肉用绵羊IncRNA特征分析及细胞水平功能验证

发布时间：2018-04-01 13:12

  本文选题：绵羊　切入点：胎儿　出处：《中国农业科学院》2015年硕士论文

【摘要】：绵羊是重要的经济动物,肉用性状选育已成为绵羊育种的主要方向。不同品种绵羊生长调控机制存在差异,对其开展研究有助于发现未知基因调控作用。Lnc RNA是一类长度大于200nt的非编码RNA分子,在哺乳动物肌肉发育具有重要调控作用,且时空差异表达明显。本研究选用发育明显差异的两品种乌珠穆沁和特克赛尔绵羊,利用二代测序技术分别对妊娠85d、120d、初生和一周岁腓肠肌进行分析,从lnc RNA方面揭示两品种肉用性能差异的内在机制。同时在细胞水平对筛选到的TCONS_00102859进行试验分析。主要试验结果如下:1.绵羊lnc RNA测序及描述型分析测序数据经筛选处理后平均每个样本获得2.25 Gb数据,其中85日龄胎儿数据量最小,过滤数据质量可靠。过滤数据回帖基因组,整体比对率≥89%,组装后进行lnc RNA筛选,共获得2002条候选lnc RNA。Lnc RNA在绵羊染色体上不存在偏好性,长度跨越大,但78.5%的lnc RNA长度2000nt。候选lnc RNA平均外显子数目为2.8个,90%的lnc RNA外显子数少于4个。比对后发现1967条为新获得转录本。2.候选lnc RNA靶基因注释靶基因GO注释分析发现,筛选到的lnc RNA可能与结合和催化作用、代谢过程、色素沉着和繁殖性能等相关。KEGG注释分析发现,信号转导、易感疾病、癌症、转录和翻译等通路注释到最多基因。3.特克赛尔和乌珠穆沁相同时期差异lnc RNA分析不同品种相同发育阶段差异lnc RNA分析发现,每个特定阶段两个品种差异表达lnc RNA靶基因富集结果有明显差异。85日龄胎儿富集到MAPK信号通路、Wnt信号通路等胚胎肌肉发育、肿瘤发生和其他相关通路;120日龄阶段富集到心脏机能相关通路;初生阶段富集到磷脂酰肌醇信号系统和钙信号等通路;成年阶段富集到烟酸和烟酰胺代谢通路。所有发育阶段两个品种都有差异表达的lnc RNA筛选到10个。其中有三个lnc RNA,TCONS_00051148、TCONS_00079077和TCONS_00078553,对应到基因内部,对应的基因分别为ANKRD10、FGF2和LOC101105780;TCONS_00102859只在特克赛尔表达且表达量逐渐升高。4.TCONS_00102859在绵羊成肌细胞功能初步分析构建TCONS_00102859慢病毒过表达载体,在绵羊原代成肌细胞上初步验证其作用。发现GV287-TCONS_00102859对原代成肌细胞具有较高的转染效率,且可以显著提高TCONS_00102859在细胞内表达量。转染细胞中Myo D、Myo G和Mef2c表达上调,MSTN表达下调,初步证明TCONS_00102859可能对成肌细胞的增殖和分化均有一定作用。本研究进行的绵羊lnc RNA转录组的特征分析与细胞水平功能初步分析,将进一步促进绵羊lnc RNA功能和基因调控机制的研究。
[Abstract]:Sheep is an important economic animal and breeding of meat traits has become the main direction of sheep breeding. It is helpful to find that the unknown gene regulation. LNC RNA is a class of noncoding RNA molecules which are longer than 200nt and play an important role in the regulation of mammalian muscle development. In this study, two different breeds of sheep, Wuzhimuqin and Texer, were used to analyze the gastrocnemius muscle of 85 days gestation at 120 days after pregnancy, using the second generation sequencing technique. The internal mechanism of meat performance difference between the two breeds was revealed from the aspect of lnc RNA. At the same time, the selected TCONS_00102859 was tested and analyzed at the cell level. The main results were as follows: 1.The sequencing data of sheep lnc RNA and descriptive analysis were screened. After processing, an average of 2.25 GB data per sample was obtained, Among them, 85 day-old fetuses had the smallest amount of data and the quality of filtered data was reliable. After filtering the genome, the overall comparison rate was 鈮，

本文编号：1695717