基于驴基因组的可变剪切及驴皮转录组初步研究

发布时间：2018-04-13 18:13

本文选题：驴 + 全基因组　；参考：《山东师范大学》2017年硕士论文

【摘要】：驴(Equus asinus)作为役用家畜,具有抗病力强、体质强健、耐粗饲和适应力强等特点。如今它在交通不便的地区仍是主要的交通工具,其肉、奶、皮均具有较高的营养价值。可变剪切对于真核生物的分化、生长、信号转导等多种生物学现象有着较为重要的意义。当前国内外对于家畜的可变剪切的研究主要集中在毛色、体尺等表型性状和抗病性状及产奶产肉等经济性状方面,但迄今仍未见驴全基因组可变剪切的报道。驴皮作为阿胶的原材料,目前对其研究主要集中在驴皮的蛋白水平和微量元素的鉴定及阿胶的真伪鉴定上,而对其转录组水平的研究比较少。鉴于此,本课题拟在前期驴基因组测序、组装基础上,通过对44个组织的转录组数据分析,对驴进行全基因组范围内的可变剪切的研究;同时深入对比研究了德州驴和新疆驴的驴皮转录组数据,旨在解析驴皮的免疫保护、特殊功效的分子机制。首先是利用Illumina Hiseq 2000测序平台对德州驴和新疆驴两个品种的15个驴皮组织进行了RNA-seq测序。经皮尔森相关性分析后去除2个组织样品,从13个驴皮组织中共鉴定出35429个转录本。前100的高表达基因功能主要富集在维持生命活动的各个通路及抗病免疫相关的代谢通路中。通过cuffcompare评估、蛋白功能预测、与其他数据库比对以及与参考注释文件比较,共获得379个新转录本,其中363个转录本在KEGG中有注释结果,功能富集显示这些新转录本主要集中在蛋白质、免疫抗病相关功能上。鉴定的新转录本可用来补充驴基因组的注释。通过对lncRNA常规筛选及四种蛋白编码能力预测软件的预测,共检测出1148个linc RNAs,同时发现这些linc RNAs的功能主要集中在氨基酸合成代谢相关的通路中。从德州驴和新疆驴两个品种皮中共鉴定出6008个发生可变剪切的转录本,功能主要富集在抗病、代谢相关的通路上。进一步对两个品种对应部位的驴皮组织的转录组数据进行比较分析,发现222个表达差异基因,且这些基因功能多集中在免疫代谢通路中。最后利用同源基因将驴和其他物种进行比较,发现了皮组织中驴特异性表达的基因,其功能富集主要集中在能量代谢、免疫和抗病性上。对驴皮转录组水平的研究将会为驴皮功效、驴地方品种的形成及环境适应性提供分子遗传基础资料。驴全基因组范围内可变剪切的研究。利用astalavista-3.2对RNA测序所得的15种组织44个样品进行可变剪切的鉴定,发现驴全基因组范围内有50.73%的基因发生了可变剪切,且外显子跳跃为主要可变剪切模式,发生可变剪切事件的基因主要参与抗病、免疫及能量代谢相关的通路。对15种组织进行组织间剪切事件的比较分析,发现肉和血液两个组织发生可变剪切事件数目少于其他组织;在44个组织中只有130个基因均发生了AS事件,它们的功能主要集中在维持生命活动的各个通路中;还发现在130个保守剪切基因中42个基因的剪切模式完全一致。比较分析了发生可变剪切的基因和未发生可变剪切的基因结构,发现此两种类型的基因在基因长度、外显子长度和数目、表达量等方面均存在显著差异,表明两种类型的基因在基因结构上存在显著差异。为研究发生可变剪切体基因的组织特异性,对驴、牛、山羊、绵羊及马五个物种通过同源基因进行比较分析,获得了五个物种间各个组织保守的剪切基因及驴特异于其他物种的剪切基因。综上所述,本研究运用高通量测序及生物信息学的方法,首次对驴进行了全基因组范围内的可变剪切图谱鉴定、可变剪切的基因组织特异性分析及不同食草性物种间可变剪切比较分析;同时对德州驴和新疆驴两个品种的驴皮组织从可变剪切、表达量等方面进行了分析,并预测出了新转录本和linc RNAs,丰富了驴的全基因组注释结果。
[Abstract]:The donkey (Equus asinus) as working animals, with strong disease resistance, strong physique, strong adaptability and resistance characteristics of forage. It is now in inaccessible areas is still the main means of transport, the nutritional value of meat, milk, skin have high differentiation., alternative splicing for eukaryotic growth, has the important significance of signal transduction and other biological phenomena. The research on variable shear livestock at home and abroad mainly focus on the hair color, body size and other phenotypic traits and disease resistance traits and milk production of meat and other economic traits, but so far no donkey genome reported. The variable shear donkey skin as a raw material gelatin the authenticity of the identification and identification of gelatin protein levels and trace elements of its current research mainly focus on the donkey skin, but little research on the transcriptional level. In view of this, this article in the early donkey genome sequencing The assembly, on the basis of the transcriptome data of 44 tissue analysis, study alternative splicing in genomic region of donkey donkey skin; transcriptome data and in-depth comparative study of Dezhou and Xinjiang donkey, immune protection aims to reveal the molecular mechanism of donkey skin, special effects. The first is the use of Illumina Hiseq 2000 Dezhou and Xinjiang donkey sequencing platform of two varieties of 15 donkey skin tissue were analyzed by RNA-seq sequencing. The Pearson correlation analysis after removal of 2 tissue samples from 13 donkey skin tissue identified 35429 transcripts. The high expression of gene function is mainly concentrated in the top 100 of each pathway and life sustaining activities immune disease related metabolic pathways. Through