金梅, 张丽娟, 曹倩, 郭鑫英辽宁师范大学生命科学学院/辽宁省生物技术与分子药物研发重点实验室,辽宁大连 116029

The Screening and Identification of LncRNA Related to Villus Growth in Liaoning Cashmere Goats by MT and FGF5

JIN MEI, ZHANG LIJUAN, CAO QIAN, GUO XinYingLiaoning Normal University School of Life Sciences, Liaoning Provincial Key Laboratory of Biotechnology and Molecular Drug Development, Dalian 116029, Liaoning

收稿日期:2018-09-3接受日期:2018-12-3网络出版日期:2019-02-16

基金资助:

国家自然科学基金.31772557 辽宁省自然科学基金.20170540577

Received:2018-09-3Accepted:2018-12-3Online:2019-02-16
作者简介 About authors
金梅,E-mail： jm6688210@163.com






摘要
【目的】筛选出辽宁绒山羊皮肤成纤维细胞中与绒毛生长相关的LncRNA,为绒毛生长相关LncRNA的功能及机制研究提供基础性数据。【方法】提取MT和FGF5处理的辽宁绒山羊皮肤成纤维细胞总RNA,通过样品总RNA电泳检测、测序数据质量评估、Mapping比对、样品间相关性检查对提取的总RNA进行质量检测。筛选出差异表达的LncRNA并预测其靶基因,通过GO和KEGG富集分析,筛选出与绒毛生长相关的LncRNA,并通过Real-time PCR对目标LncRNA进行表达验证。【结果】（1）样品总RNA质量检测结果显示：RNA 完整性良好、GC含量相对较高,序列较稳定、样品间表达水平相关性均较高、符合测序要求。（2）差异表达LncRNA的筛选结果显示：1.0g·L ^-1 24h组差异表达LncRNA有32个,其中4个表达上调,28个表达下调;0.2g·L ^-1 24h组差异表达LncRNA 有10个,其中4个表达上调,6个表达下调;0.2g·L ^-1 72h组差异表达LncRNA有113个,其中5个表达上调,108个表达下调。10 ^-4g·L ^-1 24 h组差异表达LncRNA有164个,其中有70个上调,94个下调;10 ^-4g·L ^-1 72 h差异表达LncRNA 有189个,其中有78个上调,111个下调;10 ^-6g·L ^-1 24 h组差异表达的LncRNA有123个,其中有27个上调,96个下调 。（3） 靶基因GO富集分析结果显示：1.0g·L ^-1 24h组差异表达LncRNA靶基因富集在GO的negative regulation of transcription from RNA polymerase II promoter;0.2g·L ^-1 24h组无差异表达LncRNA靶基因富集的GO term;0.2g·L ^-1 72h组差异表达LncRNA靶基因富集在GO的cellular metabolic process biological_process,binding molecular_function,FGF5处理组中只有10 ^-4g·L ^-1 72 h组差异表达LncRNA靶基因富集在cell cellular_component、cell part cellular_component、intracellular cellular_component、binding molecular_function等6个条目。（4）靶基因KEGG富集分析结果显示：1.0g·L ^-1 24h组差异表达LncRNA靶基因富集在Steroid biosynthesis pathway;0.2g·L ^-1 24h组无差异表达LncRNA靶基因富集的Pathway term;0.2g·L ^-1 72h组差异表达LncRNA靶基因富集在Cell cycle,DNA replication,Steroid biosynthesis,TNF,Nod-like receptor,NF-kappa B等信号通路,其中TNF和NF-kappa B信号通路与绒毛生长相关。FGF5处理组中,10 ^-4g·L ^-1 72 h组差异表达的LncRNA靶基因显著富集到Fanconi anemia pathway,Huntington's disease,Metabolic pathway,Aminoacyl-tRNA biosynthesis等9个pathway term,其中Metabolic信号通路与绒毛生长相关;10 ^-4g·L ^-1 24 h组差异表达的LncRNA靶基因无显著富集的pathway term;10 ^-6g·L ^-1 24 h组差异表达的LncRNA靶基因只富集在Taste transduction pathway。（5）NF-κB和TNF两个信号通路中富集的靶基因TNFα、TNFAIP3（A20）、NFKBIA（IkBα）、NFKB2、IL8所对应的LncRNA有2个,分别为（Gene ID）：XLOC_005914;XLOC_018763;Metabolic信号通路中靶基因所对应的LncRNA有4个,分别为（Gene ID）：XLOC_011424、XLOC_009522、XLOC_009063、XLOC_01115。Real-time PCR结果显示：LncRNA XLOC_011424、XLOC_011157、LncRNA XLOC_005914和XLOC_018763与高通量测序结果一致。 【结论】LncRNA XLOC_011424、XLOC_011157、LncRNA XLOC_005914和XLOC_018763可能通过调控与绒毛生长相关的NF-κB 、TNF或Metabolic信号通路,提高羊绒密度和长度,进而提高辽宁绒山羊绒产量及品质。
关键词： 辽宁绒山羊;褪黑激素;FGF5;LncRNA;RNA-seq;信号通路

Abstract
【Objective】 The aim of this study was to screen out the LncRNA associated with villus growth in Liaoning cashmere goat skin fibroblasts, and provide basic data for the study of the function and mechanism of LncRNA related to villus growth. 【Method】 The total RNA of MT and FGF5 treated Liaoning cashmere goat skin fibroblasts was extracted, and the total RNA extracted was detected by total RNA electrophoresis detection, sequencing data quality evaluation, mapping comparison and inter-sample correlation test. The differentially expressed LncRNA was screened and its target gene was predicted. The LncRNA related to villus growth was screened by GO and KEGG enrichment analysis, and the target LncRNA was verified by Real-time PCR. 【Result】 (1) The total RNA quality of the sample showed that the RNA was in good integrity, the GC content was relatively high, the sequence was stable, and the expression level between samples was high, which met the sequencing requirements.(2) Screening of differentially expressed LncRNA showed that there were 32 differentially expressed LncRNA in 1.0g·L ^-1 24h group, 4 of which were up-regulated and 28 of which were down-regulated. There were 10 differentially expressed LncRNA in 0.2g·L ^-1 24h group, 4 of which were up-regulated and 6 were down-regulated. There were 113 differentially expressed LncRNA in the 0.2g·L ^-1 72h group, of which 5 were up-regulated and 108 were down-regulated. There were 164 differentially expressed LncRNA in the 10 ^-4g·L ^-1 24 h group, of which 70 were up-regulated and 94 were down-regulated. There were 189 differentially expressed LncRNA in the10 ^-4g·L ^-1 72 h group, of which 78 were up-regulated and 111 were down-regulated. There were 123 LncRNA differentially expressed in the 10 ^-6g·L ^-1 24 h group, among which 27 up and 96 down.(3) Target gene GO enrichment analysis showed that the 1.0g·L ^-1 24h group differentially expressed LncRNA target gene enrichment in GO's negative regulation of transcription from RNA polymerase II promoter; 0.2g·L ^-1 24h group did not differentially express LncRNA target gene enriched GO term;0.2g·L ^-1 72h group Differentially expressed LncRNA target gene enrichment in GO's cellular metabolic process biological_process, binding molecular_function, FGF5 treatment group only10 ^-4g·L ^-1 72 h group differentially expressed LncRNA target gene enriched in cell cellular_component, cell part cellular_component, intracellular cellular_component, binding molecular_function and other six items. (4) Target gene KEGG enrichment analysis showed that the differential expression of LncRNA target gene in 1.0g·L ^-1 24h group was enriched in Steroid biosynthesis pathway; in 0.2g·L ^-1 24h group, there was no differential expression of LncRNA target gene enrichment Pathway term; 0.2g·L ^-1 72h group differentially expressed LncRNA target gene enrichment in Cell cycle, DNA replication, Steroid biosynthesis, TNF, Nod-like receptor, NF-kappa B and other signaling pathways, in which TNF and NF-kappa B signaling pathways are involved in villus growth. In FGF5-treated group, differentially expressed LncRNA targets in 10 ^-4g·L ^-1 72 h group The gene was significantly enriched into nine path termes such as Fanconi anemia pathway, Huntington's disease, Metabolic pathway, Aminoacyl-tRNA biosynthesis, among which Metabolic pathway was associated with villus growth; the differentially expressed LncRNA target gene in 10 ^-4g·L ^-1 24 h group had no significant enriched pathway term;10 ^-6g·L ^-1 24 h The differentially expressed LncRNA target genes were only enriched in the Taste transduction pathway. (5) There are two LncRNA corresponding to the target genes TNFα, TNFAIP3 (A20), NFKBIA (IkBα), NFKB2 and IL8 enriched in NF-κB and TNF signaling pathways, respectively (Gene ID): XLOC_005914; XLOC_018763; There are four LncRNA corresponding to the target genes in the Metabolic pathway, namely (Gene ID): XLOC_011424, XLOC_009522, XLOC_009063, XLOC_01115. Real-time PCR results showed that LncRNA XLOC_011424, XLOC_011157, LncRNA XLOC_005914 and XLOC_018763 were consistent with high-throughput sequencing results. 【Conclusion】 LncRNA XLOC_011424, XLOC_011157, LncRNA XLOC_005914 and XLOC_018763 may increase the density and length of cashmere by regulating NF-κB, TNF or Metabolic signaling pathways related to villus growth, and thus improve the yield and quality of cashmere in Liaoning cashmere goat.
Keywords：Liaoning cashmere goat;melatonin;FGF5;LncRNA;RNA-seq;signaling pathway


PDF (4036KB)元数据多维度评价相关文章导出EndNote|Ris|Bibtex收藏本文
本文引用格式
金梅, 张丽娟, 曹倩, 郭鑫英. MT和FGF5调控辽宁绒山羊绒毛生长相关LncRNA的筛选及鉴定[J]. 中国农业科学, 2019, 52(4): 738-754 doi:10.3864/j.issn.0578-1752.2019.04.014
JIN MEI, ZHANG LIJUAN, CAO QIAN, GUO XinYing. The Screening and Identification of LncRNA Related to Villus Growth in Liaoning Cashmere Goats by MT and FGF5[J]. Scientia Agricultura Sinica, 2019, 52(4): 738-754 doi:10.3864/j.issn.0578-1752.2019.04.014

