兴义矮脚鸡匍匐性状关键基因Cp的定位、克隆及其功能研究

Xingyi Aijiao chicken, originated from Guizhou province, is a valuable genetic resource characterized with the creeper phenotype, good uniformity, and quality meat with good taste. The creeper phenotype is determined by a major gene, Cp, which is located in Chromosome 7 and has been showed as a dominant homozygous lethal gene. However, the genomic mapping, structure and function of Cp gene are unknown. In the present study, Xingyi Aijiao chicken will be used as experimental populations and segregated families will be constructed to generate full-sib progeny. Whole-genome resequencing and bioinformatics analyses will be performed for genomic mapping of Cp (Creeper) gene and identification of relevant causative mutation. The genetic effects of the Cp gene will be determined. Subsequent experiments including quantitative PCR, Western blotting, fluorescence in situ hybridization (FISH) and cell transfection will be performed to elucidate biological functions of the Cp gene. Researchers around the world have been studying the Cp gene for more than eighty years.The results of this study will decipher the genomic structure and molecular mechanism of Cp gene and develop an accurate method of identifying the causative mutation, which will contribute to conservation and selection of Xingyi and other Chinese local chicken breeds.

兴义矮脚鸡是原产于我国贵州省的地方品种，胫短身矮、体躯匀称、肉美味鲜，是一种稀有珍贵的家禽遗传资源。匍匐体型是该品种的重要特征，控制这一性状的关键基因是Cp（Creeper）基因。Cp基因位于7号染色体上，为显性纯合致死基因，但其基因定位、结构和功能尚不清楚。本项目以兴义矮脚鸡为实验材料，组建分离家系，采用全基因组重测序技术及生物信息分析，对控制该品种匍匐体型的Cp基因进行基因组定位，筛选导致匍匐体型的原因突变，并对该基因的遗传效应进行研究；通过定量表达分析、Western blotting、荧光原位杂交（FISH）、细胞转染技术对该基因的功能进行深入研究，推测该基因的生物学功能。国际上对Cp基因的研究已有80多年历史，本研究将首次从分子水平揭示Cp基因的结构和功能特点，并建立准确的Cp基因原因突变检测方法，有助于对兴义矮脚鸡和我国其他地方鸡品种资源的保种和改良工作。

鸡匍匐性状是由常染色体上显性纯合致死基因Cp所控制，其主要特征为胫短身矮、翅膀短小。Cp作为常染色体上显性半致死基因，被很多遗传学和分子生物学教科书所引用。本研究以兴义矮脚鸡为实验素材，组建Cp基因的分离家系。组建Cp基因型分离家系，选择3个全同胞或半同胞家系中个体，保证每个家系有2只致死型胚胎、2只匍匐型个体、2只野生型个体，共计18个个体。所有个体进行全基因组重测序，在全基因组范围内进行各种变异类型分析。研究发现，7号染色体上21798705到21810600区域缺失，该缺失区域仅存在于匍匐型与致死型胚胎。该区域内仅含唯一的功能基因是印度刺猬因子(IHH)，因此，确定IHH基因缺失是导致鸡匍匐表型的遗传原因，这也在Cp基因型群体分离实验中得到证实。多重PCR分析表明，致死型个体仅扩增224bp条带，野生型个体仅扩增438bp条带，匍匐型个体扩增出224bp和438bp两条带，由此建立了Cp基因型诊断的分子标记。利用CRISPR/Cas9系统在鸡DF-1细胞系中，构建IHH基因缺失纯合型细胞系，并利用转录组比较敲出IHH前后基因表达谱的变化，进一步确认了IHH相关通路基因的变化。利用qPRC验证，确认野生型相比，匍匐型个体IHH、PTCH1、Smo、HIP以及下游转录因子Gli1、Gli2和靶基因ColⅡ、OPN表达水平均显著降低，进一步证实IHH缺失通过调控该信号通路基因的表达水平而影响鸡匍匐表型。