marcks蛋白家族在斑马鱼背腹轴形成中的作用及机制研究

MARCKS is a widely distributed calmodulin- and actin- binding protein substrate of protein kinase C (PKC) in all organisms and has been used as a marker of PKC activation in vivo. MARCKS has been implicated in cellular migration and adhesion, in endo-, exo-, and phagocytosis, and in neurosecretion and mucin secretion by cross-linking with actin. MARCKS has emerged as a potential therapeutic target for the amelioration of mucin secretion and inflammation in patients with chronic obstructive pulmonary disease. In the present study, we used a phage display technique to screen differentially expressed proteins from zebrafish post-gastrula embryos. We found that the high expression of marcksb in the post-gastrula embryos. The marcksb is one of the four members of Marcks protein family identied by us.The whole mount in situ hybridization studies demonstrated that the zebrafish marcks genes family displayed distinct patial and temporal expression patterns during early development. The misexpression studies suggested that members of marcks gene family participated in dorsal and ventral patterning in zebrafish. Zebrafish is important model organism and the formation of axis is the critical events during the early development of vertebrate. Study entirely on the function of zebrafish marcks protein family by modern biological technology and discover the mechanism of embryo developments, will play a role to understand the process of embryo developments and complete the function of MARCKS.

豆蔻酰化的富含丙氨酸的蛋白激酶底物，即MARCKS蛋白，是蛋白激酶（PKC）的重要底物。MARCKS蛋白家族在组织中广泛表达，通过与肌动蛋白的相互作用，功能涉及到细胞黏附和移动，神经递质传导和粘液的分泌。MARCKS蛋白与呼吸道的高分泌性和炎症的关系使其成为治疗慢性阻塞性肺部疾病的候选因子。申请人在淘选斑马鱼早期胚胎发育相关基因的过程中，克隆鉴定了斑马鱼的marcksb基因。通过研究，斑马鱼的marcksb基因是我们鉴定的Marcks蛋白家族的四个成员之一，各成员在胚胎发育过程中具有时空表达的特异性。过表达和表达敲低研究提示与细胞命运决定和胚胎背腹轴的建立密切相关。而体轴的形成是脊椎动物早期发育的关键事件，利用现代生物学技术对于斑马鱼Marcks蛋白家族进行全面深入的研究，阐明其影响胚体发育的相关机制，将有助于了解鱼类胚胎早期发育过程，并完善对MARCKS蛋白功能的解读。

豆蔻酰化的富含丙氨酸的蛋白激酶MARCKS蛋白，是蛋白激酶（PKC）的重要底物。申请人在利用噬菌体展示技术淘选斑马鱼早期胚胎发育相关基因的过程中，克隆鉴定了在原肠后期高表达的斑马鱼的marcksb基因。通过本项目的研究，克隆并鉴定了斑马鱼Marcks蛋白家族的四个成员。斑马鱼Marcks蛋白家族成员均有促进斑马鱼胚胎腹侧细胞命运形成的功能。其中marcksa的腹部化效应最强，marcksl1的腹部化效应最弱。marcksb 的功能是斑马鱼胚胎腹侧命运的发育所必需的。斑马鱼Marcks蛋白家族其他成员与marcksb在早期胚胎发育过程中的功能存在冗余，因此，基因敲除后表型不明显。marcksb影响胚胎背腹轴发育可能与chd和bmp2b有关。marcksb基因的表达受细胞因子IL8的调节。在研究的过程中，发现斑马鱼marcksb影响中性粒细胞和巨噬细胞的发育和运动和斑马鱼眼部的发育。将marcksb 敲低胚胎与野生型胚胎进行去核糖体法转录组测序：mRNA差异表达的基因222个，上调表达的151种，下调表达71种。差异表达基因最为明显的基因主要涉及到肌肉收缩；中性粒细胞和免疫系统功能调节；造血；心脏和血管的发育；脊索的发育；晶状体的发育；瘦素、甲状腺激素、甲状旁腺激素等内分泌激素的合成；细胞凋亡；离子的结合。差异基因的KEGG分析结果显示markcsb的靶基因主要富集的生物通路有：p53 信号通路、细胞周期和核糖体生物合成等。差异基因的GO基因功能富集分析显示marcksb的靶基因富集于核仁、90s前核糖体和细胞外区域；marksb的靶基因功能主要富集于G蛋白受体结合、激素受体结合、RNA结合和糖胺多糖结合区域。获得一系列marcksb基因作用靶点：背腹轴发育（tnni 1a；tcap；ankrd1a；cox66）；血管生成（cdkn1a；rspo1）；激素活性（leptin；pthla；slc5a5；sultlst4）；中性粒细胞功能（ms4a17.10；egln3）；晶状体的发育（crygm2d16；crygm2d28；crygmxl1；griffin等）；脊索的发育（emilin3a）。综上所述，marcksb基因对背腹轴发育的影响广泛涉及多种组织发育和多类细胞内生物过程。