藏绵羊不同毛色皮肤组织miRNA表达谱及靶基因分析

doi:10.3864/j.issn.0578-1752.2018.02.014

摘要/Abstract

摘要： 【目的】藏绵羊又称藏系绵羊，是中国三大粗毛羊品种之一，主要分布在西藏及其毗邻的高寒牧区，如青海、甘肃、四川、云南等地。藏绵羊由于其被毛纤维粗长，毛质细腻且保暖性好，是制造藏式地毯的上好原料。而毛色作为一种重要的经济性状，同时制约羊毛的经济价值。然而，目前对于绵羊毛色的分子调控机制尚不明确，以藏绵羊为研究对象，对其不同颜色皮肤组织（黑色和白色）进行转录组测序，旨在探讨miRNA在藏绵羊不同毛色皮肤组织转录后水平中发挥的作用及其可能参与的调控通路。【方法】选取2只具有黑白花毛色的健康藏绵羊，减去体侧被毛使皮肤完全裸露并用75%酒精消毒处理，屠宰后快速切取皮肤组织并保存在组织样品保存液中防止RNA降解，提取RNA后通过miRNA测序和生物信息学分析，获得藏绵羊不同毛色（黑色和白色）皮肤组织miRNA表达谱，筛选组织差异表达miRNA并预测相关靶基因。通过靶基因分析，获得与调控藏绵羊毛色性状可能相关的信号通路。【结果】黑色和白色皮肤组织共获得85.76兆条原始读长，经过滤后得到85.08兆条clean reads；共鉴定出334个已知的miRNA和59个新的miRNA；从中获得23个差异表达miRNA，其中14个差异表达miRNA在白色皮肤组织中表达显著上调，9个表达下调。miR-2284b和miR-744仅在藏绵羊白色皮肤中表达，而miR-23b、miR-411a-5p、miR-30c、miR-423-3p和miR-324-5p则仅在藏绵羊黑色皮肤中表达，但表达量相对较低。表达量最高的miRNA为miR-10a，而倍数差异最大的为miR-23b。其中，miR-10a可参与调节多种信号通路，其作用涉及增殖、分化、凋亡、细胞粘附等各个方面，目前并无调控绵羊毛色方面的相关报道，但其功能及作用机制的研究仍在不断扩展。miR-23b则与Wnt、Notch等信号通路有关，这些信号通路中的部分基因也和黑色素的生成有密切关系。结合倍数差异和miRNA表达量分析，miR-411a-5p、miR103和miR-200b可能也和毛色调控密切相关。本研究共预测出981个靶基因可能参与毛色调控，多数基因和WNT信号通路、MAPK通路、EDNRB信号通路及黑素原生成通路（melanogenesis pathway）相关，其中黑素原生成通路显示和WNT信号通路、KIT信号通路及EDNRB信号通路相互关联。黑素原生成通路显示藏绵羊不同毛色可能最终通过MITF基因调控下游的TYR基因（酪氨酸酶基因）影响黑色素的合成。同时，筛选7个差异表达miRNA进行定量验证，结果表明，5个miRNA在黑色皮肤组织中上调表达，2个miRNA在白色组织中上调表达，定量结果与测序结果一致。【结论】获得了藏绵羊皮肤组织miRNA表达谱，并通过生物信息学分析得到可能和调控毛色性状相关的miRNA和信号通路。这些结果表明，藏绵羊毛色性状可能受到多种miRNA的调控并涉及多个信号通路，这有助于更进一步了解毛色转录后水平的调控过程，并对进一步的功能验证奠定基础。

关键词: 藏绵羊, 皮肤组织, 高通量测序, miRNA

Abstract: 【Objective】 Tibetan sheep is one of three coarse wool sheep breeds in China, distributing mainly in Tibet and its adjacent alpine pastoral areas, such as Qinghai, Gansu, Sichuan, Yunnan. The wool of Tibetan sheep, felt smooth and warm duo to the long and thick fibers, is good raw material for manufacture of Tibetan carpet. Coat color is an important economic traits. However, at present, the mechanism of molecular regulation of sheep coat color is not clear. In this study, the transcriptome of skin tissues of different color (black and white) was sequenced with a aim to explore the role of miRNAs at post-transcriptional levels in different coat color skin tissues and possible regulatory pathways. 【Method】Healthy sheep with black and white coat color was sacrificed to provide skin tissues for RNA extraction and miRNA analysis. Through miRNA sequencing and bioinformatics analysis, miRNA expression profiles of skin tissues of black and white wool color were obtained. Then the differentially expressed miRNAs were screened and the related target genes were predicted. Through the target gene analysis, the signal pathway which is related to Tibetan coat color skin traits were proposed. 【Result】 A total of 85.76 million original reads and 85.08 million clean reads were obtained from the analyzed tissues. Among the clean reads were 334 known miRNAs and 59 newly identified miRNAs and 23 of them were differentially expressed between white and black color tissues. In the 23 differentially expressed miRNAs, 14 and 9 were up-regulated and down-regulated in white skin tissues respectively. miR-2284b and miR-744 were the type that is expressed only in white Tibetan sheep skin, and miR-23b, miR-411a-5p, miR-30c, miR-423-3p, miR-324-5p were the miRNAs that expressed only in black skin. Among them, miR-10a can participate in the regulation of a variety of signaling pathways, involving proliferation, differentiation, apoptosis, cell adhesion. MiR-23b is associated with Wnt, Notch and other signal pathways, which may related to melanin production. MiR-411a-5p, miR103 and miR-200b may also closely relate to regulation of hair color. A total of 981 target genes were predicted to be involved in coat color control, most of which were related to WNT signaling pathway, MAPK pathway, EDNRB signaling pathway and Melanogenesis pathway. The melanogenesis pathway was found to be associated with the WNT signal pathway, the KIT signal pathway, and the EDNRB signal pathway. The synthesis of melanin may be regulated by downstream TYR gene (tyrosinase gene) through MITF gene. Quantitative analysis showed that five miRNAs were up-regulated in black skin tissue, and two miRNAs were up-regulated in white tissues. The quantitative results were consistent with the sequencing results. 【Conclusion】 The miRNA expression profile of the skin of Tibetan sheep were obtained and the miRNAs and signal pathways which may be related to control of coat color were obtained by bioinformatics analysis. These results indicated that the color traits could be regulated by many miRNAs and involved many signal pathways. This may help understand the level of post-transcriptional regulation of the process, and lay the foundation for further functional verification.

Key words: Tibetan sheep, skin tissue, high throughput sequencing, miRNA

吴震洋，唐晓惠，付玉华，王昇，章程，李京津，余梅，杜小勇. 藏绵羊不同毛色皮肤组织miRNA表达谱及靶基因分析[J]. 中国农业科学, 2018, 51(2): 351-362.

WU ZhenYang, TANG XiaoHui, FU YuHua, WANG Sheng, ZHANG Cheng, LI JingJin, YU Mei, DU XiaoYong. Profiles of miRNAs and Target Gene Analysis with White and Black Skin Tissues of the Tibetan Sheep[J]. Scientia Agricultura Sinica, 2018, 51(2): 351-362.

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