羊毛品质性状相关基因CCNY的验证和分析

Verification and Analysis of Wool Quality Traits-associated CCNY Gene

旨在验证Cyclin Y(CCNY)基因与羊毛品质性状的相关性,并探索其可能的作用机制。本研究以744只中国美利奴羊(新疆军垦型)为试验材料,采用Sanger法混池测序寻找多态性位点;利用MALDI-TOF-MS方法进行个体基因型鉴定;利用real-time RT-PCR检测基因表达;利用免疫荧光染色技术分析蛋白定位;利用CCK-8方法检测细胞的增殖;利用荧光素酶报告基因技术检测Wnt/β-catenin信号通路的活性。结果表明,本研究鉴定出中国美利奴羊CCNY基因的3个SNPs位点,分别命名为CCNYSNP1(rs613860955)、CCNYSNP2(rs622396422)和CCNYSNP3(rs611417387)。关联分析显示,这3个SNPs及其构建的单倍型均与羊毛细度显著相关(P<0.05);组织表达谱检测分析显示,CCNY基因在睾丸和肺中有较高的表达,在皮肤等组织中有中等程度的表达;免疫荧光分析显示,CCNY主要定位于毛囊的内根鞘和毛母质细胞;CCK-8细胞增殖检测分析显示,过表达CCNY基因显著促进人表皮永生化细胞(HaCaT)和绵羊胎儿成纤维细胞(SFF)的增殖(P<0.05);报告基因分析结果显示,过表达CCNY基因极显著促进Wnt/β-catenin信号通路的活性(P<0.01)。以上结果显示,CCNY是控制羊毛细度的一个重要基因,所鉴定出的CCNY基因3个SNPs有望用于细毛羊的分子辅助选育。

The aim of this study was to verify whether CCNY gene was associated with sheep wool production and quality traits, and to explore its possible mechanism. Seven hundred forty-four Chinese Merino sheep （Junken type） were used in the present study. SNPs were identified by Sanger sequencing of the pooled genomic DNA samples, and genotyped by MALDI-TOF-MS. Real-time RT-PCR was used to analyze the gene expression, and immunofluorescence staining was used to localize the protein. CCK-8 method was used to detect cell proliferation. Luciferase reporter gene assay was used to assess the activity of Wnt/β-catenin signaling pathway. The results showed that a total of 3 SNPs（CCNYSNP1, CCNYSNP2, CCNYSNP3） were identified in CCNY gene in Chinese Merino sheep. Association analysis displayed that the 3 identified SNPs and their haplotypes were significantly associated with wool fiber diameter （P〈0.05）. Tissue expression analysis showed that CCNY gene was highly expressed in testicle and lung, and mod- erately expressed in several tissues including skin. Immunofluorescence staining showed that CCNY was localized in inner root sheath and hair matrix of hair follicle. CCK-8 assay results showed that CCNY overexpression significantly promoted proliferation of HaCaT and SFF cells （P〈0.05）. Reporter gene analysis showed that CCNY overexpression significantly increased the activity of Wnt/β-catenin signaling pathway（P〈0.01）. These data suggest that CCNY is a crucial factor governing fiber diameter trait, and 3 identified CCNY SNPs might be used in molecular marker-assisted selection for fine wool sheep breeding.