绵羊HGTP基因家族在羊毛发育中的功能解析

The study of wool fiber growth and development are the basis of fine wool sheep breeding, and the content and distributions of high glycine-tyrosine protein (HGTP) had significant difference between fine wool and coarse wool. HGTP is an important ingredient in sheep wool fiber that affects the wool finess and crimp, which includes KAP6, KAP7 and KAP8, but their molecular mechanisms in the wool development are not well understood. After preliminary studies of KAP6, KAP7 and KAP8 gene expressions and DNA polymorphisms in Chinese Merino fine wool sheep and coarse wool sheep, we supposed that the difference for gene expression and regulator elements would play a key role in the wool development. So, the aim of this program is to determine and find the HGTP gene family promoter core regulatory regions and further to investigate the transcriptional regulation mechanisms. By cloning the promoter sequences and especial promoter sequences for Chinese Merino fine wool sheep and coarse wool sheep, to predict regulatory elements and find core promoter. By using PGL3-basic dual luciferase report gene vector to construct different length fragments, and then are transfected into ovine fibroblast to determine the promoter activity and tissue specificity. By using EMSA to find cis-acting element and determine its affinity to trans-acting factor. By using Chromatin Immunoprecipitation (ChIP), RNAi and Real time-PCR to further investigate the effects of trans-acting factor on cis-acting element and gene expression level. The last, comprehensive analysis of the above results will help to determine the regulatory mechanism of HGTP gene family in wool development and their effect mechanisms on wool physical characteristics.

羊毛生长调控研究是细毛羊育种的基础，粗细不同羊毛中存在显著的高甘氨酸-酪氨酸蛋白（HGTP）含量和分布差异，该蛋白家族包括KAP6、KAP7和KAP8，其是影响羊毛细度和弯曲的重要组分，但表达调控机制尚不清楚。申请者通过初步研究推测HGTP家族编码基因及其调控原件差异是该基因表达以及影响羊毛细度的关键。因此，本项目以HGTP基因为研究对象，以中国美利奴细毛羊和萨福克羊为材料，分析HGTP基因在羊毛发育中的调控作用。本研究拟通过克隆两个品种羊的KAP6、KAP7和KAP8基因及其上游序列，预测比较调控元件并筛选核心启动子；构建荧光素酶表达载体，分析启动子活性和组织特异性差异；EMSA分析启动子的顺式作用元件以及与转录因子的亲和性；利用CHiP、转录因子抑制和RNAi研究转录因子与启动子顺式元件的作用以及对基因表达的影响。综合分析以阐明HGTP基因家族在羊毛发育中的作用以及对羊毛性状的影响。

羊毛是重要的畜产品之一，其细度和卷曲决定了其实用性和经济价值。研究表明，粗毛和细毛成分的差异之一在于HGTP的含量和分布不同，研究还证明其家族编码基因多态性影响了羊毛性状。绵羊HGTPs是一个多基因编码的角蛋白家族，主要包括KAP6、KAP7和KAP8三种不同的角蛋白联合蛋白基因。本研究以中国美利奴细毛羊和萨福克羊为细毛羊和粗毛羊代表，主要比较分析两种绵羊及其他品种的三个基因表达差异，并对cDNA全长克隆分析，比较基因编码区及上游调控区DNA差异，分析启动子调控元件和甲基化，从而进一步阐明KAP6、KAP7、KAP8基因在不同绵羊品种中的功能差异。.RQ-PCR分析表明KAP6、KAP7和KAP8基因在萨福克羊和中国美利奴细毛羊皮肤中的表达水平和羊毛直径高度正相关，相关系数分别为0.941（P=0.00）、0.842（P=0.01）和0.948（P=0.00），在萨福克羊皮肤组织中的表达量分别为细毛羊的4.23、5.74和5.87倍。5´-端和3´-端RACE克隆得到了三个基因全长cDNA序列，长度分别为606 bp、711 bp、559 bp，其CDS区序列长度分别为252 bp、258 bp和189 bp，5´UTR长度分别为43 bp、60 bp和50 bp，并确定了三个基因的转录起始点及其上游序列。对比分析了三个基因CDS区在四个绵羊群体的多态性，KAP6基因获得六种多样性序列，KAP7基因检测到两种多样性序列，KAP8基因检测到五种多样性序列，基因多态性可能影响羊毛特性。采用TAIL-PCR分别克隆获得三个基因上游区未知序列片段，测序得到三个基因编码区上游未知序列1877bp、3224bp和1826bp的DNA信息，分析得到了一些调控元件的结合位点，发现了KAP8上游一个可以用于细毛羊育种标记的STR位点，为进一步阐明KAP6、KAP7和KAP8特异性表达及其活性和功能奠定了基础。