0 引言

【研究意义】辽宁绒山羊是中国代表性的绒山羊品种,其体型较大,适应能力强,遗传稳定性好。辽宁绒山羊是绒肉兼用的地方良种,该种绒山羊产绒量高,绒毛品质好,其绒毛是珍贵的纺织原料。因此,研究如何提高辽宁绒山羊羊绒产量及品质尤为重要。【前人研究进展】毛囊是皮肤中的附属结构,可分为初级毛囊和次级毛囊,初级毛囊产毛,次级毛囊产绒^[3]。哺乳动物被毛的生长替换是一个复杂的生理过程,被毛的替换与毛囊的周期性生长密切相关,一般一个生长周期内毛囊要经历生长期、退行期和休止期三个阶段^[4]。环境、代谢水平和基因调控等因素都可影响绒山羊毛囊的周期性生长过程,有研究表明,很多的信号分子都在毛囊的形态发生过程中具有很重要的作用,褪黑激素(melatonin,MT)、催乳素(prolactin)、成纤维细胞生长因子5（fibroblast growth factor 5, FGF5）、甲状腺素(thyroxine)等 ^[5,6]。MT是由松果体分泌出来的一种高度保守的吲哚类激素^[7]。它在很多细胞、组织和器官中都起到重要作用^[8]。IBRAHEEM 等研究发现,催乳素和褪黑激素对次级毛囊的毛干伸长具有刺激作用^[9]。LOGAN等发现,褪黑激素能够抑制由α-黑素细胞刺激素（MSH）引起的黑素生成的增加^[10]。有****发现,皮肤组织是除松果体外,MT合成与代谢的又一重要的场所^[11]。近年来,很多研究表明MT可能在毛发生理学中起重要作用,其受体（MT2和RORα）以毛发周期依赖性方式在小鼠皮肤中表达,抑制角质形成细胞凋亡^[12,13]。另外,MT能够改变山羊中羊绒生长周期的时间,外源性的MT能够促进毛囊从休止期向生长初期转变,延长生长初期^[14,15]。有****发现,褪黑激素可作为自由基清除剂和DNA修复诱导剂,代谢和增殖活性高的毛发生长初期毛球也可以利用褪黑素合成作为自身细胞保护策略^[16]。FGF5是目前所发现的一种与绒毛生长有直接关系的基因之一。最早发现的安哥拉鼠被毛增长就是由于FGF5基因突变所致^[17]。此后,很多研究者们开始了一系列有关FGF5与毛发生长的研究。2007年JAMES等人利用家猫作为实验动物进行研究,结果显示FGF5是影响家猫毛发长度的主要因素^[18]。SUZUKI等利用体外注射蛋白产物的方法进行了验证实验,结果表明FGF5的蛋白产物在毛囊生长的不同时期都具有影响^[19]。此外,有些****发现FGF5在毛发生长及某些老鼠的脱毛过程中也具有调节作用^[20]。KREGE等人发现FGF5能够通过影响Sox2的表达从而对皮肤毛囊的再生起到非常重要的影响^[21]。笔者一直致力于如何提高辽宁绒山羊的绒毛产量及品质的研究,通过大量的试验,最终筛选出了MT和FGF5这两种药物,其中MT处理组中1.0g·L^-1 24h、0.2g·L^-1 24h、0.2g·L^-1 72h三个处理条件最有利于辽宁绒山羊皮肤成纤维细胞的增殖与生长,FGF5处理组中10^-4 g·L^-1 24 h、10^-4 g·L^-1 72 h、10^-6 g·L^-1 24 h三个处理条件最适宜辽宁绒山羊皮肤成纤维细胞的增殖与生长。长链非编码RNA(long noncoding RNA, LncRNA)是由RNA聚合酶Ⅱ转录的无蛋白质编码功能的调节性非编码RNA,是一类新型的真核生物转录物^[22]。目前,人们将其分为：正义LncRNA、反义LncRNA、双向LncRNA、基因内LncRNA和基因间LncRNA 5种主要类型^[23]。很多****研究发现lncRNA在多种生命过程中发挥重要作用,并且在细胞及生物体中的调节方式有多种,例如某些LncRNA可作为转录调控因子（或共调控因子）上调或下调某些基因的表达^[24]。某些LncRNA在一定程度上对细胞的分化与增殖、生长发育、器官生成、免疫应答及肿瘤发生等多个生命活动都有一定的调节作用^[25,26]。REN等通过将幽州黑山羊和渝东白山羊作为实验动物进行研究,他们对色素沉着早期阶段皮肤细胞中的LncRNA进行结构和表达分析,筛选出差异表达的LncRNA,并对LncRNA进行顺式和反式靶基因的预测^[27]。ZHU等发现,长链非编码RNA H19转录物与毛囊重建的真皮乳头细胞的诱导能力有关,在次生毛囊中,lncRNA-H19转录物在毛发生长初期阶段的相对表达显着高于毛发生长终期阶段和毛发生长中期阶段,表明lncRNA-H19转录物可能在山羊绒绒纤维的形成和生长中起重要作用^[28]。LIN等在毛乳头细胞中发现了有助于毛发生长相关基因表达的LncRNA的表达 ^[29]。有****通过高通量测序技术对绵羊的LncRNA进行了生物信息学分析, 对绵羊基因组的研究有重大帮助^[30]。【本研究切入点】近年来,关于如何提高羊绒产量及品质的研究越来越多。但是,有关MT和FGF5两种药物能否通过影响相关LncRNA的表达进而提高羊绒产量及品质的研究非常少。【拟解决的关键问题】本试验以辽宁绒山羊为研究对象,分别用MT和FGF5处理辽宁绒山羊皮肤成纤维细胞,通过高通量测序技术筛选出差异表达的LncRNA,预测差异表达LncRNA的靶基因,并通过GO和KEGG富集分析筛选出与绒毛生长相关的LncRNA并进行Real-time PCR验证。

1 材料与方法

1.1 细胞培养与药物处理

试验于2016年在辽宁师范大学生命科学学院,辽宁省生物技术与分子药物研发重点实验室进行。辽宁绒山羊皮肤成纤维细胞原代培养,用不同浓度及时间的MT和FGF5进行处理, 处理的条件分别为：MT处理组 1.0g·L^-1 24h（M1_24H)、0.2g·L^-1 24h（M2_24H)、0.2g·L^-1 72h（M2_72H); FGF5处理组,10^-4 g·L^-1 24 h（F4_24H）、10^-4 g·L^-1 72 h（F4_72H）、10^-6 g·L^-1 24 h（F6_24H）。

1.2 提取RNA

参照宝生物工程(大连)有限公司的 DNase I(RNase Free)使用说明进行操作。

1.3 RNA-Seq文库测序

由北京诺禾致源生物信息科技有限公司提供测序服务,测序平台为 Illumina Hiseq^TM2500,以PE125的测序策略进行测序。利用fastx_toolkit(v0.0.14)软件对得到的Raw reads进行分析。应用Illumina Casva1.8软件通过Q_Phred=-10log₁₀(e)公式对碱基的质量进行检测。

1.4 Mapping以及样品相关性检验

利用Tophat 2(V2.0.9)对过滤后的测序序列与山羊参考基因组进行比对分析,应用cufflinks和scripture软件进行转录本的拼接。

1.5 差异表达LncRNA的筛选

首先通过五步筛选法进行基本的筛选,然后利用CPC、CNCI、pfam蛋白结构域及PhyloCSF分析方法进行编码潜能的筛选,这几种方法筛选的交集为候选的LncRNA。然后从候选LncRNA中筛选出差异表达的LncRNA

1.6 差异表达的LncRNA靶基因预测

LncRNA是通过与其靶基因mRNA相互作用来发挥作用的,因此采用Pearson相关系数法分析各样本中LncRNA与蛋白编码基因的表达量相关性和共表达分析方法来预测其靶基因。使用cuffdiff（http://cufflinks.cbcb.umd.edu/manual.html#cuffdiff）软件对筛选所得到的LncRNA进行定量分析,从而得到各样品中LncRNA的FPKM信息。

1.7 差异表达LncRNA靶基因的功能富集分析

将差异表达LncRNA的cis和trans靶基因分别做GO和KEGG富集分析。

1.8 目标LncRNA的表达验证

首先培养辽宁绒山羊皮肤成纤维原代细胞,用0.2g·L^-1的MT和10^-4 g·L^-1的FGF5分别处理细胞72h后,提取RNA并反转录成cDNA,接下来通过Real-time PCR对差异表达的LncRNA进行验证,引物见表1。

Table 1
表1
表1RT-PCR引物序列
Table 1RT-PCR Primer sequence

基因 Gene	引物 Primer	引物序列（5'→3'） Primer sequence(5'→3')	产物 Products
β-actin	β-actin -F β-actin -R	GATGGCTACTGCTGCGTCG GGCATACAGGTCCTTTCGG	208bp
LncRNA XLOC_005914	LncRNA XLOC_005914 -F LncRNA XLOC_005914-R	CAGACCTTGCCACACCGA CAGTCCCGAACAAAATCAGC	143bp
LncRNA XLOC_018763	LncRNA XLOC_018763 -F LncRNA XLOC_018763-R	CAGCAGAGAAAGGAGAAAATGG GTGATGTAGGTGAGAGGCGAG	299bp
LncRNA XLOC_011424	LncRNA XLOC_011424 -F LncRNA XLOC_011424-R	AAAAGCACATTAGAGGCTGGAG GCAAGCAAATTCATGGCACA	116 bp
LncRNA XLOC_009522	LncRNA XLOC_018763 -F LncRNA XLOC_018763-R	GGGCGTGACATTTTCAGATT TCAGGGGACCTTTGGACTTG	289 bp
LncRNA XLOC_009063	LncRNA XLOC_009063-F LncRNA XLOC_009063-R	GCATCATTTCTTCCGCATTC TGGGTTACCATTTCCTTCTCC	220 bp
LncRNA XLOC_011157	LncRNA XLOC_011157-F LncRNA XLOC_011157-R	CTTTGGAAATGGTTCTGTGCTG GGAAGTAGGAGATGCTGGGTAA	140 bp

新窗口打开|下载CSV

2 结果

2.1 样品总RNA电泳检测

经过 DNase I(RNase Free)处理,得到辽宁绒山羊皮肤成纤维细胞总RNA样品,然后对各组样品进行琼脂糖凝胶电泳。结果显示试验中提取的辽宁绒山羊皮肤成纤维细胞样品的总RNA 琼脂糖凝胶电泳检测条带清晰,无拖尾和降解现象,表明提取的 RNA 完整性良好,可以用于后续分析（图1）。

图1

新窗口打开|下载原图ZIP|生成PPT
图1样品中总RNA电泳检测

图A为MT处理组,图B为FGF5处理组。图A中泳道1、2、3、4分别为M2_72H组、M2_24H组、M1_24H组和对照组C;图B中泳道1、2、3、4分别为F4_24H组、F6_24H组、F4_72H和对照组C
Fig. 1The electrophoresis results of total RNA in samples

Panel A is the MT treatment group and Figure B is the FGF5 treatment group. In Figure A, lanes 1, 2, 3, and 4 are M2_72H group, M2_24H group, M1_24H group, and control group C; in Figure B, lanes 1, 2, 3, and 4 are F4_24H group, F6_24H group, and F4_72H group, and control group C.

2.2 测序数据质量评估

对RNA-Seq测序得到的数据质量进行检测的结果如表2.1所示。通过表中数据可知,Error rate表示测序错误率,它与碱基质量有关,同时也受测序仪本身、测序试剂、样品等多个因素共同影响,由表中数据可知碱基错误率较低。Q = -10log₁₀p,其中p值是由Phred计算出,它表示一个碱基被识别错误的可能性,Q 值为 10 表示这个碱有90%的概率是正确的, 20 就是 99%。各个样品中绝大部分reads的Q值均大于20。基GC含量相对较高,表明测序序列较稳定。综上,我们认为各个样品的测序序列都具有较高的质量,可以进行后续分析 。

Table 2
表2
表2RNA-Seq数据一览表
Table 2RNA-Seq data list

样本 Sample	原始序列 Raw reads	过滤后序列 Clean reads	错误率 Error rate (%)	Q20 Q20 (%)	Q30 Q30 (%)	GC含量 GC content(%)
C_1	48045300	46275212	0.03	96.86	93.68	48.39
C_2	48045300	46275212	0.04	94.99	90.67	48.37
M2_72H_1	42277089	40464850	0.03	96.34	92.55	51.77
M2_72H_2	42277089	40464850	0.04	93.42	87.84	51.95
M2_24H_1	50302056	48536611	0.03	96.75	93.50	48.03
M2_24H_2	50302056	48536611	0.03	95.04	90.73	48.03
M1_24H_1	48057476	46336801	0.03	96.80	93.55	48.90
M1_24H_2	48057476	46336801	0.04	94.99	90.66	48.92
F4_24H_1	51394058	49453178	0.03	96.82	93.65	47.67
F4_24H_2	51394058	49453178	0.03	95.31	91.23	47.57
F4_72H_1	49418892	47713121	0.03	96.72	93.27	52.04
F4_72H_2	49418892	47713121	0.04	94.71	90.09	52.26
F6_24H_1	46346088	44739804	0.03	96.88	93.73	48.58
F6_24H_2	46346088	44739804	0.04	94.92	90.55	48.55

Sample_name: _1 represents reads of the left side, _2 represents reads of the right side; Raw reads: The original sequence data; Clean reads: The filtered sequencing data; Clean bases: The product of the number of sequencing sequence and the length of sequencing sequence; Error rate: Base error rate; Q20, Q30: The percentage of the bases that phred values are greater than 20 or 30 and all the bases; GC content: The percentage of the number of G and C bases and all the bases
样品名称：_1代表左端的reads,_2代表右端的reads;Raw reads：统计原始序列数据;Clean reads：过滤后的测序数据;Clean bases：测序序列个数与其长度的积;Error rate：碱基错误率;Q20、Q30：Phred 数值大于20、30的碱基与总碱基数之比;GC含量：含有G和C的碱基数量与占总碱基数之比

新窗口打开|下载CSV

2.3 Mapping

通过Tophat 2软件将试验中样品的clean reads分别与NCBI中山羊参考基因组进行比对分析,发现试验中所产生的测序序列定位百分比均低于70%,其中具有多个定位的测序序列占总体的百分比也均低于10%,表明试验中参考基因组选择合适,不存在污染。而且所有样品中Unique Mapping Rate均为80%以上,因此可进行下一步分析。从图2中可以更加直观看出染色体长度和reads总数的关系,染色体的长度与定位到该染色体内reads总数呈正相关,MT处理组和FGF5处理组样品比对到山羊1号、2号染色体上的reads相对来说都比较多。

图2

新窗口打开|下载原图ZIP|生成PPT
图2Reads在染色体上的密度分布图

横坐标：染色体的长度信息（以百万碱基为单位）;纵坐标：log₂（reads的密度的中位数）;绿色为正链,红色为负链
Fig. 2Density distribution of Reads on chromosome

Abscissa: the length of a chromosome (in millions of bases); ordinate: log2 (the median of reads density); green is a positive chain, and red is a negative chain

2.4 样品间相关性检查

样品间表达水平相关性是检验试验可靠性和样本选择是否合理的重要指标。如图3所示,可知各处理组与对照组相比较,R²值均大于0.8, 表明样品间表达水平相关性均较高。因此本试验所选择的样品均符合测序要求,可以继续进行下一步分析。

图3

新窗口打开|下载原图ZIP|生成PPT
图3各处理组中样品间相关性检查

Fig. 3Correlation between samples in each treatment group

2.5 差异表达的LncRNA的筛选

筛选的条件是P-adjust <0.05,log₂(Fold change) >1。由图4可知,在M1_24H与C进行比较,获得32个差异表达的LncRNA,其中有4个LncRNA上调,28个下调;M2_24H与C组进行比较,获得10个差异表达的LncRNA,其中有4个LncRNA上调,6个下调;M2_72H与C组中进行比较,获得113个差异表达的LncRNA,其中有5个LncRNA上调,108个下调;F6_24H与C进行比较,获得123个差异表达的LncRNA,27个上调,96个下调;F4_24H与C进行比较,获得164个差异表达的LncRNA,70个上调,94个下调;F4_72H与C组中进行比较,获得189个差异表达的LncRNA,其中有78个上调,111个下调。

图4

新窗口打开|下载原图ZIP|生成PPT
图4差异表达LncRNA的分布

有显著性差异表达的转录本用红色点（上调LncRNA）和绿色点（下调LncRNA）表示;横坐标代表LncRNA表达水平变化;纵坐标代表LncRNA表达变化的统计学意义
Fig. 4Differentially expressed LncRNA distribution

Red dots represent the up-regulated LncRNA and green dots represent the down-regulated LncRNA; Abscissa represents expression level of LncRNA; Ordinate represents statistical significance of LncRNA expression level.


