光控增绒技术影响绒山羊长绒相关miRNA调控机制研究

Cashmere goat is economically important animals for its cashmere, however, cashmere growth has strong seasonality, and most of goats cannot produce cashmere in hair follicle telogen (from May to August). The technology, promoting cashmere growth by light control, could promote the secondary follicles of goat to transmit from telogen to anagen, which increase average cashmere yield up to 50% and above each in a whole year. In this study, combining high-throughput-sequencing with bioinformatics, is aiming to deep sequence MicroRNA(miRNA) of Cashmere goats under artificial light control and natural daylight, then to excavate miRNA transcriptome expression profile changes of Cashmere goat hair follicle after light control. Furthermore, we will do miRNA sequence analysis on hair follicle and skin micro-environment, build network construction of the interaction between miRNA and candidate genes, verify expression and location of specific miRNA and target mRNA, and detect regulation function of miRNA in skin micro-environment during Cashmere goat hair follicle growth, preliminarily clarify the hair follicle miRNA regulation mechanism under light control can promote cashmere growth. It can also establish a theoretical foundation for selecting genes related to the quality of cashmere growth，molecular breeding and generalizing the light control promoting cashmere growth technology for industrialization in cashmere main producing areas.

绒山羊是重要的绒用经济动物，其绒毛生长具有较强的季节性，大多数绒山羊在非生绒期（5-8月份）不长绒。应用光控增绒技术可以促进绒山羊毛囊提前进入兴盛期长绒，显著提高产绒量50%以上。本项目通过高通量测序技术并结合生物信息学，对光控增绒和自然放牧条件下绒山羊非生绒期皮肤毛囊miRNA进行深度测序，研究光控增绒处理的绒山羊皮肤毛囊miRNA转录组表达谱的变化特点。进一步进行毛囊及其皮肤微环境miRNA序列分析、miRNA与候选基因间相互作用的网络构建、特异miRNA和靶mRNA的定量定位表达，探究皮肤微环境中miRNA在绒山羊毛囊生长发育中的调控作用，初步阐明光控增绒技术促进绒山羊非产绒期长绒的毛囊miRNA调控机制。为筛选绒毛生长及品质相关的基因及分子育种提供依据，并为内蒙古绒山羊绒山羊主产区进一步产业化推广示范光控增绒技术奠定理论基础。

内蒙古绒山羊毛与绒的生长周期具有典型的季节性生长特点，并与实际的生产密切相关。研究表明，光周期为影响羊绒生长的因素之一。本项目采用光控增绒技术处理试验羊，通过测序平台对毛囊生长及其皮肤微环境相关mRNA、miRNA及甲基化进行深度测序，成功构建内蒙古白绒山羊皮肤组织中差异表达的miRNA、mRNA和甲基化表达谱，发现14个已知miRNA（P<0.05）和56个mRNA（P<0.01）显著性差异表达，对显著差异表达的miRNA与mRNA做了关联分析获得2个显著差异表达miRNA与8个差异表达基因相对应。KEGG通路富集在PI3K-Art signaling pathway 和prolactin signaling pathway上。甲基化测序显示不同光周期下的甲基化水平基本一致，不同序列环境下（CG、CHG和CHH）甲基化水平无显著差异。我们在CG、CHG和CHH序列环境中分别鉴定了9700个差异甲基化区域(DMRs)和4187个差异甲基化基因(DMGs)。KEGG功能注释显著富集到circadian entertainment信号通路。实时荧光定量PCR法检测显著差异表达miR-107及ADCK5、DSBC1、ARSA、FMAP4、RHBDF2和ALDH3A2。双荧光素酶报告基因实验显示，miR-107与BSDC1、ARSA、RHBDF2、ALDH3A2具有靶向关系。发现三种品系的绒山羊毛囊在形态特征上基本一致，但在同一时期生长规律不同，miR-107和RHBDF2基因在不同品系绒山羊皮肤毛囊不同发育周期的表达量呈相反趋势。miR-107和RHBDF2基因表达水平均与皮肤毛囊性状显著相关(P<0.05)，说明miR-107和RHBDF2基因均为三品系绒山羊皮肤毛囊生长发育的重要影响因子。ALDH3A2基因的表达量在皮肤毛囊休止期至兴盛期的过程中逐渐降低，由退行期至休止期的过程中逐渐增高。ALDH3A2基因与皮肤毛囊各性状均显著相关（P＜0.05），表明高表达的ALDH3A2可能抑制三品系绒山羊毛囊的生长发育，是调控皮肤毛囊生长发育的重要影响因子。光控增绒实验中差异表达的miRNA及甲基化差异通过调控mRNA形成复杂的调控网络，在毛囊的发生发展中发挥重要作用，为羊绒生长