羊毛品质性状相关基因CCNY的验证和分析

发布时间：2018-04-13 04:26

本文选题：中国美利奴绵羊 + CCNY基因　；参考：《畜牧兽医学报》2017年08期

【摘要】：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(P0.05);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(P0.05);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(P0.01)。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。
[Abstract]:The aim of this study was to verify the correlation between Cyclin YC gene and wool quality traits, and to explore its possible mechanism of action.In this study, 744 Chinese Merino sheep (Xinjiang Army Reclamation) were used to search for polymorphic loci by Sanger mixed pool sequencing, individual genotypes were identified by MALDI-TOF-MS method, and gene expression was detected by real-time RT-PCR.The protein localization was analyzed by immunofluorescence staining, the proliferation of cells was detected by CCK-8 method, and the activity of Wnt/ 尾 -catenin signaling pathway was detected by luciferase reporter gene technique.The results showed that three SNPs loci of the CCNY gene of Chinese Merino sheep were identified and named as CCNYSNP1 rs613860955) and CCNYSNP3 rs611417387 respectively.Correlation analysis showed that the three SNPs haplotypes and their haplotypes were significantly correlated with wool fineness (P0.05A), and the expression of CCNY gene was higher in testis and lungs and moderately expressed in skin and other tissues.Immunofluorescence analysis showed that CCNY was mainly located in the inner root sheath of hair follicles and the proliferation of CCK-8 cells in hair follicles.Overexpression of CCNY gene significantly promoted the proliferation of human epidermal immortalized cells (HaCaT) and sheep fetal fibroblast (SFF), and the results of report gene analysis showed that overexpression of CCNY gene significantly promoted the activity of Wnt/ 尾 -catenin signaling pathway.The results showed that CCNY was an important gene to control wool fineness, and the three SNPs genes identified in this study were expected to be used in molecular assisted selection of fine wool sheep.
【作者单位】：农业部鸡遗传育种重点实验室;黑龙江省动物细胞与遗传工程重点实验室;新疆农垦科学院畜牧兽医研究所;
【基金】：国家转基因专项课题(2014ZX08009-002;2009ZX08009-160B)
【分类号】：S826.2

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