HGTP基因在羊毛发育中的调控作用

The previous data of arrangement and ingredients of wool fiber showed that the content of high glycine tyrosine protein (HGTP) in fine wool was significantly higher than that of coarse wool in sheep. HGTP belongs to wool keratin associated proteins (KAPs) and comprises KAP6, KAP7 and KAP8 genes in sheep. HGTP was highly expressed with specificity and which formed the wool curvature. So it is suggested that the regulation and specific expression pattern of HGTP encoding gene have a strong correlation with the wool diameter and it can be provided guidance for fine wool sheep breeding. In this application, sheep KAP6, KAP7, KAP8 genes and their specific promoters will be analyzed in fine wool sheep and coarse wool sheep as cotrol. The aim is to analyze the differences in gene polymorphisms, gene expression levels, promoter activity and their associations with wool fiber diameter with the methods include Real-time PCR, HRM, EMSA and differential expression analysis. Furthermore, the HGTP promoter activity and its affinity with transcroption factors will be analyzed by the way of electrophoretic mobility shift assay (EMSA) and constructing vector, tranfecting cells and detecting the expression of reporter genes. The effects of polymorphisms of HGTP gene and its promoter region on combination between regulating factor and combining site in control region will be analyzed, including the effects of promoter region and control region on the expression levels of the objective gene. The polymorphisms detection can be used Real-time PCR, high resolution melting (HRM) and PCR-SSCP. The DNA microarray is used for gene expression profile. The last, the objective is to demonstrate the role of HGTP gene, the effects of polymorphisms and promoter activity on wool fiber development, which provide some guidance for fine wool sheep breeding.

羊毛细度是决定羊毛品质的主要影响因素。研究表明羊毛的细度与弯曲有显著相关性，且羊毛纤维正副皮质中高度特异性表达的高甘氨酸酪氨酸蛋白（HGTP）是决定羊毛性状的重要因素。因此，申请者认为研究HGTP基因的表达调控是阐明羊毛发育机制和细毛羊育种的前提基础。本研究以绵羊HGTP编码基因及其启动子为研究对象，采用Real-time PCR、HRM、EMSA和DNA芯片等技术分析比较粗毛羊和细毛羊在基因多态性、基因表达和启动子活性差异以及与羊毛细度之间的关联性。利用HRM和PCR-SSCP等分析基因多态性；通过构建载体并检测报告基因分析启动子活性及特异性；利用EMSA分析启动子及调控区与转录因子的亲和性以及对基因表达的影响；利用定量PCR和DNA芯片分析基因表达差异。综合上述研究，阐明绵羊HGTP基因在羊毛发育中的调控作用以及基因多态性对羊毛发育的影响，项目的实施可为细毛羊育种提供基础。

完成了绵羊HGTP基因（KAP6.1、KAP7和KAP8）及其上游序列和启动子的筛选、克隆，包括引物设计和条件优化、序列测定和比对；依据预测启动子区域，设计并构建不同长度的表达载体，转染皮肤成纤维细胞，并以3T3细胞系做对照，进行细胞培养以及启动子活性检测。获得的两个启动子元件都能驱动外源基因在体外细胞水平表达。以中国美利奴羊（军垦型）5只作为实验组，萨福克羊5只作为对照组，采集背部毛样检测其纤维直径和自然长度；并且采集肩胛部皮肤组织提取RNA，利用实时荧光定量的方法分析了KAP6.1、KAP7和KAP8.1在两品种绵羊皮肤组织中的表达情况，结果发现3 个基因在萨福克羊皮肤组织中的表达水平极显著或显著高于在中国美利奴皮肤组织中的表达水平；利用PCR-SSCP方法分析了3个基因在中国美利奴羊、湖羊、罗姆尼羊和美利奴-湖羊杂交羊4个群体中的多态性分布，筛选到与羊毛品质有关联的位点。结合HGTP基因的表达水平，分析了基因表达与羊毛纤维和自然长度间的相关，结果表明HGTP基因表达水平与羊毛纤维直径都存在着极显著的高度正相关，与羊毛自然长度呈现显著的中等负相关；羊毛直径与自然长度之间存在极显著的高度负相关。项目撰写并发表研究论文4篇，其中SCI 2篇。培养研究生4人，其中2人毕业。