cuffcompare evaluation, prediction of protein function, and other database and compared with the reference annotation file, received a total of 379 new transcripts, 363 of them turn This book in the KEGG annotation results show that these new functional enrichment transcripts mainly concentrated in protein, immune related functions. The identification of new transcription annotation can be used to supplement the donkey genome. The lncRNA routine screening and four protein encoding capacity prediction software prediction, detected a total of 1148 Linc RNAs at the same time, found that these Linc RNAs mainly focuses on the synthesis of amino acid metabolism pathway. From Dezhou and Xinjiang donkey skin two varieties identified 6008 transcripts have variable shear function, mainly enriched in disease resistance, metabolic pathway. The transcriptome data of two varieties of the corresponding part Lvpi organization to carry on the comparative analysis, found 222 differentially expressed genes, and these genes are more concentrated in the immune metabolism. The homologous genes and other species than Is found in skin tissue, the expression of donkey specific gene, the functional enrichment focuses on energy metabolism, immunity and disease resistance. The research will be on the donkey skin transcriptome level for skin effect, formation and environmental adaptability of donkey breeds to provide molecular genetic basis. The research of donkey genome wide variable shear. The identification of 44 samples of 15 kinds of tissues by astalavista-3.2 RNA sequencing of the variable shear, found a variable shear donkey genome-wide 50.73% gene and exon skipping is the main variable shear mode, variable shear events occur mainly involved in disease resistance gene, immune and metabolic pathway related. Comparative analysis of 15 kinds of shear event organization organization, found that the flesh and blood of two groups of variable shear events number less than other organizations; only 1 in 44 tissues 30 genes have undergone AS events, each path of their functions mainly focus on life sustaining activities; also found in shear mode 130 conserved gene shear 42 genes are identical. A comparative analysis of the occurrence of alternative splicing genes and no variable shear gene structure, found that the two types of the genes in the gene exon length, length and number, expression etc. there were significant differences, indicating two types of genes have significant differences in gene structure. For tissue specificity, of variable shear body gene on donkeys, cattle, goats, sheep and horse five species by homologous gene comparative analysis of shear gene obtained five species each organization conservative shear gene and other species specific to the donkey. In summary, this study used high-throughput sequencing and bioinformatics method, for the first time The donkey was spliced identification of genome wide comparative analysis, analysis of species specific gene organization and different herbivorous variable shear between the variable shear; while the Dezhou donkey and Xinjiang donkey two varieties of donkey skin tissue from the variable shear, expression and other aspects of the analysis, and to predict new RNAs and Linc transcripts, enrich the whole genome annotation results of a donkey.

【学位授予单位】：山东师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S822

【参考文献】