根据不同样品中差异表达的LncRNA表达水平的高低,进行层次聚类（hierarchical clustering）分析,从而判断这些LncRNA在不同试验条件下的表达模式（图5）。结果显示在F4_24H组和M2_72H中差异表达的LncRNA表达水平相对较高。

图5

新窗口打开|下载原图ZIP|生成PPT
图5差异表达LncRNA聚类图

每列代表一个样品,每行代表一种基因;红色代表高表达LncRNA,蓝色代表低表达LncRNA
Fig. 5Differentially expressed LncRNA clusters

Each column represents a sample and each row a gene; High relative expression of LncRNA is indicated by red and low relative expression of LncRNA by green.

2.6 差异表达的LncRNA靶基因Gene Ontology功能显著性富集分析

分别根据LncRNA临近位置的（上下游10k /100k）蛋白编码基因和LncRNA与蛋白编码基因的表达量相关性分析或共表达分析方法来预测其cis/trans靶基因。再对靶基因分别进行cis和trans的GO富集分析,分别从生物过程（biological process,BP）、分子功能（molecular function,MF）和细胞组分（cellular component,CC）三个层面对靶基因进行GO注释。结果如表3、4所示：M2_24H vs C、F4_24H vs C和 F6_24H vs C组中差异表达的LncRNA均无显著富集的条目;M1_24H vs C组中差异表达LncRNA的trans靶基因无显著富集的GO term,其cis靶基因只在BP中有一个显著富集的GO term,即negative regulation of transcription from RNA polymerase II promoter;M2_72H vs C和F4_72H vs C组中差异表达的LncRNA的cis靶基因均无显著富集的GO term,而M2_72H vs C组trans靶基因主要富集在BP的cellular metabolic process biological_process、nitrogen compound metabolic process biological_process;MF中的binding molecular_function、catalytic activity molecular_function;CC中的membrane- bounded organelle cellular_component、F4_72H vs C组中差异表达的LncRNA的 rans靶基因主要富集在BP的cellular metabolic process biological_process、cellular macromolecule metabolic process biological_process,CC的cell cellular_component、cell part cellular_component、intracellular cellular_component,MF的binding molecular_ function中。

Table 3
表3
表3M2_72H vs C组差异表达LncRNA靶基因GO term分类
Table 3M2_72H vs C group differential expression LncRNA target gene GO term classification

条目类型 Term_type	功能 Features	过表达的P值 Over_represented P value	校正的P值 Corrected P value
生物过程 Biological_process (BP)	细胞代谢过程 Cellular metabolic process	7.15E-06	0.003923
	氮化合物代谢过程 Nitrogen compound metabolic process	8.48E-06	0.0039252
	细胞氮化合物代谢过程 Cellular nitrogen compound metabolic process	1.67E-05	0.0063413
	有机氮化合物代谢过程 Organonitrogen compound metabolic process	8.12E-05	0.019891
	细胞芳香化合物代谢过程 Cellular aromatic compound metabolic process	3.26E-05	0.0097043
	杂环代谢过程 Heterocycle metabolic process	5.36E-05	0.013956
	有机环状化合物代谢过程 Organic cyclic compound metabolic process	3.84E-05	0.01066
	染色体组织 Chromosome organization	9.55E-05	0.022102
	含核碱基的化合物代谢过程 Nucleobase-containing compound metabolic process	0.00011221	0.024598
	染色质修饰 Chromatin modification	0.00025719	0.04581
	细胞成分组织或生物发生 Cellular component organization or biogenesis	0.00026397	0.04581
细胞组分Cellular_component (CC)	核 Nucleus	1.48E-06	0.0031426
	膜结合的细胞器 Membrane-bounded organelle	3.52E-06	0.003923
	细胞内膜结合细胞器 Intracellular membrane-bounded organelle	4.25E-06	0.003923
	染色体 Chromosome	0.00022214	0.042055
分子功能Molecular_function (MF)	核酸酶活性 Nuclease activity	1.51E-06	0.0031426
	腺苷酸核苷酸结合 Adenyl nucleotide binding	5.49E-06	0.003923
	腺苷核糖核苷酸结合 Adenyl ribonucleotide binding	7.54E-06	0.003923
	ATP结合 ATP binding	6.43E-06	0.003923
	蛋白质结合 Protein binding	1.65E-05	0.0063413
	细胞因子活性 Cytokine activity	3.00E-05	0.0096034
	水解酶活性,作用于酯键 Hydrolase activity, acting on ester bonds	0.00012043	0.02508
	催化活性 Catalytic activity	0.00021879	0.042055
	结合物 Binding	1.90E-05	0.0065865

新窗口打开|下载CSV

Table 4
表4
表4F4_72H vs C组差异表达LncRNA靶基因的GO term分类
Table 4Fnc_72H vs C group differentially expressed LncRNA target genes GO term classification

条目类型 Term_type		功能 Features	过表达的P值 Over_represented P value	校正的P值 Corrected P value
生物过程Biological_process （BP）		细胞代谢过程 Cellular metabolic process	1.22E-06	0.002285
		生物合成过程 Biosynthetic process	3.29E-06	0.002285
		有机物生物合成过程 Organic substance biosynthetic process	6.96E-06	0.0034462
		细胞生物合成过程 Cellular biosynthetic process	8.88E-06	0.0034462
		细胞大分子代谢过程 Cellular macromolecule metabolic process	9.10E-06	0.0034462
		基因表达 Gene expression	1.87E-05	0.0051872
		细胞蛋白质代谢过程 Cellular protein metabolic process	2.03E-05	0.0052866
		翻译 Translation	3.43E-05	0.0071461
		高分子生物合成过程 Macromolecule biosynthetic process	7.00E-05	0.013255
		细胞大分子生物合成过程 Cellular macromolecule biosynthetic process	8.41E-05	0.015233
		有机环状化合物代谢过程 Organic cyclic compound metabolic process	9.81E-05	0.017021
		细胞氮化合物代谢过程 Cellular nitrogen compound metabolic process	0.0001207	0.019468
		细胞芳香化合物代谢过程 Cellular aromatic compound metabolic process	0.00012153	0.019468
		细胞芳香化合物代谢过程 Nitrogen compound metabolic process	0.00013872	0.021399
		蛋白质定位 Protein localization	0.00015944	0.023716
		杂环代谢过程 Heterocycle metabolic process	0.00018062	0.025736
		DNA复制,合成RNA引物 DNA replication, synthesis of RNA primer	0.00023573	0.031671
		含核碱基的化合物代谢过程 Nucleobase-containing compound metabolic process	0.00038174	0.045427
胞组分Cellular_component (CC)	细胞内部分 Intracellular part		9.95E-08	0.00041428
	细胞质 Cytoplasm		1.93E-06	0.002285
	细胞内膜结合细胞器 Intracellular membrane-bounded organelle		2.49E-06	0.002285
	膜结合的细胞器 Membrane-bounded organelle		3.16E-06	0.002285
	细胞 Cell		7.88E-06	0.0034462
	细胞部分 Cell part		7.88E-06	0.0034462
	细胞内 Intracellular		1.34E-05	0.0046662
	细胞质部分 Cytoplasmic part		1.65E-05	0.0049423
	核 Nucleus		1.66E-05	0.0049423
	细胞内细胞器 Intracellular organelle		2.56E-05	0.0061674
	细胞器 Organelle		2.69E-05	0.0061674
	大分子复合物 Macromolecular complex		2.81E-05	0.0061674
	核部分 Nuclear part		4.41E-05	0.0087498
	膜封闭的管腔 Membrane-enclosed lumen		0.00018537	0.025736
	细胞器腔 Organelle lumen		0.00025929	0.032725
	细胞内细胞器腔 Intracellular organelle lumen		0.00025929	0.032725
	内膜系统 Endomembrane system		0.00031133	0.038138
	蛋白质复合物 Protein complex		0.00047111	0.049055
	核腔 Nuclear lumen		0.00048565	0.049335
分子功能Molecular_function (MF)	连接酶活性,形成碳 - 氧键 Ligase activity, forming carbon-oxygen bonds		0.00040555	0.045652
	连接酶活性,形成氨酰-tRNA和相关化合物 Ligase activity, forming aminoacyl-tRNA and related compounds		0.00040555	0.045652
	腺苷酸核苷酸结合 Adenyl nucleotide binding		0.00043007	0.047138
	结合物 Binding		0.00046	0.049055

新窗口打开|下载CSV

2.7 差异表达的LncRNA靶基因Pathway显著性富集分析

通过KEGG数据库,对样品中差异表达LncRNA的靶基因进行通路富集分析,得到各比较组合中显著性富集（Qvalue<0.05）的pathway term。结果如表5—9所示：M2_24H vs C和F4_24H vs C组中均无显著富集的pathway term;F6_24H vs C组中差异表达的LncRNA的trans靶基因无显著富集的pathway term,其cis靶基因只富集在Taste transduction通路;M2_72H vs C、M1_24H vs C和F4_72H vs C组中差异表达的LncRNA的cis靶基因均无显著富集的pathway term,M2_72H vs C 组差异表达的LncRNA的trans靶基因显著富集到15个pathway term,其中TNF和NF-kappa B 信号通路与毛囊发育及绒毛周期性生长相关,其通路中差异表达的靶基因、M1_24H vs C组差异表达的LncRNA的trans靶基因只富集在Steroid biosynthesis通路、F4_72H vs C组中差异表达的LncRNA的trans靶基因显著富集到9个pathway term,其中只有Metabolic信号通路与毛囊发育及绒毛周期性生长相关,其通路中差异表达的靶基因。

Table 5
表5
表5M2_72H vs C组Pathways富集数据表
Table 5Pathways enrichment data for M2_72H vs C group

通路 Pathway	通路ID Pathway ID	样本编号 Sample number	背景编号 Background number	校正的P值 Corrected P value
细胞周期 Cell cycle	chx04110	62	116	1.30736872661e-07
DNA复制 DNA replication	chx03030	26	34	1.97822480896e-07
范可尼贫血症 Fanconi anemia	chx03460	29	50	0.000199893004074
错配修复 Mismatch repair	chx03430	15	22	0.00320771571904
赖氨酸降解 Lysine degradation	chx00310	25	50	0.0157486842455
癌症中的微小RNA MicroRNAs in cancer	chx05206	50	127	0.0399507008006
阿尔茨海默氏病 Alzheimer's disease	chx05010	61	164	0.0467892160603
类固醇生物合成 Steroid biosynthesis	chx00100	12	20	0.0467892160603
TNF	chx04668	39	97	0.0467892160603
Nod样受体 Nod-like receptor	chx04621	22	47	0.0467892160603
嘧啶代谢 Pyrimidine metabolism	chx00240	37	91	0.0467892160603
RNA转运 RNA transport	chx03013	54	144	0.0467892160603
柠檬酸循环 Citrate cycle	chx00020	16	31	0.0467892160603
同源重组 Omologous recombination	chx03440	14	26	0.0467892160603
NF-kappa B	chx04064	32	77	0.0467892160603

