本文選題：大黃魚　切入點：轉(zhuǎn)錄組　出處：《華中農(nóng)業(yè)大學》2017年碩士論文

【摘要】：大黃魚是我國特有的經(jīng)濟魚類,位于四大海洋捕撈魚類之首。雖然大黃魚養(yǎng)殖產(chǎn)業(yè)已經(jīng)形成了由人工授精到成魚養(yǎng)殖完整的體系,但對于大黃魚胚胎發(fā)育時期各種基因的表達變換尚不清楚。因此有必要對大黃魚胚胎發(fā)育各個時期轉(zhuǎn)錄組進行研究。本研究采集受精卵受精后0.5h、1h、1.5h、2h、3h、7.5h、14.5h和22h(對應1-cell、2-cell、8-cell、16-cell、256-cell、囊胚期、原腸期、咽囊期)的胚胎,提取總RNA后經(jīng)反轉(zhuǎn)構建cDNA文庫。質(zhì)檢合格后利用HiSeq2000測序平臺進行深度測序。最終獲得53,004,265 reads,其中有70.69%-73.46%比對到基因組上。隨后利用主成分分析(PCA)和層次聚類的方法分析發(fā)現(xiàn)在8個樣本聚為兩類,1-cell期到囊胚期的6個時期聚為一類;原腸期和咽囊期聚為一類,說明胚胎在囊胚期和原腸期之間的基因表達出現(xiàn)了較大的變化。在隨后的對28個組合的樣品進行了差異基因的比對和統(tǒng)計結果與PCA聚類分析結果一致,在囊胚期和咽囊期差異基因最多,我們推測這是由于合子基因的啟動和母源因子的降解造成的。然后使用基因共表達分析(WGCNA)的方法,根據(jù)基因的表達模式將其劃分為13個模塊,并對其進行KEGG富集分析,發(fā)現(xiàn)只有四個模塊出現(xiàn)顯著富集,且其基因呈現(xiàn)特定時期表達的特點。結合已有的胚胎發(fā)育知識,討論了母源因子的調(diào)控、能量的代謝以及器官發(fā)生相關基因的表達特點。通過分析母源基因的表達發(fā)現(xiàn)母源因子參與調(diào)控了多種胚胎發(fā)育事件,包括細胞周期的調(diào)節(jié)、母源基因的降解、合子基因的啟動、胚層的分化、免疫系統(tǒng)的發(fā)育等。在對氨基酸、脂肪酸、三羧酸循環(huán)(TCA)及氧化磷酸化相關基因的分析之后,發(fā)現(xiàn)胚胎在分裂時期消耗能量較少。首先利用了氨基酸及脂肪酸代謝的能量,之后胚胎分化耗能增多三羧酸循環(huán)及氧化磷酸化就被加強了。針對器官發(fā)生相關基因表達分析發(fā)現(xiàn)肌肉組織發(fā)育略晚于眼部發(fā)育,而不同于我們所觀察到的肌節(jié)的產(chǎn)生早于眼點,大黃魚胚胎中肌肉的發(fā)育可能是在26對肌節(jié)形成之后才開始。為了驗證轉(zhuǎn)錄組結果的準確性,以β-actin和GAPDH作為雙內(nèi)參對隨機選取的16個差異基因進行實時熒光定量PCR驗證,檢測結果與轉(zhuǎn)錄組數(shù)據(jù)基本相符。這些結果相對全面地展示了大黃魚從受精卵到咽囊期的早期胚胎發(fā)育過程為日后大黃魚尋找發(fā)育相關基因及繁育工作提供了重要的分子資源。
[Abstract]:Pseudosciaena Crocea is a unique economic fish in China, located in the first of the four major marine fishing fish.Although a complete system from artificial insemination to adult fish culture has been formed in the industry of large yellow croaker, the expression and transformation of various genes in the embryonic development of Pseudosciaena Crocea is still unclear.Therefore, it is necessary to study the transcriptome in all stages of embryonic development of Pseudosciaena Crocea.HiSeq2000 sequencing platform was used to carry out deep sequencing after qualified quality control.Finally, 53004265 RDS was obtained, of which 70.69%-73.46% were compared to the genome.Then, by using the method of principal component analysis (PCA) and hierarchical clustering, it was found that the 8 samples were clustered into one group in the six stages of the two groups from 1-cell stage to blastocyst stage, and that in the euenteric phase and pharyngeal sac stage, they were clustered into one class.The results showed that the gene expression between blastocyst stage and proto-intestinal stage was changed greatly.The comparison and statistical results of the differentially expressed genes in 28 combinations were consistent with the results of PCA cluster analysis. The difference genes in blastocyst and pharyngeal sac were the most.We speculate that this is due to the initiation of zygote genes and the degradation of maternal factors.Then, by using the method of gene coexpression analysis (WGCNA), the gene was divided into 13 modules according to the gene expression pattern, and the KEGG enrichment analysis showed that only four modules showed significant enrichment, and its gene showed the characteristics of expression in a specific period.The regulation of maternal factors, the metabolism of energy and the expression of genes related to organogenesis were discussed.By analyzing the expression of maternal genes, it was found that maternal factors were involved in many embryonic development events, including the regulation of cell cycle, the degradation of maternal genes, the initiation of zygote genes, the differentiation of embryo layer, the development of immune system, and so on.After the analysis of amino acid, fatty acid, TCA and oxidative phosphorylation related genes, it was found that the embryo consumed less energy during the division period.The energy of amino acid and fatty acid metabolism was first utilized, then the energy consumption of embryo differentiation increased the tricarboxylic acid cycle and oxidative phosphorylation.Based on the analysis of organogenesis related gene expression, it was found that the muscle tissue developed a little later than the eye, which was different from the formation of muscle ganglion earlier than the eye spot.Muscle development in large yellow croaker embryos may not begin until 26 pairs of sarcomere are formed.In order to verify the accuracy of transcriptome results, 16 randomly selected differentially selected genes were verified by real-time fluorescence quantitative PCR using 尾 -actin and GAPDH as double internal parameters. The results were basically consistent with the transcriptional data.These results show that the early embryonic development from fertilized eggs to pharyngeal sac provides an important molecular resource for the later search for developmental genes and breeding work of large yellow croaker (Pseudosciaena Crocea).
【學位授予單位】：華中農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S917.4

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