约克夏猪和金华猪甲状腺组织mRNA、lncRNA和miRNA测序及功能分析

发布时间：2019-07-02 14:20

【摘要】：猪的脂肪代谢是一个十分复杂的过程,涉及多个组织器官的调控,其中甲状腺分泌的甲状腺激素在脂质代谢过程中发挥着重要的作用。目前对甲状腺激素调控的研究主要集中在DNA和mRNA层面,对非编码RNA介导的基因表达调控的研究还比较匮乏。长链非编码RNA (long non-coding RNA, lncRNA)和microRNA都是重要的调控性非编码RNA。约克夏猪是瘦肉型猪,生长快且脂肪率低,而金华猪是脂肪型猪,生长慢但脂肪率高,它们在脂肪代谢方面有很大的差异。本研究以阉割的120日龄的约克夏猪和金华猪为研究对象,比较它们的血清甲状腺激素水平,并利用RNA-seq和small RNA-seq技术筛选出了大量在约克夏猪和金华猪甲状腺组织中差异表达的mRNA、lncRNA和miRNA,并对差异表达的mRNA及差异表达的lncRNA和miRNA的靶基因进行了功能注释和通路分析,利用荧光定量PCR技术对筛选出的部分差异基因进行了验证,此外,还将获得的RNA-seq结果和small RNA-seq结果进行了联合分析。主要研究内容和结果如下：(1)约克夏猪的体重和甲状腺重均显著高于金华猪(P0.05),但两者比值不变。约克夏猪的血清TT4和TT3水平显著高于金华猪(P0.05),约克夏猪的血清FT4和FT3水平高于金华猪,但差异不显著(P0.05)。(2) RNA-seq共获得约351 M (million)高质量的读长为125 nt的双末端原始读段,其中约83%可以比对到猪参考基因组。共鉴定到22,435个已知mRNA在猪甲状腺组织中表达,其中分别有1,492、2,479个在约克夏猪和金华猪特异性表达。并筛选得到1,018个lncRNA,包括760个lincRNA,140个intronic lncRNA和118个anti-sense lncRNA,此外还筛选到1189个新mRNA。本研究鉴定到的lncRNA与猪已知的蛋白编码基因相比,lncRNA具有更短的转录本和ORF长度、更少的外显子和更低的序列保守性。差异表达分析共筛选出492个mRNA的表达量在两个品种间存在显著差异,其中275个mRNA在约克夏猪甲状腺中高表达,217个在金华猪甲状腺中高表达,差异基因被注释到酪氨酸代谢、钙离子信号通路、甲状腺激素合成等生物学通路,主要富集在细胞周期和基于微管的过程。差异表达分析还筛选出48个lncRNA在约克夏猪和金华猪的甲状腺中差异表达,其中10个经cis预测找到13个靶基因。在猪甲状腺组织中共发现了12类可变剪切,其中TSS(转录起始位点)与TTS(转录终止位点)两种剪切类型所占的比例最大,分别约占38%和30%的百分比。(3) small RNA-seq共获得约44 M (million)高质量的单末端原始读段,其中约74%可以比对到猪参考基因组。共鉴定到293条已知的miRNA在猪甲状腺组织中表达,对应266个miRNA的前体,属于146个miRNA家族,此外还鉴定到60个新miRNA.差异表达分析共筛选出18个miRNA在两个品种间差异表达,其中12个miRNA在约克夏猪甲状腺中高表达,6个在金华猪甲状腺中高表达,生物信息学分析显示差异miRNA参与甲状腺激素调控相关通路,如内吞、甲状腺激素合成、钙离子信号通路、过氧化物酶体、溶酶体等。(4)荧光定量PCR检测了18个差异基因在约克夏猪和金华猪甲状腺组织的表达情况,结果表明与测序结果基本一致,说明本实验的分析结果基本可以反映猪甲状腺组织内基因的真实表达情况。(5)对差异表达的mRNA、lncRNA和miRNA进行联合分析,结果得到的网络调控图中包括237个mRNA、18个miRNA和1个lncRNA。
[Abstract]:The fat metabolism of the pig is a very complex process involving the regulation of multiple tissue organs, in which the thyroid hormone secreted by the thyroid plays an important role in the process of lipid metabolism. At present, the research on the regulation of thyroid hormone is mainly focused on the level of DNA and mRNA, and the research on the regulation of non-coding-RNA-mediated gene expression is still scarce. Long-chain non-coding RNA (lncRNA) and microRNA are important regulatory non-coding RNAs. Yorkshire is a lean-type pig with a rapid growth and low fat rate, while the Jinhua pig is a fat-type pig with slow growth but high fat rate, which has a great difference in fat metabolism. In this study, about 120-day-old Yorkshire and Jinhua pigs were studied and their serum thyroid hormone levels were compared, and the mRNA, lncRNA and miRNA in the thyroid tissues of Yorkshire and Jinhua pigs were selected by RNA-seq and small RNA-seq techniques. In addition, the results of the RNA-seq and the results of the small RNA-seq were also analyzed. The main contents and results were as follows: (1) The weight of the Yorkshire pig and the weight of the thyroid were significantly higher than that of the Jinhua pig (P 0.05), but the ratio of the two was the same. The serum TT4 and TT3 levels of Yorkshire pigs were significantly higher than that of Jinhua pigs (P0.05). The levels of serum FT4 and FT3 in Yorkshire pigs were higher