温度驯化对斑马鱼发育影响的表观遗传学研究

发布时间：2018-05-11 02:25

本文选题：温度驯化 + 斑马鱼　；参考：《上海海洋大学》2017年博士论文

【摘要】：环境温度对生物的影响贯穿其整个生活史,包括生长、发育、繁殖、运动等多个重要的生物学过程。有气象预测显示由于全球变暖引起的极端高温和由于北极冰盖融化引起的寒潮将会频繁侵袭我国。鱼类生活在水环境中,水温的变化对鱼类的行为和生理生化活动影响巨大。极端温度会使水产养殖业遭受重大的损失,使养殖鱼类品质下降或导致大面积死亡。因此,研究温度对鱼类的影响就显得至关重要。斑马鱼是一种重要的水生生物模型,具有拼接完整的基因组和可信度较高的基因注释结果,可以满足转录组、甲基化组等高通量测序数据分析的需要。近年来的研究结果对斑马鱼在低温下的调控网络有比较透彻的概述,同时发现了斑马鱼与罗非鱼耐寒能力差异的分子机制。为我们选择以斑马鱼作为研究对象奠定了坚实的基础。近年来多项研究表明,生物体在弱于极端温度的条件下,将会产生温度驯化效应,使其耐受极端温度的能力上升。另外有一些研究表明环境因素对生物体产生的影响可以持续很长的时间,甚至会影响其后代的生存能力。然而这些研究工作主要集中在植物领域,对动物尤其是鱼类的研究还比较少。因此,研究模式鱼类——斑马鱼对温度驯化的响应机制就显得至关重要。为了从多角度探究斑马鱼对温度驯化的响应机制,我们分别设计了三个实验进行研究。首先,将斑马鱼ZF4细胞(正常培养温度28°C)进行短期低温驯化(18°C/5d)和长期低温驯化(18°C/30d),探究在没有神经调节和激素调节的情况下,鱼类细胞本身是否能够获得温度驯化效应。实验发现,将常温(28°C)、短期驯化(18°C/5d)、长期驯化(18°C/30d)的ZF4细胞放入细胞致死低温(10°C/3d)后,通过细胞存活率检测和alamar blue还原性检测实验发现,不管短期驯化和长期驯化的ZF4细胞的耐低温能力都增强,说明其都获得了温度驯化效应。利用MeDIP-seq的方法研究常温(28°C)、短期驯化(18°C/5d)、长期驯化(18°C/30d)的ZF4细胞的甲基化的状态。研究发现寒冷压力可以改变ZF4细胞DNA甲基化模式,短期低温驯化后DNA甲基化的水平增加,而在长期低温驯化后DNA甲基化的水平有所恢复,且稍稍低于对照组。通过分析找到了1024个低温驯化后启动子区域发生明显改变的基因,并对这些基因做了GO富集和KEGG富集分析,结果显示短期低温驯化后参与叶酸合成通路的相关基因的启动子区域发生了显著的欠甲基化,由于叶酸可以作为5mC的甲基供体,因此推测叶酸合成通路的激活可能与短期驯化后整体甲基化水平升高有关。利用MeDIP-qPCR和BSP验证生物信息分析得到的差异甲基化区域(DMRs),结果显示MeDIP-qPCR和BSP的实验结果与MeDIP-seq的测序结果基本相符。结果显示,在没有神经调节和激素调节的条件下,斑马鱼细胞本身就可以对温度驯化产生反应。其次,为了研究温度驯化是否能够对斑马鱼产生持续的影响,并进一步探究产生持续性影响的分子机制。我们将斑马鱼受精卵分别放入不同胚胎发育温度(22°C,27°C,32°C)中发育出膜,简称TE22、TE27、TE32,然后于常温(27°C)下饲养8个月,以BS-seq的方法检测各组斑马鱼成鱼肌肉中DNA甲基化水平。通过分析gene、peseudogene、lincRNA、CGI、miRNA、rRNA及其上下游2Kb区域的甲基化状态发现,在gene body区域可以观察到TE32的甲基化水平稍微高于TE22和TE27,而在上游和下游2K的区域,TE32的甲基化水平稍微低于TE22和TE27,在CpG岛区域,TE27的甲基化水平略高于TE22,而TE32的甲基化水平最低。在miRNA和rRNA区域并没有看到甲基化水平明显降低的区域,但是其本身的甲基化水平变化比较剧烈。methylKit分析发现,TE22和TE27、TE32和TE27之间存在大量DMCs位点,并且差异甲基化位点在每个染色体上比例大致相同。DMRs分析发现TE22 vs TE27之间的差异甲基化区域(DMRs)为1915个,其中超甲基化区域为990个,欠甲基化区域为925个。TE32 vs TE27之间的差异甲基化区域(DMRs)为2646个,其中超甲基化区域为1506个,欠甲基化区域为1140个。结果显示,斑马鱼不同胚胎发育温度(22°C,27°C,32°C)可以导致斑马鱼成鱼的肌肉中特定区域的甲基化水平发生变化,证明温度驯化可以对斑马鱼肌肉的DNA甲基化状态产生持续性的影响。最后,为研究斑马鱼中低温驯化效应是否能够遗传,将在常温(27°C)下饲养6个月的32对斑马鱼分成两组,每组16对,一组继续在常温(27°C±0.5°C)下饲养,编号TA27,另一组放置于低温(22°C±0.5°C)下饲养,编号TA22,驯化时长为4个月。分别配对TA27和TA22两组斑马鱼,在产卵后的30min内收集受精卵,放入培养皿中,将TA27组和TA22组的受精卵分为两份,分别放置于27°C与20°C,统计四个培养皿中受精卵的出膜率,评估斑马鱼受精卵的低温耐受能力。分别取TA27、TA22斑马鱼卵巢和在低温20°C处理0h、2h、4h、6h、8h的受精卵,进行转录组测序,进而进行差异表达基因、GO和KEGG分析,并对表观酶类的表达水平进行分析。结果发现经过22°C驯化的斑马鱼的后代在致死温度下(20°C)可以正常发育,并顺利完成卵裂期,进入下一发育时期。而未驯化的斑马鱼在低温下,会导致卵裂异常的表型,例如卵裂时细胞间隙过大、出现两个分裂球,更有甚者会出现卵破碎的现象。将非驯化组斑马鱼所产的卵置于常温下发育一定时间,再放置于低温下,可以发现在常温发育3h以内的受精卵不能完成胚胎发育,而在常温发育3.5h以后的受精卵则可以顺利完成胚胎发育,进而出膜。这证明胚胎发育的前三个小时是易受到温度影响的敏感期,而对亲代的低温驯化可以使其所产的受精卵度过这段时期,正常完成卵裂。GO富集分析显示大量与信号通路激活、细胞间黏连的相关的生物学过程被激活。涉及大量与离子相关的生物学过程,例如离子通道活性、钙离子结合、钙离子通道复合体。涉及较多信号传导相关的生物学过程,例如G蛋白偶联受体活性、G蛋白受体信号通路、细胞间的信号传导、信号传导活性、Rho蛋白的信号传导。涉及细胞移动和固着等过程,例如细胞固着、细胞移动、细胞生长等过程。与生长繁殖有关的过程,例如,成纤维细胞生长因子受体信号通路、细胞因子受体结合、干细胞分化、发育的负调节作用、胚胎器官发育等过程。KEGG富集分析发现钙离子信号通路富集程度较高。GPCR信号通路可以调控Ca~(2+)信号通路的激活。由此可以进一步推测GPCR信号通路首先感知温度降低,然后通过IP3影响Ca~(2+)信号通路,使Ca~(2+)信号通路激活。因此,斑马鱼低温驯化下产生的耐寒能力可能就是通过激活Ca~(2+)信号通路而产生的。通过对多种组蛋白修饰相关酶类的表达量的统计分析,发现多种赖氨酸的甲基化和去甲基化酶类表达量发生了显著的变化,证明还有其他表观标记参与了低温驯化的过程。结果显示,温度驯化效应可以遗传给下一代,推测这个现象与GPCR调控下的Ca~(2+)通路有关。