Sample number: The number of comment the differentially expressed genes in this pathway;Background number: The number of all genes in this pathway
样本编号：已注释到该条通路中,同时在表达水平上有统计学意义的基因总数;背景编号：该条通路中所有的基因数

新窗口打开|下载CSV

Table 6
表6
表6M2_72H vs C组差异表达基因富集的绒毛生长相关信号通路
Table 6Differential genes enriched in signaling pathways related cashmere growth between M2_72H and C group

通路 Pathway	基因ID Gene ID	基因名称 Gene name	方式 Style
TNF NF-kB	100861232 102169137 102172758 102186424 100861171 102182977 100860785 102188524 100861236 102185051 100861232 102182977 102169137 102191581 102178438	TNFα NFKBIA PIK3R5 NOD2 MT1MMP TNFAIP3 IL6 LIF IL18R1 CSF1 TNFα TNFAIP3（A20） NFKBIA（IKBα） NFKB2 IL8	UP UP UP UP UP UP UP UP UP UP UP UP UP UP UP

新窗口打开|下载CSV

Table 7
表7
表7FGF处理组,KEGG富集分析
Table 7FGF treatment group,KEGG enrichment analysis results

样本名称 Sample name		显著富集的信号通路 Significantly enriched signaling pathway	Q值 Qvalue
F4_24H vs C	cis靶基因 cis target gene	无 No	-
	trans靶基因 trans target gene	无 No	-
F4_72H vs C	cis靶基因 cis target gene	无 No	-
	trans靶基因 trans target gene	核糖体 Ribosome RNA转运 RNA transport 范可尼贫血途径 Fanconi anemia pathway 亨廷顿氏病 Huntington's disease 代谢途径 Metabolic pathway 氨酰基-tRNA生物合成 Aminoacyl-tRNA biosynthesis 柠檬酸循环（TCA循环） Citrate cycle (TCA cycle) 阿尔茨海默氏病 Alzheimer's disease 泛素介导的蛋白水解 Ubiquitin mediated proteolysis	0.001 0.018 0.018 0.018 0.025 0.029 0.033 0.033 0.037
F6_24H vs C	Cis靶基因 Cis target gene	味觉转导 Taste transduction	0.022
	Trans靶基因 Trans target gene	无 No	-

新窗口打开|下载CSV

Table 8
表8
表8FGF处理组,Pathways显著性富集数据表
Table 8FGF treatment group,the statistical significance enrichment of pathways data

通路 Pathway	通路ID Pathway ID	样本编号 Sample number	背景编号 Background number	校正后的P值 Corrected P-value
核糖体 Ribosome	chx03010	36	129	0.001
RNA转运 RNA transport	chx03013	34	144	0.018
范可尼贫血途径 Fanconi anemia pathway	chx03460	16	50	0.018
亨廷顿氏病 Huntington's disease	chx05016	38	171	0.018
代谢途径 Metabolic pathway	chx01100	178	1126	0.025
氨酰基-tRNA生物合成 Aminoacyl-tRNA biosynthesis	chx00970	14	44	0.029
柠檬酸循环（TCA循环） Citrate cycle (TCA cycle)	chx00020	11	31	0.033
阿尔茨海默氏病 Alzheimer's disease	chx05010	35	164	0.033
泛素介导的蛋白水解 Ubiquitin mediated proteolysis	chx04120	28	124	0.037
味觉转导 Taste transduction	chx04742	2	35	0.022

Sample number: The number of comment the differentially expressed genes in this pathway;Background number: The number of all genes in this pathway
样本编号：已注释到该条通路中,同时在表达水平上有统计学意义的基因总数;背景编号：该条通路中所有的基因数

新窗口打开|下载CSV

Table 9
表9
表9F4_72H vs C组差异表达基因富集的绒毛生长相关信号通路
Table 9Differential genes enriched in signaling pathways related cashmere growth between F4_72H and C group

通路 Pathway	基因ID Gene ID	基因名称 Gene name	方式 Style
氨基酸代谢通路 Metabolic pathway	102172231	CTH	UP
	102190784	PSAT1	UP
	102178128	PHGDH	UP
	102184920	NAPRT1	UP
	102187590	GCNT1	UP
	102179919	SHMT2	UP
	102186540	FDPS	UP
	102171848	DHCRT	UP
	102184667	CBS	UP

新窗口打开|下载CSV

2.8 目标LncRNA的表达验证

M2_72H组LcRNA靶基因富集的NF-κB信号通路中靶基因TNFα、TNFAIP3（A20）、NFKBIA（IkBα）、NFKB2、IL8所对应的LncRNA有两个,分别为（Gene ID）：XLOC_005914;XLOC_018763。F4_72H 组靶基因富集的Metabolic 信号通路中靶基因所对应的LncRNA有4个,分别为（Gene ID）：XLOC_011424、XLOC_009522、XLOC_009063、XLOC_01115。通过Real-time PCR对筛选出的6个LncRNA进行验证,结果如图7所示,MT作用相关的两个LncRNA在RNA- Seq中的表达量上调,Real-time PCR检测结果与RNA- Seq测序结果一致,进一步验证了RNA-Seq测序结果的准确性。FGF5处理辽宁绒山羊皮肤成纤维细胞后,Real-time PCR结果为LncRNA XLOC_011424和XLOC_011157表达量下调;LncRNAXLOC_009063表达量上调; LncRNA XLOC_009522表达量无显著性差异。结合RNA-Seq测序中4个LncRNA表达量均下调的结果,表明LncRNA XLOC_011424、LncRNA XLOC_011157与前期结果一致。

图7

新窗口打开|下载原图ZIP|生成PPT
图7Real-time PCR验证RNA-Seq中相关的LncRNA表达水平

Fig. 7Real-time PCR verifies correlated LncRNA expression in RNA-Seq

3 讨论

本试验通过KEGG富集分析,共筛选出了3个与绒毛生长相关的信号通路,分别为TNF、NF-κB和Metabolic信号通路。

KLOEPPERT等研究发现,NF-κB在维持人类毛囊的生长期阶段具有功能重要性。人类毛发生长初期,头皮毛囊快速增殖的毛发基质上皮中,NF-κB活性非常显著,关键的毛发生长调节剂如TNFα和IL-1通过调控NF-κB信号通路从而影响绒毛生长^[35]。核因子（NF）-κB途径参与毛囊的形态发生,Gilon M、Sher N、Cohen S和 Gat U通过瞬时转染技术分析了p65 / RelA（一种NF-kB效应子）对毛发角蛋白（HK）调节区的影响,结果表明p65能够诱导人和小鼠来源的几种酸性毛发角蛋白5（Ha5）的转录激活,p65与Ha5基因调节区域中的NF-κB/ RelA结合位点直接结合^[36]。NF-κB/ Rel转录因子和IkappaB激酶（IKK）参与骨形态发生,皮肤增殖和分化等过程。另外,SCHMIDT-ULLRICH等研究发现,抑制NF-κB的小鼠会出现毛囊缺陷^[37]。皮肤干细胞可以再生表皮附属物。然而,由于受伤而损失的毛囊几乎没有再生。WANG等研究显示,伤口中的巨噬细胞激活毛囊干细胞,导致伤口周围的毛囊在休止期向生长期过渡,毛囊再生等过程,主要通过TNF信号传导调控^[38]。LAURIKKALA等发现,外异蛋白（ED1）和外异蛋白A受体（EDAR）作为新的TNF配体-受体对的鉴定表明,TNF信号在胚胎形态发生中的作用,另外他们认为ED1/EDAR信号传导也调节毛囊的形态发生^[39]。

蛋白质是生命活动的主要承担者,而氨基酸是构成蛋白质分子的基本单位。L-半胱氨酸目前被认为是条件必需的硫氨基酸,不仅是角蛋白的关键组分,还可以促进许多生物途径^[40]。角蛋白相关蛋白8.1基因（KAP8.1）是一种负责羊绒的结构基因。KAP8.1蛋白含有高甘氨酸和酪氨酸,参与基质结构纤维的调节。ZHAO等认为KAP8.1基因的多态性可能与纤维直径有关^[41]。TONG等发现,原代培养物中的角蛋白17（K17）无效时,皮肤角质形成细胞对TNFα选择性更敏感。K17与TNF受体1（TNFR1）相关的死亡域蛋白（TRADD）相互作用,这是一种必需的死亡适配体TNFR1依赖性信号传递,而且NF-κB（TNFα的下游靶标）的活性在K17无效皮肤中增加^[42]。Wnt信号通路是毛囊发育中重要的途径之一,次级毛囊中成纤维细胞生长因子21和酪蛋白激酶是Wnt途径中β-连环蛋白的重要调节因子。天冬酰胺和丝氨酸可能在初级毛囊生长过程中具有重要作用^[43]。由此可见,Metabolic、NF-kB 、TNF三个信号通路对绒毛生长发育有着十分重要的作用。

LncRNA是影响绒毛生长的重要因素之一。近几年,关于LncRNA影响绒毛生长的研究越来越多。BAIA等研究表明,LncRNA（LncRNA-599618、-599556、-599554、-599547、-599531和-599509）在毛发生长初期阶段的表达量显着高于毛发生长终期阶段^[31]。CAI 等发现,LncRNA5322能够通过靶向毛囊干细胞中miR-21介导的PI3K-AKT信号传导途径来促进毛囊干细胞的增殖和分化^[32]。ZHOU 等在山羊皮肤中鉴定了1 122种已知的和403种新的LncRNA,其中173种在毛发生长初期和退化期之间差异表达。另外他们发现,LncRNA和miRNA在毛囊生长转变中协同作用,并且退行期诱导因子（TGFβ1和BDNF）在miRNA-miRNA-mRNA网络中由miR-873和Lnc108635596调节^[33]。SONG等研究表明,LncRNA XLOC_539599,XLOC_556463,XLOC_015081,XLOC_1285606,XLOC_297809和XLOC_764219对原发性羊毛毛囊诱导具有潜在的重要性,且差异表达的LncRNA的潜在靶基因在NF-κB信号通路显著富集^[34]。

本试验,利用高通量测序和Real-time PCR技术在Metabolic、NF-kB 、TNF 3个信号通路靶基因对应的LncRNA中共筛选出4个与辽宁绒山羊绒毛生长相关的LncRNA,分别为：LncRNA XLOC_ 011424、XLOC_011157、XLOC_005914、XLOC_ 018763。因此,可以认为MT和FGF5两种药物处理,可通过影响某些相关LncRNA的表达,进而影响绒毛生长。

4 结论

LncRNA XLOC_011424、XLOC_011157、XLOC_ 005914和XLOC_018763可能通过增加羊绒密度及长度,进而提高辽宁绒山羊羊绒产量及品质。其中,前两个LncRNA通过调节其上游或下游的靶基因,调节TNF或NF-kB信号通路,进而影响绒毛的生长。LncRNA XLOC_011424和XLOC_011157通过调节其与Metabolic pathway相关的靶基因从而影响绒毛生长。笔者所选择的两种药物中,MT更能引起某些与绒毛生长相关LncRNA的差异表达从而影响绒毛生长。但是这4种LncRNA具体的功能和作用机制尚不清楚,后续试验研究将集中探讨LncRNA促进绒毛纤维生长的作用机制。

（责任编辑 林鉴非）

参考文献 View Option 原文顺序
文献年度倒序
文中引用次数倒序
被引期刊影响因子

[1] YANG M, SONG S, DONG K, CHEN X, LIU X, ROUZI M, ZHAO Q, HE X, PU Y, GUAN W, MA Y, JIANG L . Skin transcriptome reveals the intrinsic molecular mechanisms underlying hair follicle cycling in Cashmere goats under natural and shortened photoperiod conditions
Scientific Reports, 2017,7(1):135.
DOI:10.1038/s41598-017-00185-wURLPMID:28273933
Cognitive dysfunction in fibromyalgia patients has been reported, especially when increased attentional demands are required. Transcranial direct current stimulation (tDCS) over the dorsolateral prefrontal cortex (DLPFC) has been effective in modulating attention. We tested the effects of a single session of tDCS coupled with a Go/No-go task in modulating three distinct attentional networks: alertness, orienting and executive control. Secondarily, the effect on pain measures was evaluated. Forty females with fibromyalgia were randomized to receive active or sham tDCS. Anodal stimulation (165mA, 2065min) was applied over the DLPFC. Attention indices were assessed using the Attention Network Test (ANT). Heat pain threshold (HPTh) and tolerance (HPTo) were measured. Active compared to sham tDCS led to increased performance in the orienting (mean difference [MD]65=6514.63) and executive (MD65=6521.00) attention networks. There was no effect on alertness. Active tDCS increased HPTh as compared to sham (MD65=651.93) and HPTo (MD65=651.52). Regression analysis showed the effect on executive attention is mostly independent of the effect on pain. DLPFC may be an important target for neurostimulation therapies in addition to the primary motor cortex for patients who do not respond adequately to neurostimulation therapies.

