羊毛品质性状相关基因CCNY的验证和分析

doi:10.11843/j.issn.0366-6964.2017.08.003

摘要/Abstract

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（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。

Abstract:

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 moderately 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.

中图分类号:

S826.2

张文建, 马广伟, 张潇飞, 褚衍凯, 刘静, 杨华, 闫晓红, 李玉茂, 李辉, 王宁. 羊毛品质性状相关基因CCNY的验证和分析[J]. 畜牧兽医学报, 2017, 48(8): 1389-1400.

ZHANG Wen-jian, MA Guang-wei, ZHANG Xiao-fei, CHU Yan-kai, LIU Jing, YANG Hua, YAN Xiao-hong, LI Yu-mao, LI Hui, WANG Ning. Verification and Analysis of Wool Quality Traits-associated CCNY Gene[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2017, 48(8): 1389-1400.

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