[Abstract]:The impact of environmental temperature on life throughout its whole life history, including growth, development, reproduction, and sports, and other important biological processes. Weather forecasts show that the extreme heat caused by global warming and the cold wave caused by the melting of the Arctic ice cover will frequently attack China. The behavior and physiological and biochemical activities of the class are greatly affected. Extreme temperatures can cause the aquaculture industry to suffer major losses and cause the quality of fish to decline or cause large areas of death. Therefore, it is essential to study the effects of temperature on fish. Zebrafish is an important aquatic biological model, with a complete genome and credible splice. The results of high degree gene annotation can meet the needs of high flux sequencing data analysis, such as the transcriptional group and the methylation group. The recent research results have a thorough overview of the regulation network of zebrafish at low temperature. At the same time, we found the molecular mechanism of the difference in the cold tolerance of zebrafish and tilapia. Studies have laid a solid foundation. In recent years, a number of studies have shown that living organisms will produce temperature taming effects under extreme temperatures and increase their ability to tolerate extreme temperatures. In addition, some studies have shown that the effects of environmental factors on organisms can continue for a long time, and may even affect their offspring. However, these studies are mainly concentrated in the plant field, and there are few studies on animals, especially fish. Therefore, it is very important to study the response mechanism of model fish, zebrafish, to the temperature domestication. In order to explore the response mechanism of zebrafish to temperature domestication from multiple angles, we have designed three facts respectively. First, a short term low temperature acclimatization (18 degree C/5d) and long term low temperature domestication (18 degree C/30d) of zebrafish ZF4 cells (normal culture temperature 28 C) were carried out to investigate whether the fish cells could obtain temperature domestication in the absence of nerve regulation and hormone regulation. After a long tamed (18 C/30d) ZF4 cell was put into a cell lethal low temperature (10 degree C/3d), the cell survival rate and the Alamar Blue reducibility test showed that the low temperature tolerance of the ZF4 cells, regardless of the short-term domestication and the long-term domestication, showed that the temperature acclimation effect was obtained. The normal temperature (28 degree C) was studied by the MeDIP-seq method. The methylation status of ZF4 cells in short-term domestication (18 C/5d) and long-term acclimatization (18 C/30d) showed that cold pressure could change the DNA methylation pattern of ZF4 cells, and the level of DNA methylation increased after short term low temperature domestication, and the level of DNA methylation after long term low temperature domestication was recovered, and slightly lower than that of the control group. 