Functional analyses of vertebrate tissue-globins in zebrafish

(血球蛋白)是生物科学中研究最多的蛋白质之一，但仍带来惊喜。我们的研究导致在颌骨脊椎动物中发现了六种新的珠蛋白，大多数功能仍不明确。本研究旨在研究细胞珠蛋白(Cygb1和Cygb2)、脑红蛋白(NGB)和珠蛋白X(GBX)在斑马鱼体内的功能。我们将使用CRISPR/Cas9方法来产生珠蛋白基因的敲除。该方法已在实验室中通过产生Cygb1基因敲除斑马鱼品系而建立。珠蛋白基因敲除将测试发育缺陷、氧气消耗的变化、低氧耐受性和反应、对活性氧物种(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)将有助于确定球蛋白参与的相应代谢途径。根据我们的假设，NGB可能参与O2的供应或ROS的分解，而GBX可能对细胞膜起保护作用。在本应用程序中，我们将专门关注Cygb1和Cygb2。与其他脊椎动物不同，硬骨鱼有两个截然不同的Cygb基因。我们假设它们是通过基因复制后的亚功能化而分化的。复制的基因可能在胶原合成的氧气供应、保护ROS或在NO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.