朊蛋白基因（PRNP）23bp和12bp插入/删除位点可否作为非家养牛疯牛病抗性的分子标记？

Recent attempts to discover genetic factors affecting cattle resistance/susceptibility to bovine spongiform encephalopathy (BSE) have led to the identification of the 23bp insertion/deletion (Indel) within the promoter and the 12bp indel within the intron 1 of the prion protein gene (PRNP), showing a significant association with the occurrence of classical form of the disease. In the present study, the Tibetan Yellow cattle (Bos taurus) will be selected as the control group while the Chinese yakow (Bos taurus x Bos grunniens) and yak (Bos grunniens) under the same circumstance will be assigned to the experimental groups for answering the scientific question that do the non-domestic cattle (non-Bos taurus) have the similar characteristics on resistance to BSE with cattle (Bos taurus). After collecting samples (330 heads/type), the PRNP gene frequencies of the 23bp and the 12bp indels polymorphism from Tibetan Yellow cattle, Chinese yakow and yak will be detected and compared their distribution between healthy and BSE-affected cattle based on published papers. The model effect and characteristics of the 23bp and 12bp indels in the PRNP gene on the resistance to BSE between non-Bos taurus cattle and normal cattle (Bos taurus) will be compared and solved, according to evaluate the promoter activity by the dual luciferase reporter gene assay, to identify binding activity of transcription factors SP1 and RP58 to the PRNP promoter with different haplotypes by electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) methods, to analyze PRNP mRNA expression in the medulla oblongata by real-time PCR technique based on samples with different the 23bp and 12bp indels polymorphisms and haplotypes. Finally, our results conducting from the present study will provide noticeable theory significance and practice value on improving non-Bos taurus cattle quality and reducing potential BSE threaten in future.

最近的研究表明家养牛朊蛋白基因（PRNP）23bp和12bp的插入/删除多态与疯牛病（BSE）抗性有显著关联。为探索非家养牛与家养牛在疯牛病方面是否有类似的规律这个科学问题，本项目巧妙地以同一生态环境下含不同家养牛血源的藏黄牛、犏牛和牦牛为对象，通过大量样品（各330头），检测PRNP基因23bp和12bp的插入/删除多态，统计各位点的频率，与报道的健康牛和BSE病牛比较，分析之间的差异；利用双荧光素酶报告基因评价不同插入/删除多态对PRNP基因启动子表达活力的影响；通过凝胶阻滞迁移和染色质免疫共沉淀技术评价转录因子（RP58和SP1）与不同单倍型DNA的结合能力；最后测定不同单倍型的3种牛PRNP基因mRNA在延髓的表达量，比较分析获得非家养牛PRNP基因23bp和12bp的插入/删除多态与BSE抗性的作用模式与特点。对提高非家养牛品质和降低潜在的BSE威胁有重要的理论意义和实践价值。

为全面探索非家养牛和家养牛在疯牛病抗性机理方面的差异，我们以藏黄牛、犏牛和牦牛为研究对象，通过采集中甸的562头牦牛、377头犏牛和368头黄牛组织DNA和RNA样品，对其PRNP基因多态性、mRNA表达量及其分子机理进行了研究。.三种牛的23bp等位基因以缺失为主（频率分别为0.910、0.588和0.594），12bp等位基因以插入为主（频率分别为0.869、0.830和0.764）；三种牛的23bp的基因型均以缺失/缺失（-/-）为主（频率分别为0.838、0.467和0.399），12bp以插入/插入(+/+)为主（频率分别为0.767、0.714和0.621）；中甸牦牛和中甸犏牛单倍型均以23-12+为主（频率为0.794和0.444），而中甸黄牛以23+12-为主（频率为0.434）。三种牛23bp和12bp的不同基因型和二倍型之间的mRNA表达量差异均不显著（p>0.05），但牦牛的23bp和12bp及犏牛和黄牛的23bp均表现为插入基因型的mRNA表达量低于相关缺失基因型，同时牦牛的二倍型也是--/--的表达量较高（2.88±0.62）。表明对于牦牛于23bp和12bp多态性对PRNP的表达与家养牛基本一致。.双荧光素酶报告基因检测不同单倍体启动子活性顺序为：23-/12- > 23-/12+ > 23+/12- > 23+/12+，差异均显著（p<0.05）；但是三种牛的相同基因型或单倍型间差异不显著（P>0.05）。EMSA实验结果表明PRNP基因23bp+和12bp+型可以和转录因子AP1(RP58)和SP1进行特异性结合。说明转录子RP58和SP1参与了PRNP基因启动子转录的进行，从而调控转录和mRNA的表达。说明牦牛与家养牛在启动子表达活性上有相同的趋势，即RP58和SP1可能对PRNP基因的表达有负反馈调节作用。这种作用对于牦牛、犏牛和黄牛具有相同作用机制。因此PRNP的23bp和12bp多态位点可以作为非家养牛疯牛病抗性的分子标记。.综上，本文首次系统研究了大样本中甸牦牛、中甸犏牛和中甸黄牛PRNP基因的启动子和内含子1中的Indel多态，为今后将PRNP基因作为各种牛BSE抗性性状分子遗传标记提供了参考。已发表SCI论文5篇，参加国内学术会议3次，参与指导培养硕士和博士研究生2人和1人，1人晋升高一级技术职称，团队实力加强。