1024 genes which have obviously changed in the promoter region after 1024 low temperature acclimation have been enriched and analyzed by KEGG enrichment. The results showed that the promoter region of the related genes involved in the folic acid synthesis pathway was significantly under methylation after the short term low temperature domestication, as folic acid could be used as the methyl donor of 5mC, thus speculating on the leaves Activation of acid synthesis pathway may be associated with elevated level of overall methylation after short-term domestication. Using MeDIP-qPCR and BSP to verify the differential methylation area (DMRs) obtained by bioinformatics analysis, the results show that the experimental results of MeDIP-qPCR and BSP are basically consistent with the sequencing results of MeDIP-seq. The results show that there is no neuromodulation and hormone regulation. Under the conditions, zebrafish cells themselves can react to temperature domestication. Secondly, in order to study whether temperature acclimation can have a sustained effect on zebrafish and further explore the molecular mechanism that produces persistent effects, we develop zebrafish fertilized eggs in different embryonic development temperatures (22 C, 27 C, 32 C). Membrane, called TE22, TE27, TE32, and then fed at normal temperature (27 degree C) for 8 months. The methylation level of DNA in the muscle of zebrafish fish was detected by BS-seq method. The methylation status of gene, peseudogene, lincRNA, CGI, miRNA, rRNA and its upper and lower regions could be observed. Higher than TE22 and TE27, and in the upstream and downstream 2K regions, the methylation level of TE32 is slightly lower than TE22 and TE27. In CpG island region, the methylation level of TE27 is slightly higher than TE22 and TE32 methylation level is the lowest. In miRNA and rRNA regions, there is no visible region of methylation level, but its own methylation levels are compared. Severe.MethylKit analysis showed that there were a large number of DMCs sites between TE22 and TE27, TE32 and TE27, and the difference methylation sites on each chromosome were roughly the same as.DMRs analysis found that the difference methylation region between TE22 vs TE27 (DMRs) was 1915, of which the hypermethylation region was 990, and the under methylation region was 925.TE32. The difference methylation area (DMRs) was 2646, in which the hypermethylation area was 1506 and the methylation area was 1140. The results showed that the different embryonic development temperature (22 C, 27 degree C, 32 C) of zebrafish could lead to the change of the methylation level in the specific region of the muscle of zebrafish. It was proved that the taming of the zebrafish could be used to the zebrafish muscle. The DNA methylation status of meat has a continuous effect. Finally, in order to study whether the effects of low temperature acclimatization in zebrafish can be inherited, 32 pairs of zebrafish for 6 months at normal temperature (27 C) are divided into two groups, each group is 16 pairs. One group continues to be kept under the normal temperature (27 C + 0.5 degrees C) and TA27, and the other is kept under low temperature (22 [C + 0.5 [C]). The number of TA22 and taming for 4 months. Two zebrafish groups of TA27 and TA22 were paired respectively. The fertilized eggs were collected in the 30min after spawning. The fertilized eggs of the TA27 and TA22 groups were divided into two portions, respectively, and placed in 27 C and 20 degree C, respectively, to calculate the film rate of the fertilized eggs in four Petri dishes, and to evaluate the low temperature tolerance of the zebrafish fertilized