[2] JIN M, GUO C L, HU J H, GAO W B, WANG W . Correlation Analysis of Economic Traits in Liaoning New Breed of Cashmere Goats Using Microsatellite DNA Markers
Yi Chuan Xue Bao, 2006,33(3):230-235.
DOI:10.1016/S0379-4172(06)60045-0URLPMID:16553211
The economic traits of Liaoning new breed of cashmere goats, a special Chinese genetic resource, were analyzed in 150 animals by typing 11 microsatellite loci. The association between three economic traits (body weight, cashmere yield and fineness) in this new breed of goats and the marker genotypes were analyzed. The results show that: and at LSCV13, at IDVGA64 and at BMS2782 were favorable genotypes for body weight. at LSCV13, and at CSSM11, and at IDVGA64, and at BMS2782 were favorable genotypes for cashmere yield. at CSSM11, and at IDVGA64, at BMS2782 were favorable genotypes for cashmere fineness.

[3] ZHANG C Z, SUN H Z, LI S L, SANG D, ZHANG C H, JIN L, ANTONINI M, ZHAO C F . Effects of photoperiod on nutrient digestibility, hair follicle activity and cashmere quality in Inner Mongolia white cashmere goats
Asian-Australasian Journal of Animal Sciences, 2018,27.
[本文引用: 1]

[4] ZHANG Q L, LI J P, CHEN Y, CHANG Q, LI Y M, YAO J Y, JIANG H Z, ZHAO Z H, GUO D . Growth and viability of Liaoning Cashmere goat hair follicles during the annual hair follicle cycle
Genetics And Molecular Research, 2014 , 13(2):4433-4443.
DOI:10.4238/2014.June.16.2URLPMID:25036348 [本文引用: 1]
Abstract Here, we studied hair follicle development of Liaoning Cashmere goats. Every month for 1 year, skin samples were collected from five 1.5-year-old female goats, and made into paraffin sections. A number of parameters were measured of primary and secondary hair follicles via microscopic observation including follicle depth, hair bulb width, dermis and epidermis thickness, changes in follicle activity, and histology. The results showed the presence of three phases in the annual hair cycle: anagen, catagen, and telogen. Primary and secondary hair follicle depth varied across the months; however, no significant difference was obtained between adjacent months (P>0.05). Primary hair follicles had a bigger hair bulb width compared to secondary hair follicles; however, this difference declined during hair follicle developed in anagen. As hair follicle growth slowed, the hair bulb broadened, and hair root depth became shallower. During the entire hair cycle, hair follicle depth and dermis thickness were positively correlated; however, this relationship was not significant (P>0.05) for primary and secondary hair follicle density and the ratio of secondary hair follicle density and primary hair follicle density (S/P ratio). In addition, new and old primary hair follicles coexisted with secondary hair follicles. Finally, secondary hair follicles had a higher activity rate compared to primary hair follicle in adult Liaoning Cashmere goats in certain months.

[5] YU F, LIU Z, JIAO S, ZHANG X, BAI C, ZHANG J, YAN S . A nonsense mutation in the FGF5 gene is associated with the long- haired phenotype in domestic guinea pigs (Cavia porcellus).
Animal Genetics, 2018 , 49(3):269.
DOI:10.1111/age.12656URLPMID:29603294 [本文引用: 1]

[6] 付绍印, 赵宏丽, 郑竹清, 李金泉, 张文广 . 褪黑激素对绒山羊皮肤中毛囊周期相关 miRNAs表达模式的影响
遗传, 2014,36(12):1235-1242.
DOI:10.3724/SP.J.1005.2014.1235URLMagsci [本文引用: 1]
褪黑激素(Melatonin, MT)和miRNAs在毛囊的生长发育过程中发挥重要的作用, 但MT对绒山羊皮肤毛囊miRNAs表达模式的影响尚未见报道。为探索MT从miRNAs层次影响山羊绒生长的机制, 文章在内蒙古绒山羊中实施了褪黑激素埋植试验：5只青年母羊作为实验组埋植褪黑激素, 另外5只青年母羊作为对照。利用荧光定量PCR检测褪黑激素埋植前后毛囊周期相关miRNAs的表达变化。结果表明, 埋植MT明显改变了6个绒毛相关miRNAs的表达规律：在一个绒毛周期内, 除let-7a外, miR-203、miR-205、miR-96、miR-183和miR-199a的表达量均发生3次跃迁;埋植MT改变了miRNAs之间的共表达模式。对照组各miRNA之间相关系数范围为0.87~0.99(P<0.01)。与对照组相比, 埋植组中let-7a与miR-96、miR-199a、miR-205, miR-203与miR-96、miR-199a, miR-96与miR-183, miR-183与miR-199a之间的相关系数被明显消弱;MT通过下调6月份埋植组各miRNA的表达量提早诱发二次生绒。
FU S Y, ZHAO H L, ZHENG Z Q, LI J Q, ZHANG W G . Melatonin regulating the expression of miRNAs involved in hair follicle cycle of cashmere goats skin
YiChuan., 2014,36(12):1235-1242. (in Chinese)
DOI:10.3724/SP.J.1005.2014.1235URLMagsci [本文引用: 1]
褪黑激素(Melatonin, MT)和miRNAs在毛囊的生长发育过程中发挥重要的作用, 但MT对绒山羊皮肤毛囊miRNAs表达模式的影响尚未见报道。为探索MT从miRNAs层次影响山羊绒生长的机制, 文章在内蒙古绒山羊中实施了褪黑激素埋植试验：5只青年母羊作为实验组埋植褪黑激素, 另外5只青年母羊作为对照。利用荧光定量PCR检测褪黑激素埋植前后毛囊周期相关miRNAs的表达变化。结果表明, 埋植MT明显改变了6个绒毛相关miRNAs的表达规律：在一个绒毛周期内, 除let-7a外, miR-203、miR-205、miR-96、miR-183和miR-199a的表达量均发生3次跃迁;埋植MT改变了miRNAs之间的共表达模式。对照组各miRNA之间相关系数范围为0.87~0.99(P<0.01)。与对照组相比, 埋植组中let-7a与miR-96、miR-199a、miR-205, miR-203与miR-96、miR-199a, miR-96与miR-183, miR-183与miR-199a之间的相关系数被明显消弱;MT通过下调6月份埋植组各miRNA的表达量提早诱发二次生绒。

[7] YANG Q, DAI S, LUO X, ZHU J, LI F, LIU J, YAO G, SUN Y . Melatonin attenuates postovulatory oocyte dysfunction by regulating SIRT1 expression
Reproduction, 2018,156(1):81-92.
DOI:10.1530/REP-18-0211URLPMID:29752296 [本文引用: 1]

[8] FISCHER T W, SWEATMAN T W, SEMAK I, SAYRE R M, WORTSMAN J, SLOMINSKI A . Constitutive and UV-induced metabolism of melatonin in keratinocytes and cell-free systems
Faseb Journal, 2006,20(9):1564-1566.
DOI:10.1096/fj.05-5227fjeURLPMID:16793870 [本文引用: 1]
Melatonin, which can be produced in the skin, exerts a protective effect against damage induced by UV radiation (UVR). We have investigated the effect of UVB, the most damaging component of UVR, on melatonin metabolism in HaCaT keratinocytes and in a cell-free system. Four metabolites were identified by HPLC and LC-MS: 6-hydroxymelatonin, N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK), 2-hydroxymelatonin (the main intermediate between melatonin and AFMK), and 4-hydroxymelatonin. Concentrations of these photoproducts were directly proportional to UVR-dose and to melatonin substrate content, and their accumulation was time-dependent. The UVR-dependent increase of AFMK and 2-hydroxymelatonin was also detected in keratinocytes, where it was accompanied by simultaneous consumption of intracellular melatonin. Of note, melatonin and its two major metabolites, 2-hydroxymelatonin and AFMK, were also detected in untreated keratinocytes, neither irradiated nor preincubated with melatonin. Thus, intracellular melatonin metabolism is enhanced under exposure to UVR. The additional biological activity of these individual melatonin metabolites increases the spectrum of potential actions of the recently identified cutaneous melatoninergic system.

[9] IBRAHEEM M, GALBRAITH H, SCAIFE J, EWEN S . Growth of secondary hair follicles of the Cashmere goat in vitro and their response to prolactin and melatonin
Journal of Anatomy, 1994 , 185(1):135-142.
[本文引用: 1]

[10] LOGAN A, WEATHERHEAD B . Post-tyrosinase inhibition of melanogenesis by melatonin in hair follicles in vitro.
Journal of Investigative Dermatology, 1980,74(1):47-50.
DOI:10.1111/1523-1747.ep12514608URLPMID:6766170 [本文引用: 1]
Abstract In short-term (48 hr) culture hair follicles of the Siberian hamster retain both tyrosinase activity and the capacity to produce melanin. The addition of melatonin to such cultures at concentrations between 10-6 M and 10-10 M brings about a dose-related inhibition of melanogenesis but tyrosinase activity is unaffected. The use of a series of melatonin analogues and blockers suggests that the hair follicle melanocytes possess melatonin receptors, although their location remains to be determined. Melatonin also inhibits the increase in melanogenesis brought about by alpha-melanocyte-stimulating hormone (MSH) but again it has no effect upon the increased levels of tyrosinase which accompany this MSH response. It is suggested that melatonin inhibits melanogenesis through a mechanism which operates at some post-tyrosinase step in the melanin biosynthetic pathway.

[11] GE W, WANG S H, SUN B, ZHANG Y L, SHEN W, KHATIB H, WANG X . Melatonin promotes Cashmere goat (Capra hircus) secondary hair follicle growth: a view from integrated analysis of long non-coding and coding RNAs.
Cell Cycle, 2018,17(10):1255-1267.
DOI:10.1080/15384101.2018.1471318URLPMID:29895193 [本文引用: 1]

[12] FISCHER T W . The influence of melatonin on hair physiology
Hautarzt, 2009,60(12):962-972.
DOI:10.1007/s00105-009-1817-yURLPMID:19957072 [本文引用: 1]
Abstract Melatonin, the pineal gland hormone and a strong antioxidant, has long been known, particularly in animal-experiment based research and the wool-producing industry, to be a potent regulatory neuroendocrine substance in relation to hair growth, hair color and hair cycle, depending on light periods, seasonal rhythms, environmental factors and reproductive rhythms. Nevertheless, the biological mechanisms of this extremely versatile hormone, especially with regard to human hair follicles, are not fully understood. In recent years, however, essential knowledge has been gained on the melatoninergic system of the skin, melatonin levels in keratinocytes and hair follicles, extrapineal intrafollicular melatonin synthesis and noradrenalin-induced increase in synthesis, as well as hair cycle-dependent expression of the membrane-bound melatonin receptor MT2 and the nuclear receptor RORalpha. Functional data on the growth of human hair both in vitro and in vivo show that melatonin might play an essential role in hair physiology.

[13] KOBAYASHI H, KROMMINGA A, DUNLOP T W, TYCHSEN B, CONRAD F, SUZUKI N, MEMEZAWA A, BETTERMANN A, AIBA S, CARLBERG C, PAUS R . A role of melatonin in neuroectodermal-mesodermal interactions: the hairfollicle synthesizes melatonin and expresses functional receptors
Faseb Journal, 2005,19(12):1710-1712.
DOI:10.1096/fj.04-2293fjeURLPMID:16030176 [本文引用: 1]
Abstract Since mammalian skin expresses the enzymatic apparatus for melatonin synthesis, it may be an extrapineal site of melatonin synthesis. However, evidence is still lacking that this is really the case in situ. Here, we demonstrate melatonin-like immunoreactivity (IR) in the outer root sheath (ORS) of mouse and human hair follicles (HFs), which corresponds to melatonin, as shown by radioimmunoassay and liquid chromatography/tandem mass spectrometry (LC/MS/MS). The melatonin concentration in organ-cultured mouse skin, mouse vibrissae follicles, and human scalp HFs far exceeds the respective melatonin serum level and is significantly increased ex vivo by stimulation with norepinephrine (NE), the key stimulus for pineal melatonin synthesis. By real-time PCR, transcripts for the melatonin membrane receptor MT2 and for the nuclear mediator of melatonin signaling, retinoid orphan receptor alpha (ROR)alpha, are detectable in murine back skin. Transcript levels for these receptors fluctuate in a hair cycle-dependent manner, and are maximal during apoptosis-driven HF regression (catagen). Melatonin may play a role in hair cycle regulation, since its receptors (MT2 and RORalpha) are expressed in murine skin in a hair cycle-dependent manner, and because it inhibits keratinocyte apoptosis and down-regulates ERalpha expression. Therefore, the HF is both, a prominent extrapineal melatonin source, and an important peripheral melatonin target tissue. Regulated intrafollicular melatonin synthesis and signaling may play a previously unrecognized role in the endogenous controls of hair growth, for example, by modulating keratinocyte apoptosis during catagen and by desensitizing the HF to estrogen signaling. As a prototypic neuroectodermal-mesodermal interaction model, the HF can be exploited for dissecting the obscure role of melatonin in such interactions in peripheral tissues.

