisms of 23 bp and 12 bp I/D loci of the PRNP gene in Zhongdian Yellow cattle (Bos taurus) and select the homogeneous and ideal RNA samples. Then, RNA-sequencing will be used high throughput to explore the genes expression profile in medulla oblongata with four haplotypes (23I-12I, 23I-12D, 23D-12I and 23D-12D). Thus, the differential expression genes molecular network within different haplotypes will be established. With the application of the Northern hybridization, real-time PCR and protein expression level analysis, the differential expression genes will be confirmed and screened. Moreover, according to the related physiological and biochemical knowledge, two importantly differential expression genes and additional two important genes with coding SNP mutation will be selected to clone and acquire their full length. The mutations in the coding and regulatory regions will be check and predict the related regulating factors by using the cis-trans regulatory elements mutual experiment and the reporter gene assay. All attempts will help to select the relative gene or molecular marker which could affect, judge, control or convert to BSE.. Finally, our results conducting from the present study will provide noticeable theory significance and practice value on improving cattle quality and reducing potential BSE threaten for beef industry in future.

我们和国外独立研究均表明Bos属各种牛朊蛋白基因（PRNP）23bp和12bp插入/缺失（I/D）4种不同单倍型间其mRNA表达量有显著差异，那么它们在执行后续生物学功能过程中如何进一步影响或调节相关基因表达从而与疯牛病（BSE）抗性相关联了？.本项目通过检测中甸黄牛PRNP23bp和12bpI/D基因型遴选均质样品，采用转录组测序高通量检测和比较4种单倍型的延髓基因表达谱，构建差异表达基因分子调控网络；通过Northern杂交、定量PCR和蛋白表达验证，挖掘出网络中有代表性的关键基因；结合差异表达基因的理化功能，克隆2个关键差异表达基因和2个重要cSNP所在基因全序列，确认和检测其编码区和调控区突变，预测相关调控因子并进行顺反式调控元件互作和报告基因分析，从而筛选到提高疯牛病抗性甚至影响、衡量、控制或转化为疯牛病的基因或分子标记。对提高牛品质和降低BSE威胁有重要的理论意义和实践价值。

为筛选牛海绵状脑病（BSE）抗性以及影响控制BSE的新基因或分子标记，提高牛品质和降低BSE威胁。因前期发现中甸黄牛PRNP基因四种相关单倍型频率较为均匀，故以它作为研究对象开展研究，主要结果如下：.1.检测了来自中甸的921头黄牛的mtDNA D-loop高变区，遴选出Bos taurus属中甸黄牛655头，对其PRNP基因的23-12bp indel鉴定，23I-12I、23D-12D、23D-12I和23I-12D单倍型的频率分别为0.433、0.168、0.377和0.022。.2.筛选到23I-12I和23D-12D两个极端单倍型下性别、年龄、毛色原则上均一的各12列中甸黄牛样品，对其延髓进行mRNA和miRNA测序，mRNA测序获得108个差异基因，其中50个上调、58个下调；miRNA测序获得42个差异miRNA，其中24个上调、18个下调。以PRNP基因为核心，重点解析了两个单倍型之间的差异表达基因分子调控的四个网络：mRNA-miRNA网络、ceRNA网络、蛋白质-蛋白质相互作用（PPI）网络、TF-mRNA调控网络。对调控网络中的基因分别与其他基因计算相关性，成功获得7个关键基因PCDH19、MYL2、TRPV6、LEPR、CDKN2B、CNGB1、DNAH11。.3.通过RT-PCR测定部分差异基因和miRNA的表达量，对比mRNA和miRNA测序结果，获得相同的表达趋势。生物信息学软件预测了候选基因与差异miRNA之间的靶向关系，将PRNP与bta-miR-196b、bta-miR-2368-3p，PCDH19与bta-miR-196b，APOBEC2与bta-miR-122、bta-miR-499a进行双荧光素酶报告基因检测，发现均对靶基因有靶向负调控。对PCDH19基因进行5’调控区分子克隆，得到6个突变位点，共构成12个单倍型，其中GTCACC为优势单倍型。在五大数据库进行转录因子结合位点预测，进一步确认突变位点的影响。.综上，本项目通过大样本运用mRNA和miRNA测序，系统发掘了以PRNP基因为核心下抗BSE性状的新基因及其分子标记，为牛BSE抗性性状分子遗传标记提供了新见解。已发表论文3篇（其中SCI论文两篇），参加国内学术会议6次，参与指导培养研究生3人，1人晋升教授职称，团队实力加强。