Functional analyses of vertebrate tissue-globins in zebrafish

血红蛋白是生物科学中研究得最好的蛋白质之一，但仍能带来惊喜。我们的研究导致在颌类脊椎动物中发现了六种新的珠蛋白，其中大多数功能仍然不明确。本研究拟研究细胞珠蛋白（Cygb1和Cygb2）、神经珠蛋白（Ngb）和珠蛋白X （GbX）在斑马鱼斑马鱼体内的功能。我们将采用CRISPR/Cas9方法产生珠蛋白基因的敲除。该方法已在实验室中通过产生Cygb1敲除斑马鱼系建立。这些珠蛋白敲除将用于检测发育缺陷、氧气消耗、缺氧耐受性和反应的变化、对活性氧（ROS）和一氧化氮的保护以及对膜脂的保护。此外，比较RNA-Seq方法（基因敲除与野生型）将有助于确定与珠蛋白相关的相应代谢途径。根据我们的假设，Ngb可能参与O2供应或ROS分解，而GbX可能保护细胞膜。在这个应用程序中，我们将特别关注Cygb1和Cygb2。与其他脊椎动物不同，硬骨鱼拥有两种不同的Cygb基因。我们假设它们在基因复制后通过亚功能化而分化。这些重复的基因可能在胶原合成的氧气供应、对活性氧的保护或一氧化氮代谢中发挥作用。这些结果有望揭示Ngb、Cygb1、Cygb2和GbX在斑马鱼中的生理作用，并有助于更好地了解这些珠蛋白在脊椎动物中的功能多样性。

(Hemo-)globins are among the best-investigated proteins in biological sciences but still offer surprises. Our studies led to the discovery of six novel globins in gnathostome (jawed) vertebrates, mostly with still poorly defined functions. Here we propose to study the functions of cytoglobin (Cygb1 and Cygb2), neuroglobin (Ngb), and globin X (GbX) in the zebrafish Danio rerio. We will employ the CRISPR/Cas9 method to generate knockouts of the globin genes. The method has been established in the lab by generating a Cygb1 knockout zebrafish line. The globin knockouts will be tested for developmental defects, changes in O2 consumption, hypoxia tolerance and response, protection from reactive oxygen species (ROS) and nitric oxide, and protection of membrane lipids. Further, the comparative RNA-Seq approach (knockout vs. wildtype) will help to identify the corresponding metabolic pathways in which the globins are involved. According to our hypothesis, Ngb may be involved in O2 supply or ROS decomposition, while GbX possibly protects the cell membrane. In this application, we will specifically focus on Cygb1 and Cygb2. In contrast to other vertebrates, teleosts harbor two distinct Cygb genes. We hypothesize that they diverged by subfunctionalization after gene duplication. The duplicated genes may play a role in the supply of O2 for collagen synthesis, protection from ROS, or in the NO metabolism. The results are expected to reveal the physiological roles of Ngb, Cygb1, Cygb2, and GbX in zebrafish in particular, and help to better understand the functional diversity of these globins in vertebrates in general.