[14] FOLDES A, HOSKINSON R M, BAKER P, MCDONALD B J, MAXWELL C A, RESTALL B J . Effect of immunization against melatonin on seasonal fleece growth in feral goats
Journal of Pineal Research, 1992,13(2):85-94.
DOI:10.1111/j.1600-079X.1992.tb00059.xURLPMID:1453313 [本文引用: 1]
Abstract: Four vaccination protocols were utilized to investigate the effects of immunoneutralizing circulating melatonin on the annual cashmere growth cycle and cashmere production in Australian feral goats. A fluctuating anti-melatonin antibody response, achieved by repeated booster vaccinations, resulted in an acceleration of the growth cycle in goats which exhibited a significant immune response, compared to sham-immunized controls. Responding goats showed two cycles of cashmere length growth in the first 16 months and increased annual cashmere production in the first year. However, in the second year, these effects were no longer apparent, suggesting either some form of desensitization to melatonin, or a diminished response due to declining antibody titre. The effects of immunization were observed in both sexes; the effect on cashmere length was greater in wethers than in does. Cashmere fibre growth in response to a continuously declining plane of specific antibody showed increased cycle frequency, albeit with a decreased amplitude; guard hair growth cycles were affected to a much lesser extent. Small transient peaks of specific immunity at the summer or winter solstice were without significant effect on cashmere growth. Immunization to provoke a persistent anti-melatonin antibody response at the winter solstice resulted in significantly increased greasy fleece weight, % cashmere yield, and mass of cashmere produced, but no change in fibre diameter in both sexes. Thus the timing of cashmere growth cycles in goats may be, at least transiently, altered by appropriately timed immunization against melatonin. The mechanism of pineal-mediated regulation of cashmere growth cycles may involve (i) entrainment of an endogenous rhythm by melatonin, or (ii) seasonal alteration of cashmere follicle sensitivity to the effect of melatonin.

[15] NIXON A J, CHOY V J, PARRY A L, PEARSON A J . Fiber growth initiation in hair follicles of goats treated with melatonin
Journal of Experimental Zoology Part A-Ecological Genetics and Physiology, 1993,267(1):47-56.
DOI:10.1002/jez.1402670108URLPMID:8376951 [本文引用: 1]
The sequence of structural changes in goat hair follicles was investigated using melatonin implants to advance and synchronize spring hair growth. Ten pasture fed cashmere wethers each received a controlled release formulation of 70 mg of melatonin on September 1 1989, and showed plasma melatonin elevated above physiological levels over 14 days post-treatment (914 ± 154 pg/ml [mean ± SEM] on day 14). In ten untreated animals, daytime plasma melatonin was 19.9 ± 4.7 pg/ml. Histological examination of skin biopsies taken over the 14 days from the start of the experiment showed that primary hair follicles of goats with manipulated hormone levels had initiated fiber growth (entered proanagen), whereas primary follicles of untreated goats largely remained in the quiescent phase (telogen). A standardized terminology was used to describe the sequence of events during induced proanagen. Structural reorganization of follicles began in treated animals between days 6 and 12 post-treatment, and emergent fibers grew by day 24. Advancement of spring fiber growth was associated with a suppression of the normal rise in plasma prolactin concentration. Prolactin levels in untreated goats increased from 7.4 ± 1.8 ng/ml on day 1 to 12.8 ± 1.6ng/ml on day 14, but declined in treated goats from 6.3 ± 2.3 ng/ml to 2.2 ± 0.8 ng/ml over the same period. 08 1993 Wiley-Liss, Inc.

[16] FISCHER T W, SLOMINSKI A, TOBIN D J, PAUS R . Melatonin and the hair follicle
Journal of Pineal Research, 2008,44(1):1-15.
[本文引用: 1]

[17] HéBERT J M, ROSENQUIST T, GǒTZ J, MARTIN G R . FGF5 as a regulator of the hair growth cycle: evidence from targeted and spontaneous mutations
Cell, 1994,78(6):1017-1025.
DOI:10.1016/0092-8674(94)90276-3URLPMID:7923352 [本文引用: 1]
Abstract Fibroblast growth factor 5 (FGF5) is a secreted signaling protein. Mice homozygous for a predicted null allele of the Fgf5 gene, fgf5neo, produced by gene targeting in embryonic stem cells, have abnormally long hair. This phenotype appears identical to that of mice homozygous for the spontaneous mutation angora (go). The fgf5neo and go mutations fail to complement one another, and exon 1 of Fgf5 is deleted in DNA from go homozygotes, demonstrating that go is a mutant allele of Fgf5. Expression of Fgf5 is detected in hair follicles from wild-type mice and is localized to the outer root sheath during the anagen VI phase of the hair growth cycle. These findings provide evidence that FGF5 functions as an inhibitor of hair elongation, thus identifying a molecule whose normal function is apparently to regulate one step in the progression of the follicle through the hair growth cycle.

[18] KEHLER J S, DAVID V A, SCH?FFER A A, BAJEMA K, EIZIRIK E, RYUGO D K, HANNAH S S, O'BRIEN S J, MENOTTI- RAYMOND M .L . Four independent mutations in the feline fibroblast growth factor 5 gene determine the long-haired phenotype in domestic cats
Journal of Heredity, 2007,98(6):555-566.
DOI:10.1093/jhered/esm072URLPMID:17767004 [本文引用: 1]
To determine the genetic regulation of "hair length" in the domestic cat, a whole-genome scan was performed in a multigenerational pedigree in which the "long-haired" phenotype was segregating. The 2 markers that demonstrated the greatest linkage to the long-haired trait (log of the odds > 6) flanked an estimated 10-Mb region on cat chromosome B1 containing the Fibroblast Growth Factor 5 (FGF5) gene, a candidate gene implicated in regulating hair follicle growth cycle in other species. Sequence analyses of FGF5 in 26 cat breeds and 2 pedigrees of nonbreed cats revealed 4 separate mutations predicted to disrupt the biological activity of the FGF5 protein. Pedigree analyses demonstrated that different combinations of paired mutant FGF5 alleles segregated with the long-haired phenotype in an autosomal recessive manner. Association analyses of more than 380 genotyped breed and nonbreed cats were consistent with mutations in the FGF5 gene causing the long-haired phenotype in an autosomal recessive manner. In combination, these genomic approaches demonstrated that FGF5 is the major genetic determinant of hair length in the domestic cat.

[19] SUZUKI S, OTA Y, OZAWA K, IMAMURA T . Dual-mode regulation of hair growth cycle by two FGF5 gene products
Journal of Investigative Dermatology, 2000,114(3):456-463.
DOI:10.1046/j.1523-1747.2000.00912.xURLPMID:10692103 [本文引用: 1]
Abstract As the result of alternative mRNA splicing, Fgf-5, the gene encoding fibroblast growth factor-5, translates to both long and short forms of the protein, respectively, designated fibroblast growth factor-5 and fibroblast growth factor-5S. We previously showed that localization of fibroblast growth factor-5 and the level of fibroblast growth factor-5S in murine skin are hair-cycle dependent. In this study, we examined the effect of fibroblast growth factor-5 and fibroblast growth factor-5S on the hair growth cycle in mice. Once the anagen phase of the hair growth cycle was induced in the dorsal skin by depilation during telogen, and effects of subcutaneous injection of fibroblast growth factor-5 and fibroblast growth factor-5S into the affected region were analyzed. We found that fibroblast growth factor-5 inhibited hair growth during anagen and promoted the transition from anagen to catagen. Interestingly, whereas fibroblast growth factor-5S alone exerted no effect on hair growth, it significantly inhibited the catagen-promoting activity of fibroblast growth factor-5 when the two proteins were injected simultaneously. Because neither fibroblast growth factor-5 nor fibroblast growth factor-5S affected skin thickness, it is postulated that changes in skin thickness during hair cycle are separately regulated by factors other than those regulating hair and follicle growth. The present results, together with our earlier findings that fibroblast growth factor-5-producing cells gather around dermal papillae during catagen, whereas fibroblast growth factor-5S is abundantly expressed in the hair follicles only during the latter half of anagen, suggests that the mouse hair growth cycle is regulated by the two Fgf-5 gene products acting in concert: fibroblast growth factor-5 induces catagen, whereas fibroblast growth factor-5S antagonizes this activity during anagen.

[20] KONYUKHOV B V, MARTYNOVA M Y, NESTEROVA A P . Gene angora as a modifier of the mouse hairless gene
Genetika-Belgrade, 2007,43(2):254-260.
DOI:10.1134/S1022795407020147URLPMID:17385325 [本文引用: 1]
The interactions between mouse angora-Y (Fgf5go-Y) and hairless (hr) genes have been studied. Homozygous mutant gene Fgf5go-Y increases hair length starting on day 14 after birth. We obtained mice with genotypes +/+ hr/hr F2, +/Fgf5go-Y hr/hr and Fgf5go-Y/Fgf5go-Y hr/hr. Both +/Fgf5go-Y hr/hr and +/+ hr/hr mice began to loose hair from their heads on day 14. This further extended on the whole body. On day 21 the mice were completely deprived of hair. Therefore a single dose of gene Fgf5go-Y does not affect alopecia mice homozygous for hr. However in double homozygotes Fgf5go-Y/Fgf5gO-hr/hr alopecia started 4 days later, namely on day 18. It usually finished 10-12 days after detection of first bald patches. On days 28-30 double homozygotes have lost all the hair. Hair loss in double homozygous mice was 1,5-fold slower than in +/+ hr/hr mice. This resulted from a significant extension of anagen phase induced by a mutant homozygous gene Fgf5go-Y in morphogenesis of the hair follicle. In contrast, hr gene was expressed only at the transmission phase from anagen to catagen. Our data shows that the angora gene is a modifier of the hairless gene and this results in a strong repression of alopecia progression in double homozygous mice compared to +/+ hr/hr animals.

[21] JOHNSTON A P, NASKA S, JONES K, JINNO H, KAPLAN D R, MILLER F D . Sox2-Mediated Regulation of Adult Neural Crest Precursors and Skin Repair
Stem Cell Reports, 2013,1(1):38-45.
DOI:10.1016/j.stemcr.2013.04.004URLPMID:24052940 [本文引用: 1]
Nerve-derived neural crest cells are essential for regeneration in certain animals, such as newts. Here, we asked whether they play a similar role during mammalian tissue repair, focusing on Sox2-positive neural crest precursors in skin. In adult skin, Sox2 was expressed in nerve-terminal-associated neural crest precursor cells (NCPCs) around the hair follicle bulge, and following injury was induced in nerve-derived cells, likely dedifferentiated Schwann cell precursors. At later times postinjury, Sox2-positive cells were scattered throughout the regenerating dermis, and lineage tracing showed that these were all neural-crest-derived NCPCs. These Sox2-positive NCPCs were functionally important, since acute deletion of Sox2 prior to injury caused a decrease of NCPCs in the wound and aberrant skin repair. These data demonstrate that Sox2 regulates skin repair, likely by controlling NCPCs, and raise the possibility that nerve-derived NCPCs may play a general role in mammalian tissue repair.

[22] ULITSKY I, BARTEL D P . lincRNAs: genomics, evolution and mechanisms
Cell, 2013,154(1):26-46.
DOI:10.1016/j.cell.2013.06.020URLPMID:23827673 [本文引用: 1]
Long intervening noncoding RNAs (lincRNAs) are transcribed from thousands of loci in mammalian genomes and might play widespread roles in gene regulation and other cellular processes. This Review outlines the emerging understanding of lincRNAs in vertebrate animals, with emphases on how they are being identified and current conclusions and questions regarding their genomics, evolution and mechanisms of action.