than that of Jinhua pigs, but the difference was not significant (P0.05). (2) RNA-seq has a total of about 351 M (milion) of a high-quality, double-terminal, original reading segment of 125 nt, of which about 83% can be compared to the pig reference genome. A total of 22,435 known mRNAs were expressed in porcine thyroid tissue, of which 1,492, 2,479 were specifically expressed in Yorkshire and Jinhua pigs, respectively. A total of 1,018 lncRNA was screened, including 760 lincRNA,140 ttronic lncRNA and 118 anti-sense lncRNA, and 1189 new mRNAs were also screened. The lncRNA identified in this study has a shorter transcript and ORF length, fewer exons and lower sequence conservative compared to the known protein-encoding genes of pigs. The differential expression analysis showed that the expression of 492 mRNA was significantly different among the two varieties, of which 275 mRNA was highly expressed in the thyroid of Yorkshire,217 in the thyroid of Jinhua pig, and the difference gene was noted to the tyrosine metabolism and calcium ion signal pathway. The biological pathways, such as the synthesis of thyroid hormone, are mainly enriched in the cell cycle and the microtubule-based process. The differential expression analysis also screened out the differential expression of 48 lncRNA in the thyroid of Yorkshire and Jinhua pigs,10 of which were predicted to find 13 target genes. A total of 12 variable shear were found in the porcine thyroid tissue, where the ratio of the TSS (transcription initiation site) to the TTS (transcription termination site) was the largest, with a percentage of about 38% and 30%, respectively. (3) The small RNA-seq is a total of about 44 M (million) high-quality, single-terminal, original reading, of which about 74% can be compared to the pig reference genome. A total of 293 known miRNAs were identified in porcine thyroid tissue, corresponding to a precursor of 266 miRNAs, belonging to 146 miRNA families, in addition to the identification of 60 new miRNAs. The differential expression analysis showed that 18 miRNAs were differentially expressed in two varieties, of which 12 miRNAs were highly expressed in the thyroid of Yorkshire,6 of them were highly expressed in the thyroid of Jinhua pig, and the bioinformatics analysis showed that the differentially expressed miRNAs were involved in the regulation of thyroid hormone regulation, such as endocytosis, Thyroid hormone synthesis, calcium ion signal pathway, peroxidase, lysosome, etc. (4) The expression of 18 differential genes in the thyroid tissues of Yorkshire and Jinhua pigs was detected by the fluorescence quantitative PCR, and the results showed that the results of the analysis were basically consistent with the results of the sequencing. (5) The mRNA, ncRNA and miRNA expression of the differentially expressed mRNA, ncRNA and miRNA were analyzed, and 237 mRNAs,18 miRNAs and 1 lncRNA were included in the resulting network control chart.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S828

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