糖原贮积症Ⅴ型斑马鱼疾病模型的构建及其表型分析  

Construction and phenotypic analysis of zebrafish disease model of glycogen storage disease typeⅤ

作　　者：黄姣 霍锦倩 刘姣 祖尧 张庆华[1,2,3] 李伟明 任建峰 HUANG Jiao;HUO Jinqian;LIU Jiao;ZU Yao;ZHANG Qinghua;LI Weiming;REN Jianfeng(International Research Center for Marine Biosciences,Ministry of Science and Technology,Shanghai Ocean University,Shanghai 201306,China;Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources,Ministry of Education,Shanghai Ocean University,Shanghai 201306,China;National Demonstration Center for Experimental Fisheries Science Education,Shanghai Ocean University,Shanghai 201306,China;Department of Fisheries and Wildlife,Michigan State University,East Lansing 48824,USA)

机构地区：[1]上海海洋大学科技部海洋生物科学国际联合研究中心,上海201306 [2]上海海洋大学水产种质资源发掘与利用教育部重点实验室,上海201306 [3]上海海洋大学水产科学国家级实验教学示范中心,上海201306 [4]密歇根州立大学渔业与野生动物系,密歇根东兰辛48824

出　　处：《上海海洋大学学报》2021年第1期11-20,共10页Journal of Shanghai Ocean University

基　　金：上海市科学技术委员会国际科技合作项目(15410723300)。

摘　　要：建立肌型糖原磷酸化酶基因pygma和pygmb突变体斑马鱼疾病模型,对人类PYGM基因突变导致的糖原贮积症Ⅴ型(glycogen storage disease typeⅤ,GSDⅤ)的研究具有重要意义。利用CRISPR/Cas9技术成功敲除了斑马鱼pygma和pygmb基因,分别获得pygma^((-11bp)-/-)和pygma^((+14bp)-/-)两种纯合突变体,以及pygmb^((+1bp)-/-)和pygmb^((-2bp)-/-)两种纯合突变体。过碘酸希夫糖原染色(periodic acid Schiff stain)实验显示pygma^(-/-)和pygmb^(-/-)纯合突变体心脏和肌肉组织出现明显的糖原累积。对野生型斑马鱼15个早期发育时间点以及12个成鱼组织中pygma和pygmb基因表达分析显示,pygma和pygmb早期发育阶段表达模式相似,从24 hpf到125 hpf,两基因表达量总体呈上升趋势,且pygmb的上升幅度大于pygma的上升幅度。在检测的12个组织中,pygma和pygmb只在少数几个组织中有明显表达。pygma在肌肉组织中表达最高,其次为心脏和皮肤组织;pygmb在心脏、脑、眼睛3个组织中高表达,在肌肉组织中也有表达,但表达量相对较低。斑马鱼pygma^(-/-)和pygmb^(-/-)突变体具有GSDⅤ病肌肉糖原累积的典型特征,该疾病模型的建立为进一步研究糖原贮积症的发病进程、详细机制和药物筛选等治疗策略提供基础数据。It is of great significance to study the human glycogen storage disease type V(GSDⅤ)caused by PYGM mutation through constructing zebrafish mutants of muscle glycogen phosphorylase gene pygma and pygmb.Two homozygous mutants were successfully obtained using CRISPR/Cas9 genome editing technique.The periodic acid Schiff staining experiment showed that there was obvious glycogen storage in the muscle and heart of pygma^(-/-)and pygmb^(-/-)compared with those of wild type(WT).The expression analysis of pygma and pygmb genes in WT at 15 early developmental stages and 12 adult tissues demonstrated that:the expression pattern of pygma and pygmb was similar at early stages,the expression of pygma and pygmb showed an overall upward trend and the increase of pygmb was greater than that of pygma from 24 hpf to 125 hpf.In the 12 tissues tested,pygma and pygmb were expressed only in a few tissues.pygma was mostly expressed in muscle tissue,followed by heart and skin;pygmb was highly expressed in heart,brain and eyes,and also expressed in muscle but at relatively lower level.Zebrafish pygma^(^(-/-))and pygmb^(-/-)mutants have the typical characters of GSDV with glycogen accumulation in muscle.The establishment of the zebrafish disease model provides a basis for further research on the pathogenesis,mechanism and drug screening for treatment of glycogen storage disease.

关 键 词：斑马鱼 肌型糖元磷酸化酶 糖原贮积症V型 基因敲除 疾病模型 

分 类 号：S917[农业科学—水产科学]