【摘要】：豬的脂肪代謝是一個十分復雜的過程,涉及多個組織器官的調(diào)控,其中甲狀腺分泌的甲狀腺激素在脂質(zhì)代謝過程中發(fā)揮著重要的作用。目前對甲狀腺激素調(diào)控的研究主要集中在DNA和mRNA層面,對非編碼RNA介導的基因表達調(diào)控的研究還比較匱乏。長鏈非編碼RNA (long non-coding RNA, lncRNA)和microRNA都是重要的調(diào)控性非編碼RNA。約克夏豬是瘦肉型豬,生長快且脂肪率低,而金華豬是脂肪型豬,生長慢但脂肪率高,它們在脂肪代謝方面有很大的差異。本研究以閹割的120日齡的約克夏豬和金華豬為研究對象,比較它們的血清甲狀腺激素水平,并利用RNA-seq和small RNA-seq技術篩選出了大量在約克夏豬和金華豬甲狀腺組織中差異表達的mRNA、lncRNA和miRNA,并對差異表達的mRNA及差異表達的lncRNA和miRNA的靶基因進行了功能注釋和通路分析,利用熒光定量PCR技術對篩選出的部分差異基因進行了驗證,此外,還將獲得的RNA-seq結果和small RNA-seq結果進行了聯(lián)合分析。主要研究內(nèi)容和結果如下：(1)約克夏豬的體重和甲狀腺重均顯著高于金華豬(P0.05),但兩者比值不變。約克夏豬的血清TT4和TT3水平顯著高于金華豬(P0.05),約克夏豬的血清FT4和FT3水平高于金華豬,但差異不顯著(P0.05)。(2) RNA-seq共獲得約351 M (million)高質(zhì)量的讀長為125 nt的雙末端原始讀段,其中約83%可以比對到豬參考基因組。共鑒定到22,435個已知mRNA在豬甲狀腺組織中表達,其中分別有1,492、2,479個在約克夏豬和金華豬特異性表達。并篩選得到1,018個lncRNA,包括760個lincRNA,140個intronic lncRNA和118個anti-sense lncRNA,此外還篩選到1189個新mRNA。本研究鑒定到的lncRNA與豬已知的蛋白編碼基因相比,lncRNA具有更短的轉錄本和ORF長度、更少的外顯子和更低的序列保守性。差異表達分析共篩選出492個mRNA的表達量在兩個品種間存在顯著差異,其中275個mRNA在約克夏豬甲狀腺中高表達,217個在金華豬甲狀腺中高表達,差異基因被注釋到酪氨酸代謝、鈣離子信號通路、甲狀腺激素合成等生物學通路,主要富集在細胞周期和基于微管的過程。差異表達分析還篩選出48個lncRNA在約克夏豬和金華豬的甲狀腺中差異表達,其中10個經(jīng)cis預測找到13個靶基因。在豬甲狀腺組織中共發(fā)現(xiàn)了12類可變剪切,其中TSS(轉錄起始位點)與TTS(轉錄終止位點)兩種剪切類型所占的比例最大,分別約占38%和30%的百分比。(3) small RNA-seq共獲得約44 M (million)高質(zhì)量的單末端原始讀段,其中約74%可以比對到豬參考基因組。共鑒定到293條已知的miRNA在豬甲狀腺組織中表達,對應266個miRNA的前體,屬于146個miRNA家族,此外還鑒定到60個新miRNA.差異表達分析共篩選出18個miRNA在兩個品種間差異表達,其中12個miRNA在約克夏豬甲狀腺中高表達,6個在金華豬甲狀腺中高表達,生物信息學分析顯示差異miRNA參與甲狀腺激素調(diào)控相關通路,如內(nèi)吞、甲狀腺激素合成、鈣離子信號通路、過氧化物酶體、溶酶體等。(4)熒光定量PCR檢測了18個差異基因在約克夏豬和金華豬甲狀腺組織的表達情況,結果表明與測序結果基本一致,說明本實驗的分析結果基本可以反映豬甲狀腺組織內(nèi)基因的真實表達情況。(5)對差異表達的mRNA、lncRNA和miRNA進行聯(lián)合分析,結果得到的網(wǎng)絡調(diào)控圖中包括237個mRNA、18個miRNA和1個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.
【學位授予單位】：浙江大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：S828

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