IHH基因缺失对鸡匍匐性状的作用及分子机制

The Creeper trait is a classical phenotype of chickens, which affects the hatchability and uniformity of growth and development in indigenous chicken breeds.This trait is controlled by an autosomal dominant semi-lethal gene (Cp) and has been cited in the genetics and molecular biology textbooks for illustrating autosomal inheritance for decades. Researchers around the world have been investigating the phenotype and the Cp gene over ninety years. Our previous studies showed that the deletion of Indian hedgehog (IHH) caused the Creeper phenotype by resequencing data analysis and this deletion was fully linked with the Creeper trait by large scale segregation analysis. However, the role and molecular mechanism of IHH deletion on the Creeper trait remains unknown. In this study, the IHH deletion cell lines will be constructed from chicken DF-1 cell by CRISPR/Cas9 system, and the monoclonal cell of IHH deletion will be screened using the flow cytometry. The downstream signaling pathways and target genes regulated by the IHH gene will be identified by RNA sequencing and subsequent experiments will be performed to elucidate the functions of target genes. In addition, the constructed segregation families will be used to separate primary chondrocytes carrying the Creeper mutation. Moreover, RNA interference(overexpression and knockdown of the IHH gene in primary chondrocytes) will be conducted to observe the changes of cell phenotypes (such as cell morphology, cell cycle, proliferation and differentiation) and examine expression profiles of genes associated with cell proliferation and differentiation. The results will reveal the role and molecular mechanism of IHH gene deletion on the Creeper trait in chickens from different perspectives and provide a theoretical basis for the conservation and utilization of local chicken breeds.

匍匐性状是影响地方鸡种孵化率和生长发育整齐度的一种重要的表型，其由常染体上显性纯合致死基因Cp所控制，并被很多遗传学和分子生物学教科书所引用。国际上对匍匐性状及基因的研究有着90多年的历史。申请人前期研究发现，IHH基因缺失导致鸡匍匐表型，IHH缺失与鸡匍匐性状紧密关联，但其作用及分子机制尚不清楚。因此，本项目通过CRISPR/Cas9系统在鸡DF-1细胞中构建IHH基因缺失型细胞株，利用流式细胞术筛选IHH基因缺失型单克隆细胞，通过转录组测序鉴定受IHH基因调控的下游信号通路及靶基因；利用已组建的匍匐型分离家系，分离缺失型软骨细胞，干预IHH表达对缺失型软骨细胞表型及与细胞增殖分化相关基因表达的影响。研究结果将从不同角度揭示IHH基因缺失对鸡匍匐性状的作用及分子机制，为地方鸡品种资源的保护和利用提供理论依据。

匍匐性状是其由常染体上显性纯合致死基因Cp所控制，并被很多遗传学和分子生物学教科书所引用。申请人前期研究发现，IHH基因缺失导致鸡匍匐表型，IHH缺失与鸡匍匐性状紧密关联，但其作用及分子机制尚不清楚。本项目分析了IHH基因结构和功能；构建了CRISPR/Cas9敲除鸡IHH细胞株模型及其表达分析；构建IHH敲低和过表达细胞模型，并研究其对原代软骨细胞增殖和分化的分子机制；筛查安徽省地方鸡种中Cp基因型以及干预IHH表达对下游靶基因和信号通路的影响。结果表明，IHH蛋白在禽类中保守性较高，具有偏碱性、强热稳性和低亲水性等特点的分泌蛋白，其在软骨组织中显著高表达。借助CRISPR/Cas9技术，经流式分选缺失型细胞、TA克隆和测序鉴定，成功构建了IHH基因缺失细胞模型。表达分析表明，与WT组相比，PTCH1、Smo、Gli1、Gli2和OPN在敲除组中的表达水平显著降低，ColⅡ表达水平显著上升。成功分离鸡原代软骨细胞，并进一步采用软骨细胞标志蛋白ColⅡ进行鉴定。构建IHH敲低和过表达细胞模型，与对照组和si-NC组相比，si-IHH组中ALP、ColⅩ、TGF-β1、Wnt11、PTHrP、JAK2表达显著降低，ColⅡ表达量显著增加；与对照组和空载体组（OV-NC）相比，OV-IHH组（IHH过表达组）ALP、ColⅩ、Wnt5a、Wnt11、PTHrP、JAK2表达量显著上调，ColⅡ的表达量显著下调。基因分型表明，安徽省7个地方鸡种中未检测到Cp基因型。高通量测序分析表明，si-NC组与si-IHH组相比，差异表达基因305个，其中上调基因152个，下调基因153个，差异基因主要富集于TGF-beta和Wnt等信号通路。本项目进一步解析鸡匍匐性状潜在的分子机制，为地方鸡品种资源的保护和利用提供重要参考。