大黄鱼（Larimichthys crocea）胚胎发育转录组分析

发布时间：2018-04-08 18:08

本文选题：大黄鱼　切入点：转录组　出处：《华中农业大学》2017年硕士论文

【摘要】：大黄鱼是我国特有的经济鱼类,位于四大海洋捕捞鱼类之首。虽然大黄鱼养殖产业已经形成了由人工授精到成鱼养殖完整的体系,但对于大黄鱼胚胎发育时期各种基因的表达变换尚不清楚。因此有必要对大黄鱼胚胎发育各个时期转录组进行研究。本研究采集受精卵受精后0.5h、1h、1.5h、2h、3h、7.5h、14.5h和22h(对应1-cell、2-cell、8-cell、16-cell、256-cell、囊胚期、原肠期、咽囊期)的胚胎,提取总RNA后经反转构建cDNA文库。质检合格后利用HiSeq2000测序平台进行深度测序。最终获得53,004,265 reads,其中有70.69%-73.46%比对到基因组上。随后利用主成分分析(PCA)和层次聚类的方法分析发现在8个样本聚为两类,1-cell期到囊胚期的6个时期聚为一类;原肠期和咽囊期聚为一类,说明胚胎在囊胚期和原肠期之间的基因表达出现了较大的变化。在随后的对28个组合的样品进行了差异基因的比对和统计结果与PCA聚类分析结果一致,在囊胚期和咽囊期差异基因最多,我们推测这是由于合子基因的启动和母源因子的降解造成的。然后使用基因共表达分析(WGCNA)的方法,根据基因的表达模式将其划分为13个模块,并对其进行KEGG富集分析,发现只有四个模块出现显著富集,且其基因呈现特定时期表达的特点。结合已有的胚胎发育知识,讨论了母源因子的调控、能量的代谢以及器官发生相关基因的表达特点。通过分析母源基因的表达发现母源因子参与调控了多种胚胎发育事件,包括细胞周期的调节、母源基因的降解、合子基因的启动、胚层的分化、免疫系统的发育等。在对氨基酸、脂肪酸、三羧酸循环(TCA)及氧化磷酸化相关基因的分析之后,发现胚胎在分裂时期消耗能量较少。首先利用了氨基酸及脂肪酸代谢的能量,之后胚胎分化耗能增多三羧酸循环及氧化磷酸化就被加强了。针对器官发生相关基因表达分析发现肌肉组织发育略晚于眼部发育,而不同于我们所观察到的肌节的产生早于眼点,大黄鱼胚胎中肌肉的发育可能是在26对肌节形成之后才开始。为了验证转录组结果的准确性,以β-actin和GAPDH作为双内参对随机选取的16个差异基因进行实时荧光定量PCR验证,检测结果与转录组数据基本相符。这些结果相对全面地展示了大黄鱼从受精卵到咽囊期的早期胚胎发育过程为日后大黄鱼寻找发育相关基因及繁育工作提供了重要的分子资源。
[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).
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S917.4

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