[23] KAPRANOV P, CHENG J, DIKE S, NIX D A, DUTTAGUPTA R, WILLINGHAM A T, STADLER P F, HERTEL J, HACKERMüLLER J, HOFACKER I L, BELL I, CHEUNG E, DRENKOW J, DUMAIS E, PATEL S, HELT G, GANESH M, GHOSH S, PICCOLBONI A, SEMENTCHENKO V, TAMMANA H, GINGERAS T R . RNA maps reveal new RNA classes and a possible function for pervasive transcription
Science, 2007,316(5830):1484-1488.
DOI:10.1126/science.1138341URL [本文引用: 1]

[24] FEJES-TOTH K, SOTIROVA V, SACHIDANANDAM R, ASSAF G, HANNON GJ, KAPRANOV P, FOISSAC S, WILLINGHAM A T, DUTTAGUPTA R, DUMAIS E, GINGERAS T R . Post- transcriptional processing generates a diversity of 59-modified long and short RNAs
Nature, 2009,457(7232):1028-1032.
DOI:10.1038/nature07759URL [本文引用: 1]

[25] CESANA M, CACCHIARELLI D, LEGNINI I, SANTINI T, STHANDIER O, CHINAPPI M, TRAMONTANO A, BOZZONI I . A long noncoding RNA controls muscle differentiation by functioning as a competing endogenous RNA
Cell, 2011,147(2):358-369.
DOI:10.1016/j.cell.2011.09.028URLPMID:22000014 [本文引用: 1]
A long noncoding RNA, linc-MD1, governs muscle cell differentiation by competitively binding to microRNAs that target muscle-specific transcription factors. linc-MD1 functions not only as a decoy RNA but also as a precursor pri-microRNA.

[26] KLATTENHOFF C A, SCHEUERMANN J C, SURFACE L E, BRADLEY R K, FIELDS P A, STEINHAUSER M L, DING H, BUTTY V L, TORREY L, HAAS S, ABO R, TABEBORDBAR M, LEE R T, BURGE CB, BOYER LA . Braveheart, A long noncoding RNA required for cardiovascular lineage commitment
Cell, 2013,152(3):570-583.
DOI:10.1016/j.cell.2013.01.003URLPMID:23352431 [本文引用: 1]
Braveheart (Bvht) is a heart-associated long noncoding RNA that is necessary for cardiovascular lineage commitment in embryonic stem cells. Its interaction with the polycomb complex suggests that it may regulate the cardiac program epigenetically.

[27] REN H, WANG G, CHEN L, JIANG J, LIU L, LI N, ZHAO J, SUN X, ZHOU P . Genome-wide analysis of long non-coding RNAs at early stage of skin pigmentation in goats (Capra hircus)
BMC Genomics, 2016,17:67.
DOI:10.1186/s12864-016-2365-3URLPMID:26785828 [本文引用: 1]
Background: Long noncoding RNAs (lncRNAs) play roles in almost all biological processes; however, their function and profile in skin development and pigmentation is less understood. In addition, because lncRNAs are species-specific, their function in goats has not been established. Result: We systematically identified lncRNAs in 100-day-old fetal skin by deep RNA-sequencing using the Youzhou dark goat (dark skin) and Yudong white goat (white skin) as a model of skin pigmentation. A total of 841,895,634 clean reads were obtained from six libraries (samples). We identified 1336 specific lncRNAs in fetal skin that belonged to three subtypes, including 999 intergenic lncRNAs (lincRNAs), 218 anti-sense lncRNAs, and 119 intronic lncRNAs. Our results demonstrated significant differences in gene architecture and expression among the three lncRNA subtypes, particularly in terms of density and position bias of transpose elements near the transcription start site. We also investigated the impact of lncRNAs on its target genes in cis and trans, indicating that these lncRNAs have a strict tissue specificity and functional conservation during skin development and pigmentation. Conclusion: The present study provides a resource for lncRNA studies in diseases involved in pigmentation and skin development. It expands our knowledge about lncRNA biology as well as contributes to the annotation of the goat genome.

[28] ZHU Y B, WANG Z Y, YIN R H, JIAO Q, ZHAO S J, CONG Y Y, XUE H L, GUO D, WANG S Q, ZHU Y X, BAI W L . A LncRNA-H19 transcript from secondary hair follicle of Liaoning cashmere goat: Identification, regulatory network and expression regulated potentially by its promoter methylation
Gene, 2018, 30, 641:78-85.
[本文引用: 1]

[29] LIN C M, LIU Y, HUANG K, CHEN X C, CAI B Z, LI H H, YUAN Y P, ZHANG H, LI Y . Long noncoding RNA expression in dermal papilla cells contributes to hairy gene regulation
Biochemical And Biophysical Research Communications, 2014,453(3):508-514.
DOI:10.1016/j.bbrc.2014.09.119URL [本文引用: 1]

[30] BAKHTIARIZADEH M R, HOSSEINPOUR B, AREFNEZHAD B, SHAMABADI N, SALAMI S A . In silico prediction of long intergenic non-coding RNAs in sheep
Genome, 2016,59(4):263-275.
DOI:10.1139/gen-2015-0141URLPMID:1011392015014139882003 [本文引用: 1]
Long non-coding RNAs (lncRNAs) are transcribed RNA molecules >200 nucleotides in length that do not encode proteins and serve as key regulators of diverse biological processes. Recently, thousands of long intergenic non-coding RNAs (lincRNAs), a type of lncRNAs, have been identified in mammalians using massive parallel large sequencing technologies. The availability of the genome sequence of sheep (Ovis aries) has allowed us genomic prediction of non-coding RNAs. This is the first study to identify lincRNAs using RNA-seq data of eight different tissues of sheep, including brain, heart, kidney, liver, lung, ovary, skin, and white adipose. A computational pipeline was employed to characterize 325 putative lincRNAs with high confidence from eight important tissues of sheep using different criteria such as GC content, exon number, gene length, co-expression analysis, stability, and tissue-specific scores. Sixty-four putative lincRNAs displayed tissues-specific expression. The highest number of tissues-specific lincRNAs was found in skin and brain. All novel lincRNAs that aligned to the human and mouse lincRNAs had conserved synteny. These closest protein-coding genes were enriched in 11 significant GO terms such as limb development, appendage development, striated muscle tissue development, and multicellular organismal development. The findings reported here have important implications for the study of sheep genome.

[31] BAI W L, ZHAO S J, WANG Z Y, ZHU Y B, DANG Y L, CONG Y Y, XUE H L, WANG W, DENG L, GUO D, WANG S Q, ZHU Y X, YIN R H . LncRNAs in Secondary Hair Follicle of Cashmere Goat: Identification, Expression, and Their Regulatory Network in Wnt Signaling Pathway
Animal Biotechnology, 2018,29(3):199-211.
DOI:10.1080/10495398.2017.1356731URLPMID:28846493 [本文引用: 1]
Long noncoding RNAs (lncRNAs) are a novel class of eukaryotic transcripts. They are thought to act as a critical regulator of protein-coding gene expression. Herein, we identified and characterized 13 putative lncRNAs from the expressed sequence tags from secondary hair follicle of Cashmere goat. Furthermore, we investigated their transcriptional pattern in secondary hair follicle of Liaoning Cashmere goat during telogen and anagen phases. Also, we generated intracellular regulatory networks of upregulated lncRNAs at anagen in Wnt signaling pathway based on bioinformatics analysis. The relative expression of six putative lncRNAs (lncRNA-599618, -599556, -599554, -599547, -599531, and -599509) at the anagen phase is significantly higher than that at telogen. Compared with anagen, the relative expression of four putative lncRNAs (lncRNA-599528, -599518, -599511, and -599497) was found to be significantly upregulated at telogen phase. The network generated showed that a rich and complex regulatory relationship of the putative lncRNAs and related miRNAs with their target genes in Wnt signaling pathway. Our results from the present study provided a foundation for further elucidating the functional and regulatory mechanisms of these putative lncRNAs in the development of secondary hair follicle and cashmere fiber growth of Cashmere goat.

[32] CAI B, ZHENG Y, MA S, XING Q, WANG X, YANG B, YIN G, GUAN F . Long non-coding RNA regulates hair follicle stem cell proliferation and differentiation through PI3K/AKT signal pathway
Frontiers in Physiology, 2018,17(4):5477-5483.
DOI:10.3892/mmr.2018.8546URLPMID:29393477 [本文引用: 1]
Abstract Long non-coding RNAs (lncRNAs) are defined as non-coding transcripts (>200 nucleotides) that serve important roles in the proliferation and differentiation of stem cells. Hair follicle stem cells (HFTs) have multidirectional differentiation potential and are able to differentiate into skin, hair follicles and sebaceous glands, serving a role in skin wound healing. The aim of the present study was to analyze the regulatory role of lncRNA AK015322 (IncRNA5322) in HFTs and the potential mechanism of IncRNA532262mediated differentiation of HFTs. The results demonstrated that lncRNA5322 transfection promoted proliferation and differentiation in HFTs. It was identified that lncRNA5322 transfection upregulated the expression and phosphorylation of phosphoinositide 362kinase (PI3K) and protein kinase B (AKT) in HFTs. It was also observed that lncRNA5322 transfection upregulated microRNA (miR)6221 and miR6221 agonist (agomir6221) eliminated lncRNA532262induced expression and phosphorylation of PI3K and AKT. The present study also demonstrated that agomir6221 blocked IncRNA532262induced expression and phosphorylation of PI3K and AKT in HFTs. The results indicated that agomir6221 transfection also suppressed the IncRNA532262induced proliferation and differentiation of HFTs. In conclusion, the results of the present study suggest that lncRNA5322 is able to promote the proliferation and differentiation of HFTs by targeting the miR622162mediated PI3K62AKT signaling pathway in HFTs.

[33] ZHOU G, KANG D, MA S, WANG X, GAO Y, YANG Y, WANG X, CHEN Y . Integrative analysis reveals LncRNA-mediated molecular regulatory network driving secondary hair follicle regression in cashmere goat
BMC Genomics. 2018,19(1):222.
DOI:10.1186/s12864-018-4603-3URL [本文引用: 1]
Cashmere is a keratinized product derived from the secondary hair follicles (SHFs) of cashmere goat skins. The cashmere fiber stops growing following the transition from the actively proliferating anagen stage to the apoptosis-driven catagen stage. However, little is known regarding the molecular mechanisms responsible for the occurrence of apoptosis in SHFs, especially as pertains to the role of non-coding RNAs (ncRNAs) and their interactions with other molecules. Hair follicle (HF) degeneration is caused by localized apoptosis in the skin, while anti-apoptosis pathways may coexist in adjacent HFs. Thus, elucidating the molecular interactions responsible for apoptosis and anti-apoptosis in the skin will provide insights into HF regression. We used multiple-omics approaches to systematically identify long non-coding RNAs (lncRNAs), microRNAs (miRNAs) and mRNAs expressed in cashmere goat skins in two crucial phases (catagen vs. anagen) of HF growth. Skin samples were collected from three cashmere goats at the anagen (September) and catagen (February) stages, and six lncRNA libraries and six miRNA libraries were constructed for further analysis. We identified 1122 known and 403 novel lncRNAs in the goat skins, 173 of which were differentially expressed between the anagen and catagen stages. We further identified 3500 gene-encoding transcripts that were differentially expressed between these two phases. We also identified 411 known miRNAs and 307 novel miRNAs, including 72 differentially expressed miRNAs. We further investigated the target genes of lncRNAs via both cis- and trans-regulation during HF growth. Our data suggest that lncRNAs and miRNAs act synergistically in the HF growth transition, and the catagen inducer factors (TGF尾1 and BDNF) were regulated by miR-873 and lnc108635596 in the lncRNA-miRNA-mRNA networks. This study enriches the repertoire of ncRNAs in goats and other mammals, and contributes to a better understanding of the molecular mechanisms of ncRNAs involved in the regulation of HF growth and regression in goats and other hair-producing species. The online version of this article (10.1186/s12864-018-4603-3) contains supplementary material, which is available to authorized users.

[34] NIE Y, LI S, ZHENG X, CHEN W, LI X, LIU Z, HU Y, QIAO H, QI Q, PEI Q, CAI D, YU M, MOU C . Transcriptome reveals long non-coding RNAs and mRNAs involved in primary wool follicle induction in carpet sheep fetal skin
Frontiers in Physiology, 2018,9:446.
DOI:10.3389/fphys.2018.00446URL [本文引用: 1]
Murine primary hair follicle induction is driven by the communication between the mesenchyme and epithelium and mostly governed by signaling pathways including wingless-related integration site (WNT), ectodysplasin A receptor (EDAR), bone morphogenetic protein (BMP), and fibroblast growth factor (FGF), as observed in genetically modified mouse models. Sheep skin may serve as a valuable system for hair research owing to the co-existence of sweat glands with wool follicles in trunk skin and asynchronized wool follicle growth pattern similar to that of human head hair follicles. However, the mechanisms underlying wool follicle development remain largely unknown. To understand how long non-coding RNAs (lncRNAs) and mRNAs function in primary wool follicle induction in carpet wool sheep, we conducted high-throughput RNA sequencing and revealed globally altered lncRNAs (36 upregulated and 26 downregulated), mRNAs (228 elevated and 225 decreased), and 80 differentially expressed novel transcripts. Several key signals in WNT (WNT2BandWNT16), BMP (BMP3, BMP4, andBMP7), EDAR (EDARandEDARADD), and FGF (FGFR2andFGF20) pathways, and a series of lncRNAs, including XLOC_539599, XLOC_556463, XLOC_015081, XLOC_1285606, XLOC_297809, and XLOC_764219, were shown to be potentially important for primary wool follicle induction. GO and KEGG analyses of differentially expressed mRNAs and potential targets of altered lncRNAs were both significantly enriched in morphogenesis biological processes and transforming growth factor-, Hedgehog, and PI3K-Akt signaling, as well as focal adhesion and extracellular matrix-receptor interactions. The prediction of mRNA-mRNA and lncRNA-mRNA interaction networks further revealed transcripts potentially involved in primary wool follicle induction. The expression patterns of mRNAs and lncRNAs of interest were validated by qRT-PCR. The localization of XLOC_297809 and XLOC_764219 both in placodes and dermal condensations was detected byin situhybridization, indicating important roles of lncRNAs in primary wool follicle induction and skin development. This is the first report elucidating the gene network of lncRNAs and mRNAs associated with primary wool follicle early development in carpet wool sheep and will shed new light on selective wool sheep breeding.

