线虫中水感受基因筛选及神经通路的初步研究

发布时间：2018-10-10 15:45

【摘要】：水是地球上广泛存在的物质,是生物体重要的组成成分,也是所有生命体赖以生存的重要条件。生物体具备感知内外界环境中水含量及其变化的能力即水感受,感觉信号在神经系统中进行分析与整合,使机体做出相应的生理与行为反应,以适应环境变化,维持生理活动的动态稳定及生存。在生物体对水感受的研究中,秀丽隐杆线虫(Caenorhabditis elegans)可以作为一个理想的模式生物。不论在野生还是人工培养环境中,线虫都生存在含有水膜的介质表面上,线虫需要借助水的张力在介质表面移动,水膜太厚会给线虫运动带来很大的阻碍,而水膜太薄会让线虫失水而亡,所以介质表层的水含量对于线虫至关重要。线虫能够感受多种刺激,具备完善的感觉器官和神经系统,这让线虫在面临水刺激时能够做出相应的反应。然而,目前线虫水感受的分子机制和神经回路还未研究清楚。本研究在我们发展的楔形圆柱体琼脂模型和琼脂糖四相板模型的基础上进行优化,设计了6%-2%琼脂糖二相扩散模型和钠离子均等四相模型,从而排除了钠盐离子及其他水溶性离子的干扰,只引入水含量这一个变量。通过优化模型我们验证了野生型线虫的避水行为,对GPCR G蛋白 DAF-11 cGMP TAX-2/TAX-4信号通路的关键基因进行逐一验证,并证实这一通路作用于带纤毛的感觉神经元中。在找寻参与线虫水感受神经元的过程中,采用空气和水交替刺激模式对感觉神经元进行钙成像。发现在tax-4(ks28)突变型的背景下,用特异性启动子在神经元ASER中回复tax-4基因,能够恢复ASER神经元对水刺激的钙信号,这说明ASER参与水感受。然而daf-11基因并不在ASER中表达,于是对其他在ASER神经元中表达的受体型鸟苷酸环化酶进行基因筛选,综合钙成像结果发现gcy-22基因参与水感受。在gcy-22(tm2364)突变型背景下,用特异性启动子在ASER神经元中回复gcy-22基因能够恢复钙信号,这证明了GCY-22作用于ASER神经元来介导避水行为。本实验对线虫水感受基因及神经通路进行了初步研究,为探索完整的神经回路提供了基础。
[Abstract]:Water is a ubiquitous substance on earth, an important component of organisms, and an important condition for all living things to survive. Organisms have the ability to perceive water content and its changes in the internal and external environment, that is, water perception. Sensory signals are analyzed and integrated in the nervous system in order to make the body make corresponding physiological and behavioral responses to adapt to the changes in the environment. Maintain the dynamic stability and survival of physiological activities. In the study of water sensitivity of organisms, (Caenorhabditis elegans) can be used as an ideal model organism. Both in the wild and in the artificial culture environment, nematodes live on the surface of the medium containing water film, and the nematodes need to move on the medium surface with the help of the tension of water. The thick water film will cause great obstacles to the movement of nematodes. The water film is too thin to kill nematode, so the water content in the media surface is very important for nematodes. Nematodes can sense multiple stimuli, have perfect sensory organs and nervous system, which allows nematodes to respond to water stimuli. However, the molecular mechanisms and neural circuits of nematode water sensitivity have not been studied. Based on the wedge cylinder Agar model and agarose four-phase plate model developed by us, the 6-2% agarose two-phase diffusion model and the sodium ion equalization four-phase model were designed. Therefore, the interference of sodium salt ion and other water-soluble ions is eliminated, and only one variable, water content, is introduced. By optimizing the model, we verified the water repellent behavior of wild nematodes. The key genes of DAF-11 cGMP TAX-2/TAX-4 signaling pathway of GPCR G protein were verified one by one, and it was proved that this pathway acted on the sensory neurons with ciliates. In the process of finding water sensing neurons of nematode, calcium imaging of sensory neurons was performed by alternating air and water stimulation mode. It was found that under the background of tax-4 (ks28) mutation, the recovery of tax-4 gene in neuron ASER by specific promoter could restore the calcium signal of ASER neurons to water stimulation, which indicated that ASER was involved in water perception. However, the daf-11 gene was not expressed in ASER, so the other genes expressed in ASER neurons were screened. The results of calcium imaging showed that gcy-22 gene was involved in water perception. In the context of gcy-22 (tm2364) mutation, restoring gcy-22 gene to ASER neurons with specific promoters can restore calcium signal, which suggests that GCY-22 acts on ASER neurons to mediate water avoidance. In this study, the water sensing genes and neural pathways of nematodes were studied, which provided the basis for exploring the complete neural circuits.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q42

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