转录组测序揭示乌骨绵羊乌质性状的功能基因及其TagSNP

The Lanping black-boned sheep (Ovis aries), which were found by our team, are identified as the first mammal with black traits by the Ministry of Agriculture in China. This proposal is based on the interesting results that the melanoblast of black-boned sheep is migrated abnormally and developed as the melanocytes which are distributed into periosteum, liver, spleen and kidney. The melanocyte can secrete more melanin for presenting black traits of black-boned sheep compared with normal sheep (Ovis aries) under the same condition. In the present study, the Lanping normal and Lanping black-boned sheep will be assigned to the control and experimental groups, respectively. The transcriptome sequencing (RNA-Seq) technology will be used high throughput to measure the differentially expressed genes, novel genes, coding SNP (cSNP) and potential splicing junctions of periosteum, liver, spleen and kidney tissues between Lanping normal and black-boned sheep at the stages of after birth and two years old. Thus, the gene network of black traits from black-boned sheep will be constructed. The key genes will be validated by the Northern hybridization and mRNA real-time PCR analysis. The full length of two important differentially expressed genes and other two genes with important cSNP will be identified, cloned and sequenced. The TagSNP will be confirmed after identifying the SNP and haplotype from the each 100 samples within two breeds. Moreover, the predominant SNP which cause the black traits will be checked and confirmed by the Sequenom MassARRAY genotype technology for analyzing the large samples from the random population of black-boned sheep and normal sheep even F2 segregated population from Lanping black-boned sheep x Suffolk. The SNPs from the coding and regulatory regions are predicted their function by using bioinformatics, reporter gene and gel shift technologies. Finally, we will retrieve and identify the major gene or molecular marker for the black traits from black-boned sheep. These results conducted from this study will provide the primary foundation for the development and utilization of black-boned sheep in future.

兰坪乌骨绵羊是我们发现并被农业部认定为第一个具有乌质性状的家畜遗传资源。本项目在乌骨绵羊成黑色素细胞于体内异向迁徙发育为黑色素细胞后反常分布于骨膜、肝脏、脾脏和肾脏并分泌黑色素形成乌质性状的研究基础上，以兰坪普通绵羊为对照，采用转录组测序（RNA-Seq）检测两品种1月龄和2岁时上述4种特定组织差异表达基因、新基因、编码SNP(cSNP)和可变剪切，构建乌质性状的基因调控网络；对关键基因进行Northern杂交和荧光定量验证；克隆2个关键差异表达基因和2个重要cSNP所在基因全序列；在两品种各100余个个体中初筛SNP，对SNP进行单倍型分析，锁定标签SNP（TagSNP）；利用MassARRAY进行大样本TagSNP检测，确立优势SNP；分析编码区和调控区SNP功能，通过报告基因、凝胶阻滞验证位点功能；从而获得乌骨绵羊乌质性状的主效基因或分子标记。研究结果为乌骨绵羊的开发和利用奠定基础。

采集了443只兰坪乌骨绵羊、298只兰坪普通绵羊和160只会泽普通绵羊的血液，发现兰坪乌骨绵羊有显著高的血浆比色、酪氨酸酶活力、碱溶性黑色素、脱黑色素含量及真黑色素/总黑色素，确认乌质性状是由酪氨酸酶催化合成了显著多的真黑色素并沉淀在不同组织器官造成的。.屠宰了2岁的兰坪乌骨绵羊和兰坪本地普通绵羊各6只，采用转录组测序技术对肝脏mRNA进行了高通量测序和全面比较，普通绵羊总得到了324,332,090条序列（reads），共32.76G的数据；乌骨绵羊得到了447,878,861条reads，共45.22G的数据；共找到表达差异基因共有为115个，其中上调基因为85个，下调基因为30个；基因本体（GO）功能富集分析共发现通路共42个，其中31个被归纳为“生物过程”（Biological process）、5个被归纳为“细胞组分”（Cellular component）、6个被归纳入“分子功能”（Molecular function）；KEGG注释相关通路349个，其中在三羧酸循环、黑色素合成、黑色素瘤合成、WNT信号通路、细胞外基质受体相互作用、P13k-akt信号通路、黏着斑途径、Toll样受体信号通路、细胞素受体互作途径中分别注释到22、29、24、61、37、27、16、23和82个基因；在兰坪乌骨绵羊和普通绵羊之间找到4个mRNA表达量有差异显著的基因，它们分别是TNFRSF6B、CRTAC1、TNFRSF12A和SPP1；实时荧光定量PCR验证也有差异显著，从而推断这4个基因与乌骨绵羊高黑色素含量有关。.首次克隆了绵羊和乌骨绵羊RAB21基因7个完整外显子及其部分侧翼区，在编码区发现了3个多态位点，分别为A90G、A207T和T237C。均为同义突变。检测了各100只羊3个位点的多态性，对3种绵羊类群进行了基因频率和基因型的X2检验，评价了RAB21基因多态性与绵羊乌质性状和毛色之间的关系，关联分析表明该基因多态性与可能对乌质性状有影响。.共培养了研究生3人，发表了1篇SCI论文和3篇中文文章，项目主持人获得云南省中青年学术和技术带头人荣誉称号。.研究成果促进了兰坪县及其毗邻的剑川县、香格里拉乌骨绵羊养殖和销售，从2014年到现在，至少由本人给当地农户带来1000万元的销售收入。在CCTV播放乌骨绵羊专题片5次，起到了非常震撼的宣传效果。