eggs. The ovaries of TA27, TA22 zebrafish and the fertilized eggs of 0h, 2h, 4h, 6h and 8h were treated at 20 degrees C at low temperature, and the transcriptional group was sequenced, then the differentially expressed genes, GO and KEGG analysis were carried out, and the expression levels of the epigenetic enzymes were analyzed. The results showed that the offspring of zebra fish after 22 degree C domestication could develop normally at the lethal temperature (20 degrees C). The undomesticated zebrafish can cause abnormal cleavage at low temperature, such as the oversize of the cleavage, the emergence of two cleavage balls, and the breakage of the eggs. The eggs produced by the non domesticated zebra fish are placed at normal temperature for a certain time and then placed under low temperature. It is found that the fertilized eggs within the normal temperature of 3H can not complete the embryo development, while the fertilized eggs after the normal temperature of 3.5H can successfully complete the embryo development and then produce the membrane. This proves that the first three hours of the embryo development are susceptible to the temperature influence, and the relative low temperature domestication can pass the fertilized eggs of the embryos. During this period, the analysis of normal complete cleavage.GO enrichment analysis showed that a large number of biological processes associated with signaling pathway activation and intercellular adhesion were activated. Many biological processes related to ion related, such as ion channel activity, calcium ion binding, calcium ion channel complex, involving many signal transduction related biological processes, such as G protein coupling receptor activity, signal transduction of G protein receptor, signal transduction between cells, signal conduction of signal, signal transduction of Rho protein, processes involving cell migration and fixation, such as cell fixation, cell movement, cell growth, and other processes related to growth and reproduction, such as fibroblast growth factor receptor signaling pathway, Cell factor receptor binding, stem cell differentiation, negative regulation of development, and embryo organ development,.KEGG enrichment analysis found that high concentration of calcium signaling pathway.GPCR signaling pathway can regulate the activation of Ca~ (2+) signaling pathway. Thus, it is further conjectured that the GPCR signaling pathway first decreases the perception temperature and then affects the IP3 pathway. The Ca~ (2+) signaling pathway activates the Ca~ (2+) signaling pathway. Therefore, the cold tolerance produced by zebrafish may be produced by activating the Ca~ (2+) signal pathway. By statistical analysis of the expression of a variety of histone modified enzymes, the methylation of several lysine and the expression of demethylation enzymes have been found. Significant changes have shown that there are other apparent markers involved in the process of low temperature acclimation. The results show that the temperature acclimation effect can be inherited to the next generation, which is related to the Ca~ (2+) pathway under the GPCR regulation.

【学位授予单位】：上海海洋大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：Q953

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