[35] KLOEPPER J E, ERNST N, KRIEGER K, BODó E, BíRó T, HASLAM I S, SCHMIDT-ULLRICH R, PAUS R . NF-κB activity is required for anagen maintenance in human hair follicles in vitro
Journal of Investigative Dermatology, 134(7):2036-2038.
DOI:10.1038/jid.2014.82PMID:24518172 [本文引用: 1]
The Journal of Investigative Dermatology publishes basic and clinical research in cutaneous biology and skin disease.

[36] GILON M, SHER N, COHEN S, GAT U . Transcriptional activation of a subset of hair keratin genes by the NF-κB effector p65/RelA
Differentiation, 2008,76(5):518-530.
DOI:10.1111/j.1432-0436.2007.00246.xURLPMID:18021261 [本文引用: 1]
Abstract The hair follicle is an intricate miniature organ dedicated to the production of the structural hair fiber, which is largely composed of hair keratin (HK) proteins. Many developmental pathways contribute to hair follicle development; however, the molecular control of HK genes is still far from being resolved. Because the nuclear factor (NF)-0202B pathway is known to be involved in the morphogenesis of the hair follicle, we explored the possibility that it may also regulate HK expression. To this end, we analyzed the effect of p65/RelA, an NF-0202B effector, on HK regulatory regions using transient transfections into tissue culture cells. Reporter assays on cells transfected with HK promoter constructs and real-time polymerase chain reaction analysis of endogenous HK gene activity demonstrated that p65 induces transcriptional activation of several HK genes of human and mouse origin, primarily that of acidic hair keratin 5 (Ha5) . Focusing on the highly responsive human Ha5 gene, we defined the major NF-0202B/RelA binding sites in its regulatory region and showed the direct binding of p65 to these sites using gel shift assays. We further show, using immunohistochemistry on human hair follicle sections, that p65 is co-expressed with HKs in the hair shaft compartment and may thus be the effector that mediates the NF-0202B pathway's activity, which recently was genetically demonstrated to be active in the same region. Thus, we provide evidence for a previously unknown function of NF-0202B in hair formation090000direct activation of HK target genes090000a function that may shed light on some of the symptoms of ectodermal dysplasias.

[37] SCHMIDT-ULLRICH R, AEBISCHER T, HüLSKEN J, BIRCHMEIER W, KLEMM U, SCHEIDEREIT C . Requirement of NF-kappaB/Rel for the development of hair follicles and other epidermal appendices
Development, 2001,128(19):3843-3853.
[本文引用: 1]

[38] WANG X, CHEN H, TIAN R, ZHANG Y, DRUTSKAYA MS, WANG C, GE J, FAN Z, KONG D, WANG X, CAI T, ZHOU Y, WANG J, WANG J, WANG S, QIN Z, JIA H, WU Y, LIU J, NEDOSPASOV SA, TREDGET EE, LIN M, LIU J, JIANG Y, WU Y . Macrophages induce AKT/-catenin-dependent Lgr5+ stem cell activation and hair follicle regeneration through TNF
Nature Communications, 2017,8:14091.
DOI:10.1038/ncomms14091URLPMID:5378973 [本文引用: 1]
Hair can be regenerated after skin wounding. Here the authors show that inflammatory macrophages produce TNF that activates Wnt signalling in hair follicle stem cells to drive this hair regeneration after wound repair in mice.

[39] LAURIKKALA J, PISPA J, JUNG HS, NIEMINEN P, MIKKOLA M, WANG X, SAARIALHO-KERE U, GALCERAN J, GROSSCHEDL R, THESLEFF I . Regulation of hair follicle development by the TNF signal ectodysplasin and its receptor Edar
Development, 2002,129(10):2541-2553.
DOI:10.1007/s00429-002-0240-2URLPMID:11973284 [本文引用: 1]
Abstract X-linked and autosomal forms of anhidrotic ectodermal dysplasia syndromes (HED) are characterized by deficient development of several ectodermal organs, including hair, teeth and exocrine glands. The recent cloning of the genes that underlie these syndromes, ectodysplasin (ED1) and the ectodysplasin A receptor (EDAR), and their identification as a novel TNF ligand-receptor pair suggested a role for TNF signaling in embryonic morphogenesis. In the mouse, the genes of the spontaneous mutations Tabby (Ta) and downless (dl) were identified as homologs of ED1 and EDAR, respectively. To gain insight into the function of this signaling pathway in development of skin and hair follicles, we analyzed the expression and regulation of Eda and Edar in wild type as well as Tabby and Lef1 mutant mouse embryos. We show that Eda and Edar expression is confined to the ectoderm and occurs in a pattern that suggests a role of ectodysplasin/Edar signaling in the interactions between the ectodermal compartments and the formation and function of hair placodes. By using skin explant cultures, we further show that this signaling pathway is intimately associated with interactions between the epithelial and mesenchymal tissues. We also find that Ta mutants lack completely the placodes of the first developing tylotrich hairs, and that they do not show patterned expression of placodal genes, including Bmp4, Lef1, Shh, Ptch and Edar, and the genes for beta-catenin and activin A. Finally, we identified activin as a mesenchymal signal that stimulates Edar expression and WNT as a signal that induces Eda expression, suggesting a hierarchy of distinct signaling pathways in the development of skin and hair follicles. In conclusion, we suggest that Eda and Edar are associated with the onset of ectodermal patterning and that ectodysplasin/edar signaling also regulates the morphogenesis of hair follicles.

[40] MINIACI M C, IRACE C, CAPUOZZO A, PICCOLO M, DI PASCALE A, RUSSO A, LIPPIELLO P, LEPRE F, RUSSO G, SANTAMARIA R . Cysteine prevents the reduction in keratin synthesis induced by iron deficiency in human keratinocytes
Journal of Cellular Biochemistry, 2016,117(2):402-412.
DOI:10.1002/jcb.25286URLPMID:26212225 [本文引用: 1]
ABSTRACT l-cysteine is currently recognized as a conditionally essential sulphur amino acid. Besides contributing to many biological pathways, cysteine is a key component of the keratin protein by its ability to form disulfide bridges that confer strength and rigidity to the protein. In addition to cysteine, iron represents another critical factor in regulating keratins expression in epidermal tissues, as well as in hair follicle growth and maturation. By focusing on human keratinocytes, the aim of this study was to evaluate the effect of cysteine supplementation as nutraceutical on keratin biosynthesis, as well as to get an insight on the interplay of cysteine availability and cellular iron status in regulating keratins expression in vitro. Herein we demonstrate that cysteine promotes a significant up-regulation of keratins expression as a result of de novo protein synthesis, while the lack of iron impairs keratin expression. Interestingly, cysteine supplementation counteracts the adverse effect of iron deficiency on cellular keratin expression. This effect was likely mediated by the up-regulation of transferrin receptor and ferritin, the main cellular proteins involved in iron homeostasis, at last affecting the labile iron pool. In this manner, cysteine may also enhance the metabolic iron availability for DNA synthesis without creating a detrimental condition of iron overload. To the best of our knowledge, this is one of the first study in an in vitro keratinocyte model providing evidence that cysteine and iron cooperate for keratins expression, indicative of their central role in maintaining healthy epithelia. J. Cell. Biochem. 117: 402 412, 2016. 2015 Wiley Periodicals, Inc.

[41] ZHAO M, CHEN H, WANG X, YU H, WANG M, WANG J, LAN X Y, ZHANG C F, ZHANG L Z, GUO Y K, ZHANG B, HU S R . aPCR-SSCP and DNA sequencing detecting two silent SNPs at KAP8.1 gene in the cashmere goat
Molecular Biology Reports, 2009,36(6):1387-1391.
DOI:10.1007/s11033-008-9325-1URLPMID:18670906 [本文引用: 1]
Keratin-associated proteins 8.1 gene (KAP8.1) is a structural gene responsible for the cashmere. KAP8.1 protein contains high glycine and tyrosine, which concerns regulation and function of the matrix structure fiber. In this study, the polymorphism of KAP8.1 gene was detected by methods of aPCR-SSCP (asymmetric polymerase chain reaction single-strand conformation polymorphism) and DNA sequencing in 791 individuals from two breeds. The results showed that there were two mutations in this gene. The mutations were described as c.63 T>G and c.66 C>G, which would result in two synonymous mutations in KAP8.1 protein. The findings go against previous research, in which there was not polymorphism at KAP8.1 gene. The reasons might be that different cashmere breeds were detected in two studies. Further analysis of results leads us to believe that the polymorphism of KAP8.1 gene might be relevant to fiber diameter.

[42] TONG X, COULOMBE P A . Keratin 17 modulates hair follicle cycling in a TNF alphadependent fashion
Genes & Development, 2006,20(10):1353-1364.
DOI:10.1101/gad.1387406URL [本文引用: 1]
Mammalian hair follicles cycle between stages of rapid growth (anagen) and metabolic quiescence (telogen) throughout life. Transition from anagen to telogen involves an intermediate stage, catagen, consisting of a swift, apoptosis-driven involution of the lower half of the follicle. How catagen is coordinated, and spares the progenitor cells needed for anagen re-entry, is poorly understood. Keratin 17 (K17)-null mice develop alopecia in the first week post-birth, correlating with hair shaft fragility and untimely apoptosis in the hair bulb. Here we show that this abnormal apoptosis reflects premature entry into catagen. Of the proapoptotic challenges tested, K17-null skin keratinocytes in primary culture are selectively more sensitive to TNFalpha. K17 interacts with TNF receptor 1 (TNFR1)-associated death domain protein (TRADD), a death adaptor essential for TNFR1-dependent signal relay, suggesting a functional link between this keratin and TNFalpha signaling. The activity of NF-kappaB, a downstream target of TNFalpha, is increased in K17-null skin. We also find that TNFalpha is required for a timely anagen-catagen transition in mouse pelage follicles, and that its ablation partially rescues the hair cycling defect of K17-null mice. These findings identify K17 and TNFalpha as two novel and interdependent regulators of hair cycling.

[43] DONG Y, XIE M, JIANG Y, XIAO N, DU X, ZHANG W, TOSSER-KLOPP G, WANG J, YANG S, LIANG J, CHEN W, CHEN J, ZENG P, HOU Y, BIAN C, PAN S, LI Y, LIU X, WANG W, SERVIN B, SAYRE B, ZHU B, SWEENEY D, MOORE R, NIE W, SHEN Y, ZHAO R, ZHANG G, LI J, FARAUT T, WOMACK J, ZHANG Y, KIJAS J, COCKETT N, XU X, ZHAO S, WANG J, WANG W . Sequencing and automated whole-genome optical mapping of the genome of adomestic goat (Capra hircus).
Nature Biotechnology, 2013,31(2):135-141.
DOI:10.1038/nbt.2478URLPMID:23263233 [本文引用: 1]
We report the similar to 2.66-Gb genome sequence of a female Yunnan black goat. The sequence was obtained by combining short-read sequencing data and optical mapping data from a high-throughput whole-genome mapping instrument. The whole-genome mapping data facilitated the assembly of super-scaffolds >5x longer by the N50 metric than scaffolds augmented by fosmid end sequencing (scaffold N50 = 3.06 Mb, super-scaffold N50 = 16.3 Mb). Super-scaffolds are anchored on chromosomes based on conserved synteny with cattle, and the assembly is well supported by two radiation hybrid maps of chromosome 1. We annotate 22,175 protein-coding genes, most of which were recovered in the RNA-seq data of ten tissues. Comparative transcriptomic analysis of the primary and secondary follicles of a cashmere goat reveal 51 genes that are differentially expressed between the two types of hair follicles. This study, whose results will facilitate goat genomics, shows that whole-genome mapping technology can be used for the de novo assembly of large genomes.